THE EFFECT OF BONE MORPHOGENETIC PROTEIN-7 ON THE REGENERATION OF RAT

PERIODONTIUM

D haarmini Raj shankar

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Cellular and Molecular Pathology University of Toronto

O Copyright by Dhaarmini Rajshankar ( 1 997)

National Library 1*1 of Canada Bibliothèque nationale du Canada

Acquisitions and Acquisitions et Bibliographic Services services bibliographiques

395 Wellington Street 395, nie Weltington ottawaON KiAON4 Ottawa ON K1A ON4 Canada Canada

The author has granted a non- exclusive licence allowing the National Library of Canada to reproduce, loan, distribute or seU copies of th s thesis in microfom, paper or electronic formats.

The author retains ownership of the copyright in this thesis. Neither the thesis nor substantial extracts &om it may be printed or othewise reproduced without the author's permission.

L'auteur a accordé une licence non exclusive permettant a la Bibliothèque nationale du Canada de reproduire, prêter, distribuer ou vendre des copies de cette thèse sous la forme de microfiche/film, de reproduction sur papier ou sur format électronique.

L'auteur conserve la propriété du droit d'auteur qui protège cette thèse. Ni la thèse ni des extraits substantiels de celle-ci ne doivent être imprimés ou autrement reproduits sans son autorisation.

Abstract

Periodontal diseases are among the most prevalent diseases of humankind, affecting

the integrity and composition of soft and mineraiized connective tissues that surround the

tooth. Bone morphogenetic proteins (BMPs) are known for their ability to induce new bone

formation locally at the site of application. Consequently, we have used BMP-7. incorporated

into a collagen vehicle, to assess its effects on healing of wounded rat periodontium in a rat

periodontal window wound modei at vanous time points (days 2, 5, 10. 2 1 & 60). Controls

included wounds filled with collagen vehicle alone or unfilled wounds. 'H-thymidine was

given by injection to al1 animais one hour before sacrifice to permit autoradiographic

assessrnent of cycling cells. Two stage-specific bone markers, osteopontin (OPN) and bone

sialoprotein (BSP) were used to evaluate early and late osteogenic differentiation stages.

respectively. BMP-7 did not affect the overall % of proliferating cells, but did induce clonal

growth of presumptive osteogenic cells as early as day 10. Increased bone formation was

evident at day 21 in BMP-7 treated samples and there was a 3-fold increase (pc0.00 1) in the

width of bone on the buccal side. There was an about 30% increase (pc0.00 1) in the

expression of OPN and BSP on the outer surface of nascent bone whereas in controls bone

formation ceased at day 21. PL width was unchanged in wounded and intact areas, regardless

of the treatment . The initial BMP-induced increase in proliferative activity in regenerating

bone-related areas retumed to basal levels at day 60. BMP appeared to act locally as the

tissues far away corn the drill site exhibited no increase in proliferation or increased OPN or

BSP staining. 1 conclude that BMP-7 promotes the proliferation and differentiation of

putative osteoçenic progenitor cells, and induces bone formation, but does not dismpt PL

homeostasis.

1 dedicate this book to my loving husband Rajshankar, for helping me achieve my goal by being more than patient, supportive and understanding, and to my wonderfûl family, ammah. appah, Kalyani and Thillainadesan, for keeping me motivated whenever 1 felt like giving up, through their endless wealth of love and affection. Without your guidance. encouragement. unconditional support and unfailing faith in me, I could have never accomplished this task. 1 am everything 1 am, because you love me!

Fust of d l , 1 wish to express my sincere gratitude to my supervisor, Dr. Howard C.

Tenenbaum, for his excellent guidance, support and constructive criticisms throughout my

graduate studies. Secondly, I am deeply indebted to my CO-supervisor, Dr. Predrag C. Lekic,

for his enthusiasm, encouragement and continued support, even from Winnipeg. 1 am indeed

very lucky to be supervised by such a dynamic pair of scientists.

1 would also like to thank Drs. Christopher A.G. hlcCuIloch and Jaro Sodek for their

intellectuai guidance and valuable suggestions. 1 really appreciate them finding the time in

their busy schedule to assist me. 1 am aiso very grateful to Dr. Kuber T. Sampath for

generously donating BMP-7 impregnated in coliagen and the collagen ody vehicle. The

critical appraisals and constructive suggestions of Drs. Rita Kandel and Marc Grynpas, during

the course of this project are highly appreciated.

1 am forever grateful to Mr. Balram Sukhu, for lending me a helping hand whenever

1 needed it the most and for being a "wise" fiend. My gratitude also extends to Kam-Ling

Yao and Maria Mendez for their expert technical assistance. Above ail, 1 thank Ms. Violetta

Tapia for her secretanal assistance and skilful organization of my cornmittee meetings. Last

but not least, me sincere gratitude are also due to my colleagues, Paul D'Auost and Peter

Fritz, for helping me out in the lab and for making my graduate study an enjoyable and

memorable expenence.

The expenments involved in this project were financially supported by the Medical

Research Council of Canada.

........................................................................................................... Abstract ii Acknowledgements ....................................................................................... iv

.................................................................................................. List of Figures vii ... ........................................................................................ List of Abbreviations wir

Chapter 1: Introduction ...................................................... I . 1 The biology of the penodontium

1 . l a Cementum ......................................................................... ......................................................... 1 . l b Penodontal ligament

1 . lc Alveolar bone ............................ .... ............................... .......*.................... ......... 1 -2 Regulation of periodontal homeostasis ..

.............................................................. 1.3 Pathology of periodontium ......................................... 1.4 Promotion of penodontal wound healing

................................................................................ 1.5 Growth factors ................................................................... 1.6 Mode of BMP-7 action

........................................... ............................ 1.7 Mode1 systems ..... ....................................... 1 -8 Periodontal tissue differentiation markers

................................................................ 1 -9 S tatement of the problem ...................................................................................... 2.0 Hypothesis

Chapter 2: Materials and Methods .................................... 2.1 Periodontal window wounding .... ............ 22

.............................................................. 2.2 Preparation of the implants 22 ........................................................................... 2.3 Tissue preparation 24

.................................................................... 2.4 Immunohistochemistry 24 ............................................................................. 2.5 Autoradiography 26

............................................................... 2.6 Morphometric assessrnent 28 .................................................. ................... 2.7 Statistical analyses .. 28

Chapter 3: Results ......................................................................... 3.1 Effect of wounding 29

...................................... 3.1 a Mitogenic and clonogenic activity 29 .......................... 3.1 b Assessrnent of the type of bone formation 29

3 . l c Evaluation of bone and fibroblastic markers ....................... 30 3 . l d Morphometnc assessment of regeneratinç and intact

....................................................................................... tissues 30

............................................................................ 3.2 Effect of collagen ...................................... 3.2a Mitogenic and clonogenic activity

........................... 3.2b Assessment of the type of bone formation ...................... 3 . 2 ~ Evaluation of bone and fibroblastic markers ..

3.2d Morphometric assessment of regenerating and intact tissues ........................................................................................

........................................................................... 3.3 Effect of BMP-7 ....... ............................ 3.3a Mitogenic and clonogenic activi ty ....

.......................... 3.3 b Assessment of the type of bone formation ........................... .................... 3 . 3 ~ Bone and PL markers ....

3.3d Morphometric assessment of regenerating and intact tissues ................... .. ................................................................

. ................................................................. Figure legends for Figs 2- 1 1

Chapter 4: Discussion ...................... 4.1 BMP-7 effects on initial stages of periodontal healing

4.2 BMP-7 effects on PL homeostasis ................................................... 4.3 BMP-7 stimulation of bone growth in regenerating periodontal tissues ...................................................................................................

............................... 4.4 BMP-7 effects on MNCs .................... ......

. ...................... 4.5 Endochondral Vs Intramembranous bone formation ... ....................................... ................................ 4.6 Conclusions ... ..

4.7 Clinical reievance and fùture directions ...........................................

Bibliography ..................................................

Figure 1 : Diagramatic view of the surgical mode1 ...........................................

Figure 2: Graphs of proliferation profiles ......................... ... .........................

Figure 3: Graphs of clustering profiles ..........................................................

Figure 4: Histological sections showing toluidine blue staining .......................

Figure 5: Histological sections showing OPN immunolocalization ..................

Figure 6: Graphs of semi-quantitative assessments of OPN imrnunostaining ...

Figure 7: Histological sections showing BSP immunolocalization ...................

Figure 8: Graphs of semi-quantitative assessments of BSP immunostaining ......

Figure 9: Histological sections of a-SMA immunolocalization .........................

Figure IO: Graphs of morphometric assessments of PL and AB ........................

Figure I I : High magnification photornicrogrphs of proliferating cells . . . . . . . . . . . . .

Page

vii

a-SMA

AB

BMP

BSP

CI

CT

DFBA

FGF

HEBP

hr

IGF

IgG

LI

m c

OPN

OP

PBS

PDGF

PL

TGF

TRAP

Alpha-smooth muscle actin

Aiveolar bone

Bone morphogenetic protein

Bone sialoprotein

Clustenng index

Connective tissue

Demineralized freeze-dried bone allograft

Fibroblast growth factor

Ethane- l-hydroxy- 1,l-bisphosphcnate

Human recombinant

Insulin-like growth factor

Immunoglobulin G

Labelling index

MuItinucleated ce11

Osteopontin

Osteogenic protein

Phosphate buffer solution

Platelet-derived growth factor

Periodontal ligament

Transforming growth factor

Tartrate resistant acid phosphatase

Chapter 1: Introduction

1.1 The Biolow of Periodontium

The periodontium is the supporting apparatus of the tooth and is composed of

mineralized and fibrous connective tissues. The mineralized or hard connective tissues are

aiveolar bone and cementum; the fibrous or sofi connective tissues are penodontal ligament

and the lamina propria of the gingiva (Melcher and Eastoe, 1969). The penodontium anchors

the roots of the teeth to the bones of jaw and thus provides support during mechanical stress

and trauma (Williams et al., 1992a). It is also capable of renewal, provides nutrients and

contains neural elements that sense various stimuli such as temperature and pressure

(Terranova et al., 1990). Cells, originally derived from the inner layer of dental follicle and/or

the dental papilla, give rise to the cells that synthesize cementum, alveolar bone and

periodontai ligament (Palmer and Lumsden, 1987; Ten Cate et al., 197 1 ; Ten Cate and Mills,

1972; Yoshikawa and Kollar, 198 1). Alveolar bone cells are also denved from pre-existing

alveolar bone.

l . l a Cementum

Cementum is a speciaiized mineralized tissue that binds firmly to dentine and covers

the entire surface of the root (Lindhe and Karring, 1983). Although cementum has some

charactenstics of bone, it does not have blood or lymph vessels, is not innervated and does

not undrrgo to physiological resorption and remodelling (Lindhe and Kamng, 1 983).

In mammals, there are two f o m of cementum: primary or acellular cementum covers

the entire surface of the root and secondary or cellular cementum covers the apical 1/2 to 2/3

of the root surface (Melcher and Eastoe, 1969). Primary cementum is formed during the

formation of the root and tooth eruption, while the secondary cementum is formed after tooth

enrption and is slowly formed throughout Iife (Williams et al.. 1992a). The secondary

cementum is usually thicker and cellular and is covered by a layer of cementoblasts, whereas

the pnmary cementum is thinner and acellular. The thickness and composition of cementum

varies from the coronal to the apical aspects of the root dentine.

1.1 b Periodontal ligament

The periodontal ligament (PL) is the sofl connective tissue between the two hard

co~ec t ive tissues of the periodontium. A heterogenous population of cells is responsible for

the normal extracelluar matrix synthesis and maintenance of homeostasis within penodontal

tissues (McCuIloch and Bordin, 199 1). The predorninant ceIl type of the penodontal ligament

is the fibroblast (Lindhe and Kamng, 1983). Since these spindle or stellate shaped cells are

motile and contractile, they are able to assist in the structural organization of PL in

development and in regeneration (Ten Cate, 1989). They also synthesize and resorb

extracelluar matrix components, thus active1 y contributing to connect ive tissue turnover

(Beersten, 1987; Kanoza et al., 1980; Ten Cate et al., 1976).

The PL connective tissue matnx is known to be turned over quite rapidly. about five

tirnes faster than alveolar bone and fifieen times faster than dermal tissue (Williams et al.,

1992a). Moreover, investigations on ce11 population kinetics (McCulloch and Melcher. 1983a;

Roberts et al., 1975) and on extracelluar matrix (ECM) metabolism (Pearson and Gibson,

1982; Sodek 1977) show that periodontai cells and matrix molecules are turned over at a rate

of 0.5-2 Wday. PL cells exhibit a higher ceil generation rate (Leblond et al.. 1959) and faster

turnover of collagen (Sodek, 1977 & 1988) than dennal connective tissue.

However it should be noted that the turnover rate of normally functioning periodontal

tissues is quite modest compared to a rapidly renewing tissue such as srnaIl intestinal

epithelium (McCulloch and Melcher, 1983a). Further, continuous labelling with 3~-thymidine

show that almost 70% of mouse PL cells do not cycle (McCulloch and Melcher, 1983b;

Nemeth et al., 1989). This smail growth fraction may contribute to the limited regenerative

capacity of' the PL despite the high matrix turnover rate. Cells within the PL have the potential

to give rise to three different forms of synthetic cells: osteoblasts (found on the bony side),

fibroblasts (found in the body of PL) and cementoblasts (found on the cemental side)

(Melcher, 1980)There are also ot her minor populations of cells found at restricted locations

in PL, such as the epithelial ce11 rests of Malassez, macrophages, undifferentiated

mesenchymal cells, neural and vascular elements (Lekic and McCuiloch, l996a).

There are at least three groups of collagen fibres found in the PL including principal

(oblique) fibres, horizontal fibres and apical fibres. These fibre bundles help to anchor the

tooth fimly in the bone socket while accommodating minor tooth movements as a result of

mastication or onhodontic forces (Lindhe and Kamng, 1983). The principal collagen types

found in the PL are types 1, III and V (Reviewed by: Sodek and Overall, 1988). The

extracelluar matnx of the PL also includes several water binding molecules such as hyaluronic

acid. hepann sulphate and dermatan sulphate which help to provide a hydraulic cushion for

the toot h root (Williams et ai., l992a).

An interesting feature of PL is that its width is maintained at a constant level

throughout life (McCulloch and Melcher, 1983a). Even though the PL is sandwiched between

two rnineralized connective tissues, one of which (bone) is actively tumed over, the PL in

physiological conditions does not permit ankylosis (i.e. direct fusion of bone to cementum).

PL cells in physiological conditions seem to be capable of inhibiting osteogenesis thereby

preventing ankylosis and maintaining PL width (Lekic et al., 1996b). This inhibition of

osteogenesis by PL cells may be mediated by prostaglandins (Ogiso et al.. 199 1 & 1992).

1. l c Alveolar bone

The other major mineralized component of the periodontiurn is the alveolar bone

(AB), in which are inserted the Sharpey's fibres of periodontai ligament. The AB is continuous

with the basal bone of the jaw, and thus fimly attaches the tooth to the jaw. It consists of a

thin outer layer of cortical or compact bone and an inner cancellous or medullary bone

(Melcher and Eastoe, 1969). The principal synthetic cell type in the AB is the osteoblast.

Osteoblasts are involved in deposition of organic matrix (osteoid) and calcification by

regulating the formation of hydroxyapatite crystals. The cells that become trapped in the

calcified matrix dunng bone formation are terminally differentiated osteoblasts. and are

referred to as osteocytes. The other important ce11 type, the osteoclast, is involved in the

resorption of bone via acidic demineralization of the bone matrix followed by proteolytic

degradation and/or phagocytosis of the uncalcified collagenous matrix (Williams et al.,

l992a).

AB is remodelled continuously in response to local factors such as prostaglandin E2,

cytokines (eg.:IL-1, and tumour necrosis factors a. & a), and systemic factors such as

parathyroid hormone and calcitonin, as is the rest of the skeleton (Heersche, 1989). These

agents presumably contribute to bone homeostasis by acting on osteoblasts, which release

osteoclast stimulating factors in addition to collagenase thus facilitatinç resorption of bone

(Heersche, 1989). Bone formation is also thought to be controlled in pan by locally produced

factors that are stored in the bone matnx and released durinp resorption (Linkhan et al.,

1996). The factors that stimulate bone formation include transforming growth factors a &

0, insulin-like growth factors 1 & II, platelet-derived growth factor. acidic & basic fibroblast

growth factors and bone morphogenetic proteins (BMPs) (Giannobile. 1996).

