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THE ANATOMICAL RECORD 245:361-373 (1996)

Implications of Cellular and Molecular Biology Advances in


Periodontal Regeneration
SALOMON AMAR
Center for Advanced Biomedical Research, Department of Oral Biology and
Periodontology, Boston University, Boston, Massachusetts
ABSTRACT Backgmund: The mechanisms by which new periodontium
is established on root surfaces previously exposed to periodontal disease
has been an area of active research interest for the past decade.
Methods: Recently, histological examination of periodontal regeneration
has revealed a complex process orchestrated by temporo-spatial specific
cell-matrix interactions.
Results: Advances in cell and molecular biology techniques have pro-
vided invaluable tools to begin investigating the cascade of events occur-
ring during periodontal regeneration.
Conclusions: This report summarizes current understanding of the cellu-
lar and molecular aspects of periodontal regeneration and determines the
clinical relevance of these findings. o 1996 Wiley-Liss, Inc.
Key words: Periodontal wound healing, Extracellular matrix macromole-
cules, Stem cell, tissue regeneration

Perhaps the most important attribute of living or- cedes physiological competence. In regeneration, which
ganisms is their capacity for self-repair. This implies is an example of postembryonic development, the dis-
an ability continously to monitor normal tissue func- tinction between morphology and physiology is not so
tion and to respond appropriately to any deviations well defined. To be sure, nothing can function until its
from the normal state. In its most ubiquitous form, material substrate is in place, yet nature has contrived
self-repair is accomplished through the physiological that no structure shall not regenerate unless it will
adaptations of the body in an effort to maintain a become functional. Not only is regeneration a morpho-
steady state. These adaptations, which occur at virtu- logical outgrowth from the adult, it is also a functional
ally all levels of organization, are achieved by classic restoration. Thus in the very initiation of regeneration,
negative feedback loops. Imbalance at the molecular there has evolved a dependence on physiological inf lu-
level is corrected by appropriate equilibrium shifts in ences that integrates the activities of the future regen-
chemical reactions. At the cell and tissue levels, ho- erate with those of the rest of the body. This utilitarian
meostasis is maintained through a careful balance of imperative ensures that useless structures are gener-
biosynthetic and degradative processes. At higher lev- ated a t the expense of more important processes. This
els of organization, some organism are capable of re- report addresses current concepts of the cellular and
generating entire tissue and organ structures includ- molecular aspects of periodontal regeneration and dis-
ing appendages. In mammals, any injury to body parts cusses their potential clinical application.
will normally heal by repair or regeneration that is
dependent on the nature of the wounded tissue. PERIODONTAL CONSIDERATIONS TO THE
Whereas some tissues such as epithelia retain substan- REGENERATING PROCESS
tial regenerative potential throughout life, others such
as nervous or connective tissue may preferentially heal In periodontal diseases, inflammation of the support-
by a repair process, especially under the influence of ing structures of the tooth, or periodontitis, causes dis-
chronic infection. tinct changes in the periodontium, which subsequently
Although repair and regeneration are usually impact on its regenerative potential. Collagen tissues
thought of in morphological terms, there is an impor- are lost from the root surface, which becomes exposed
tant functional consideration. Thus the repaired tis- to inflammatory processes, soft and hard bacterial de-
sues must carry out the same physiological activities posits, and the oral environment. The alveolar bone
performed by the original structures. Otherwise, re- and periodontal ligament are reduced in height and
growth would be useless and an expensive drain on the
resources of the organisms. Clearly, there is an essen-
tial functional dimension to repair and regeneration, a
Address reprint requests to Salomon Amar, D.D.S., Boston Univer-
dimension inseparable from the morphological one. It sity, Center for Advanced Biomedical Research, Department of Oral
might be asked which comes first, structure or func- Biology and Periodontology, W201-C, 700 Albany Street, Boston, MA
tion? In embryogenesis, structural development pre- 02118.
0 1996 WILEY-LISS, INC.
362 S. AMAR

Initial Clot Formation ation, progenitor cell manipulation, cell exclusion (gin-
gival and epithelial), wound stabilization, and growth

