ISOLATION AND CHARACTERIZATION OF HUMAN PERIODONTAL LIGAMENT STEM CELLS

by

ISABEL C. GAY

MARY MACDOUGALL, COMMITEE CHAIR NICOLAAS GEURS

AMJAD JAVED FIROZ RAHEMTULLA

A THESIS

Submitted to the graduate faculty of the University of Alabama at Birmingham,

in partial fulfillment of the requirements for the degree of Masters in Clinical Dentistry

BIRMINGHAM, ALABAMA

2007

ISOLATION AND CHARACTERIZATION OF HUMAN PERIODONTAL LIGAMENT STEM CELLS

ISABEL C GAY

MASTERS IN CLINICAL DENTISTRY

ABSTRACT

The periodontal ligament (PDL) is thought to include progenitor cells capable of

forming fibroblasts, osteoblasts and cementoblasts. However, characterization of PDL stem

cell populations (SC) remains undetermined due in part, to the diversity of cell types that

conform to the periodontal apparatus. Objective: To isolate and characterize PDLSC and

assess their pluripotent capabilities to differentiate into bone, cartilage and adipose cell

types. Methods: PDL was scraped from human teeth, enzymatically digested, and cells

stained for STRO- 1 (SC marker) using fluorescent immunohistochemistry. Positive cells

were FACS sorted and expanded in culture. Human bone marrow SC (BMSC) served as a

positive control. PDLSC and BMSC were cultured using conditions conducive for osteo-

genic, chondrogenic and adipogenic differentiation. Osteogenic induction was assayed us-

ing alizarine red staining, and expression of alkaline phosphatase (ALP) and bone sialopro-

tein (BSP) by RT-PCR and immunohistochemistry. Adipogenic induction was assayed us-

ing Oil Red O staining and expression of PPARγ 2 and LPL (early/late specific markers) by

RT-PCR. Chondrogenic induction was immunoassayed by collagen type II expression (car-

tilage marker) and toluidine blue staining. Results: Of the PDL cells isolated, 27% were

positive for STRO-1, with 3% staining strongly. ALP expression was initially observed in

PDLSC osteogenic cultures by day-14, where BMSC showed expression by day-7. BSP

expression was detectable by day-7; with more intense staining found in PDLSC cultures.

Under adipogenic conditions both population showed positive Oil Red O staining by day-

ii

35 with expression of PPARγ 2 and LPL. By day-21 both BMSC and PDLSC chondro-

genic cultures expressed collagen type II. Control cultures showed no differentiation. Con-

clusions: Human PDL tissue contains a high percentage of STRO-1 positive stem cells.

These PDLSC remain undifferentiated until challenged with various differentiating condi-

tions having the potential to differentiate into osteoblasts, chondrocytes and adipocytes,

comparable to BMSC. Our method for obtaining stem cell populations can be utilized for

potential therapy procedures and possibly formation of a periodontal ligament around den-

tal implants.

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DEDICATION

I wish to dedicate this thesis to my mother Maria Teresa, my brother, niece and

nephew. To my beloved Larry; my colleague and best friend Martha Alicia. Also, I want

to dedicate this thesis to Dr Mary MacDougall for her continuous, exemplary support as a

friend and role model. Thanks to her wise advice, I am able reach my dream.

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ACKNOWLEDGEMENTS

I want to express my grateful acknowledgements to the many faculty and staff

members that have helped me along my tenure at UAB. I want to emphasize my appre-

ciation for my mentor, Dr. Mary MacDougall, who is a wonderful friend and a person

whom I respect and admire immensely. Drs. Nico Geurs, Firoz Rahemtulla and Amjad

Javed served as my committee members and have provided me with careful guidance and

patience with my research endeavors. I also want to express my admiration and respect

for Dr. Michael Reddy, chair of the Periodontics Department. Drs. Kent Palcanis, Tho-

mas Weatherford, Hung Wen Lee and Alvin Stevens who have provide advice and guid-

ance through my training.

I also want to acknowledge the support from my closest colleagues and friends

Drs. Shuo Chen and Juan Dong.

Finally, I wish to also thank to every person that was involved in this work for

their support.