1.2 Rep~lation of Homeostasis

The PL consists of a heterogenous population of cells (McCulloch and Bordin, 199 1 ):

some are undifferentiated proliferating precursors that are involved in regeneration (Cameron,

1970; Gould et al., 1982; Leblond et al., 1959; McCulloch and Melcher, l983a), some are

relatively differentiated progenitors that can respond to repopulating stimuli, such as

woundinç (Gould et al.. 1980 & 1983), and some do not divide or have very long cycling

times (Carneron, 1970; Gould et al., 1982; McCulloch and Melcher, 1983a, 1983b & 1983~).

There are cells located paravascularly in PL (Gould et al., 1980; McCulloch and

Melcher, 1983a) and in the endosteal spaces of alveolar bone (McCulloch, 1987) that have

some of the characteristics of early progenitor cells or stem cells, such as small size.

responsiveness to growth factors, slow cycle time (Gould, 1983; Evans and Potten, 199 1;

McCulloch, 1985; Potten and Loeffler, 1990), high basai mitotic rate and ability to proliferate

rapidly following certain stimuli (eg. wounding). Even though these precursors are closely

associated with blood vessels, their origin seems to be predominantly fibroblastic rather than

haematogenous, as suggested by electron microscopie observations made on irradiated

animals (Gould et al., 1980).

In intact periodontium, PL progenitor cells may differentiate into a variety of

phenotypes, including osteoblasts that cm synthesize bone matrix, fibroblasts that can

synthesize PL matrix, or cementoblasts that can synthesize and mineralize cemental matnx

(McCulloch and Melcher, 1983b).These cells contribute to the heterogenous cell populations

in PL (McCulloch et al., 1989).

PL fibroblasts can also synthesize mineralized tissue matrix molecules under certain,

restricted stimulations (Cho et al., 1992). Conceivably, the PL contains either rnultipotential

cells or a mixture of cells that potentially c m give nse to several ce11 types depending on the

stimulus and the local environment (Gould et al., 1980; McCullocli and Melcher. 1983a;

Melcher and Eastoe, 1969).

Wounding studies of the PL have facilitated identification of three distinct populations

of progenitors: precursors of osteoblasts that are located paravascularly adjacent to bone.

fibroblast progenitors that are aiso found paravascularly in the body of PL. and cementoblast

precursors that are observed in the vicinity of cementum, but not associated with blood

vessels (Gould et al., 1977). In healthy PL of rodents, proliferating cells and differentiated

cells appear to be kinetically separate with different cycling times and mortality rates

(Davidson and McCulloch, 1986). Ce11 kinetic experiments show that there is a steady-state

fibroblast cell system in the PL that is capable of regeneration (Gould et al., 1977 & 1980;

McCulloch and Melcher, 1983a & 1983b; Perera and Tonge. 198 1 ) and in which the

proliferation rate equals the sum of ce11 death and ceIl migration rates (McCulloch, et al.,

1989).

Cell density in the PL is maintained at a high level in reçions near the bone, cementum

and blood vessels and is lower in the remaining PL (McCulloch and Melcher, 1983a). In

contrast, the percentage of 'H-~hymidine OH-Tdr) labelled cells (Le. proliferating cells) is

highest near blood vessels, and significantly higher in the central body of PL than in the areas

close to bone or cementum (McCulloch and Melcher, 1983b). In contrast, ce11 death occurs

at a faster rate in zones of high ceIl density (McCulloch et al., 1989). Hence, the distribution

patterns of cellular density and cycling precursors are inversely related. The maintenance of

this pattern and of the steady-state is achieved by balancing cell migration within the PL

(Davidson and McCulloch, 1986; Garant and Cho, 1979; McCulloch and Melcher, 1 9 8 3 ~ )

and migration from endosteai spaces into PL (McCulloch et al., 1987). with ce11 death, which

occurs at a faster rate in zones of high ce11 density (McCulloch et ai., 1989). Migration of cells

within and into the PL compartment occurs mainly along collagen fibres (Davidson and

McCulloch, 1986; Garant and Cho, 1979).

1.3 Patholow of Periodontium

Penodontal diseases are among the most prevalent diseases in humankind (Genco,

1990). The initial lesion of the periodontiurn is gingivitis which can (but not always), lead to

periodontitis (Zambon, 1990). In periodontitis, loss of AB and PL leads to the exposure of

root cernentum and the accumulation of sofi and hard bacterial deposits (Genco. 1990). With

advanced periodontal destruction, permanent loss of affected teeth may result (Eçelberg.

1987).

Bactenal pathogens may initiate tissue destruction by acting directly or indirectly on

host connective tissue cells and which, if prolonged, may eventually bnng about a shifl in

connective tissue metaboiism to net destruction (Page and Shroeder. 198 1). Bactenal extracts

are considered to be causal factors in periodontal diseases as they express molecules that are

capable of dest roying the epithelium, degrading collagen, loosening tibrous attachment and

inhibiting osteogenesis directly and irrevenibily (Loomer et al., 1 994 & 1995). In addition,

bacterial extracts can promote bone Ioss by inducing osteoclast formation and facilitating

bone resorption (Nair et al., 1983; Norton et al., 1970). Indeed, periodontal diseases are

exemplified in large part by severe destruction of alveolar bone.

These foreign bacterial products cause inflammation and induction of host immune

responses (Carlsson, 1983). Accumulation of activated lymphocytes in response to putative

periodontal pathogens amplifies idammatory processes by secreting cytokines such as IL- 1

and TNF-a, which in tum inducc bone resorption, collagen breakdown. fibroblast

proliferation and prostaglandin synt hesis. Neutrop hils and macrophages contribute to tissue

destmction as they are attracted to the inflamed area by increased prostaglandin production

and various chernotactic factors (Genco, 1990).

Notably, even in chronically inflamed sites, repair and regenerative processes occur

in periodontal tissues. Sporadic outbursts of disease activity are followed by periods of

inactivity during which repair/remodelling occurs (Zambon, 1990). However. as indicated

above, PL ce11 populations contain well differentiated cells and the regenerative capacity of

the PL in most marnmals is Iow. Therefore the PL has a limited capacity to regenerate

(Egelberg, 1987). The ongoing presence of bactenal factors further compromises the

regenerative potential of the periodontium. Hence, lost tissues are often replaced by inflamed

connective tissue and by the downgrowth of epithelium (Page and Schroeder. 198 1).

Most current treatment regimes focus on elimination of pathogenic microorganisms

by mechanical and chemotherapeutic rneans to control or stop disease progression. However

these do not lead to restoration of destroyed penodontal tissues (Williams et al., 1992b) but

only to cessation of their destruction. Periodontal surgical procedures, involving debridernent

and root planing of the diseased root surface, gingival curettage and placement of bone grafts

and/or bone substitutes, have met with limited success in bringing about regeneration, and

generally result in repair at most (Caton and Nyman, 198 1; Forum et al., 1982; Isidor et al..

1985; Renvert and Egelberg, 1981; Steiner et al., 1981).

1.4 Promotion of oeriodontal wound healing

Our inability to regenerate lost periodontium is due in part to the lack of knowledge

with respect to basic aspects of periodontal wound healing. Healinç of periodontal tissues is

mediated by cells (Melcher, 1988). Reports on replantation of teeth (Egelberg, 1987) and

wounding procedures (Lekic, l996b; Melcher, 1977) show that cells from the neighbouring

intact PL are necessary for repopulation and establishment of new penodontal tissues.

Followinç wounding and generation of the inflammatory response, these cells migrate,

proliferate, differentiate and finally regenerate the lost tissues.

The outcome of healing processes depends primarily on the phenotype of the cells that

colonize the wound (Aukhil et al., 1990; Melcher, 1976; Nyman et al., 1983). Hence,

understanding the cellular origins of repopulating cells (Pitam et al., 1994) and being able to

manipulate them in such a way so as to achieve selective repopulation of PL cells in the

wound is important for successful regeneration (Nyman et al., 1983).

There are several current investigations that are attempting to identi& important

deteminants of periodontal regeneration such as conditioning of root surface, modulation of

precursor cells, prevention of epithelial ce11 migration ont0 the root surface. stabilization of

the flap margins and enhancement of local growth factors (Arnar, 1996). However, flap

procedures and root surface conditioning protocols have limited success due to difficulties

including healing in the presence of bacterial plaque and the complexity of cell-ceIl and cell-

matrix interactions (Froum et ai., 1983; Moore et al., 1987; Stahl et al., 1982). Investigations

on osseous grafiing techniques (Bowen et al., 1991; Mellonig, 199 1 ) and çuided tissue

regeneration procedures (Nyman et al., 1982a & 1982b. Pitaru et al., 1989) often promise for

the promotion of periodontal regeneration but reported gains are rnodest.

Other approaches have employed the use of demineralized fieeze-dned bone allografts

(DFDBA) (Helm et al., 1997; Schwartz et al., 1996; Sonis et al., 1983; Unst, 1965). as well

as polypeptide growth factors alone or in combination (Greenhalgh et al., 1990; Greenhalgh

et al., 1993; Hebda, 1988; Matsuda et al., 1992; Mustoe et al., 1987; Pierce et al., 199 1).

Since the osteoinductive ability of DFDBA was first demonstrated (Urist. 1965; Reddi and

Huggins, 1972). it has been used widely in atternpts to regenerate lost periodontium over the

last two decades (Libin et al., 1975; Quintero et al., 1982; Schallhorn and McClain, 1988;

Bowers et al., 1989). DFDBA is non-antigenic (Sonis et al., 1983). and contains several

BMPs such as BMP-2.4 and 7, in addition to bone siaioprotein (BSP), osteonectin and type

1 collagen (Becker et al., 1995; Shigeyama et ai., 1995). Despite the encouraging results

reponed by some (Bowers et al., 1989; McClain and Schallhom, 1993; Mellonig, 1996). there

have been some disturbing findings where no osteoinduction was observed (Becker et al.,

1992, 1994, 1995; PinhoIt et al., 1992).

Recent evidence shows that comrnercialiy prepared bone ailografls employed in dental

preparations contain variable levels of BMP-2, -4 and -7 (Becker et al., 1995; Schwartz et al.,

1996; Shigeyarna et al., 1995). This may explain the highly variable results obtained with

DFDB A preparations. Furthemore, differences in the bioactivity of bone grafis due to the age

of donors (Jergesen et al., 199 1; Syftestad and Urist, 1982) and previous exposure to dmg

therapies (Lian et al., 1984) have also been observed. Hence it is conceivable that other

variables such as sex, health status and genetic history of the donor could also influence the

activity of DFDBA (Somerman, 1996). Standardized protocois for the collection and

preparation of bone samples and the evaluatior. of bioactivity of DFDBA are required before

DFDBA can be used clinically (Schwanz, 1996).

1.5 Growth factors

Polypeptide growth factors are natural biological mediators that orchestrate critical

cellular events involved in regenerative processes, such as ceIl proliferation, directed

migration, differentiation, and matrix spthesis (Kiristy and Lynch, 1993; Matsuda et al.,

1992). The growth factors found in bone matnx include transforming growth factor-beta

(TGF-B), insulin-like growth factors 1 and 11 (IGF-I & -II), platelet-derived growth factor

(PDGF), acidic and basic fibroblast growth factors (a- and b-FGF) and BMPs (Graves and

Cochran, 1994; Lind, 1996).

The primary cellular sources of PDGF. FGF, TGF-O and IGF are macrophages and

osteoblasts (Giannobile, 1996). These factors are also stored in bone matrix and may be

released dunng bone remodelling, thus helping to couple bone formation to resorption

(Linkhart et ai., 1996). In bone, the main target cells of these growth factors are osteoblasts,

although some of them induce PL ce11 proliferation as well (Graves and Cochran, 1994).

BMPs are members of the TGF-B farnily, sharing a high degree of homology in their

carboxyl termini within the so called TGF-O domain that harbours 7 conserved cysteine

residues (Messague, 1990; Kingsley, 1994). Several BMPs have been identified so far, namely

BMP-1 throuçh -8 (Womey, 1995). These are produced as large precursors that are cleaved

immediately before the proteolytic signal peptide Arg-X-X-Arç (Jones et al.. 1994) to yieid

the mature, glycosylated, dimeric protein linked by disulphide bonds. The relatively short N-

terminal of the processed protein varies considerably more between species than the

corresponding C-terminai containing the TGF-t3 domain (Cook and Rueger, 1996). Notably,

the bone forming activity of the BMPs is not species-specific (Sampath and Reddi, 198 1 &

1983).

In compannç the conserved TGF-O domain of the various BMPs it appears that BMP-

2 through -8 are related to one another and can be grouped into three categories: BMP-2 &r.

-4 make up one group with a sequence similanty of 92%, BMP-5.-6.-7 and -8 make up the

second group with a sequence similarity of 82% and BMP-3 (osteogenin) by itself constitutes

the third group (Womey, 1995). BMP-7 is more related to BMP-2 and -4 (60% & 58%).

than to BMP-3 (42%) or to TGF-Bs themselves (3538%) (Sampath and Rueger, 1994).

BMPs, like other bone inducing factors, are produced by osteoblasts and are stored

in the extracelluar matnx of bone (Giamobile. 1996). BMPs are the only growth factors

known to date that act on undifferentiated pluripotential mesenchymal cells to induce de rwvo

bone fornation (Chen et al.. 199 1; Urist et al., 1983; Yamaguchi et al., 199 1 ). M e r fracture,

BMPs are releaseâ in the early stages of healing and are possibly involved in the chernotaxis

and differentiation of progenitor cells that eventually repopulate the wound site (Jin et al..

1994; Nakase et al., 1994).

BMPs also induce metabolic activities of human PL fibroblast-like cells (Gao et al..

1995). BMP-7 in particular, stimulates proliferation and differentiation of human mandibular

bone cells (Knutsen et al., 1993) and stimulates differentiation staçe-specific expression of

various proteins by fetal rat calvarial cells (Li et al., 1996). BMP-7 has also been found to be

a potent stimulator of proteoglycan (mostly aggrecans) and collagen (mostly type II)

production by human anicular chondrocytes (Flechtenmacher et al., 1996). This and other

results suggest that BMP-7 may be applicable in the treatment of not oniy bone fractures and

periodontal diseases, but diseases of articular cartilage as well. such as osteoarthrit is (Cook

et al.. 1996; Hiroshi et al., 1990).

Recombinant human OP-1 (rhBMP-7) reconstituted with collagen type 1, recruits

mesenchymal cells and induces them to proliferate, migrate and differentiate into osteoblasts.

while the collagen provides a scaffiold that d o w s the cells to attach and repopulate the wound

site (Cook and Rueçer, 1996). Furthemore, rhBMP-7 has a specific activity similar to that

of highly punfied bovine osteogenic protein (Sarnpath et al., 1 990). which is a dimer of BMP-

2 and BMP-7 (Sampath et al., 1990).

In surgically created, critical size diaphyseal segmenta1 defects. rhBMP-7 completely

restores bone volume and function in rabbits (Cook et al., 1994a), in dogs (Cook et al.,

1994b) and in non-human primates (Cook et al., 1995). whereas carrier alone or no implant

controls result in fibrous unions in al1 cases. The control used in the non-human primate

model studies, autogenous bone, did not heal the defects (Cook et al.. 1994a; Cook et al,

1995). In addition BMP-7 has been applied in a canine spinal Fusion model to achieve more

rapid and stable spinal fusion compared to the autogenous bone graft controls (Cook et al.,

1994b). Attempts to restore human adult non-union diaphyseal fractures with BMP-7 began

in 1992 and more recent data suggest it is a promising agent clinically (Cook and Rueger,

1996).

Even though the amount and rate of neo-osteogenesis Vary with the concentration of

the BMP-7 applied, they generate sirnilar results above a threshold level. which is species and

carrier dependent (Cook et al., 1994 & 1995).