1
Necrosis/ResorptiveActivity
factor enrichment.
Attempts to manipulate these biologic parameters
have led to minimal success partly because of the lack
of knowledge of basic mechanisms of molecular peri-
Granulation Tissue odontal wound healing, along with complications of the
wound healing inherent to the periodontal environ-
ment. Furthermore, reparative and regenerative pro-
cesses in the periodontium occur in anatomical sites
and under conditions that are considerably more com-
R ulation of Root Syrface plicated than for incisional wounds. Complications in-
&POPfenotypic Expression herent to periodontal wound healing include: (1) oral
microbial plaque: healing of the wound occurs in pres-
ence of a microflora deleterious to the process, (2) phys-
ical environment: variations in pH and/or temperature
often occurring in the oral environment can impair the
REPA1R REGENERATION wound healing process, (3)root surface: the contribu-
Fig. 1. Temporal pattern of healing following periodontal insult.
tion of this avascular surface to the wound healing pro-
cess is minimal, (4) multiple interacting cells: epithe-
lial and various connective tissue cells must work in
concert to reestablish the periodontal attachment
volume and are replaced by inflamed connective tissue apparatus, (5) cell-matrix interactions: the extensive
and the downgrowth of the gingival epithelium. matrix destruction occurring in periodontitis affects
It is important to consider the treatment of the in- tremendously the wound healing process, and (6) func-
flammatory response in the periodontium and the tional considerations: deleterious occlusal forces can af-
treatment of the anatomical defects produced by peri- fect wound stabilization during the healing process.
odontitis as two distinct entities. Periodontitis is be- Given all the aformentioned considerations, it is not
lieved to be a bacterial plaque-induced inflammatory surprising that various flap procedures and root sur-
process in which the supporting alveolar bone, peri- face conditioning protocols have provided only limited
odontal ligament, and the surrounding connective tis- evidence of periodontal regeneration (Listgarten and
sue are destroyed. Treatment of periodontitis involves Rosenberg, 1979; Caton et al., 1980; Stahl et al., 1982;
mechanical and chemotherapeutic procedures aimed at Froum et al., 1983; Moore et al., 1987). However, aside
eliminating pathologic microorganisms that invoke the from certain osseous grafting techniques (Hiatt et al.,
inflammatory response. This treatment, however, en- 1978; Bowers et al., 1991; Mellonig, 1991), studies on
genders a defect characterized clinically by bone and guided tissue regeneration outcomes (Fig. 3) have
periodontal ligament loss, gingival pockets, and reces- proven the most convincing histologic evidence of sig-
sion of the gingival margin. Periodontal surgery is di- nificant periodontal regeneration. Nyman and co-
rected primarily at resolving these defects, either by workers (Nyman et al., 1982a,b)were the first to report
repair or preferably through regeneration (Fig. 1). the formation of a new attachment by means of using
Periodontal repair represents the healing process af- Millipore filters to prevent gingival epithelial and con-
ter periodontal surgery that does not lead to the re- nective tissue cells to invade and perturb the periodon-
placement of the lost periodontal tissues, whereas peri- tal wound area. Subsequently, several studies (Gottlow
odontal regeneration represents a healing process after et al., 1984, 1986; Aukhil et al., 1986; Caffesse et al.,
periodontal surgery which results in the formation of 1990; Caton et al., 1992) have reported histological ev-
new cementum, new bone, and new periodontal liga- idence of the regenerative capacity of the periodontal
ment on a tooth root surface that has been previously ligament cells. Whereas the cellular and molecular cas-
exposed to the periodontal disease (Fig. 2) (Kalkwarf, cade of events associated with periodontal regeneration
1974). is not completely understood, recent data hold great
Conventional therapy for periodontal defects consists promise for the modulation and/or control of the peri-
in scaling and root planing of the diseased root surface, odontal regeneration process to achieve a predictable
gingival curettage, and various other surgical proce- outcome.
dures, with and without implants of bone and/or bone
substitutes. The rationale for many of these therapeu-
tic procedures is regeneration of the periodontium. CELLULAR ASPECTS
However, histological data supported by several labo- The process of periodontal regeneration involves a
ratories (Yukna, 1976; Listgarten and Rosenberg, number of cell phenotypes, including cementoblasts,
1979; Moskow et al., 1979; Caton and Nyman, 1981; endothelial cells, epithelial cells (sulcular and junc-
Steiner et al., 1981) indicates that repair rather than tional), fibroblasts, and osteoblasts, as well as nerve
regeneration results from conventional periodontal cells. True periodontal regeneration entails the re-es-
therapy. This knowledge has redefined the rationale tablishment of new cementum with inserting collagen
for conventional periodontal therapy and has focused fibers and supporting alveolar bone (Nyman et al.,
attention on the more basic aspects of wound healing in 1982a,b). However, the heterogeneity of the cell popu-
the hope of identifying important variables for peri- lation of the periodontal ligament, together with the
odontal regeneration, which include: root surface alter- lack of definitive cell differentiation markers, has
ADVANCES IN PERIODONTAL REGENERATION 363

Periodontal pocket with granulation tissue


and bony defect

Regeneration I Forms of New Attachment Formation (Repair)

New cementum, Long Junctional Connective & Connective & Bony ankylosis
bone & PDL fibers Epithelium partly attached non-attached Attachment
Bone-like tissue Bone-like tissue
Attachment Attachment

Fig. 2. Schematic representation of histological outcomes of periodontal therapy.