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TABLE OF CONTENTS

. Page

ABSTRACT....................................................................................................................ii-iii

DEDICATION................................................................................................................... iv

ACKNOWLEDGEMENTS.................................................................................................v

LIST OF TABLES............................................................................................................ vii

LIST OF FIGURES ......................................................................................................... viii

LIST OF ABBREVIATIONS............................................................................................ ix

INTRODUCTION ..............................................................................................................1

Embryonic and Adult Stem Cells ............................................................................2 Stem Cell Markers ...................................................................................................5 History, Characteristics and Functions of MSC ......................................................8 Extracellular matrix, Cell shape and Control of Proliferation ...............................12 Differentiation Studies ...........................................................................................13 Progenitor Cells Involved in Periodontal Wound Healing and Regeneration .......14 Stem Cells in the Periodontal Ligament ................................................................18

ISOLATION AND CHARACTERIZATION OF MULTIPOTENT HUMAN PERIO- DONTAL LIGAMENT STEM CELLS ............................................................................21

FUTURE PROSPECTS FOR PERIODONTAL REGENERATION ...............................56

SUMMARY AND CONCLUSIONS ................................................................................57

GENERAL LIST OF REFERENCES ...............................................................................61

APPENDIX: IRB APPROVAL.........................................................................................67

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LIST OF TABLES

Table Page

1. Markers commonly used to identify bone marrow stem cells and hematopoietic precursors .................................................................................... 6-7

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LIST OF FIGURES

ISOLATION AND CHARACTERIZATION OF MULTIPOTENT HUMAN PERIO- DONTAL LIGAMENT STEM CELLS

Figure Page

1. Immunohistochemistry of human BMSC and PDL cells with mesenchymal stem cell marker STRO 1...............................................................47

2. FACS sorting of STRO-1 positive PDL cells ........................................................48

3. PDLSC and BMSC cell proliferation rates............................................................49

4. PDLSC and BMSC colony forming units..............................................................50

5. Expression of mineral deposits within PDLSC and BMSC long term

cultures...................................................................................................................51

6. BSP and ALP gene expression profiles of osteogenic BMSC and PDLSC cultures .....................................................................................................52

7. Characterization of adipogenic differentiation of PDLSC and BMSC

cultures...................................................................................................................53

8. In vitro chrondogenic differentiation of PDLSC and BMSC ................................54

9. Immunohistochemistry for collagen type II expression in PDLSC and BMSC chrondrogenic induced cultures .................................................................55

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LIST OF ABREVIATIONS

PDL Periodontal Ligament

PDLSC Periodontal Ligament Stem Cells

BMSC Bone Marrow Stem cells

DPSC Dental Pulp Stem Cells

MSC Mesenchymal Stem Cells

SC Stem Cells

STRO-1 Stromal Cell Antigen

CFU Colony Forming Units

ECM Extracellular Matrix

RT-PCR Real Time Polymerase Chain Reaction

LPL Lipoprotein Lipase

PPARγ2 Peroxisome Proliferator-Activated Receptor Gamma 2

ALP Alkaline Phosphatase

BSP Bone Sialoprotein

TGF-β1 Transforming Growth Factor Beta 1

TGF-β3 Transforming Growth Factor Beta 3

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- x - x

Runx2 Runt-Related Transcription Factor 2

FACS Fluorescence Activated Cell Sorting

GAPDH Glyceraldehide-3-Phosphate Dehydrogenase

SEM Standard Error from the Media

INTRODUCTION

The periodontal ligament (PDL) is a soft, richly vascular and cellular connective tissue

which surrounds the roots of the teeth and joins the root cementum with the alveolar bone

wall. The alveolar bone surrounds the tooth to a level approximately 1mm apical to the

cemento-enamel junction. The PDL space has the shape of an hourglass, narrowest at the

mid root level. The presence of a periodontal ligament permits forces elicited through mas-

ticatory function and other tooth contacts to be distributed and absorbed by the alveolar

process. Tooth mobility is to a large extent determined by the width, height, and quality of

the PDL. Other functions include tooth nutrition, homeostasis, and repair of damaged tis-

sue.

The PDL cell population is heterogeneous, consisting of two major lineages, fibroblas-

tic and mineralizing tissues, further divided into osteoblastic and cementoblastic subsets1-4.

The fibroblasts are aligned along the principal fibers, while cementoblasts line the surface

of cementum and osteblasts cover the surface of the bone.

During wound healing, PDL cells derive from progenitors located at the paravascular

zones5. This progenitor stem cell population is enriched by precursor cell populations de-

rived from the endosteal spaces of the alveolar bone6. Cell kinetic studies during the heal-

ing process suggest that some of these progenitors migrate toward alveolar bone and ce-

mentum where they terminally differentiate into osteoblasts, cementoblasts, and fibro-

blasts6-8. As a result, repair or regeneration is regulated by a vast array of extracellular ma-

trix molecules and by cytokines that induce both selective and non-selective responses in

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the different cell lineages and their precursors. Unfortunately, our understanding concern-

ing the numerous important signaling systems is irretrievably lost after development or re-

pair is complete3.