1.6 Mode o f BMP-7 action

The way by which BMP-7 initiates and perpetuates the entire sequence of reçenerative

events, including recmitment, stimulation of proliferation and differentiation of progenitors,

is not well understood. It is conceivable that these proliferative and differentiative effects of

BMP-7 could at least in part be mediated by its augmentation of the synthesis andor action

of other known growth and differentiation factors. BMP-7 potentiates the insulin-like growth

factor (IGF) reylato~y system by reguiating the balance between stimulatory and inhibitory

IGF binding proteins (IGFBP) thus increasing the secretion of IGF-II in a time and dose-

dependent manner (Knutsen et al., 1995). Furthermore, chernotactic activities of BMP-4 and

-3(osteogenin) have been shown to be mediated in part by the up-regulation of TGF-O 1

(Cunningham et al., 1992).

The exact intracellular mechanisrns by which BMP-7 and other BMPs regulate the

expression of other polypeptide growth and daerentiation factors are still under investigation.

However, recent data show that there are BMP specific type I and type II binding sites found

on the surface of receptive ceus, analogous to TGF receptors (Yamashita et al., 1996). In the

presence of type II receptors, most BMPs bind to two types of type 1 receptors, BMP

receptor type IA (BMPR-IA, also known as activin receptor-like kinase [ALK] -3) and type

IB (BMPR-IB; Koenig et al., 1994; ten Dije et la., 1994). In addition, BMP-7(0P- 1 ). B W-2

also bind to the activin type 1 receptor (ActR-1, ALK-2; Liu et al., 1995; Yamashita et al.,

1995). Most BMPs recognke BMP type Ii receptor (BMPR-II; Liu et ai.. 1995)- while BMP-

7 (OP-1; Yarnashita et al., 1995) and -2 (Hoodless et al., 1996) have been recently shown to

recognize activin receptor type II (ActR-II) and type IIB (Act-IIB). Whether these receptors

are playing a role in the wound model used here (See below section 1.7) has yet to be

elucidated, but would seem to be an important issue.

1.7 Mode1 Svstems

Several model systems could be used to study periodontal wound healinç. h r vitro cell

culture analyses of penodontal cells allow the simplified understanding of main events in

regenerative processes, without the interference of other types of cells and factors. There have

been several studies done on remodelling (Qwarnstrom and Page, 1986), stimulation of

osteogenesis (Hughes and McCulloch, 1991) and inhibition of osteogenesis (Ogiso et al.,

199 l), using either homogenous cultures or m-cultures. However, i~z vitro investigations can

be somewhat rnisleading as they cannot recreate the events involved in regeneration and the

complex intercellular communications that may exist between the different types of

periodontal cells (McCulloch, 1993). Another alternative is to use an 111 vivo penodontal

wound healing model.

Using an Ïri vivo model of the healing periodontium is often complicated by the

presence of bacteria and other factors in the oral cavity (e-g. salivary glycoproteins). Critical

size defects, which cannot heal spontaneously during the life time of the animal, are very

usefül in identiQing the factors that cm promote wound healing extensively. Unfonunately,

there are no models for periodontal cntical size defects available yet. There are however

cranial and segmental wound models that do not heal spontaneously.

The periodontal window wound model developed by Melcher (1970) and further

refined by Gould et al. (1977) provides an excellent systern to study the ce11 kinetics of

penodontal wound healing in the absence of oral bacteria and epithelial downçrowth.

Selective deletion of parts of the PL and AB on the buccal side of the jaw is perfonned. This

is a standardized and highly reproducible model as it has been well characterised with respect

to wound size, configuration and stability (Gould et al., 1980; Lekic et al., 1996a). It also

creates synchronous cohorts of proliferating of periodontal cells in a spatially and temporally

defined manner.

Although this is an excellent model, it does have some drawbacks. One of them is that

the model does not mimic the "real" situation, due to the absence of bacterial factors (which

is also a strength of the model, in providing a simplified system). Also, there are complex

interactions of tissues involved during the regenerative processes, which complicate data

interpretation. In addition, the wound heals spontaneously over time, even in the absence of

any promoting factors. lmplementing wounding of rats further renders the model "unrealistic",

for the structure and healing pattern of the periodontium Vary between human and rat.

Nonetheless, it is a predictable and reliable rnodel and hence is reliable for elucidatinç the

effects of dmgs or growth factors in periodontal regeneration.

1.8 Periodontal tissue differentiation markers

A problem of using an ili vivo penodontal wound heaiinç model system is the absence

of well defined phenotypic markers for PL fibroblasts. However. there are several non-

collagenous rnatnx proteins, cytoskeletai proteins and membrane proteins, that can be used

in the identification of different penodontal ce11 phenotypes, such as alkaline phosphatase

(AP), a-smooth muscle actin (a-SMA), osteopontin (OPN), osteonectin (ON) and bone

sidoprotein (BSP). There are fibroblast subtypes that can be cultured from the PL which are

highly contractile and express a-SMA (Arora and McCulloch, 1994). PL cells constitutively

express AP (Groenveld et al., 1993) and therefore this enzyme may provide a good marker.

OPN, a protein that may be expressed by cells in the PL with osteogenic charecteristics, has

been imrnunolocalized in healing PL (Lekic et al., 1996b & 1996~).

AP, OPN and ON are expressed dunng early osteodifferentiation, and rnay be

expressed by osteoblast precursor cells (AP: Follis, 1949; OPN: McKee et al.. 1993 and

Kasugai et al., 1991; ON: Bianco et al., 1988). OC is detected in the areas destined for

mineralization (Bronkers et al., 1987). BSP is mostly expressed by osteoblasts at later stages

of differentiation and mineralization (Bianco et al., 199 1 ; Young et al., 1992). Hence OPN

and BSP are considered to be early and late markers of mineralized tissue differentiation,

respectively (Lekic et al., 1996b & 1996~).

OPN is a secreted glycoprotein that is highly sulphated and phosphorylated and is

enriched in sialic acid (Butler, 1989; Sodek et al., 1992). In addition to the cells of

mineralized periodontal tissues, OPN is also expressed by activated macrophages and

lymphocytes, kidney cells, cells in atherosclerotic plaques, lining epithelial cells and

ontogenically transforrned cells (Reviewed by Denhardt and Guo, 1 993 ).

BSP is a hiçhly sulphated and glycosylated phosphoprotein found in bone matrix,

mineralized CT as well as dentin and cernentum (Oldberg et al., 1988; Fisher et al.. 1990;

Chen et al., 199 1; Somerman et al., 1991). BSP can mediate ce11 attachment and bind

selectively to hydroxyapatite (Somerman et al., 1987; Gorski, 1992). Also, BSP could have

a role in initial bone matrix formation and mineralization as it is expressed early in dentine and

bone formation (Chen et al., 1993).

1.9 Statement of the ~rob lem

The siçnalling mechanisms and cellular events involved in periodontal regeneration

in some ways recapitulate developmental processes. Our lack of understanding of these

processes precludes the development of new periodontal treatments that can reproducibaly

regenerate the lost penodontal tissues. For this reason, existing treatment provides outcornes

that are only rnodest improvements.

BMPs are signalling molecules involved in ontogeny and bone formation. Of the BMP

family members, BMP-2, -3 and -7 have been studied most extensively for their application

in healing of bone fractures and bone-related diseases. Trials of bone induction in rodent

subcutaneous mode1 systems reveal that BMP-7 may be more potent than BMP-2 (Sampath

et al ., 1 992). Furthemore, atternpts to stimulate the regeneration of periodontal tissues in

humans with purified BMP-3 combined with DFDBA not only failed, but resulted in ankylosis

as well (Bowers et al., 1991). Also, treatment of canine periodontal wounds with hrBMP-2

combined with a biodegradable synthetic vehicle revealed that it strongly promoted bone and

cernentum formation. while causing four fold increase in ankylosis compared to the vehicle

only controls (Sigurdsson et al., 1995). A pilot study of non-human primates indicated that

BMP-7 induces increased bone growth (Rutherford et al., 1 992).

To be effective itl vivo, BMP-7 must be applied in a biodegradable vehicle that

facilitates the optimum spatial and temporal release of BMP-7 (Hollinger and Leong, 1996).

BMP-7 actively recruits plunpotential precursor cells toward the site of application (Sampath

et al., 1992). Coilagen on the other hand, serves as an inert scaffold that aids the invasion,

embedding, differentiation and maturation of progenitor cells in the implant, and is finally

completely replaced by new bone (Reddi et al., 1987; Urist, 1965). Hence this choice of

factor and vehicle combination facilitates bone formation by being both osteoinductive and

ostoconductive.

The periodontal window wounding system was implernented on rats primarily because

t hey have high regenerative capacity and therefore healing processes could be studied in a

relatively short period of time (Page and Schroeder, 1982). However results obtained with

a rat model should be interpreted cautiously, as the structure of human periodontiurn differs

somewhat from that of rat (eg. cellular cementum is thicker in rats. the gingival epithelium

joins the coronal portion of the junctional epithelium ofien producing stratum granulosum and

stratum corneum, the most superficial cells of which have desmosomal contact with

nondifferentiating cells of junctional epithelium; Bosshardt and Schroeder. 1996; Listgarten,

1975).

Rat periodontal window wounds have been utilized previously to resolve the sequence

of periodontal reparative events at 1, 3, 7, 10 and 21 days postoperatively followinç the

extirpation or preservation of PL (Lekic et al., 1996a & 1996b). These reports show that an

initial and transient inflammatory response occurs around day 1; migration of progenitors

toward the wound occun around day 3; increased proliferation of progenitors in the mid

wounded PL and AB is evident around day 7; regeneration of PL is achieved and that of the

AB commences around day 10; and finally AB is completely restored at day 2 1. Hence in this

study, healing was examined postoperatively at 2, 5, 10, 21 and 60 days. The 60 day

observation period was selected to determine the longer term effects of a single application

of BMP-7 on penodontal homeostasis and remodelling, since prelirninary data suçgested that

there was still considerable cellular activity at 21 days after woundinç.

In view of these data, 1 used BMP-7 reconstituted with bovine collagen vehicle in a

rat penodontal window wound model. 1 have investigated the effects of BMP-7 on

penodontal ceIl proliferation and differentiation at 2, 5 , 10, 2 1 and 60 days afler wounding.

Phenotypic markers a-SMA, OPN and BSP were utilized to characterize PL fibroblasts in

early and late mineralization periods. respectively.

2.0 Hv~othesis

BMP-7 upregulates regenerative processes in vivo by stimulating the proliferation.

clona1 growth and differentiation of progenitors in PL and AB.

$

The wounding system described by Gould et al., (1977) and Lekic et al., (1996~) was

adopted in this study. Male wistar rats (1 10- UOg) were caged in pairs in a room with 12h

darMight cycle. Under general anaesthesia (Halothane, N20:02 at 2: l ) , an incision about

lrnm in length was made over the incisor trunk or just above it. The overlying anterior

rnasseter muscle was raised to reveal the mandible and the portion of the posterior masseter

muscle which was then reflected to expose the underlying alveolar bone. Wounding was

performed with the aid of a dissecting microscope (Wild M3Z. 1 O X ) on the buccal side,

between the superior margin of the alveolar bone and the foramen mentale. lmrn away from

the anterior edge of the mandible (Fig. 1). Initially a modified end-cutting bur (0.6mm in

diameter) dnven at a low speed by a dental drill was used to remove the bone overlying the

mesial root of the first molar and to create a hole 0.6mm in diameter. Subsequently, the

remaining thin portion of bone and the underlying PL were extirpated using a sharpened

dental probe followed by the cooling of the bone with saline and removal of the debris using

wet gauze. The wound site was then filled with an implant or left empty. Finally the tissues

were closed with 4-0 Vicryl suture. Recovering animals were warmed and watched closely

for about an hour following surgery.

2.2 Preparation of the implants

Hurnan recombinant BMP-7 (hrbmp-7) was provided by Dr.Sampath (Creative Bio

Molecules; Hopkinton, Ma), as beads or particles of fibnllar collagen impregnated with

hrbmp-7. The concentration was not disclosed for proprietary reas0r.s. A paste of collagen

alone or BMP + collagen was made by mixing about O. lmg of the polymer with O. Iml of

saline, under aseptic conditions. The wound sites were either implanted with collagen alone,

with BMP + collagen, or were Ieft unfilled.

To study the temporal effects of BMP-7 on wound healing, three animals for each

experirnental condition (see above), were sacrificed by N,O asphyxiation, at 2, 5, 10, 2 1 and

60 days following surgery. One hour pnor to sacrifice, rats were injected intraperitonially with

1 pCi/g body weight 'H-thymidine (Specific activity = 20 Ci~rnmol; NEN, Oakville, ON)

diluted with PBS (Ph 7.4) to 1 .Orni.

Figure 1: A sketch of the rat mandible depicting the relative location and size of drill site @).

Mandible (M), first lower molar (LM), mesial root (MR), foramen mentale (FM) and incisor

(1). (Adapted fkom Nguyen et al., 1997).

2.3 Tissue Preparation

Following sacrifice of the rat, the mandibles were removed. cleared of any attached

sofl tissue and tnmmed at the mid-incisor and at the third molar region. Tissues were fixed

in periodate-lysine-paraformddehyde (McLean and Nakane. 1970) at pH 7.4 for 24 hours at

4OC, demineralized for 24 houn in 0.2N HCI (Hni et al., 198 1) at room temperature and later

washed in PBS for 20 hours at room temperature. Subsequently the specimens were

dehydrated by washing in graded ethanoi, cleared in toluene and embedded in parafin. These

parafiin embedded specimens were coded to prevent examiner bias in assessrnent procedures.

With the aid of a dissecting microscope, the sections close to the wound site were stored on

a tray and every fifteenth section was stained with haematoxylin and eosin to determine the

exact location of the drill site. Sections in the middie of the drill site were used in the

immuno histochemical and autoradiographical assays.

Sections adhered ont0 slides by alburnin "glue" were deparafinized with xylene (4 X

1 min) and rehydrated in a senes of graded ethanol solutions (2 X 1 min in 100% ethanol, 1

X lmin in 95% ethanol and 1 X lmin in 70% ethanol). The excess ethanol was washed off

by dipping several times in clean tap water. Sections were dipped in toluidine blue for 30

seconds, and the excess stain was removed by washing thoroughly in 3 changes of water, and

dehydrated by dipping in 5 changes of 100% ethanol. To facilitate mounting of the slides,

sections were washed in xylene (2 X 2 mins) and attached with Pennount@.

2.4 lmmunohistochemistrv

For immunohistochemical analysis, sections were adhered onto Superfrost Plus@

24

slides and dewaxed in toluene (3 X 2 min). Sections were rehydraied in a series of graded

ethanol solutions (2 X 1.5 min in 100% ethanol and 2 X 1.5 min in 95% ethanol), washed in

distilled water and incubated with 3% H,O, in methanol for 30 min protected from light. This

step enables the inactivation of the endogenous peroxidase activity. Sections were blocked

with 1% casein in phosphate buffer saline (PBS) for 1 hour in a moist chamber to decrease

non-specific background staining.

Sections were incubated with pnmary antibody for 3 hours in a moist chamber,

washed in PBS (2 X 5 min) and incubated with secondary antibody for 30 min. To detect

OPN expression 1 :600 mouse monoclonal anti-OPN antibody (Hybridoma Bank, Johns

Hopkins University, Baltimore, MD) diluted in PBS, and 1 :600 biotinylated anti-mouse IgG

(Vector, Burlingame, CA) diluted in PBS. Immunolocalization of BSP was performed with

150 rabbit polyclonal anti-BSP antibodies provideci by Dr. J. Sodek diluted in PBS and 1 :600

biotinylated anti-rabbit IgG diluted in PBS (Vector, Burlingame, CA). Detection of a-SMA

was done with 1 :25 mouse monoclonal anti-a-SMA antibody (Clone 1 A4; Sigma) diluted in

antibody diluting buffer @AKO Diagnostics Laboratones, Mississauga, ON) and 1 : 100

biotinylated anti-mouse IgG (Vector, Burlingame, CA) diluted in PBS.