These cells may have a local or systemic origin; thus,


local progenitor cells present in the periodontal mem-
brane or endosteal spaces could be activated, whereas
systemic factors released during the wound healing
process could chemotactically attract cell progenitors
from the pre-existing or invading vascular tissues to
migrate into the periodontium and differentiate respec-
tively into cementoblasts, osteoblasts and periodontal
fibroblasts.
Periodontal Progenitor/Stem Cell
The periodontium possesses the capacity for self-re-
Fig. 3. Diagrammatic representation of a diseased periodontal site newal (McCulloch et al., 1989)as evidenced by its rapid
treated by guided tissue regeneration. Following the placement the turnover. McCulloch and Melcher (1983) have demon-
elevation of a mucoperiosteal flap and thorough debridement of the strated that a significant number of periodontal cells
root surface, a porous membrane is placed over the infrabony defect
for the purpose of exclusion of gingival epithelium and fibroblasts do not enter into the cell cycle, suggesting that some of
from the wound area (from Page, 1993, J. Periodontol., p. 749). these cells may be a population of stem cells. Hemato-
logical studies (Sprangrude et al., 1988; Baum et al.,
1992; Peault et al., 1993) involving mouse bone mar-
made the task of identifying cells that participate in row cells have suggested CD34+ and Thy-l positive
the regenerating process a very difficult and compli- cells with low Rhodaminel23 as candidate stem cells.
cated one. The current belief is that cells that contrib- Furthermore, Uchida et al. (1993) demonstrated an
ute to periodontal regeneration are derived from either heterogeneity in the small population of stem cells that
a small population of multipotential stem cells or from comprised subsets of quiescent cells, capable of self-
a number of populations of committed progenitor cells. renewal and multipotancy. A similar population of
364 S. AMAR

Matrix s t i m u l a t e 7
attract Stem Cells

ROOT
SURFACE
ROOT
SURFAC
Migrating Periodontal Stem Cell undergoing Mitosis 8
Differentiation into Cementoblasf Fibroblast 8 Osteoblast
Comrnited Cernentoblasi
Fig. 4. Possible response of periodontal stem cells during periodon- Progenitor Cell 4
tal wound healing. Commited Osteoblast
Progenitor Cell

Fig. 5.Possible response of progenitor cells.