Cell migration and differentiation pathways are the basis for remodeling and healing in

periodontal tissues and it is likely that some form of signaling has evolved to orchestrate, in

a temporally and spatially appropriate manner, these repopulation and differentiation re-

sponses9. However, a detailed understanding of the biochemical and molecular events as-

sociated with the remodeling process in the PDL has not been determined due to the com-

plex diversity of cells and tissue types10. Future prospects for improved healing and regen-

eration of PDL tissue may derive from the identification and isolation of specific PDL cell

populations and PDL stem cells (PDLSC) and their informational molecules that are stored

in the connective tissue matrices. This knowledge may provide novel approaches to perio-

dontal therapies that facilitate regeneration, and potentially, formation of a true periodontal

apparatus around dental implants.

Embryonic and Adult Stem Cells.

By definition, a stem cell has the ability to proliferate in culture for indefinite time pe-

riods and give rise to specialized cells11. Embryonic stem cells, derived from the inner cell

mass of the blastocyst are termed pluripotent because they can give rise to all the cells nec-

essary for fetal development12. These cells possess unique characteristics such as:

1. Capability of undergoing an unlimited number of symmetrical mitotic events

without differentiation.

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2. Exhibit and maintain a stable diploid karyotype.

3. Give rise to differentiated cell types that are derived from all three primary germ

layers of the embryo: endoderm, mesoderm and ectoderm; capable of integrat-

ing into fetal tissues during development.

4. Clonogenic capabilities; a single stem cell can give rise to a colony of geneti-

cally identical cells or clones that have the same properties as the original cell.

5. Expression of the transcription factor Oct-4 which activates or inhibits target

genes and maintains stem cells in a proliferative, non-differentiating state.

6. Lack of the G1 checkpoint in the cell cycle, spending most of their time in the

5th phase of the cycle, during which they synthesize DNA and do not show X

chromosome inactivation13.

In contrast, unlike embryonic stem cells, adult stem cells share no such definitive

means of characterization. In fact, no one knows the origin of adult stem cells found in ma-

ture tissues. Some authors have proposed that stem cells are set aside during fetal devel-

opment and restrained from differentiation. Most current research pertaining to adult stem

cells has been performed in bone marrow tissue which has demonstrated the presence of

undifferentiated cell types, termed mesenchymal stem cells (MSC)14. There are 3 proposed

mechanisms for MSC arrival to the bone marrow:

1. They can enter the bone marrow along with the vasculature.

2. They can migrate into the bone marrow space after vascularization along the vessel

paths; for example the periosteum which has documented multipotentiality.

3. They can arrive via the blood proper, indicating the existence of circulating MSC.

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The first two mechanisms differ only with respect to the timing of MSC entrance in

to the marrow, not by the path of arrival or migration. The third mechanism differs only

with respect to which side of the capillary basement membrane the MSC use, the luminal

or abluminal. However, any of these mechanisms of MSC migration or circulation have

important implications for adult tissue repair due to the possibility to deliver reparative

stem cells via the circulation as opposed to only localized applications, especially if MSC

docking sites exist on the endothelium lining the vascular network15.

The first and second mechanisms of MSC arrival (along with the vasculature) to the

bone marrow space are supported by two different observations and experiments with vas-

cular associated cells (pericytes) and on smooth muscle cells from marrow. Pericytes are

cells that are closely associated with capillaries16 which express smooth muscle markers17

and have the potential to differentiate into osteoblasts18 and odontoblasts19.

The pericytes of the periosteum are one candidate cell type for the origin of marrow

MSC because they are in the correct anatomical region for migration into the nascent mar-

row space and also because pericytes have been shown to have the potential to differentiate

into chondrocytes, adipocytes,20,21 and odontoblasts19. In addition, pericytes express the

STRO-1 antigen21, a known marker for bone marrow colony forming cells22,23 which is ex-

pressed in multipotential MSC24 (Table1). The antibody STRO-1 has been the most useful

antibody for identifying and positively selecting for MSC in bone marrow25. This circum-

stantial evidence suggests a lineage relationship between pericytes and odontoblasts that

further, implies a logical source of marrow and dental stem cells.

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Stem Cell Markers

Researchers now know that many different types of stem cells exist, but they are

found in sparse populations in the human body. Scientists identify these rare type of cells

found in different tissues by the use of stem cell markers. Coating the surface of every cell

in the body are specialized proteins, called receptors, which have the capability of selec-

tively binding or adhering to “signaling” molecules26. There are many different types of

receptors that vary in their structure and affinity for the signaling molecules. Normally,

cells use these receptors and the molecules that bind to them as a way of communicating

with other cells and to carry out their proper functions in the body. These same cell surface

receptors are the stem cell markers27. Each cell type, for example an odontoblast, has a cer-

tain combination of receptors on their surface that makes them distinguishable from other

kinds of cells. Scientists have taken advantage of the biological uniqueness of stem cell

receptors and chemical properties of certain compounds to tag or mark cells. Much of the

success in finding and characterizing stem cells is due to the use of markers.