Following the secondary antibody incubation, sections were washed thoroughly in

PBS (2 X 5 min), incubated with Strept ABC ComplexlHRP (PK-6100, Vectastain) for 30

min in a moist chamber, thoroughly washed in PBS (2 X 5 min) and incubated with

diaminobenzidine (SK4 100, Vector, Burlingarne, CA) for 15 min. This reaction was stopped

by gentle nnsing with distilled water ovemight. The Avidin in the Strept ABC Complex has

a very high affinity for biotin conjugated to the secondary antibody. This reaction has similar

specificity as the interaction between an antigen and antibody. However. unlike antigen-

antibody interactions, the Avidin-Biotin interaction is irreversible and is very high affinity.

Hence the procedure is very sensitive and specific (Hsu et al., 1981). Strept ABC is also

complexeci with Horseradish peroxidase (HRP), which enables the colorimetric visualization

of the proteins upon addition of the substrate, namely DAB-H,O,.

The intensity and the locdization of staining were then assessed semi-quantitatively

by assigning * for normal basai level of OPNBSP staining equivalent to unwounded

adjacent bone, + for weak staining that was less than the adjacent bone and t++ for strong

staining that was more intense than adjacent bone. O indicated no staining. Assessrnent was

done in eight zones: Zone 1-wound edge PL; Zone 2-middle of regenerating PL; Zone 3-

middle of regenerating AB; Zone 4- the outer surface of regenerated bone; Zone 5-AB 250

pm below the margin of the wound; Zone 6-PL 250 Fm below the margin of the wound; Zone

7-AB in unwounded (lingual side); and Zone 8-AB surrounding the incisor (Lekic et al.,

1996b; See Figs. 6 & 8). The mean and the standard deviation for each group and at each site

were calculated. However, there was no signifcant difference observed in zones 1, 5, 6, 7 and

8 regardless of the treatrnent type. Consequently, these data were not includeded in the results

sections.

2.5 Autoradioeraphy

Sections that had been irnrnunostained were quickly dehydrated by washing in 100%

ethanol, air-dried for about an hour. Slides were then dipped in full strength Kodak TO-2

(Eastman Kodak Co. , Rochester, NY), placed randornly in light-tight, dry boxes and exposed

for 2 weeks at 4OC. Following exposure, slides were developed in D- 19 developer (Eastman

Kodak) and counter-stained with haematoxylin and eosin through the emulsion.

The analyses were camed out with the aid of a light microscope (Laborlux K,

Leitz,FRG: 250X) and an intraocular grid system with 100 squares (final magnification of

each square: 25 pm X 25 pm). The presence of more than five grains surrounding or

overlaying the nucleus was considered as behg indicative of labelling (p<O.OO 1 ) (Lekic et al.,

1996b). Cellular proliferation was determined in seven zones: Zone 1-wound edge PL; Zone

2-middle of regenerating PL; Zone 3-rniddle of regenerating AB; Zone 4-the outer surface

of regenerated bone; Zone 5-PL 250 pm below the margin of the wound; Zone 6-PL in

unwounded (lingual side); and Zone 7-connective tissue surrounding the incisor (Lekic et al,

1996b; See Figs. 2 & 3). The numbers of labelled and unlabelled cells in each of these zones

were counted for each section on al1 the slides. For total ce11 counts, haematpoietic and

endothelial cells were not included. The total counts were used to denve the labelling index

for each zone, as LI=(# of labeiied celis/# of total celis) X 100. Labelled cells that were found

in close proximity (<25pm) were designated as clustered cells and may have a common

progenitor (McCulioch and Melcher, 1983b; Lekic et al., 1996a). Clonal growth of PL

progenitors was determined by calculating the % of clustering (clustering index) in each zone,

as CI=(# of clustered tells/# of labelled cells) X 100. The means for LI and CI data were

calculated for each zone examined. The data obtained for zones 5, 6 and 7 were exchded.

from the results sections since no difference was observed between the three groups

examined.

2.6 Morphometric assessment

Morphological features of O P K and BSP-stained sections (10 sections for each

experimentai condition at each post-wounding time point) were examined using a Leitz

Metallux- 3 microscope. An IBM PC cornputer equipped with a morphometry and

densitometry program (Bioquant Meg IV system; R and M Biometrics; Nashvilie, TN) and

a drawing tube adaptor, were ernployed for image analysis. The percentage of new bone area

fomed in the wound was determined by nnt digitizing the reversal line in the bone at the cut

edges which gave an estimate of the original wound margins. Second, the OPN or BSP

stained tissue in the regenerating bone cornpartment was digitized. These two areas were then

used to cornpute the percentage as %New bone area forrned = area of OPN or BSP/area of

wound X 100 (Lekic et al., 1996b). The mean 5 standard errors of the percentages were also

calculated for each group. Likewise, the widths of AB and PL were measured and mean 2

standard errors were cornputed for each group.

2.7 Statistical analyses

Analysis of variance was performed to evaluate the differences between the three

treatment groups with respect to labelling indices, clustering indices. morphometric and

immunostaining assessments in the different sites examined. Data for each type of treatment,

post-wounding tirne and exarnined site were considered as independent samples and were then

assessed by ANOVA (Sigma Stat for multiple parameters; Iandel Scientific. Ca) using

Dunnett's test. The variance was considered significant if pcO.05.

ter 3: M

This section is organized to descnbe the eKects of wounding alone, collagen vehicle

alone or BMP-7 incorporated into coliagen on regenerating periodontal tissues, thus enabling

us to compare and contrast the experimental groups more clearly and progressively from

wounding only to collagen to collagen plus BMP-7.

3.1 Effect of wounding

3.la Mitogenic and clonogenic activity

Assessment of the proliferative activities caused by wounding alone on the intact

tissues indicated that the LI was less than or equai to 2% for al1 the time points exarnined.

Hence, this was considered as the basai proliferative rate (Fig. 2a). LI reached between 3-6%

at day 5 in both mid regenerating PL and AB (Figs. 2b & 2c). The levels however, came

down to 2% or less in the later time points. There were no proliferating cells on the outer

surface of regenerating bone at any time point exarnined (Fig. 2d).

Clonal growth of the proliferating cells was observed only in the mid regenerating

bone at day 5 post-operatively (Fig 3c). There were no clustered proliferating cells found in

the PL (Figs. 3a & 3 b).

3. tb Assessment of the type of bone formation

Metachromatic staining with toluidine blue suggestive of cartilage formation was not

observed (i.e. dark bIue/purple staining), at the vanous tirne points in healing (Fig. 4a, 4c &

4e) and hence bone regeneration likely occured via the intramembranous pathway.

Repopulation of regenerating PL was evident around day 5 post-wounding and formation of

bone was evident at the 10 day post-wounding time point in the middle of the wound site

(Fig. 4c).

3. t c Evaluation of bone and fibroblastic markers

A representative photomicrograph of the irnmunostaining pattern of OPN in

regenerating and surrounding intact tissues can be seen in Fig. 5a. Quantitative assessment

of the OPN staining trend shows that it was expressed in the PL only at day 2 posî-surgically

(Fig. 6a) and there was no OPN staining in the intact, unwounded PL. In the mid-region of

the healing bone, OPN (Fig. 6b) was evident by 10 days post-wounding, but it was not seen

on the outer surface of the regenerating wound at any of the time points (Fig. 6c).

Staining for the late osteogenic marker, BSP, and corresponding bone formation are

evident in the representative photomicrograph in Fig 7a. BSP was not observed in the PL at

any sampling time (Fig. 8a), but was evident in the mid-regenerating bone by day 10 post-

operatively (Fig. 8b). BSP, like OPN, was also not expressed on the outer surface of the

regenerating wound at any of the time points (Fig. 8c).

In regard to the expression of a-SMA within the PL, no difference was observed over

time (See Fig 9 for representative staining pattern of the PL fibroblasts and endothelial cells).

3.1d Morphometric assessment of regenerating and intact tissues

Regeneration of PL width (nonnally about 100pm) was observed at day 10 post-

operatively (Fig. 1 Oa & 1 Ob) and was not sigruficantly different from adjacent intact PL. Bone

regeneration was cornpleted within the wound by day 2 1 post-surgically (Fig. 1 Od). The new

bone was contiguous with and no wider than the surrounding bone in the intact sites (Fig.

1 Oc).

3.2 Effect of collanen

3.2a Mitogenic and clonogenic activity

No significant difference of the LI at intact sites was observed in wounding only

controls (Fig. 2a). In the rnid-regenerating PL at post-operative day 5, the LI in the presence

of collaçen was significantly lower than that observed in the wounding only control (70% less;

p<O.OOl; Fig 2b). However in the mid-regenerating bone, the LI was about two-fold higher

compared to wounding only controls (p<0.001; Fig. 2c). Labelled cells were found on the

outer surface of the regenerating bone (Fig. 2d), a finding not noted in the wounding alone

controls.

As in the wounding only controls, there was no clonal growth of labelled cells in the

PL treated with collagen (Figs. 3a & 3b). The CI was sirnilar in collagen-implanted wounds

as controls, within the mid regenerating bone at the 5 day post-wounding time point (Fig. 3c).

The proliferating cells on the outer surface of the bone were not clustered (Fig. 3d).

3.2b Assessrnent of the type of bone formation

As with wounding only controls, there was no difference in the toluidine blue staining

intensity which would suggest cartilage formation (Figs. 4b & 40. Unlike wounding only

control, there was the formation of bone occurred on the outer surface, above the partially

resorbed implants at day 10 post-surgically. Resorption replacement of collagen was

completed by day 2 1 (Fig 4f).

3 . 2 ~ Evaluation of bone and fibroblastic rnarkers

The staining pattern for OPN in the presence of collagen only at 21 days post-

wounding is shown in Fig. Sc. No temporal or spatial differences were seen in the expression

of OPN compared to wounding only controls (Fig. 6).

An example of BSP staining with collagen treatment (day 21 post-wounding), is

shown in Fig. 7c. As with OPN, no difference in immunostaining was observed in cornparison

to the wounding alone control (Fig. 8). Likewise, immunostaining for the fibroblastic marker,

a - S M 4 did not Vary compared to controls over tirne (Fig. 9a & 9b).

3.2d Morphometric assessrnent of regenerating and intact tissues

There were no significant differences in morphomettic parameters between wounding

only controls and collagen-treated wounds (Kg. IO), suggesting that collagen did not affect

the PL width or promote bone healing.

3.3 Effect of BMP-7

3.3a Mitogenic and clonogenic activity

Increased cellular proliferation (3-6%) was obsewed as early as 2 days post-wounding

in samples treated with BMP, both 250pm below the wound site and in the mid repopulating

PL (Data not shown). However, labelling indices were similar for other examined sites.

including the cut-off margins of PL (Fig. 2a).

By day 5, there was exuberant proliferative aaivity in and around wounds treated with

BMP (Fig. 2). Increased labelling indices were observed (2-fold greater than controls;

peO.00 1) in the wound edge of the PL in BMP-treated samples (Fig. 2a). Moreover, about

60% of the labelled cells at the wound edges of the PL were clustered (Fig. 3a). In contrast,

clustering which is suggestive of clonal growth, was not observed at this site for any of the

time points in either of the control groups (See sections 3. la & 3.2a).

There was a 30% decrease in the LI @<0.001) in the BMP treated group compared

to the wounding only control in the mid region of the healing PL at post-operative day 5 (Fig.

2b), but clonal growth of proliferating cells was only present in the BMP group (Fig. 3b).

Likewise, the LI at post-surgical day 5 in the rnid regenerating AB in the presence of BMP

was approximately 30% (p<0.00 1) less than the collagen only control levels (Fiç. 2c), while

the clustering index was significantly higher (1.5 fold; p<0.05 ; Fig. 3 d). In addition, there was

evidence for proliferation and clonal growth of putative osteogenic precursors on the outer

surface of the wound in the BMP administered groups (Figs. 2d & 3d).

At 10 days after wounding, the LI in BMP-treated samples in the mid- regenerating

PL retumed to basal levels, but about 60% of the labelled cells were clustered (Figs. 2b &

3b). At the margins of the wound in the PL and on the outer surface, the LI remained high

in the presence of BMP (up to 2 times higher than the controls; p<O.OOl; Figs. 2a & 2d). In

the rniddle of the regenerating bone, the LI was similar in both implant groups (Fig. 2c), but

only BMP-treated samples exhibited clonal growth of actively proliferating cells (Fig. 3c). It

was also notable that bone formation had commenced by this time (day 10 post-wounding)

in al1 the treatment groups while the number and distribution of proliferating cells was

strikingly different in the no treatment control and BMP treated sample (Fig. 1 l a & 1 Ib).

There were higher Ll's and CI'S in the presence of BMP-7. On the outer surface of the wound,

the LI were similar for both implant groups (Fig. 2d), but clustered precursors were seen ody

in BMP treated groups (Fig. 3d).

At 2 1 days post-operatively in BMP-7 treated animals, increased LI3 were measuredin

the newly formed bone (Fig. 2c) and on the outer surface of the bone (Fig. 2d) but these

increases were not seen in controls. The proliferating cells found within the endosteal spaces

of the newly formed bone on the outer surface aiso showed greatly increased clustering (Figs.

3d, 1 1c & 1 Id). Notably, clona1 distribution of proliferating cells was not observed in the

controls at any of the examined time points (Fig. 3d). At 60 days after the wounding the LI's

anad CI'S of BMP-7 treated rats were reduced to that of controls in al1 the newly healed sites

(Figs. 2b, 2c, 2c, 3b. 3c & 3d). There was no significant change in the LI's on the lingual

(non-wounded) side or in comective tissues remote £kom the drill site between or within these

three groups.

3.3b Assessrnent of the type of bone formation

During the early stages of bone regeneration there was no indication of cartilage

formation within the wound site (Fig. 4d). However, there was staining suggestive of cartilage

present in sites below the wound and on the outer surface of the new bone in the wound

(Figs. 4g & 4h). Another noticeable difference compareci to collagen only controls was related

to the slow resorption of collagen in the BMP treated groups (Figs. 4f & 4g). Collagen was

still present at post-operative day 2 1 in the BMP groups.

3 . 3 ~ Bone and PL markers

Immunostaining for OPN in the intact and regenerating tissues is shown in Figs. Sb

& 5d. Evaiuation of OPN expression within the regenerating PL of the expenmental animais

treated with BMP showed that on post-wounding days 2 and 5, the expression of the early

bone marker OPN was about 3 0% higher than the controls @V).OO 1 ; Fig. Ga). However, in

the mid areas of regenerating bone, the staining was the same, regardless of the treatment

(Fig. 6b). On the outer surface of the new bone there were high levels of OPN

irnmunostaining (post-surgical day 5-2 1) with BMP-7 treatment (Fig. 6c), but this staining

cornes d o m to basai levels by day 60 post-operatively, as the bone overgrowth on the outer

surface of the defect was no longer evident.

iinmunolocalization for BSP, at the post-wounding days 10 and 2 1, in the healing and

intact tissues in BMP treated groups is shown in Figs. 7b & 7d. There was strong expression

of BSP @os-wounding days 10-21) on the outer surface of bone in the BMP treated samples

(Fig. 8c), but by post-operative day 60, as with OPN expression, BSP staining returned to

basal levels. OPN and BSP staining did not Vary between the groups in the intact (Le. non-

wounded) areas.

There was no significant difference in the distribution or number of or-SMA positive

PL fibroblasts between the various groups (Fig. 9a & 9b). Also, smooth muscle cells

surrounding blood vessels expressed this protein (Fig. 9c) but again, there were no observable

changes induced by BMP.

3 3 d Morphometric assessrnent of regenerating and intact tissues

There was no change in the width of the PL either in the wounded or intact sites (Fig.

10). At day 21, bone area was about 2-3 times greater @<0.001) in the BMP-7 treated

sarnples compareci to the coiiagen only and no implant sarnples (Figs. 10c & 10d). However,

these measurements were reduced to basal values at day 60. There were no changes in soft

tissue and bone meanirements on the non-wounded (lingual) side nor at sites away from the

wound.

Figure 2: Proliferation profile &Ils) of the 'H-Tdr labelled cells. No detectable labelling is

denoted by *.

Key for the sites analyzed: Diagramatic longitudinal representation of the rat

periodontium showing alveolar bone (AB), periodontal ligament (PL), cementum (C), buccal

or wounded side (B), lingual or unwounded side (L) and the sites analyzed: wound edge PL

(Site l), mid regenerating PL (Site 2), mid regenerating AB (Site 3), outer surface of

regenerating AB (Site 4) & 250pm below wound in AB (Site 5).