stem cells could be envisioned in the periodontium.
Amar and Chung (1994) have discussed the issues in-
volving the characterization of the purported periodon-
tal stem cell and the possible recapitulation of some of studies (Melcher, 1970; Gould et al., 1980) have shown
the developmental morphogenesis events in the peri- that cells adjacent to site of injury are stimulated to
odontium during the process of periodontal regenera- divide and migrate toward the wound. McCulloch
tion. Studies involving tooth morphogenesis in rodents (1985) suggested that rat molar periodontal ligament
(Ten Cate et al., 1971; Osborn and Price, 1988) demon- cells adjacent to the tooth may be the source of cemen-
strated that the dental papilla and dental follicle cells toblasts, whereas periodontal ligament cells adjacent
can give rise to cementoblasts, osteoblasts, and peri- to bone may provide the source for osteoblasts. These
odontal fibroblasts. Furthermore, Cho and Garant periodontal ligament cells may belong to different qui-
(1989) demonstrated that mesenchymal cells from the escent progenitor cell populations. The location of the
dental follicle can differentiate into cementoblasts and progenitor cell populations could determine the subse-
form cementum, then subsequently detach from the ce- quent production of cementoblasts, periodontal fibro-
mental surface and contribute toward early periodon- blasts and osteoblasts (Fig. 5). It is probable that these
tal fibroblast populations. It was suggested that these progenitor cells are stimulated following injury to pro-
cells, most of which seem to remain quiescent in Go duce daughter cells that will migrate and differentiate.
phase (Gottlow et al., 1986) may retain their multitipo-
tency throughout life and form a population of stem Clinical Application
cells resident in the periodontal ligament. Various Although few studies have been carried out to date,
stimuli produced in the event of injury may be respon- the use of suitable cells seeded into periodontal wounds
sible for triggering these cells to initiate cell division, seems to be a powerful strategy to promote regenera-
producing daughter cells, some of which may proceed tion of the periodontium. Hematopoietic stem cells
along any of the predetermined paths of differentiation have been utilized for ex vivo transplantation to help
and maturation while migrating toward the wound site reconstitute the bone marrow in irradiated long bones
(Fig. 4). The same progenitor resident cell is believed to (Berenson et al., 1988). In a similar manner, the pro-
periodically divide and differentiate to assure peri- genitorlstem cell population could be harvested from
odontal homeostasis and contribute toward the peri- the periodontium and expanded in vitro for subsequent
odontal turnover and remodeling. use in ex vivo transplantation in affected periodontal
Progenitor cells also may be derived from bone mar- sites. This approach could augment local cells capable
row stroma that has been shown to contain precursor of differentiating through various lineages required to
cell population (Friedenstein et al., 1966, 1982). In the restore fully, thereby contributing toward the healing
periodontium, endosteal spaces and larger marrow process. A recent study (Van Dijk et al., 1991) utilizing
spaces in the alveolar bone have been suggested as the this concept in the beagle dog demonstrated the occur-
local source of progenitor cells involved in the regen- rence of periodontal regeneration and has paved the
eration of the periodontium (McCulloh, 1985). A num- way for further investigation involving cell seeding in
ber of studies have provided some evidence of the ex- periodontal wounds. Based on this rationale, fibro-
istence of committed progenitor cells involved in blast-coated hydroxyapatite particles (HA) particles
periodontal regeneration. Periodontal wound healing were implanted in human periodontal defects, whereas
ADVANCES IN PERIODONTAL REGENERATION 365
plain HA implanted in similar periodontal defects TABLE 1. Components of the interstitial extracellular
served as controls. Substantial periodontal regenera- matrix at different phases of wound r e D a i r
tion was observed in experimental sites, whereas con-
trol sites harbored only minimal periodontal regener- Time after injury Matrix components Sources
ation (Feng and Hou, 1992).This new avenue of ex vivo 1-3 days Fibrinogen Plasma
cell transplantation holds great promise in promoting (clot) Fibronectin Platelets
human periodontal regeneration on a predictable basis. Vitronectin
Thrombospondin
MOLECULAR BIOLOGY RESEARCH 4-6 days Fibronectin Fibroblasts
Molecular research in periodontal regeneration has (early granulation Hyaluronan Macrophages
recently brought new insights into the complex process tissue) SPARC
Tenascin
of periodontal wound regeneration. Central to the or-
chestration of the wound healing process are matrix 7-10 days Collagen Type I Fibroblasts
macromolecules that influence cellular behavior and (late granulation Collagen Type 111