Stem cell markers are given short-hand names based on the molecules that bind to the

stem cell surface receptors. For example, a cell that has the receptor stem cell antigen – 1 on

its surface is identified as Sca-1. In many cases, a combination of multiple markers is used to

identify a particular stem cell type by a combination of marker names reflecting the presence

(+) or absence (–) of them. Researchers use the signaling molecules that selectively adhere

to the receptors on the cell surface as a tool that allows them to identify stem cells by attach-

ing to the signaling molecule another molecule (or tag) that has the ability to fluoresce or

emit light energy when activated by an energy source such as an ultraviolet light or a laser

beam. Table 1 presents a list of some of the commonly used stem cell markers 28-30.

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MARKER CELL TYPE DESCRIPTION Bone morpho-genetic pro-tein receptor (BMPR)

Mesenchymal stem and progenitor cells

Important for the differentiation of commit-ted mesenchymal cell types from mesen-chymal stem and progenitor cells; BMPR identifies early mesenchymal lineages (stem and progenitor cells)

CD4 and CD8 White blood cell (WBC)

Cell-surface protein markers specific for mature T lymphocyte (WBC subtype)

CD34 Hematopoietic stem cell (HSC), satellite, endothelial progenitor

Cell-surface protein on bone marrow cell, indicative of a HSC and endothelial pro-genitor; CD34 also identifies muscle satel-lite, a muscle stem cell

CD34+Sca1+ Lin- profile

Mesencyhmal stem cell (MSC)

Identifies MSCs, which can differentiate into adipocyte, osteocyte, chondrocyte, and myocyte

CD38 Absent on HSC Present on WBC line-ages

Cell-surface molecule that identifies WBC lineages. Selection of CD34+/CD38- cells allows for purification of HSC populations

CD44 Mesenchymal A type of cell-adhesion molecule used to identify specific types of mesenchymal cells

c-Kit HSC, MSC Cell-surface receptor on BM cell types that

identifies HSC and MSC; binding by fetal calf serum (FCS) enhances proliferation of ES cells, HSCs, MSCs, and hematopoietic progenitor cells

Colony-forming unit (CFU)

HSC, MSC progenitor CFU assay detects the ability of a single stem cell or progenitor cell to give rise to one or more cell lineages, such as red blood cell (RBC) and/or white blood cell (WBC) lineages

Fibroblast colony-forming unit (CFU-F)

Bone marrow fibroblast An individual bone marrow cell that has given rise to a colony of multipotent fibro-blastic cells; such identified cells are pre-cursors of differentiated mesenchymal lineages

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Hoechst dye Absent on HSC Fluorescent dye that binds DNA; HSC ex-trudes the dye and stains lightly compared with other cell types

Leukocyte common anti-gen (CD45)

WBC Cell-surface protein on WBC progenitor

Lineage sur-face antigen (Lin)

HSC, MSC Differentiated RBC and WBC lineages

Thirteen to 14 different cell-surface proteins that are markers of mature blood cell line-ages; detection of Lin-negative cells assists in the purification of HSC and hematopoi-etic progenitor populations

Mac-1 WBC Cell-surface protein specific for mature granulocyte and macrophage (WBC sub-types)

Muc-18 (CD146)

Bone marrow fibro-blasts, endothelial

Cell-surface protein (immunoglobulin su-perfamily) found on bone marrow fibro-blasts, which may be important in hemato-poiesis; a subpopulation of Muc-18+ cells are mesenchymal precursors

Stem cell anti-gen (Sca-1)

HSC, MSC Cell-surface protein on bone marrow (BM) cell, indicative of HSC and MSC Bone Marrow and Blood cont.

Stro-1 antigen Stromal (mesenchy-mal) precursor cells, hematopoietic cells

Cell-surface glycoprotein on subsets of bone marrow stromal (mesenchymal) cells; selection of Stro-1+ cells assists in isolat-ing mesenchymal precursor cells, which are multipotent cells that give rise to adi-pocytes, osteocytes, smooth myocytes, fi-broblasts, chondrocytes, and blood cells

Thy-1 HSC, MSC Cell-surface protein; negative or low detec-tion is suggestive of HSC

VCAM-1 HSC, MSC Vascular cell adhesion molecule-1 found on HSC and MSC

SH2 HMSC Binds endoglin, a type III TGF-β receptor found on mesenchymal tissues and on macrophages on endothelial cells, indicat-ing that this antibody is not specific for MSC

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SH3 and SH4 HMSC Have been shown to specifically bind an ecto-5’-nucleotidase, CD73

Table 1. Markers commonly used to identify bone marrow stem cells and hematopoietic precursors. From these, STRO-1 sorted cells have been shown to be osteogenic, chon-drogenic, adipogenic, and hematopoietic.