(Za): LI levels within the intact wound edge PL.

(2b, 2c & 2d): LI levels in the regenerating tissues.

Figure 3: Clona1 growth of ' ~ - ~ d r labelled cells. No detectable clustering is denoted by *.

Key for the sites analyzed: See Fig. 2 above.

36

(sa): CI levels within the intact wound edge PL.

(3b, 3c & 34: CI levels in the regenerating tissues.

Figure 4: Histologicd sections stained with toluidine blue. Alveolar bone (AB); cementum

(C); dentin (D); periodontal ligament (PL). (Final rnagnification of 3a-3f l6OX, 3g: 120X

3h: 75X)

(4a): 2 day post-wounding specimen treated with no implants showing the wound site

(WS) and the cut margins of the defect (large arrow heads). Portion of PL and AB were

selectively deleted during the wounding procedure.

(4b): 2 day post-wounding, BMP treated specimen illustrating the implant (1)

occupying the wound site and the cut margins of the defect (large arrow heads).

(4c): 10 day post-wounding h m the anVnals that received no implants. Note that the

regeneration of PL has been almost cornpleted (NP) and formation of new bone (NB) is

evident in the middle of the wound site. The wound margins (large arrow heads) can be

recognized at this time point also.

(4d): 10 day post-wounding specimen from the BMP administered group, showing

also signs of complete regeneration of PL (NP) and commencing regeneration of bone (NB)

at the outer surface, above the implant 0). Here also the cut margins (large anow heads) can

be seen.

(4e): No implant, 10 day post-wounding specimen, showing fully regenerated alveolar

bone (M3) and penodontal ligament (NP).

(41): Collagen only, 21 day post-wounding sections with complete regeneration of

penodontal ligament (NP) and dveolar bone (NB).

(4g & 4h): BMP treated, 21 day post-wounding sections with cornpletely regenerated

periodontal ligament (NP) and alveolar bone (NB). Note the tremendous amount of new bone

on the outer surface (OS) that goes well beyond the normal margins (large arrow heads) of

the "old" bone. Also note the metachromatic staining suggestive of chondrocytes (small arrow

heads) and the large endosteal spaces (E) found on the outer surface.

Figure 5: Histological sections imrnunostained for OPN (small arrow heads). Note the

positive staining in bone (AB) and cementum (C), but not in dentine 0) or penodontal

ligament (PL). (Final magnification of 4a-4~: 140Y and 4d: 100X).

(Sa): No implant, 10 day post-wounding specimen. The newly formed bone (NB) is

evident in the mid wound site.

(5b): BMP treated, 10 day post-wounding specimen. Note the presence of partially

resorbed implant (1) in the wound site and the new bone (NB) on the outer surface.

(Sc): No implant., 2 1 day post-wounding specimen, illustrating com pl ete regeneration

of AB and PL. Pattern of OPN expression in the newly regenerated bone (NB) resembles the

surrounding intact tissue, and is compact.

(Sd): BMP treated, 2 1 day post-wounding specirnen, showing enonnous over growth

of new bone (NB) that is less compact and has larger number of endosteal spaces (E),

compared to the control in 4c. Also note the heavy OPN staining along the cut margins (large

arrow heads) .

Figure 6: Semiquantitative assessrnent of OPN expression. No det ectable staining is deno ted

by *.

Key for the sites analyzed: See Fig. 2 above.

(6a): Quantification of OPN expression in the healing PL.

(6b & 6c): Quantification of OPN expression in the healing AB.

Figure 7: Irnrnunolocalization of BSP (small arrow heads) is notable in bone (AB) and

acellular cementum (C), but noi in dentine @) or periodontal ligament (PL). (Final

magnification of Sa-Sc: 140% and 5d: 100X).

(7a): No implant, 10 day post-wounding specimen. The newly fonned bone (NB) is

stained for BSP at higher levels cornpared to the surrounding intact tissue.

(7b): BMP treated, 10 day post-wounding specimen. Note the presence of partially

resorbed implant (X) in the wound site and the heavy BSP expression in the newly formed

bone (NB) above the implant.

(7c): No implant, 21 day post-wounding specimen expressing basal levels of BSP in

newly regenerated bone (NB).

(7d): BMP treated, 21 day post-wounding specimen, illustrating the same features

seen in Fig. 4d. Note the increased expression of BSP on the outer surface and along the cut

margins (large arrow heads). New bone (NB); endosteal space (E).

Figure 8: Semiquantitative assessrnent of BSP expression. No detectable staining is denoted

by *.

Key for the sites analyzed: See Fig. 2 above.

@a): Quantification of BSP expression in regenerating PL .

(8b & 8c): Quantification of BSP expression in the regenerating AB.

Figure 9: Histological sections showing the immun0 localization of a-SMA (arrow heads)

in penodontal tissues. (Final magnification of 6a-6d: 400X).

(91 & 9b): PL fibroblast cells expressing a-SMA.

(9c): Smooth muscle ceils surrounding blood vessels or paravascular fibroblasts stain

positive for a-SMA.

Figure 10: Morphometnc assessrnent of the percentage ofbone formed within the wound site

and the widths of soft and rnineralized comective tissues. Absence of any detectable tissue

is denoted by *.

Key for the sites analyzed: See Fig. 2 above.

(10a): PL width measurements at the wound margin.

( lob & 10c): Width measurements of regenerating PL and AB.

(10d): Arnount of new bone fonned.

Figure 11: Hïgh magnification photornicrographs of immunostained and 'H-~dr labelled

histological sections in the middle of regenerating wound (final magnification of 2a-2d:

640X).

(1 la): 10 day post-wounding specimen corn no-implant group, immunostained for

OPN (small arrow heads). Note the newly forrned bone (NB) in the mid regenerating bone

and the radio-labeiied proliferatiing ceUs (iarge arrow heads). Alveolar bone (AB); periodontal

ligament (PL).

(1 1 b): 10 day post-wounding specimen treated with BMP-7. Immunolocalization of

OPN (small arrow heads) is evident at the cut margins of alveolar bone (AB). Note the

partially resorbed implant and strikingly higher LI and CI of proliferating cells (large amow

heads).

( l l c & l ld): 21 day post-wounding specimen from the BMP treated group,

imrnunostained for OPN (1 Id; small arrow heads) and BSP (1 Id; small arrow heads). Note

the large endosteal space (E) in the newly fomed bone (NB) and the proliferating cells (large

arrow heads) found within.

O O O O O Co

O O m .t N

(%) xapu! 6uya lsn l3

C O .- II) m Q) I a X 0

m o z

Figure 9

( 4 ; ) pauiJo4 auoq m a N

Q) a

F .E n - z o o

Chanter 4: Discussion

In this study 1 have show that BMP-7 promotes regenerative processes in the early

stages of periodontai healing and stimulates the proliferation and clonal growth of putative

osteogenic progenitors. These processes lead to extensive outgrowth of bone at the wound

side. Despite its potent osteoinductive actions, BMP-7 did not perturb PL homeostasis and

no ankylosis was observed. Also, the intact sites on the lingual side exhibited little, if any,

changes in proliferation, ceIl differentiation and morphology. 1 found that the effect of BMP-7

was transitory, as the greatly increased bone and proliferation retumed to basal levels by 60

days after wounding.

4.1 BMP-7 effects on initial stages of oeriodontal healing

Tissue regeneration often follows a sequence of cellular events that rnirnics embryonic

development (Rosen and Thies, 1992). BMPs and other hormones/growth factors act as

signalling molecules to orchestrate a wide array of fùnctions involved in reparative processes

including chemotaxis, ceIl proliferation, differentiation and increased metabolism of

repopulating cells (Graves and Cochran, 1994; Hogan, 1996).

In this investigation, the increased proliferation at 2 days after wounding in and

around the site that received BMP-7, suggest that BMP acts as a potent mitogen for

repopulating cells derived fiom the surrounding intact tissue. Previous studies have show

that when multinucleated cells (MNCs) are present, the outer surface of the collagen implant

is initiaily resorbed even without BMP (Nguyen et al., 1997). In my study, MNCs were also

observed in close association with the implants fiom days 2-10 (See also section 4.4 below).

Hence, BMP could have been released by MNC-mediatd degradation of the implants as early

as day 2 nom the outer surface of the collagen vehicle. 1 suggest that this released BMP-7

rnay have promoted the migration and proliferation of cells from surrounding intact tissues.

The increased labelling and clustering indices from day 5- 10 at the wound edge and

in the rniddle of the regenerating PL and AB indicate that BMP-7 augments repair processes

in bone by promoting proliferation of progenitor cells in these regions. Stimulated progenitor

cells may migrate ont0 the outer surface of the wounded bone and continue to exhibit clonal

growth in the presence of BMP-7 fkom day 5-2 1. Hence, BMP-7 may promote chernotaxis

and proliferation of precursors involved in bone regeneration. However, the lineage and

origins of these progeniton are not known.

4.2 BMP-7 effects on PL homeostasis

Despite the massive growth of bone induced by BMP-7. PL width was maintained.

This was interesting for at least two rasons. First, as shown by others, PL cells may actually

have the capacity to inhibit osteogenesis in vitro (Ogiso et al., 199 1 & 1992) and Nt vivo (Line

et al., 1974). and this capacity may permit the PL to maintain its domain structure. In my

studies PL width was tightly maintained even in the presence of extensive bone growth

nearby. Second, the data showed increased ce11 proliferation with strong clonal growth within

the PL in response to BMP-7. Yet, despite this strong proliferative response, PL healing

assessed morphologically took more than 10 days, a time fiame that has been reporied

previously without BMP-7 (Lekic et ai., 1996b & 1996~). This raises the question as to what

was the ultimate fate of the proliferating cells within the PL (eg. osteoblast, PL fibroblast,

cementoblast). Given the findings suggesting that PL regeneration was not stimulated by

BMP-7, I suggest that these proliferating cells did not contribute to PL healing. Moreover,

1 did not observe any changes in the number of a-SMA expressing cells fùrther suggesting

that the progeny of these proliferating cells did not contribute significantly to the PI fibroblast

population.

In addition, BMP-7 had a profound effect on LI and CI in the mid regions of

regenerating bone at 2 1 days after wounding, presumably because of the prolonged release

from the partially resorbed collagen vehicle. However by this tirne, regeneration of t he PL was

completed and proliferative activities (LI anci CI) in the mid regions of regenerated PL were

similar regardless of the treatment. Hence, even with the putative sustained release of BMP-7

from the remaining collagen vehicle, PL homeostasis was not perturbed.

Given the observed stimulation of bone formation, one might conclude that BMP-7

induced differentiation of cells in the PL that ultimately contnbuted to bone formation and

that these cells may be responsible for the high levels of staining for OPN, an early bone ce11

phenotypic marker. However, OPN is also expressed by non-osteogenic ceIl types such as

activated macrophages, transformed cells and epithelial cells (Denhardt and Guo, 1993; Sodek

et al., 1992). Furthermore, up-regulation of OPN production is observed as a part of the

wound healing process in necrotizing tissue injury and in surgically created defects, by

macrophages (McKee and Nanci, 1996; Muny et ai., 1994). In other studies using this

wound mode1 (without BMP treatment)(L,ekic et al.. 1996b & 1996c), high levels of OPN

expression were associated with proliferating cells in experimental groups in which the PL

was ~reserved during the wounding procedure (unlike what was done here). As macrophages

and lymphocytes do not contribute to the precursor cell populations in PL (Gould et al.,

1980), the increased OPN expression may be attributable to PL cell subpopulations that are

highly proliferative, but not necessarily osteogenic, at least within the PL. In my study,

increased OPN production and labelling indices were also observed in the groups

adrninistered BMP, hence it must be recognized that these data could be due to the

stimulation of the above mentioned PL ceIl subpopulation.

Overall, BMP-7 stimulated the proliferation and, differentiation of PL cells and

possibly the recmitment of macrophages to aid in the initial stages of wound healing. 1 did not

determine whether PL cells actually migrated out of the PL to contribute to bone formation

and this needs to be done (eg. pulse label cycling cells with 'H-Tdr and then following over

time to look for labelled osteoblasts). Despite the possibility that the PL rnay have contributed

some cells to adjacent bone growth, it is also notable that 1 did not observe bone formation

within the PL or reduction of PL width. Although as mentioned above, driring the early stages

of healing (days 2 & 9, there were high levels of OPN expression in regenerating PL after

treatment of BMP, there was no evidence of BSP expression at any time point and hence

there was no evidence of mineralization within the PL.

Precursor cells within the PL may be capable of forming al1 the connective tissue types

of the penodontium (eg. bone, ligament, cementum), especially during the early stages of

tooth development (Perera and Tonge, 198 1) and following wounding (Gould et al., 1977 &

1980). Hence, 1 expected that some bone formation might have occurred within the PL

treated with BMP-7, but this did not occur. Others have shown that PL cells have the capacity

to make bone-like tissue or that at least cells within the PL possess osteogenic characteristics

(Cho et al., 1992; Gould et al, 1980; McCulloch and Melcher, 1983a). Indeed under the

intluence of HEBP (Lekic et al., 1997), Pl cells will form islands of bone demonstrating that

osteogenesis by PL cells is possible. That this did not occur here suggests that BMP

stimulation might not have overcome the "anti-osteogenic" effects of PL cells. Altematively,

the BMP responsive cells may, as suggested above, have migrated away frorn the site to form

bone extemal to PL. In any event, while HEBP seems to be able to stimulate proliferation and

osteogenesis in quiescent osteogenic precursors in the PL (McCulloch and Melcher, l983a;

McCulloch, 1989), under the conditions used here BMP-7 couid not do this.

4.3 BMP-7 stimulation of bone ~ r o w t h in reeeneratine periodontal tissues

Since the advent of recombinant DNA technology, BMP-7 (OP-1) has been studied

extensively for it's bone inducing activity. BMP-7 has been s h o w to have a potent bone

inducing effect in several sites including non-osteogenic (intramuscular: Sampath et al., 1992)

and osteogenic sites (periodontium: Ripamonti et al., 1994; long bones: Cook et al., 1995a

& 1995b). In this study, bone formation began in al1 the groups at 10 days after wounding.

Nascent bone was clearly evident in the middle of the wound site in the no implant controls,

whereas it was found above the collagen in the implanted groups. However, there was no

significant difference in the amount of new bone fomed or in the expression ofbone proteins

in the wound area, between the groups at day 1 0.

On the other hand, remarkable differences became evident at 2 1 days d e r wounding.

At this time point bone formation seemed to have ceased in both the wntrol groups, as shown

in previous studies (Lekic et al., 1996b & 1996~). However in the BMP groups, about 50%

of the proliferating precursors were clustered on the outer surface of the wound, while

actively expressing higher levels ofbone proteins (OPN & BSP) compared to the surrounding

intact tissue. This suggests that BMP was still acting as a powefil mitogen for selective

progenitor ce11 populations that appeared to be comrnitted to the osteoblastic ce11 lineage, thus

leading to the rather extensive bone formation (3 times as much as the controls) observed in

BMP treated wounds.

Interestingly, toluidine blue staining showed that the collagen implants were fûlly

resorbed in the vehicle alone controls at day 21, but in the BMP-7 treated groups, the

implants were only partially resorbed and were surrounded by nascent bone. It appears as

though replacement and resorption of the implant was taking place in these specirnens.

Furthemore, the presence of implant residues fùrther suggests the possibility of ongoing slow

release of BMP, even at day 21, that could cause a sustained stimulation of proliferating

precursors within the endosteal spaces of the newly forrned bone.

The stimulatory effect of BMP-7 wore off sometime between post-wounding days 2 1

and 60 and the less compact, "new" woven bone that was formed at 21 day post-wounding,

was then resorbed by MNCs followed by the deposition ofmature compact bone. which was

then remodelled to maintain the normal boundary of the buccal side AB, by post-wounding

day 60.