tissue) Fibronectin
cytokines with autocrine and/or paracrine properties. Proteoglycans
The complexity of cell-cell and cell-matrix interactions SPARC
occurring in the periodontal wound healing compart- Tenascin
ment is attributed in part to the pleiotropic effects of
growth factors and cytokines on various cells and tis-
sues. The use of immunoprobes, and recombinant DNA 1988). Additional binding domains for gelatin and
technologies has provided a better understanding of heparan sulphate, as well as fibrin have been identi-
the molecular events associated with wound healing. fied. Thus, FN may serve as a biological “glue” between
ligands (Stanford and Keller, 1991). TSP and SPARC
Extracellular Matrix (osteonectin) (Raugi et al., 1987; Sage and Bornstein,
The extracellular matrix (ECM) differs in structure 1991) are only transiently expressed by rapidly grow-
and composition depending on the nature of the tissue ing and remodelling tissue that are undergoing either
as well as its status. ECM serves a number of impor- embryonic morphogenesis, healing, repair, or osteo-
tant functions during periodontal wound healing. genic differentiation. TSP, which has been shown to
Following injury, a cascade of events is initiated bind calcium ions, matrix molecules including FN,
beginning with the formation of a fibrin clot that in- laminin, types I 8z V collagen, plasminogen, fibrinogen
corporates plasma proteins and bioactive molecules (Majack and Bornstein, 1987) osteonectin (Robey,
mainly released by platelet cells (Table 1).The fibrin 1989), and hydroxyapatite (McCulloch, 1985) inhibits
clot serves as a stable framework, linked to collagen angiogenesis (Good et al., 1990) and possesses the abil-
fibers on the root surface (Polson and Proye, 1983), ity to modulate fibrinolysis (Mosher et al., 1992). Its
which contributes toward the formation of the ECM by multifunctional capacity is due in part to an ability to
cells (Melcher, 1970; Gould et al., 1977; Ighaut et al., interact with different cell-surface receptors and to its
1988) that migrate onto the clot and take part in the existence in four homologous forms (TSP1, 2, 3, 4)
organization of the healing granulation tissue. Fibro- (Bornstein, 1992). It has been postulated that TSP has
blast and macrophages are the principal cellular source roles in cell adhesion, growth, proliferation, and migra-
of matrix components constitutive of the healing gran- tion (Mosher, 1990).
ulation tissue (Table 1).Aside from the role of ECM as Bone sialoprotein (BSP) is a highly sulphated and
a structural scaffold, isolation and characterization of glycosylated phosphoprotein found in bone matrix,
the components associated with wound healing have mineralized connective tissue as well as dentin and
revealed a multifunctional role for the ECM that in- cementum (Oldberg et al., 1988; Fisher et al., 1990;
cludes regulation of cellular function and activity. Chen et al., 1991; Sommerman et al., 1991). Although
Biochemically, the ECM components that appear to be its exact function is unknown, some studies (Sommer-
actively involved in healing events include glycopro- man et al., 1987; Gorski, 1992) have demonstrated that
teins-fibronectin (Grinell et al., 1981), thrombospon- it can mediate cell attachment and bind selectively to
din (McCullochand Melcher, 1983)tenascin (Mackie et hydroxyapatite. BSP expression during early dentin
al., 1988), SPARC (Murphy-Ullrich et al., 1991), bone and bone formation When et al., 1993) suggested a role
sialoprotein (Chen et al., 1992), osteopontin, proteogly- in initial bone matrix formation and mineralization.
cans (decorin, biglycans) (Young et al., 1992), and the Studies (Bianco et al., 1991; Young et al., 1992) have
collagenous proteins. show that BSP is primarily derived from osteoblasts at
late stages of differentiation, suggesting it to be a
Glycoproteins “late” marker of bone differentiation. Tenascin (Mac-
Matrix glycoproteins have been shown to play mul- kie et al., 1988) another extracellular matrix glycopro-
tifunctional roles. (Fig. 6) Both fibronectin (FN) (Seppa tein, demonstrates strong transient expression in heal-
et al., 1981; Terranova, 1987) and thrombospondin ing wounds, suggesting a role in the development of
(TSP) (Frazier, 1987; Bornstein, 1992) are present in granulation tissue. The initial fibrin clot formation
the early fibrin clot and seem to serve as adhesion mol- may serve as the trigger to a complex cascade of inter-
ecules with chemotactic and mitogenic properties for acting healing events regulated through autocrine
fibroblasts and other cells. Thus, FN is a large dimeric andior paracrine factors secreted by residents and/or
protein linked by disulphide bonds near the C-termi- recruited cells.
nus and within which is located a 75 kDa cell binding The expression of specific matrix glycoproteins, os-
domain that contains the RGD sequence (Ruoslahti, teonectin, bone sialoprotein (Amar et al., 19951, in
366 S.AMAR