History, Characteristics, and Functions of MSC

The presence of mesenchymal progenitor cells within bone marrow has been docu-

mented from the late 19th century by the work of Goujon31. He was the first to show the

osteogenic potential in heterotopic transplants of rabbit marrow, later confirmed in transplan-

tation experiments by Biakow and Danis32. They showed that whole marrow itself was os-

teogenic, and not just an inductive factor or chemoattractant for osteogenic cells, by placing

marrow tissue within a diffusion chamber, implanting this in an animal host, and observing

the results by histological methods. Similar confirmatory experiments were conducted by

other groups33-35 wherein the formation of cartilage and bone within the diffusion chamber

was observed, demonstrating that bone marrow had at least the potential to form bone and

cartilage. Friedenstein36 showed that the osteogenic potential of bone marrow was a feature

of a specific subgroup of cells termed the CFU (colony forming units), which made up a very

small percentage of the total marrow cell population. Friedenstein37 later, showed that CFU

were formed from single cells and some of these CFU were able to form both bone and the

microenvironment necessary for the formation of hematopoietic elements38,39.

The first formal presentation of the concept of a stem cell residing in the bone mar-

row was in a publication by Owen in 1978 40 wherein the marrow stroma was hypothesized to

consist of a lineage analogous to the hematopoietic lineage41. Owen, in 198542, expanded on

this hypothesis and proposed a model for the stromal lineage that contained “stem cell”,

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“committed progenitor”, and “maturing cell compartments”. He designed a lineage diagram

for “stromal stem cells” that included “reticular”, “fibroblastic”, “adipocytic”, and “osteo-

genic” cells as end stage phenotypes. In 1991, Caplan43 proposed the existence of a MSC

having the capacity to differentiate into multiple mesenchymal phenotypes including adipose,

tendon, ligament, muscle and dermis 44. Years later, in 2002, Gronthos described the pres-

ence of undifferentiated cell types in human dental pulp45.

Formal proof of the multipotentiality of MSC or CFU came from experiments with

clonal populations. It was demonstrated that approximately 30% of the isolated CFU colo-

nies were able to form bone alone or bone with marrow in open transplants37 and about

40% of rabbit marrow cells, isolated by limiting dilution or by cloning rings, were able to

form bone46. Multipotential mesenchymal cells were also identified in studies using MSC

isolated from the marrow of transgenic mice containing a gene for conditional immortal-

ity47,48. In that study, multiple colonies were isolated by cloning rings and limiting dilution

while under immortalized culture conditions and then tested for differentiation potential

under standard culture conditions or in vivo. Mouse marrow derived mesenchymal pro-

genitor cells contain a mixture of cells having various differentiation potentials47, ranging

form monopotential to quadripotential48. Similar results were reported for MSC isolated

from human marrow, wherein three of six isolated and expanded MSC colonies were able

to differentiate into bone, adipose and cartilage, while all six were able to differentiate into

bone49.

Adult stem cells, like all stem cells, share at least 2 characteristics. First, they possess

the ability to clone themselves for long periods of time; this ability to proliferate is referred to

as long term self renewal. Second, they can give rise to mature cell types that have tissue

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specific morphology and function50. Typically, adult stem cells generate an intermediate cell

type or types before they reach their fully differentiated state. The intermediate cell type is

called the precursor or progenitor cell51. Precursor or progenitor cells are partly differenti-

ated cells in fetal or adult tissues. Such cells are usually committed to differentiate under a

particular cellular development pathway, although, this characteristic is not as definitive as it

was once thought. Primary functions of adult stem cells include maintenance of steady state

cell function, or homeostasis, and to replace cells that die due to injury or disease52. Adult

stem cells are dispersed throughout the tissues of the adult human or animal and behave very

differently depending on the local microenvironment 53. In solid tissues with specialized

form, stem cell differentiation is accompanied by progressive remodeling of preexisting

extracellular matrix (ECM) scaffolds, and it is the coordination between these processes in

both time and space that ensures correct pattern formation. Maintenance of tissue architec-

ture throughout life also requires fine control over both tissue mass and ECM pattern integ-

rity, which are provided through spatially coordinated growth and differentiation of tissue

specific stem cells 54.