4.4 BMP-7 effects on MNCs

It has been found that BMP-7 increases the formation of tartrate-resistant acid

phosphatase (TRAP) positive, MNCs in a dose dependent rnanner, in vitro (Hentunen et al.,

1995). But the residual remains ofBMP-7 impregnated collagen implant even at 2 1 days f i e r

the wounding indicate that the activity of MNCs was probably retarded in the presence of

BMP. Notwithstanding BMP-7's effect on the development and activity of multinucleated,

TRAP positive cells, MNC forrnztion occurred in collagen implants that did not contain

BMP-7 (Nguyen et al., 1997). Thus it is probable that the collagen vehicle, and not the BMP-

7, induced the formation of or attracted multinucleated cells to the site. In any case, these

MNCs were not labelled with 'H-Tdr. An attempt was made to evaluate the effect of BMP-7

on such cells in vivo using TRAP staining, but was unsuccesstùl. This was possibly due to the

fixation a d o r embedding protocols used here.

Prolonged rernodelling and slow resorption of the collagen implant has also been

described previously by Todescan et al. (1 994) and by Nguyen et al. (1 997). Overall, BMP-7

down-regulates the differentiation of specialized ce11 types that are incapable of dividing,

while up-regulating the proliferation of specific populations of putative osteoprogenitors.

4.5 Endochondral Vs. Intramernbranous bone formation

Previous reports on BMPs show that they usually induce endochondral bone

formation (Urist, 1965; Sarnpath and Reddi, 198 1). However, recent studies conducted with

BMP-7 (OP- 1) show that it induces both endochondral and intramembranous bone formation

by causing primitive mesenchymal precursors to diflerentiate into chondroblasts followed by

the development of osteoblasts (Asahina et al., 1993 & 1996). Also in the presence of BMPs,

wound healing in calvarial and periodontal defects in other animai models apparently occurred

via intrarnembranous bone formation (Ripamonti et al., 1994; Sigurdsson et al., 1995). As

s h o w here, the regeneration of bone within the defect likely came about largely by the

intrarnembranous ossification pathway as elucidated by toluidine blue staining.

However, there was evidence for ectopic cartilage formation at sites located posterior

to the wound, by the incisor tmnk. This cartilage began to resemble calcified cartilage (day

2 l), and was later remodelled to form mature bone (day 60). This interesthg effect of BMP-7

on differentiation has also been observed in periodontal fenestration defects treated with

BMP-2 (King et al., 1997). This further reiterates the conclusions of Asahina et ai., (1 993 &

1996), who found that BMP-7 stimulates the proliferation and diflerentiation of both

osteoprogenitors (differentiate into osteoblasts, directly), as well as primitive mesenchymal

precursor cells (differentiate into chondroblasts, first).

In my studies, the cartilage that formed distant to the implant site was suggestive of

endochondral bone formation (or simply induction of cartilage islands) and could possibly

have been due to BMP-7 that "spilled-over" during surgical implantation. However, the fact

that King et al. (1997) also made similar observations may suggest another explmation. It

could be speculated that BMP-7 difises out fiom the collagen vehicle in al1 directions. The

molecules that difise out and accumulate inside or toward the wound may act upon the more

"specialized" or osteogenic precursors thought to exist within the PL (Cho et al., 1992; Gould

et al, 1980; McCulloch and Melcher. 1983a) and surrounding bone, and may thus eiicit

intramembranous ossification. The BMP molecules that difised away fiom the PL and bone

may attract more undifferentiated mesenchymal progenitors fiom the muscular layers that lie

over the mandible, thus leading to cartilage formation analogous to extra-osseous implant or

test sites (eg. subcutaneous or intra muscular) used by others (Reddi et al., 1987; Urist,

1965). Hence, the effects of BMP-7 could be site specific. In this regard, when BMP is placed

in a site that could contain "pre-prograrnrned" potentially osteogenic cells, such as the PL or

caharium, a sequence of intramembranous ossification might ensue. Aiternatively, when BMP

is placed in a site where bone formation will not ordinady occur (eg. intra-muscular and sub-

cutaneous sites), endochondral bone formation or cartilage formation may occur. These data

suggest then that BMP-7 as used here. may be acting on different target cells resident in the

different sites. Alternatively, it is also possible that this could be a concentration dependent

phenornenon. BMP concentration distant fiom the site would be lower than right at the

surgical site and at that low concentration may preferentially induce chondrogenesis.

However, in light of others' data (Ripomonti et al., 1994; Sigurdson et al., 1995) this is iess

likely .

Even though no significant increase in the expression of OPN or BSP was observed

in the BMP-7 treated groups versus the controls, BMP-7 must have promoted differentiation

of osteoblasts because of the trernendous amount of bone formed in BMP-7 treated group.

Nonetheless, a recent report on the effect of BMP-7 on fetal rat calvarid cells in vitro showed

that BMP-7 stimulates the expression of various bone rnarkers including OPN and BSP,

several fold higher and the failure to see higher levels here is somewhat puuling (Li et al..

1996). In any case, along with other polypeptide growth factors, BMPs are known to initiate

differentiation of osteoprogenitor cells (Knutsen et al., 1993; Reddi et al., 1987), besides

stimulating proliferation of these cells.

4.6 Conclusions

Signifiant levels of OPN staining and elevated proliferative levels were evident in the

healing PL treated with BMP-7, but this neither accelerated healing nor lead to osteogenesis

within PL (PL width was maintained and no BSP expression was observed within the PL).

At least two deductions could be made from these observations. First, BMP promoted the

initial wound healing stages by stimulating chemotaxis, mitogenesis and differentiation.

Second, these cells did not contribute to PL regeneration, but could have rnigrated out into

the alveolar cornpartment and participated in the massive over-growth of bone there.

As mentioned earlier, proliferation and clonal growth were elevated within the

regenerating PL and alveolar compartments in the presence of BMP-7, but the effect was

much pronounced in the latter, leading to the exuberant bone forming activity that was

continuous even at post-wounding day 2 1, while not altering the physiological domains of PL.

This massive induction of bone growth reverted to basal levels at 60 days f i e r wounding.

Hence, the BMP-7 induced excess bone formation was only temporary. This underscores the

biological tendency for tissues to maintain their own domains in order not to "invade"

adjacent temtory, thereby maintaining homeostasis.

4.7 Clinical relevance and Future directions

Current penodontal therapy mainly focuses on the removal of etiological factors and

halting of disease progression. Complete regeneration of lost periodontal tissues and

restoration of their function has not been achieved yet. This may be because in part,

endogenous growth factors are present in insufficient quantities to optimally induce

penodontal regeneration. This is one of the rationales for the exogenous application of

polypeptide hormones or other agents that might augment the effects of naturaiiy occumng

growth and differentiation factors.

The severe bone destruction around teeth caused by periodontal disease may be

analogous to a "critical size defect" that does not spontaneously regenerate. Others have

attempted to develop an experimental critical size periodontal defect (Nguyen et al., 1997)

and this or other such systems may be used to determine whether BMP (or other factors)

would actually be usefùl for regenerative treatment following the arrest of penodontal disease.

However, while BMP-7 may be usefùl for induction of bone formation, it rnay not stimulate

growth and differentiation of other tissues within the periodontium. Nonetheless the profound

effects on bone regeneration suggest that the use of BMPs (in particular, BMP-7) may be a

promising adjunct to conventional and regenerative penodontal therapy.

Amar, S. 1996. Implications of cellular and rnolecular biology advances in penodontal regeneration. The Anatomicai Record. 245: 3 6 1-3 73.

Arora, P. and C.A.G. McCulloch. 1994. Dependence of collagen remodelling on a a- smooth muscle actin expression by fibroblasts. J. Cell Physiol. 159: 16 1 - 175.

Asahina , i., T.K. Sampath, 1. Nishimura and P.V. Hauschka. 1993. Human Osteogenic protein- 1 induces both chondroblastic and osteoblastic differentiation of osteoprogenitor cells derived fiom newbom rat calvaria. J. ceIl Biol. l23:92 1-933.

Asahina , L, T.K. Sampath and P.V. Hauschka. 1996. Human Osteogenic Protein-1 induces chondroblastic, osteoblastic and adepocytic differentiation of clonal murine target cells. Exp. ceIl Res. 222:38-47.

Aukhil, L, K. Nishimura and W. Fernyhagh. 1990. Experimental regdation of the periodontium. Crit. Rew. Oral Biol. Med. 1: 10 1-105.

Baxter, RC. 1991. Insulin-like growth factor (IGF) binding proteins: the role of semm IGFBPs in regulating IGF availability. Acta. Paed. Scand. (Suppl) 372: 107-1 14.

Becker, W., S.E. Lynch and U. Lekholm. 1992. A comparison of ePTFE members alone or in combination with platelet denved growth factor and insulin-like growth factor-1 or demineralized fieeze-dned bone allograft in promoting bone formation around immediate extraction socket implants. J. Penodontol. 63:929-940.

Becker, W., B.E. Becker and R Caff'se. 1994. A comparison of demineralized fieeze- dried bone and autologous bone to induce bone formation in human extraction sockets. J. Periodontol. 65: 1 128-1 133.

Becker, W., M.R Urist, LM. Tucker, B.E. Becker and C. Ochsenbein. 1995. Human demineralized freeze-dried bone: Inadequate induced bone formation in athmic mice. A prelirninary report. J. Priodontol. 66: 822-828.

Beersten, W. 1987. Collagen phagocytosis by the fibroblast in the periodontal ligament of mouse molar during the initial phase of hypofùnction. J. Dent. Res. 42: 1708- 17 12.

Bianco, P., G. Siberstrini, J.D. Termine and E. Bonucci. 1988. Immunohistochemical localization of in developing human and calfbone using monoclonal antibodies. Calcif. Tissue. Int. 43: 155-161.

Bianco, P., L.W. Fisher, M.F. Young, J.D. Termine and P.G. Robey. 1991. Expression

of bone siaioprotein (BSP) in deveioping human tissues. Calcif Tiss. Int. 49:42 1 -426.

Bosshardt, D.D. and H.E. Shroeder. 1996. Cemetogenesis reviewed: A cornparison between human premolars and rodent moiars. Anat. Rec. 245(2):267-292.

Bowers, G., B. Chadroff, R Camevale and J. Mellonig. 1989. Histologic evaluation of new hurnan attachment apparatus in humans, Part m. J. Periodontol. 60:683-693.

Bowers G., F. Felton, C. Middleton, D. Glynn, S. Sharp, J. Mellonig, R Corïo, J. Emerson, S. Park, J. Suzuki, S. Ma, E. Romberg & A.H. Reddi. 1991. Histologic cornparison of regeneration in human intrabony defects when osteogenin is cornbined with dernineralized freeze-dried bone allograft & with purified bovine collagen. J. Periodontol. 62:690-702.

Bronckers, A.L.J.J., S. Gay, R D . Finkelman and W.T. Butler. 1987. Developmental appearance of Gla proteins (OC) and alkaline phosphatase in tooth gems and bones of the rat. Bone. Miner. 2:36 1-366.

Butler, W.T. 1989. The nature and significance of OPN. Comect. Tiss. Res. 23: 123-136.

Cameron, I.L. 1970. Ce11 renewal in the organs and tissues of non-growing adult mouse. Texas Reports of Biology and Medicine 28: 203-248.

Carlsson, J. 1983. Microbiology of plaque. In: Lindhe, I. (ed.), Text book of clinical periodontology. Munksgaard, W .B. Saunders, Philadelphia, pp. 125- 147.

Caton, J. and S. Nyman. 198 1. Histornetric evalution of periodontal surgery III. The effect of bone resection on the comective tissues attachrnent level. J. Periodontal. Res. 52: 405- 409.

Chai, Y., A. Mah, C. Crohin, S. Groff, P. Bringas Jr.,T. Le, V. Santos and H.C. Slavkin. 1994. Specific transfoming growth factor P subtypes regulate embryonic mouse Meckel's cartilage and tooth development. Dev Biol. 162: 85- 103.

Chen, J., E.S. Shapiro, J.L. Wrana, S. Reimers, J.N.M. Heersch and J. Sodek 1991 - Localization of bone sialoprotein ( BSP ) expression to the site of rnineralized tissue formation in fetal rat tissue by in situ hybridization. Matnx. 11: 133- 143.

Chen, P., J.L. Carrington, R.G.J. Hammonds and A.E. Reddi. 199 1. Stimulation of chondrogenesis in limb bud mesoderm celis by recombinant human bone morphogenetic protein-2B (BMP-2B) and modulation by transforming growth factor4 1 and 02. Exp. Ce11 Res. 195: 509-5 1 5.

Cben, JO, C.A.G. Mcculloch and J. Sodek 1993. Bone sialoprotein in developing porcine dental tissues:Cellular expression and cornparison of tissue localization with osteopontin and osteonectin. Arch. Oral Biol. 38:24 1-249.

Cho, M-I.,, W-L. Lin, A. Moshier and P.R Ramakrishnan. 1992. In vitro formation of rnineralized nodules by penodontal ligament cells fiom the rat. Calcif Tiss. Int. 50:459-467.

Cook, S.D., J.E. Dalton, E.H. Tan, T.S. Whitecloud and D.C. Rueger. 1994. in vivo evaluation of recombinant human osteogenic protein-l implants as bone gr& substitute for spinal fusions. Spine. 19(15): 1655- 1663.

Cook, S.D., M.W. Wolfe, S.L. Salkeld and D.C. Rueger. 1995. Recombinant human osteogenic protein- 1 (rh OP-1) heals segmental defects in non-human primates. J. Bone Joint Surg. 77A:734-750.

Cook, S.D., S.L. Salkeld and D.C. Rueger. 1996. Use of recombinant human osteogenic protein-1 for repair of osteochondral defects. J. Long-Term Effects Med. Implants ( In Press

-

Cook, S.D. and D.C. Rueger. 1996. Osteogenic protein-l : Biology and applications. Clin. Orthop. Rel. Res. 324:29-3 8.

Davidsan, D. and C.A.G. McCulloch. 1986. Proliferative behaviour ofperiodontal ligament ce11 populations. J. Period. Res. 21: 414-428.

Denhardt, D.T. and X. Guo. 1993. Osteopontin: A protein with diverse functions. FASEB J. 7: 1475- 1482.

Ebeling, P.R, J.D. Jones, W.M. O'FalIon, C.B. Jones and B.L. Riggs. 1993. Short t e m effects of recombinant human insulin-like growth factor-1 on bone tum over in normal women. J. Clin. Endocrinol. Metab. 77: 1384- 13 87.

Egelberg, J. 1987. Regeneration and repair of penod tissues. J. Period. Res. 22: 233-242.

Evans, G.S., C.S. Potten. 199 1. Stem cells and the elixir of life. Bioassays. 13: 13 5- 13 8.

Fernley, H.N. 197 1. Mammalian alkaline phosphatase. In Boyer. P.D. (ed.): The enzymes. Vol 4, New York, Acadernic Press pp. 4 17-447.

Fisher, L.W., O.W. McBride, J.D. Termire and M.F. Young. 1990. Human bone sialoprotein. J. Biol. Chem. 2652347-325 1.

Flechtenmacher, J., K. Huch, E. J., M.A. Thonar, J.A. Mollenhauer, S .R Davies, T.M.

Schimid, W. Puhl, T.K. Sampath, M.B. Aydetle and K.A. Kue Hner. 1996. Recombinant human osteogenic protein-1 is a potent stimulator of the synthesis of cartilage proteoglycans and collagen by human articular chondrocytes. Arthritis and Rheumatism. 39(11): 1 896- 1904.

Follis, R H . Jr. 1949. Studies on the chemicai differentiation of developing cartilage and bone. 1 General method for alkaiine phosphatase activity. Bull. Johns Hopk. Hosp. 85:360- 369.

Froum, S.J., M. Coran, B. Thaller, L. Kushner, LW. Scopp and S. Stahl. 1982. Periodontal healing following open debridement flap procedures 1. Clinical assessrnent of soft tissue and osseous repair. J. Periodontal. Sk8- 14.

Froum, S., L. Kushner and S. Stahl. 1983. Healing responses in human intraosseous lesions following the use of debridement, grafhg and citric acid root treatment: II. Clinical and histologie observations, one year post-surgery. J. Periodontal. 54: 325-338.

Gao, Y., L. Yang, Y.-R Fang, M. Mon, K. Kawahara and A. Tanaka. 1995. The inductive efFect of bone morphogenetic protein on human periodontai ligament fibroblast-like cells in vitro. J. Osaka Dent. Univ. 29(1): 9- 17.

Garant, R and M.I. Cho. 1979. Autoradiographic evidence of the coordination of the genesis of Sharpey's fibres with new bone formation in the periodontium of the mouse. J. Period. Res. 14: 107-1 14.