PhySlCOChemlCal
Matrix Lysis
structurestrength)
COLLAGEN THROMBOSPONDIN
FIBRIN

2 /
EXTRACELLULAR
/MATRIX FUNCTIONS

Chemotactic for Cells


\ Mitogenic for Cells
FIERONECTIN FIBRONECTIN
THROMBOSPONDIN THROMBOSPONDIN
COLLAGEN

Fig. 6. Interactions between extracellular m a t r i x and cells during e a r l y periodontal wound healing.

healing periodontal tissues, which grew beneath the the bone matrix and are characterized by their ability
expanded polytetrafluoroethlylene (ePTFE) mem- to induce cartilage and bone formation in vivo. To date
brane, further suggests that under these conditions the seven molecules, which have been named BMP-1
healing tissue expresses specific macromolecules that through BMP-7, have been identified and their corre-
may be important in periodontal regeneration. These sponding molecular clones obtained using fragmentary
multifunctional matrix glycoproteins found in the fi- amino acid sequence information from bone-inductive
brin clot have been shown not only to regulate cellular extracts derived from bovine bone (Celeste et al., 1990;
activity of migration, division, and differentiation but Ozkaynak et al., 1990; Wozney et al., 1990). These
also to influence the lysis, synthesis, and maintenance amino acid sequences allowed the design of multiple
of the ECM. Taken together, these results suggest that oligonucleotide probes, which were initially used to ob-
matrix glycoproteins may have a significant influence tain bovine genomic or complementary DNA (cDNA)
from the earliest phase of healing as adhesive mole- clones for each of the BMPs (Wozney et al., 1988).
cules for cell attachment and chemoattraction of cells These bovine clones were used to isolate human cDNA
into the wound area to the late phase of differentiation clones that contained the entire coding sequences for
and maturation of the wounded tissue. each of the individual BMPs. The human cDNAs for
BMP-1, BMP-2, BMP-4, BMP-5, and BMP-7 were each
Growth factors and cytokines derived from libraries constructed from the human os-
The ECM also serves as a reservoir of growth factors teoblastlike osteosarcoma cell line U-2 OS, the BMP-3
(FGF, Rifkin and Moscatelli, 1989;TGF-P, McCaffrey et cDNAs were derived from a cDNA library of the H128
al., 1989),cell-ceI1adhesion molecule (NCAM, Sanes et small cell lung carcinoma cell line, and clones for
al., 1986), and proteases (Silverstein et al., 1986) that BMP-6 were initially derived from U-2 0s cDNA li-
can be released during the temporo-spatial differenti- braries and subsequently from human placenta and
ation and maturation of the wound. Recent research brain cDNA libraries. Other reports have indicated the
have revealed an increasing number of bioactive mol- cloning of BMP-7, also called osteogenic protein-1 or
ecules including various growth factors-PDGF, (Mat- OP-1, from hippocampus and placenta libraries (Oz-
suda et al., 1992; Oates et al., 1993), TGF-P (Ignotz and kaynak et al., 1990).
Massague, 1986; Roberts et al., 1986; Overall et al., From the primary amino acid sequences of the hu-
19891,IGF-1 (Blom, 1992), BMP (Wozney, 1989; Wang, man BMPs derived from these molecular clones, six out
1993), FGF (Sanes et al., 1986)-and cytokines (IL-1, of the seven BMPs were found to be related to each
LIF) that have profound effects on various cell pheno- other and to be member of the TGF-p superfamily. An
type (Table 2). However, the regulatory mechanisms alignment of these amino acid sequences indicated that
that govern their functional interactions have yet to be significant amino acid identity exists among all the
fully elucidated. Transforming growth factor-p (TGF- BMPs in the carboxy-terminal region of the proteins.
P), which together with its homologues form a family of This region contains seven cysteine residues, whose
cytokines within the TGF-P superfamily of related mac- presence and relative positions are conserved among
romolecules exhibits numerous pleiotropic effects on all reported members of the TGF-P superfamily. Simi-
connective tissue cells. Tissue-specificeffects have been lar to the other members of the TGF-P family, the
demonstrated for TGF-P, particularly in regulating cell BMPs are synthesized within the cell in a precursor
activity and function in the process of bone metabolism form. Analysis of the primary amino acid sequences of
and wound healing, (Noda and Rodan, 1986; Centrella the six related BMPs allows them to be grouped into
et al., 1987;Kasperk et al., 1990; Wergedal et al., 1990). three separate sets. One set consists of BMP-2 and
Bone morphogenetic proteins (BMPs) are structur- BMP-4, which are 92% identical in the seven-cysteine
ally related to the TGFPs and form an other group of region; in fact, BMP-4 was originally isolated by cross-
cytokines within the TGF-f3 superfamily. BMP is a hybridization to a BMP-2 probe. BMP-5, BMP-6, and
term first used by Marshal Urist (1965) and now refers BMP-7 are also closely related to one another, possess-
to a family of growth factors that can be isolated from ing an average of 89% amino acid identity in their
ADVANCES IN PERIODONTAL REGENERATION 367
TABLE 2. Pleotropic cytokines
Cytokine Origin Function References
BMP-2 Osteoblast, bone, Induction of cartilage and bone; Wozney et al. (1990); Thies et al.
Deriodontal healinn
+
. differentiation of osteoblasts from (1992)
iissue progenitor cells
IGF Plasma, bone, Wound healing, increased mitogenesis, McCarthy et al. (1989); Lynch et
macrophage, osteoblast chemotaxis & synthesis of noncollagenous al. (1991a,b)
protein in fibroblasts; bone formation
FGF Bone, osteoblast, Angiogenic; mitogenic & chemotactic for Canalis et al. (1991); Basilico et
macrophage PDL fibroblasts; proliferation of al. (1992); Frenkel et al.
osteoblastic cells (1992)
PDGF Platelet; endothelial, Mitogenic toward fibroblastic and Terranova and Wikesio (1987);
mesenchymal cells, bone osteoblastic cells; stimulate bone matrix Centrella et al., 1989; Piche
& macrophage formation; stimulate secretion of and Graves (1989); Graves &
fibronectin & collagenase Cochran (1990); Zhang et al.
(1991)
TGF-P Bone matrix, platelet, Regulate osteoblastic and fibroblastic cell Ignotz & Massague (1986);
macrophage, osteoblast replication, differentiation & matrix Roberts et al., 1986;
synthesis; chemotactic & weekly mitogenic Postlethwaite et al. (1987);
for osteoblasts & fibroblasts; stimulate Canalis et al. (1988);
angiogenesis Kingsworth & Slavin, 1991
LIF Bone marrow stromal cells Stimulate growth & proliferation of Rodan et al., 1990; Verfaillie
multipotential progenitor cells; inhibit and McGlave (1991); Escary et
differentiation & maintain stem cells; al. (1993)
stimulate alkaline phosphatase in
presence of retinoic acid in osteoblastic
precursors
BSP Cells involved in Mediate initial stages of connective tissue Chen et al. (1992); Young et al.
formation of mineralized mineralization; may be marker of bone (1992)
matrix, bone, dentin, differentiation
cementum