The term “plasticity” has been defined in stem cell research as the ability of a cell to

transdifferentiate or dedifferentiate and redifferentiate into other cell types and has been

found to be more common than previously thought. For example, studies have shown that

blood stem cells (derived from mesoderm) may be able to generate both skeletal muscle

(also derived from mesoderm) and neurons (derived from the ectoderm)53. That realization

triggered a flurry of papers reporting that stem cells derived from one adult tissue can

change their appearance and assume characteristics that resemble those of differentiated

cells from other tissues. The term plasticity means that a stem cell from one adult tissue

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can generate the differentiated cell types of another tissue. At this time, there is no for-

mally accepted name for this phenomenon in the scientific literature, so it continues to be

referred to as plasticity or “transdifferentiation”52,54. To be able to claim that adult stem

cells demonstrate plasticity, it is first important to show that such cell populations exists in

the primary tissue that has the identifying features of stem cells. Then, it is necessary to

show that the adult stem cells give rise to cell types that normally occur in a different tis-

sue55. Neither of these criteria is easily met. Simply proving the existence of an adult stem

cell population in a differentiated tissue is a laborious process. It requires that the candi-

date stem cells are shown to be self-renewing and that they can give rise to the differenti-

ated cell types that are characteristic of that tissue. It is also important to demonstrate that

those cells have adopted key structural and biochemical characteristics of the new tissue

that they are claimed to have generated. Ultimately—and most importantly—it is neces-

sary to demonstrate that the cells can integrate into their new tissue environment, survive in

the tissue, and function like the mature cells of the tissue56-59.

Pluripotent stem cells that can differentiate into various cell types have been isolated

and some can spontaneously assemble into structures that resemble mature tissues60. At the

same time, ECM molecules have been shown to have potent effects on cell proliferation

and differentiation and to direct assembly into three dimensional tissues in vitro61, 62.

The future challenge in stem cell research is to understand how these cells can spon-

taneously generate 3D functional tissue architecture. To address this challenge we must

answer some fundamental questions. First, what is the nature of the signals conveyed by

both soluble and insoluble factors that govern where and when cells are triggered to organ-

ize into tissues and for tissues to organize into organs? Second, how do these cells process

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multiple simultaneous inputs to produce a single coordinated cell fate response? To answer

these questions we should focus first on how individual cells chemically and physically in-

teract with their local tissue environment, as well as, how cells use regulatory networks to

process information provided by multiple environmental cues.

Extracellular Matrix, Cell Shape and Control of Proliferation

The common view of mitogenic stimulation holds that cooperative stimulation of

cell-surface growth factor receptors is orchestrated by soluble mitogens and transmembrane

adhesion receptors, known as integrins63,64. This dual requirement is the basis for the con-

cept of “anchorage dependence” for growth of nontransformed cells. However, it has been

long suggested that cell shape or mechanical distortion also controls the switch between

quiescence and growth65, 66. Thus, cell ECM interactions have two components: a chemical

component, which is mediated by the ligand-induced activation of integrins and associated

biochemical signaling cascades, and a mechanical component, which is mediated by the

integrins role in physically resisting cell generated tractional forces. This later structural

effect of ECM binding to integrins is responsible for the rearrangement of the actin cy-

toskeleton and ensuing cell shape changes that are observed in response to cell adhesion to

the ECM. However, in living tissues, integrins can also modulate cell and cytoskeleton

form based on their ability to transmit external mechanical loads from the ECM to the cy-

toskeleton67, 68.

Taken together, the switch between growth, differentiation, and quiescence is gov-

erned by a set of inputs from the cell environment that includes soluble mitogens, insoluble

adhesive cues from ECM, cell-cell adhesion molecules, and mechanical forces that can

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produce cytoskeleton distortion and modulate cell shape. Any of these signals alone is not

sufficient to decide cell fate; behavioral control is an integrated response that simultane-

ously takes all of these signals into account.

Differentiation Studies

Evidence of stem cell plasticity comes from analysis of the expression of markers

for differentiation of specific cell phenotypes as well as differentiated tissue like character-

istics. In this segment we will describe what is known about stem cell qualities, of putative

postnatal dental pulp stem cells, understand the basis of research performed with dental re-

lated cell lines, and set the framework for our research design.