Genco, RJ. 1990. Patogenesis and host response in periodontal disease. In: Genco R.J., H.M. Goldman and D.W. Cohen (eds.), Contemporary periodontics. C.V. Mosby, Toronto, pp. 184-193.

Giannobile, W. V. 1996. Periodontal tissue engineering by growth factors. Bone 19(Suppl 1): 23s-37s.

Giannopoulou, C. and G. Ciamasoni. 1996. Functional characteristics of gingival and periodontal ligament fibroblasts. J. Dent. Res. (USA). 75(3): 895-902.

Gorski, J.P. 1992. Acidic phosphoproteins fom the bone matrix: A structural rationalization of their role in biornineralization. Calcif Tiss. Int. 50;39 1-396.

Gould, T.RL., A.H. Melcher and D.M. Brunette. 1977. Location of progenitor cells in periodontal ligament stimulated by wounding. Anat. Rec. 188: 1 3 3 - 1 4 1.

Gould, T.RL., A.A. Melcher and D.M. Brunette. 1980. Migration of progenitor ce11 population in periodontal ligament after wounding. J. Periodont. Res. 1520-42.

Gould, T.RL., D.M. Brunette and J. Dorey. 1982. Ce11 tumsver in the periodontal ligament determined by continuous infusion of 'H-thymidine using osmotic minipumps. J. Periodont. Res. 17: 662.668.

Gould, T.RL 1983. Ultrastmctural characteristics of progenitor ce11 populations in penodontai ligament. J. Dent. Res. 62(8): 873 -876.

Graves, D.T. and D.L. Cochmn. 1994. Periodontal regeneration with polypeptide growth factors. Curr. Opin. Periodontol. 1994: 178-1 86.

Greenhalgh, D.G., K.H. Spmgel, M.H. Murray and R Ross. 1990. PDGF and FGF stimulate wound healing in genetically diabetic mouse. Am. J. Pathol. 136: 123 5- 1246.

Greenhalgh, D.G., RP. Eamei, S. Albertson, M.P. Breeden. 1993. Synergistic actions of platelet derived growth factor and the insulin like growth factor in vivo. Wound Rep. Reg. 1 :69-8 1.

Groenevdd, M.C., V. Everts and W. Beertsen. t 993. A quantitative enzyme histochemical analysis of the distribution of AK activity in the periodontal ligament of the rat incisor. J. Dent. Res. 72: 1344-1350.

Eebda, P.A. 1988. Stimulating effects oftransforming growth factor43 and epithelial ceIl out- growth fiorn porcine skin explant cultures. J. Invest. Dennatol. 91:440-445.

Hentunen, T.A., P.T. Lakkakorpi, J. Tuukkanen, P.P. Lehenkan, T.K. Sampath and B.K. Vaananen. 1995. Effects of recombinant osteogenic protein-1 on the differentiation ofostoeclast-like cells and bone resorption. Biochem. Biophys. Res. Comm. 209(2):433-443.

Hiroshi, N., O. Yoshihiro and A. Oohira. 1990. Bioassay of chondrocyte differentiation by Bone morphogenetic protein. Clin. Orthop. 258:295-299.

Hollinger, J.O. and K.Leong. 1996. Poly (a-hydroxy acids): m i e r s for bone morphogenetic proteins. Biornaterials 17: 187- 194.

Hoodless, P.A., T. Haerry, S. Abdollah, M. Stapleton, M.P. OqConnor, L. A. Hisano and LC. Wrana. 1996. MADRI, a MAD-related protein that functions in BMP-2 signaihg pathways. Cell. 85:489-500.

Hughes, F.J. and C.A.G. McCulloch. 199 1. Stimulation of differentiation of osteogenic rat bone marrow stroma1 cells by osteoblast cultures. Lab. Invest . 64: 6 1 7-622.

Isidor, E, R Altstrom and T-Karring. 1985. Regeneration of alveolar bore following surgical periodontal treatment. J. Clin. Penodontal. 12: 687-696.

Jin, Y., L. Yang and F.H. Whitc 1994. An irnrnunohistochemicd shidy of bone morphogenetic proteins in experimentai tiacture hding of the rabbit mandible. Chin. Med. Sci. J. 9:9 1-95.

Jones, W.K., E.A. Richmond and K. White. 1994. Osteogenic protein- 1 expression and processessing in Chinese hamster ovary cells: Isolation of a soluble complex containhg the mature and pre-domain of OP- 1. Growth factors 11: 2 1 5-225.

Kanoza, RJ-Je, L. Keleher., J. Sodek 1980. A biochemical analysis of the effect of hypofiinction on collagen metabolism in the rat molar periodontal ligament. Arch oral. Biol(1980). 25: 663-668.

Kasugai, S., R Todescan, T. Nagata, K.L. Yao, W.T. Buttier and J. Sodek. 1991. Expression of bone rnatrk proteins associated with mineralized tissue formation by adult bone marrow cells in vitro: inductive effects of dexarnethasone on osteoblastic phenotype. J. Ce11 Physiol. 147: 1 1 1- 120.

King, G.N., King N., Cnichley A.T., Wozney J.M. and Hughes F.J. 1997. Recombinant human Bone morphogenetic protein-2 promotes wound healing in rat penodontal fenestration defects. J. Dent. Res. 76(8): 1460- 1470.

Kingsley, D.M. 1994. The TGF-13 superfamily: New members, new receptors and new genetic tests of function in different organs. Genes Dev. 8: 133- 146.

Kiristy, C.P. and S.E. Lynch. 1993. Role of growth factors in cutaneous wound healing: a review. Cnt. Rev. Oral Biol. Med. 4: 729-760.

Knutsen, R, J.E. Wergedal, T.K. Sampath, D.J. Baylink and S. Mohan. 1993. Osteogenic protein- 1 stimulates pioliferation and differentiation of human bone cells in &o. Biochem. Biophys. Res. Comm. 194(3): 13 52- 13 58.

Knutsen, R, Y. Bonda, D.D. Strong, T.K. Sampath, D.J. Baylink and S. Mohan. 1995. Regulation of insulin-like growth factor System components by osteogenic protein-l in human bone cells. Endocnnology. 136(3):857-865.

Koenig, B.B., J.K. Cook, D.H. Wolsing, JO Ting, J.P. Tiesrnan, P.E. Correa, C.A. Olson, A.C. Pecquet, F. Ventura, R A . Grant, G.-X. Chen, J.L. Wrana, J. Massague and J.K. Rosenbaum. 1994. Characterization and cloning of a receptor for Bone morphogenetic protein-2 and Bone rnorphogenetic protein-4 form NE? 3T3 cells. Mol. ce11 Biol. 145961- 5974.

Kurosaka, D., Y. Muraki, M. Inoue and A. Katsura. 1996a. TGF-B2 increases a-SMA in bovine retinal pigment epithelial cells. Curr. Eye Res. (England). 15(11): 1 144-1 147.

Kurasaka, D., K. Kato and T. Nagamoto. 1996. Presence of a-SMA in lens epithelial cells of aphakie rabbit eyes. Br. J. Opthalmol. (England). 80(10): 906-9 10.

Leblond, C.P., B. Messier and B. Kopnwa. 1959. Thymidine-H~ as a tool for the investigation of the renewai of ce11 populations. Lab. Invest. 8:296-306.

Lekic, P., J. Sodek, and C.A.G. McCulloch. l996a. Periodontal ligament ce11 populations: The central role of fibroblasts in creating an unique tissue. Anat. Rec. 24450-58.

Lekic, P., J. Sodek and C.A.G. McCulloch. 1996b. Relationship of cellular proliferation to expression of osteopontin and bone sialoprotein in regenerating rat periodontium. Cell and Tiss. Res. 285(3):49 1-500.

Lekic, P., J. Sodek and C.A.G. McCulloch. 1996c. Osteopontin and bone sidoprotein expression in regenerating rat periodontal ligament and alveolar bone. Anat. Rec. 24450-58.

Lekic, P., 1. Rubbino, F. Krasnoshtein, S. Chiefetz, C.A.G. McCulloch and H. Tenenbaum. 1997. Bisphosphonate modulates proliferation and differentiation of rat periodontal ligament cells dunng wound heaiing. Anat. Rec. 247:329-340.

Li, I.W.S., Cheiftz S., C.A.G. McCulloch, K.T. Sampath and J.Sodek. 1996. Effects of osteogenic protein- 1 ( OP- 1 BMP-7) on bone matrix protein expression by fetal rat calvarial cells are differentiation stage specific. J. Cell. Physiol. 169: 1 15- 125.

Libin, B.M., H.L. Ward, and L. Fishman. 1975. Decalcified, lyophilized bone allografts for use in human periodontal defects. J. Periodontol. 463 1-56.

Lind, M. 1996. Growth factors: Possible new clinical tools. Acta. Orthop. Scand. 67(4):407- 4 17.

Lindhe, J. and T. Kamng. 1983. The anatomy of periodontium. In: Lindhe, J. (ed.), Text book of clinical penodontology. Munksgaard, W.B. Saunders, Philadelphia, pp. 19-65.

Line, S.E., A.M. Polson and H.A. Zander. 1974. Relationship between periodontal injury, selective repopulation and ankylosis. J. Penodontol. 45:725-730.

Linkhart, T.A., S. Mohan and D.J. Baylink. 1996. Growth factors for bone growth and repair: IGF, TGF-û and BMP. Bone 19(Suppl 1): 1s-12s.

Listgarten, M.A. 1975. Sirnilarity of epithelial relationships in gingiva of rat and man. J. Periodontol. 46: 677-680.

Liu, F., F. Ventura, J. Doody and J. Massague. 1995. Human type II receptor for bone

morphogenetic proteins ( BMPs ) : Extension of the Zkinase receptor mode1 to the Bone morphogenetic proteins. Mol. Cell. Biol. 15:34793486.

Loemer, K.U., T. Kivela, 8. Borgman and 8. Witschel. 1996. Malignant tumour of the retinal pigment epithelium with extraoccular extension in a phthisical eye. Graefes. Arch. Clin. Exp. Ophthalmol. (Germay). 234(suppl 1): S70-S75.

borner, P.M., B. Sigusch, B. Sukhu, RP. Ellen and H.C. Tenenbaum. 1994. Direct effects of metabolic products and sonicated extracts of Porphyomom.s gigivalis 256 1 on osteogenesis in vitro. Infect. Immun. 62: 1289- 1297.

b o m e r , P.M., RP. Ellen and H.C. Tenenbaum. 1995. Characterimion of inhibitory effms of suspected periodontopathogens on osteogenesis in vitro. Infect. Immun. 63:3287- 3296.

Matsuda, N., W.-L. Lin, N.M. Kumar, MA. Chai and RJ. Genco. 1992. Mitogenic, chernotactic and synthetic responses of rat PL fibroblast cells ti polypeptide growth factors in vitro. J. Penodontol. 635 15-525.

McClain, P., R Sehallhom. 1993. Long-term assessment of cornbined osseous composite grafting, root conditioning and guided tissue regeneration. Int. J. Penodontics Restorative Dent. 13:9-27.

McCulIoch, C.A.G. and A.H. Melcher. 1983a. Ce11 density and cell generation in penodontal ligament of mice. Am. J. Anat. 167:43-58.

McCulloch, C.A.G., and A.H. Melcher. l983b. Ce11 migration in the periodontal ligament of mice. J. Periodont. Res. 18:339-352.

McCulIoch, C.A.G., and A.H. Melcher. 1983c. Continuous labelling of periodontal ligament of mice. J. Periodont. Res. l8:Z 1-24 1.

McCulloch, C.A.G. 1985. Progenitor ce11 populations in the penodontal ligament of mice. Anat. Rec. 21 1:258-262.

McCulloch, C.A.G., E. Nemeih, B. Lowenberg and A.E. Melcher. 1987. Paravascualr cells in endosteal spaces of alveolar bone contribute to periodontal ligamnet ce11 population. Anat. Uec. 2 l9:2S3 3-2242.

McCulloch, C.A.G., U. Barghava and A.H. Melcher. 1989. Ce11 death and the regulation of populations of cells in the periodontal ligament. Cell Tiss. Res. 255: 129- 138.

McCulloch, C.A.G. and S. Bordin. 199 1. Role of fibroblasts subpopulations in periodontal

physiology and pathology J. Peridont. Res. 26: 144-154.

McCulloch, C.A.G. 1993. Basic considerations in periodontal wound healing to achieve regeneration. Periodontal2000. 1 : 16-25.

McKee, M.D., M.C. Farach-Carson, W.T. Butler, P.V. Hauschka and A, Nanci. 1993. Ultrastructural irnrnunolocalization of non-collagenous (osteopontin and osteocalsin) and plasma (albumin and a-2YS-glycoprotein proteins in rat bone. J. Bone Miner. Res. 8:485- 496.

McKee, M.D. and A. Nanci. 1995. post-embedding colloida1 gold immunocytochemistry of non-collagenous extracellular matrix proteins in mineraiized tissue. Microsc. Res. Tech. 31: 44-62.

McKee, M.D. and A. Nanci. 1996. Secretion of OPN by macrophages and its accumulation at tissue surfaces at tissue surfaces during wound healing in mineralized tissues: A potential requirement for macrophage adhesion and p hagocytosis. Anat. Rec. 245:3 94-409.

Melcher, A.H. and J.E. Eastoe. 1969. The comective tissues ofthe periodontium. In: A.H. Melcher and W.H. Bowen (eds.), Biology of the pet-iodontium. Academic Press, New York, ~167-328.

Melcher, A.H. 1970. Repair of wounds in the periodontium of rat. Muence of periodontal ligament on osteogenesis. Arch. Oral Bio. 15: 1 183-1 204.

Melcher, A.A. 1976. On the repair potential of the penodontal tissues. J. Penodontol. 47:256-260.

Melcher, A.E. 1980. Penodontal ligament. Ch 7. Orban's oral histology and embryology, 9th Ed. S.N. Bhaskar, ed. Toronto. Mosby. pp: 204-239.

Mellonig, J.T. 1991. Freeze-dned bone allografts in penodontal reconstructive surgery. Dent. Clinics. N. Am. 35: 505-520.

Mellonig, J.T. 1996. Bone allografts in penodontal therapy. Clin. Orthop. 324: 1 16- 125.

Messague, J., 1990. The transfoming growth factor4 family. Ann. Rev. Cell Biol. 6: 597- 641.

Moore, J., F.P Ashley and C.A. Waterman. 1987. The effect on heading of application of citnc acid during replaced flap surgery. J. Clin. Periodontol. 54: 130- 13 5 .

Muny, C.E., C.M. Giachelli, S.M. Schwartz and R Vracko. 1994. Macrophages express

OPN during repair of myocardial necrosis. Am. J. Pathol. 145: 1450- 1462.

Mustoe, T.A., G.F. Pierce, A. Thompson, P. Gramated, M.P. Sporn and T.F. Deuel. 1987. Accelerated wound healing of incisionai wounds in rats inducd by transforrning growth factor-B. Science. 237: 1333- 1336.

Nair, B.C., W.R Maybeny, R Dziak, P.B. Chen, M.J.Levine and E. Eausmann. 1983. Biological effects of Iipopolysaccharide from Bacterordes gingivalis. J. Periodontol. Res. 18:40-49.

Nakase, T., S. Nomura, 8. Yoshikmwa, J. Eashimota, S. Hirota, Y. Kitamura, Oikawa, K. Ono and K. Tahoka. 1994. Transient and localized expression of bone morphogenetic protein messenger RNA dunng fiacture healing. J. Bone Min. Res. 9:65 1-659.

Nguyen, L., P. Lekic and C.A.G. McCulloch. 1997. Collagen implants do not preserve the periodontal ligament homeostasis in periodontal wounds. J. Penod. Res. 32:4 10-429.

Norton, L.A., W.R Proffit and RR Moore. 1970. In vitro bone growth inhibition in the presence of histamines and endotoxins. J. Periodontol. 4 1: 1 53- 157.

Nyrnan, S., J. Gottolow and T. Karring. 1982a. The regenerative potential of periodontal ligament: An experimental study in the monkey. I. Clin. Periodontol. 9: 257-265.