corresponding regions, and define a second BMP sub- sue to form hard tissues. The recent demonstration of
set. BMP-3 is the sole member of the third subset iden- BSP, osteonectin and BMP-2 (Chung et al., 1994; Amar
tified to date. The BMP-2/BMP-4 and BMP-S/BMP-6/ et al., 1995) in human healing periodontal soft tissue
BMP-7 subsets are more closely related to one another grown beneath ePTFE membranes suggests that this
than they are to BMP-3. wound tissue contains cells and matrix macromole-
BMPs, like all the members of the TGF-P family, act cules required for hard tissue formation. Along the
through specific receptors. A single report described same line of ideas, we have examined the expression of
the presence of high-affinity binding sites for BMP-4 on bone macromolecules in primary cultures of cells which
MC3T3-El and NIH3T3 cells. By cross-linking exper- outgrew from human tissue explants retrieved after
iments, binding proteins of 200 and 70 kDa are present barrier-membrane (ePTFE) placement onto periodon-
on MC3T3-El cells, whereas proteins of 200 and 90 tal defects following the concept of guided tissue regen-
kDa are present an NIH3T3 cells (Paralkar et al., eration. Control cells were obtained from the out-
1991). It is perhaps surprising that there are receptors growth of gingival connective tissues harvested after
of different sizes for the same protein on two cell types flap replacement procedure. Cells (control and experi-
of the same species. No competition by TGF-P was mental) were plated on coverslips and fixed according
found for the BMP-4 binding proteins, nor was compe- to Amar et al. (1991). Cells were immunostained at
tition by BMP-4 for the TGF-p receptors observed con- different timepoints of culture against BMP-2 (gra-
sistent with most of the activity data on cells in vitro. ciously provided by Dr. Wozney, Genetics Institute),
Several BMPs, including BMP-2 and BMP-3 (osteo- osteonectin (OTN), and bone sialoprotein (BSP) (gra-
genin), have been shown to induce ectopic bone forma- ciously provided by Dr. Larry Fisher NIH-NIDR), and
tion in vivo (Wang et al., 1990; Ripamonti et al., 1993a) BMP-7 (graciously provided by Dr. Bruce Rutherford,
by initiating local differentiation of mesenchymal cells University of Michigan). Samples were incubated for 2
into bone-forming cells. However, most studies (Mar- hours a t room temperature. An affinity purified, per-
den et al., 1993; Ripamonti et al., 1993a,b) seem to oxidase-labeled goat antimouse (BMP-2) and antirab-
suggest that BMPs requires the combination with a bit (OTN, BSP) IgG antibody was used as the secondary
carrier matrix for effective bone induction. BMP-7 also antibody (Elite Vectastain ABC kit) following the in-
has been demonstrated to induce terminal differentia- structions of the manufacturer (Vector Laboratories,
tion of chondroblasts and osteoblasts within the con- Burlingame, CA). Peroxidase activity was developed
text of endochondral bone ossification (Sampath et al., using 0.05%3.3' diamino-benzidine (Sigma Chemical,
1992; Asahina et al., 1993; Knutsen et al., 1993). St. Louis, MO) in Tris-HC1 buffer (pH 7.6) containing
The presence of these specific macromolecules in the 0.001% HZOp.
ECM has been used as markers to determine the in- As shown in Figure 7, cells harvested from tissues
trinsic potential of the periodontal healing wound tis- growing under the membrane barrier (ePTFE) and cul-
368 S. AMAR

Fig. 7. Cells harvested from tissues growing under the membrane barrier and culturedfor 5 days were
immunostained against BMP-2 at a dilution of 1/50 in PBS (a),Bone Sialoprotein(BSP) at a dilution of
1/40 in PBS (c),and Osteonectin(OTN)at a dilution of 1/40 in PBS (d).High levels of BMP-2(a)and BSP
(c) are expressed in the cytoplasm,whereas moderate levels of OTN are observed. (b)Gingival fibroblast
cells immunostained against BMP-2 (similar dilution as above); note the lack of staining. Similar ob-
servations were made when gingival fibroblasts were stained against BSP (not shown). (inverted micro-
scope x35)