During development, interactions between epithelial and mesenchymal cells from

the dental papilla lead to the differentiation of ameloblasts and odontoblasts. Each deposit

unique mineralized matrices, termed enamel and dentin, respectively. Once formed, these

matrices do not undergo further remodeling. However, after tooth eruption, dentinal dam-

age caused by mechanical trauma, exposure to chemicals, or disease processes, induces the

formation of reparative dentin. It is thought that progenitors are recruited from the dental

pulp to develop both supportive connective tissue and differentiated odontoblasts. To de-

termine the existence of such cells in dental pulp, Gronthos et al69 applied methodology

that had been previously developed for the isolation and characterization of bone marrow

stem cells (BMSC). By using a colony forming efficiency essay, they demonstrated that a

minor population within the adult human dental pulp is clonogenic. Those colonies oc-

curred in higher frequency in comparison to BMSC. Also, they were able to demonstrate

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that dental pulp stem cells (DPSC) exhibited a higher proliferation rate when compared to

the well characterized BMSC. These results were further confirmed in subsequent publica-

tions45, 70, 19. DPSC differentiation towards a tissue type was examined in cells exposed to

ascorbic acid, dexamethasone, and β-glycerophosphate and evaluated by the expression of

alkaline phosphatase activity in vitro as well as the presence of calcium deposits, with 2%

alizarine red stain. These results were compared to BMSC under the same circumstances.

It is important to highlight that the bone matrix protein, bone sialoprotein, was absent in

DPSC cultures and present in low levels in BMSC69.

The potential of DPSC to differentiate into adipocytes and neural cells, in analogy

to what has been demonstrated in BMSC, was published by Gronthos et al in 200245. After

5 weeks of culture with an adipogenic- inductive cocktail, Oil red O positive lipid clusters

were identified in DSPC. This correlated with an upregulation in the expression of two

adipocye-specific transcripts, PPARγ2 and lipoprotein lipase, detected by RT-PCR.

The chondrogenic potential of DPSC has not been elucidated, whereas an extensive

body of literature describes BMSC differentiation towards a cartilage like tissue that ex-

presses collagen type II, a specific marker of cartilage, as well as toluidine blue staining, a

metachromatic stain for ECM glycosaminoglycans24.

Progenitor Cells Involved in Periodontal Wound Healing and Regeneration

The functional periodontal attachment apparatus anchors the tooth and consists of

periodontal fibers that run between alveolar bone and cementum lining the root surface.

Furthermore, gingival connective tissues and epithelium overlay the alveolar bone and

form a dentogingival junction at the interface with the tooth. The complex structure of the

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periodontium, which consists of the soft tissues of the gingiva and PDL, as well as the min-

eralized tissues cementum and bone, makes periodontal wound healing a unique process.

Following conventional periodontal therapy involving debridement of the root sur-

face, periodontal tissues heal by repair and migration of the epithelium along the previously

contaminated root surface (long junctional epithelium), which prevents connective tissue

attachment to the root surface71. Regeneration of this destroyed attachment apparatus has

long been the goal of periodontal therapy. Periodontal regeneration requires new connec-

tive tissue attachment to the root surface, a process that involves the regeneration of perio-

dontal fibers and the insertion of these fibers into newly formed cementum. Unfortunately,

current available regeneration techniques are clinically unpredictable, resulting in only par-

tial regeneration at best72. From a biological perspective, current and future prospects for

improved regeneration of periodontal tissues are dependent on the ability to facilitate the

repopulation of the periodontal wound by cells capable of promoting regeneration. From

this perspective, the periodontal ligament has been shown to be of critical importance in the

regenerative process. It has been demonstrated that only the periodontal ligament, but not

gingival connective tissue, contains cells capable of establishing new attachment fibers be-

tween cementum and bone73,74. It has also been shown that gingival fibroblasts grown in

vitro failed to contribute to regeneration of the PDL around teeth implanted in dogs, while

periodontal ligament fibroblasts were observed to either contribute to regeneration or at

least not inhibit regeneration75. The ability of periodontal ligament cell populations to

achieve regeneration implies that progenitor cells exist within the PDL.

Although it is clear that cells residing in the PDL can achieve regeneration, this

population is heterogeneous76 and it is not known which subpopulations are capable of

- 15 - 15

achieving regeneration. Indeed, cells derived from regenerating defects were found to have

specific properties, such as increased proliferation rates, representative of a regenerative

phenotype that is different from periodontal ligament cells77. Therefore, in order to identify

progenitor and/or stem cells from the periodontium, it will be useful to establish where

these cells reside in the tissue, as well as identify markers that can be used to distinguish

these types of cells.