Nyrnan, S., T. Karring, J. Lindhl and S. Platen. 1982b. New attachment following surgical treatment of periodontal disease. J. Clin. Periodontol. 6: 290-296.

Nyman, S., J. Lindhe and T Karring. 1983. Reattachment - new attachent. In: Lindhe, J. (ed), Tea book of clinical penodontology. Copenhagen: Munksgaard, p4 10-432.

Ogiso, B., F.J. Hughes, A.H. Melcher and C.A.G. McCulloch. 1991. Fibroblast inhibit rnineralized bone nodule formation by rat bone marrow stromai cells in vitro. J. Ce11 Physiol. 146:442-450.

Ogiso, B., F.J. Hughes, J.E. Davies and C.A.G. McCulloch. 1992. Fibroblastic regdation of osteoblast function by prostaglandins. Ce11 Signal. 4: 627-63 9.

Oldberg, A., A. Franzen and D. Heinegard. 1988. Pnmary structure of bone sialoprotein. J. Biol. Chem. 263: 19430- 19432.

Ozkaynak, E., D.C. Rueger and E.A. Drier. 1990. OP-I encodes osteogenic protein in the TGF-O family. EMBO 1. 9:2085-2093.

Page, RC., and H.E. Shroeder. 198 1. Current status of host response in chronic marginal

periodontics. J. Perodontol. 52:477-49 1.

Page, RC., and H.E. Shroeder. 1982. Penodontics in other mammalian animds. In: Periodontics in man and other anirnals. Karger, New York, pp:7 1 - 107.

Palmer, RM., A.G.S. Lumsden. 1987. Development of periodontal ligament and alveolar bone in homografled recornbinations of enamel organ and papillaq, pulpal and follicular mesenchyme in mouse Arch Oral Biol. 23: 281-289.

Pearson, C.B., and G.H. Gibson. 1982. Proteoglycans of bovine periodontal ligament and skin. ûccurrence ofdzerent hybrid-sulphated galactoMminoglycans in distinct proteoglycans. Bio. Chem. J. 201:27-37.

Perera, K.A.S., C.H. Tonge. 198 1. Fibroblast ce11 population kinetics in the mouse molar periodontal ligament and tooth eruption. J. Anat. 133: 28 1-3 00.

Pierce, G.F., J. Vande Berg, R Rudolph, J. Tarpley and J.A. Mustoe. 1991. Platelet- derived growth factor-fil) and transforming growth factor-B selectively modulate glycosaminoglycans, collagen and myofibroblasts in excisional wounds. Am. J. Pathol. 138: 629-646.

Pinholt, E.M., H . R Haanaes, M. Roervik, M. Donath and G. Bang. 1992. Alveolar ridge augmentation by osteoinductive materiais in goats. Scand. J. Dent. Res. 100:36 1-365.

Pitaru S., 8. Tal, M. Soldinger and M. Noff. 1989. Ce11 membranes prevent apical migration of epithelium and support new co~ect ive tissue attachement dunng periodontal wound healing in dogs. J. Periodont. Res. 24:247-253.

Pitaru S., C.A.G. McCulloch and S.A. Narayanan. 1994. Cellular origins and differentiation control mechanisms during periodontal development and wound healing. J. Periodont. Res. 29: 8 1-94.

Potten, C.S., and M. Loeffler. 1990. Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and fiom the crypt. Development 1 10: 1 00 1 - 1020.

Qwarnstrom, E. and RC. Page. 1986. Development of a three dimensional extracellular matnx synthesized by human diploid fibroblasts in vitro. J. Ce11 Sci. 84: 183-200.

Quintero, G., J.T. Mellonig, V.M. Gambill and G.B. Pelleu. 1982. A six-month clinical evaluation of decalcified, fieeze-dned bone allografts in periodontal osseous defects. J. Periodontol. 53: 726-730.

Redii, A.H. and C. Huggins. 1972. Biochemical sequences in the transformation of nomal fibroblast in adolescent rats. Proc. Natl. Acad. Sci. (USA). 69: 1601 - 1605.

Reddi, A.H., S. Weintroub and N. Muthukumaran. 1987. Biological principles of bone

induction. Orthop. Clin. North Am. 18:207-2 12.

Reddi, A.H. and N.S. Cunningham. 1993. Initiation and promotion of bone differentiation by bone morphogenetic proteins. J. Bone Miner. Res. 8:S499-S502.

Renvert, S. and J. Egelberg. 1 98 1. Healing f i e r treatment of periodontal intraosseous defects II. Effects of citric acid conditioning of root surface. J. Clin. Periodontal. 8: 459-473.

Ripamonti, U., M. Heliotis, B.van den Heever and A.H. Reddi. 1994. %one rnorphogenetic proteins induce periodontal regeneration in baboons (Papio uninus) J. Periodontal. Res. 29:439-445.

Roberts, W.E. 1975. Cell kinetic and diumal penodicity of the rat periodontal ligament. Arch. Orai Biol. 20:465-469.

Rosen, V. and RS. Thies. 1992. The Bone rnorphogenetic protein proteins in bone formation and repair. Trends Genet ( England ). 8(3):97- 102.

Rutherford RB., T.K. Sampath, D.C. Rueger and T.D. Taylor. 1992. Use of bovine osteogenic protein to promote rapid osseointegration of endosseous dental implants. Int. J. Oral Maxillofac. Implants. 7:297-30 1.

Rutherford, RB., M.E. Ryan, J.E. Kennedy, M.M. Tucker and M.F. charette. 1993. Platelet-derived growth factor and dexamethasone combined with collagen matrix induce regeneration of the periodontium in monkeys. J periodontol. 20537-544.

Sampath T.K. & A.H. Reddi . 1981. Dissociative extraction and reconstitution of extracellular matrix components involved in local bone differentiation. Proc. Natl. Acad Sci. (USA) 78(12):7599-7603.

Sampath, T.K. and A.H. Reddi. 1983. Hornology of bone inductive proteins fiom human, monkey, bovine, and rat extracellular matrix. Proc. Natl. Acad. Sci. 8O:659 1-6595.

Sampath, T.K., J.E. Coughlin and RM. Whetstone. 1990. Bovine osteogenic protein is composed of dimers of OP- 1 and BMP-24 two mernbers of the transfomiing growth factor4 superfamily. J. Biol. Chem. 265: 13 198- 1320%

Sampath, T.K., J.C. Maliakal, P.V. Hauschka, W.K. Jones, 8. Sasak, RE Tucker, K. White, J.E. Coughlin, M.M. Tucker, RB.L Pang, C. Corbett, E. Ozkaynak, H. Opperman and D.C. Rueger. 1992. Recombinant human osteogenic protein-l (OP-1) induces new bone formation in vivo with specific activity comparable with naturai bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro. J. Biol. Chem. 267: 20352-20362.

Sampath, T.K. and D.C. Rueger. 1994. Structure, function and orthopaedic applications of osteogenic protein- 1 (OP- 1). Compl. Orthop. Winter 10 1 - 107.

SchalIhom, RG. and P.K. McClain. 1988. Combined osseous composite grafting, root conditioning and guided tissue regeneration. Int. J. Penodontics Restorative Dent. 8(4):8-3 1.

Schultz, G.S., M. White, R Mitchell, G. Brown, J. Lynch, D.R Twardzik and G.J. Todaro. 1987. Epithelial wound healing enhanceci by TGF-a and vaccinia growth factor. Science. 235: 3 50-3 52.

Schwartz, z., J.T. Mellonig, D.L. Cames Jr., J. De La Fontaine, D.L. Cochnn, D.D. Dean and B.D. Boyan. 1996. Ability of commercial demineralized fieeze-dried bone allograft to induce new bone formation. J. Periodontol. 67:918-926.

Selvig KA., U.M.E. Wikesjo, G.C. Bogle and RD. Finkelman. 1994. Impaired early bone formation in penodontal fenestration defects in dogs following application of insulin-like growth factor II, basic fibroblast growth factor beta- 1. J. Clin. Periodontol. 2 l:3 80-3 85.

Shigeyama, y., J.A. D'Emca, R Stone and M.J. Sommerman. 1995. Comrnercially- prepared allograft material has biological activity in vitro. J. Periodontol. 66:478-487.

Sigurdsson T. J., M.B. Lee, K. Kubota, T.J. Turek, J.M. Wozney and U.M.E. Wikesjo. 1995. Periodontal repair in dogs: recombinant human bone morphogenetic protein-2 significantly enhances penodontal regeneratioo. J. Penodontal. 66: 13 1 - 138.

Socransky, S.S., and A.D. Haffajee 1992. The bacterial etiology of destmctive periodontal disease: curent concepts. J. Periodontol. 63(suppl. 4):322-33 1.

Sodek, J. 1977. A companson of the rates of synthesis and tum-over of collagen and non- collagen proteins in adult rat periodontal tissue and skin using a microassay. Arch. Oral Biol. 22:655-665.

Sodek, J., and C.M. Ovenli. 1988. Matrix degradation in hard and sofi comective tissues, p.303-3 1 1. In 2. Davidovitch (ed.), The biological mechanisms of tooth eruption and root resorption.

Sodek, J., J. Chen, S. Kasugai, T. Nagata, Q. Zhang, M.D. McKee and A. Nanci. 1992. Elucidating the functions of bone sialoprotein and osteopontin in bone formation. In: Chem and biology of mineralized tissue. H. Slavkin and P. Price, eds. Elsevier Science Publishers, Amsterdam. pp297-306.

Somerman, M.J., C.W. Prince, W.T. Butler, RA. Foster and J.J. Sauk 1987. Ce11 attachment activity of the 44 kilodalton phosphoprotein is not restncted to the bone cells. Matrix 9:49-54.

Somerman, M.J., J.J. Sauk, R.A. Foster, K. Norris and K. Dickerson. 1991. Ce11 attachment activity of cementum: Bone siloprotein 11 identified in cementum. J. Periodont. Res. 26: 10-16.

Somerman, M.J. 1996. Is there a role for DFDBA in periodontal regeenrative therapy?[editorial; comment]. J. Periodontol. 67(9):9 18-926.

Sonis, S.T., LB. Kaban and J. Glowacki. 1983. Clinical trial of demineralized bone powder in the treatment of periodontd defects. J. Oral Med. 38(3): 11 7- 122.

Stahl, S., S. Froum and L. Kushner. 1982. Periodontal healing of open flap debridement flap procedures II. Histologic observations. J. Periodontal. Res. 53: 15-2 1.

Stopak, D. and A.K. Barris. 1982. Connedive tissue rnorphogenesis by fibroblast traction. 1. Tissue cuIture observations, Dev. Biol. 90:383-398.

Stcinener, S., M. Crigger and J. Egeiberg. 198 1. Co~ec t ive tissue's regeneration to periodontally diseased teeth II. Histologic observations of cases following replaced flap surgery. J. Periodontal. Res. 16: 109-1 16.

Heersche, J.N.M. 1989. Bone cells and bone turnover - The basis for pathogenesis. In: Tarn, C.S., J.N.M. Heersche and T.M. Murray (eds.), Metabolic bone disease: Cellualr and tissue mechanisrns. CRC Press, Fiorida, pp: 2- 13.

Ten Cate, A.R, C. Milis, G. Solomon. 1971. The development of the penodontium: A transplantation and autoradiographic study. Anat. Rec. 170: 365-379.

Ten Cate, A.R, C. Mills. 1972. The development of the periodontium: The origin of alveolar bone. Anat. Rec. 173: 69-77.

Ten Cate, A.R, D.A. Deporter and E. Freeman. 1976. The role of the fibroblast in the remodelling of the penodontal ligament during physiologie tooth movement. Am. J. Orthod. 69: 155-168.

Ten Cate, A.R 1989. The fibroblast and its products. In: Ten Cate, A.R. (ed). Oral Histology: development, structure and fùnction. Toronto: C.V. Mosby. pp: 90-105.

Ten Diye, P., A. Yamashita, T.K. Sampath, A.H. Reddi, M. Esteva , O. Riddle, H. Lchijo, Ce-H. Heldin and K. Miyazono. 1994. Identification of the type 1 receptors for osteogenic protein- 1 and Bone morphogenetic protein-4. J. Biol. Chem. 269: 16985-16988.

Tenenbaum, H.C., N. Kamalia, B. Sukhu, 8. Limeback and C.A.G. McCulloch. 1995. Probing glucocorticoid-dependent osteogenesis in rat and chick cells by »t vitro specific blockade of osteoblastic differentiation with progesterone and RU38486. The Anat. Rec. 242:200-2 1 O.

Terranova, V.P. and U.M.E. Wikesjo. 1986. Extracellular matrices and polypeptide growth factors as mediators of cells of the periodontium. J. Periodontol. 58(6):37 1-380.

Temnova V.P., H.M. Goldrnan and M.A. Listgarten. 1990- The periodontal attchment

apparatus: structure, fùnction, and chernistry. In: Genco R.J.. H.M. Goldman and D.W. Cohen (eds.), Contemporary periodontics. C.V. Mosby, Toronto, pp. 33-54

Todescan R, J. Sodek and J.E. Davies. 1994. Reconstituted collagen produces different heaiing reactions in bony and soft tissue peri-implant compartments. Int. J. Oral Maxilofac. Implants. 9:298-304.

Tonumi DoMe, HoS. Kotler, D.R Luxenberg, M.E. Eoltrop and E.A. Wang. 1991. Mandibular reconstruction with recombinant bone inducing factor: Functional, histologic and biomechanical evaluation. Arch. Otolaryngol. Head Neck Surg. 117: 1 10 1 - 1 1 12.

Urist, M.R 1965. Bone formation by autoinduction. Science 150:893-899.

Urist, M.R., RJ. De Lange and G.A.M. Finerman. 1983. Bone ceIl differentiation and growth factors. Science. 220:680-686.

Wahl, S.M. 1994. T ransfonning growth factor-0: the good, the bad, and the ugly. J. Exptl. Med. 180: 1587- 1590.

Williams, D.M., F.J. Hughes, E.W. Odell and P.M. Farthing. 1992a. The normal periodontium. In: Pathology of priodontal diseases. Oxford university press, New York, pp: 17-30.

Williams, D.M., F.J. Hughes, E.W. Odell and P.M. Farthing. 1992b. Host defences against microbial plaque. In: Pathology of pnodontal diseases. Oxford university press, New York, pp:67-96.

Wozney, J.M. 1995. The potential role of bone morphogenetic proteins in penodontal reconstruction. J. Periodontol. 66: 506-5 10.

Yamaguchi, A., T. Katagiri, T.Ikeda, J.M. wozney, V. Rosen, E.A. Wang, A.J. Kahn, Te Suda and S. Yoshiki. 1991. Recombinant human bone morphogenetic protein-2 stimulates osteoblasts maturation and inhibits myogenic differentiation in vitro. J. Cell Biol. 1 l3:68 1-687.

Yoshikawa, D.K., E.J. Kollar. 198 1. Recombination experts on odontogenic roles ofmouse dental papilla and dental sac tissues in ocular grafts. Arch. Oral Biol. 26: 303-307.

Yamashita, H., P. Ten Diya, D.Huylebroeck, T.K. Sampath, M. Andries, J.C. Smith, CPH. Heldin and K. Miyazono. 1995. Osteogenic protein-1 binds to activin type II receptors and induces certain activin-like effects. J. Cell. Biol. l3O:2 1 7-226.

Yamashita He, P. Ten Dijke, Ce-H. Heldin and K. Miya Zoneo . 1996. Bone morphogenetic protein receptors. Bone. 19(6): 569-574.

Young, M.F., J.M. Kerr, K. Ibaraki, A.M. Heinegaard and P.G. Robey. 1992. Structure,

expression and regulation of major non-collagenous matnx proteins. Clin. Orthop. Rel. Res. 28:275-294.

Zambon, J.J. 1990. Microbiology o f periodontal disease. In R.J. Genco, H.M. Goldman, and D.W. Cohen (eds.), Cotemprory periodontics. C.V. Mosby, St. Louis, p147-160.

IMAGE EVALUATION TEST TARGET (QA-3)

APPLIED IMAGE. lnc - = 1653 East Main Street - -- , , Rochester. NY 14609 USA -- -- - - Phone: 71 6i482-0300 -- -- - - Fax: 716i288-5989

O 1993. Applied image. inc . Ali Rights Reçarvea