tured for 5 days expressed high levels of BMP-2, OTN, mechanisms orchestrating cell-matrix interactions in
and BSP, whereas gingival fibroblast cells expressed the periodontal wound environment are needed to shed
only low levels of OTN (data not shown). Gingival fi- more light into these complex events.
broblast cells did not express BMP-2 (Fig. 7) or BSP
Collagen
(data not shown). Cells harvested from periodontal soft
tissue grown under the membrane barrier (ePTFE) and Type I collagen is the major structural component of
left in culture for 21 days in DMEM medium enriched the periodontal ligament fibres and the matrix of bone
in 10%serum aggregated to form nodules corroborat- and cementum. In fact, 70%of the total protein in the
ing previous observations using periodontal fibroblasts periodontal membrane is collagenous in nature and
(Arceo et al., 1991).These cell cultures heavily stained 85% (Narayanan and Page, 1983) is type I collagen.
for BMP-2 and BMP-7. None of the gingival cell cul- Type I collagen (Grinnell and Bennett, 1978;Postleth-
tures developed any nodules nor expressed BMP-2 or waite et al., 1978; Nishikawa et al., 1987; Hyder et al.,
BMP-7 to the limit of our detection method (Fig. 8). 1992) is known to harbor chemotactic and mitogenic
These preliminary data strongly suggest that cells activity for fibroblasts in vitro as well as being toler-
growing from tissue explants retrieved after barrier- ated by host tissue following in vivo implantation in
membrane placement express in culture tissue-specific combination with growth factors. It has also been
phenotypes associated with skeletal tissue formation. shown to be a weak immunogen (Cooperman and
Further studies aimed at elucidating the various Machaeli, 1984; Johns et al., 1993). Local hemostatic
ADVANCES IN PERIODONTAL REGENERATION 369

Fig. 8. Cells harvested from periodontal soft tissue grown under the membrane barrier and left in
culture for 21 in DMEM medium enriched in 10%serum aggregatedto form nodules (a, c). These cell
cultures heavily stained for BMP-2 at a dilution of 1/50 in PBS (a,c) and BMP-7 at a dilution of 1/50 in
PBS (d).(b)Gingival cell cultures immunostained against BMP-2 (same dilution as above);note absence
of nodule and of any staining. Similar observations were made when gingival fibroblasts were stained
against BMP-7 (not shown). (inverted microscope x 35)

properties in humans of type I collagen have been re- regeneration. Several studies (Pitaru, 1987, 1989;
ported (Stein et al., 1985). Type I collagen represents YafTe, 1987) reported utilizing various forms of type I
the predominant matrix macromolecule of the late collagen as a membrane barrier for guided tissue re-
granulative tissue. generation procedures. Clinical human studies (Chung
et al., 1990; Anderson, 1991) have also demonstrated
Extracellular Matrix in Clinical Application positive results with type I collagen membranes in the
A number of characteristics of matrix macromole- treatment of periodontal defects using guided tissue
cules that may have beneficial effects in clinical appli- regeneration techniques. Mendieta and Williams
cation have been recognized from recent research. This (1994)highlighted in their review the potential of cross-
has led to several reports investigating the use of var- linked type I collagen together with polylactic acid as a
ious components of the ECM to serve as barriers in suitable absorbable membrane. Recently, Pitaru et al.
guided tissue regeneration or biological response mod- (1991) utilized bilayered collagen membrane barriers
ifiers to enhance and/or modulate the healing process enriched with fibronectin and heparan sulphate in bea-
in the treatment of periodontal disease. gle dogs to enhance chemoattraction of healing cells and
Collagen, a major structural component of the ECM, subsequent repopulation of denuded root surfaces while
have been fabricated into suitable absorbable mem- preventing epithelial downgrowth. The next generation
brane barriers that offer considerable advantage over of barrier-membranes used in guided tissue regenera-
the eFTFE membrane advocated in guided tissue re- tion will probably take advantage of the ability of col-
generation procedures. The favorable biological char- lagen to bind growth factors.
acteristics of collagen has led to investigations on its As mentioned earlier, in vitro studies with FN sug-
clinical application in procedures related to periodontal gest several beneficial properties of FN in the ECM
370 S. AMAR

that could be useful in enhancing wound healing. How- Amar, S., P. Petrungaro, A. Amar, and T.E. Van Dyke 1995 Immu-
ever, controversial results have been reported. Animal nolocalization of bone matrix macromolecules in regenerating
human periodontal tissues. Arch. Oral Biol., 40:653-661.
studies (Caffese et al., 1985; Nasjleti et al., 1986; Ryan Amar S., B. Sires, B. Sabsay, J. Clohisy, and A. Veis 1991 The isola-
et al., 1986) showed significantly more connective tis- tion and partial characterization, of a rat incisor dentin matrix
sue reattachment in the treatment of periodontitis polypeptide with in vitro chondrogenic activity. J. Biol. Chem.,
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Anderson, H.H. 1991 The effectiveness of a collagen membrane bar-
with FN. However, no advantage was observed in ap- rier in achieving new attachment in class I1 furcations. J. Perio.,
plication of exogenous fibronectin above plasma. More 62:718(orban abstract).
research is needed fully to evaluate the influence of Arceo, N., J.J. Sauk, J. Moehring, R.A. Foster, and M.J. Somerman
exogenous fibronectin application on the healing pro- 1991 Human periodontal cells initiate mineral-like nodules in
vitro. J Periodontol, 62(8):499-503.
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