Periodontal regeneration during wound healing is likely to share many common

biological processes with those of periodontal development. Aside from the gingival epi-

thelium, the PDL is almost entirely derived from ectomesenchyme, the embryonic connec-

tive tissue derived from the neuroectoderm. MSC are precursors for a diverse group of

connective tissue cells including smooth muscle cells, endothelial cells, chondroblasts,

pericytes, adipocytes, osteoblasts and fibroblasts. At the initiation of tooth formation in

fetal jaws, MSC cells are induced by the overlying oral epithelium towards odontogenic

specificity. Initially, this interaction results in the formation of a tooth crown comprising

the ectoderm (oral epithelium) derived enamel and the mesenchyme derived dentin. Sub-

sequently, during root formation and the development of the periodontal attachment appa-

ratus, epithelial root sheath cells derived from the enamel organ fragment to allow the at-

tachment of MSC on the exposed root dentine. These MSC proceed to deposit cementum

and establish an attachment apparatus. In order to understand the cellular origins of the

developing periodontal apparatus, transplanted tooth buds were used to show that the mes-

enchymal derived dental follicle surrounding the developing tooth root is the source of pro-

genitor cells for cementum, alveolar bone, and PDL78,79.

- 16 - 16

Further evidence that cells from the dental follicle are precursors to cementoblasts

has been shown by direct cell migration studies in rat molars80,81. More recently, the dental

follicle associated with third molars has been shown to contain precursor cells which are

clonogenic and have the ability to differentiate under in vitro conditions to a membrane-

like structure containing calcified nodules82.

The source of postnatal progenitor cells which may be capable of regenerating the

periodontium has been investigated for a number of years. Cell kinetic experiments in

mice and rats 83, 84 have shown that PDL fibroblast populations represent a steady state re-

newal system, with the number of new cells generated by mitosis equal to the number of

cells lost by apoptosis and migration. This capacity for self renewal, which is further evi-

denced by the rapid turnover of the PDL, supports the notion of progenitor/stem cell popu-

lations. Furthermore, a significant number of periodontal cells do not enter the cell cycle

85, suggesting that these cells may act in a similar manner to quiescent, self-renewable, and

multipotent stem cells.

The relationship between progenitor cells in regenerating tissues and normally func-

tioning (steady-state) tissues has been investigated in studies performed in normal mouse

PDL 10, wounded mouse ligament 86, normal rat gingival tissues 87and inflamed monkey

gingival tissues 88. These studies have identified a common paravascular location of fibro-

blast progenitors which exhibit some of the classical cytological features of stem cells, in-

cluding small size, responsiveness to stimulating factors, and slow cycle time 86, 83, 10. Fur-

thermore these paravascular cells exhibit spatial clustering which suggests a possible clonal

distribution of progenitors and their progeny 10. Other possible sources of osteoblasts and

- 17 - 17

cementoblast precursors are the endosteal spaces of alveolar bone, from which cells have

been observed to adopt a paravascular location in the periodontal ligament of mice6.

Stem Cells in the Periodontal Ligament

More than any other tissue, the complexity of the periodontium, along with the un-

derstanding that only certain components are capable of achieving regeneration, makes

identification of stem cells important in the understanding of normal wound healing

events. The inherent properties of stem cells, including their ability to form different tissue

types and self renewal capabilities make their presence within the healing periodontal de-

fect desirable in order to facilitate periodontal regeneration. Therefore, the ability to iden-

tify, characterize and manipulate stem cells within the periodontium is of considerable

clinical significance, especially in terms of developing novel mechanisms of achieving

periodontal regeneration.

Although it is established that precursor cells might exist within the PDL, stem cell

properties such as self-renewal, clonogenicity, and multi-tissue differentiation have only

begun to be elucidated. For these properties to be demonstrated, it is necessary to isolate

stem cells in vitro using available markers for the identification of MSC. However, al-

though the use of stromal cell markers and/or the absence of stromal cell markers has been

successful in isolating cells from the bone marrow, additional challenges have been en-

countered when trying to extract MSC from connective tissues composed largely of fibro-

blastic cells, such as the PDL.

Single specific markers for MSC and also a combination of markers have been used

to obtain enriched MSC cell populations. A single marker that has been extensively used

- 18 - 18

to isolate dental progenitor cells is STRO-1 antibody; results claim the specificity of this

marker to target undifferentiated phenotypes in dental pulp tissues 89,19. A methodology for

isolating MSC from the PDL should be based on techniques shown to successfully isolate

BMSC and stem cells in dental pulp tissues 69,45, 90.

MSC from postnatal dental pulp express the stem cell marker STRO-1, as well as

CD 44, integrin β1, VCAM-1, CD 146/MUC-18, and alkaline phosphatase. When dental

pulp stem cells were transplanted into immunocompromised mice, they generated a dentin

like structure lined with odontoblast like cells that surround a pulp like interstitial tissue.

These DPSC were also capable of multi-lineage differentiation into adipocytes and neural-

like cells 69, 45.

Stem cells have also been isolated from the pulp of human exfoliated deciduous

teeth (SHED)70 . Immunohistochemical analyses of these cells have shown that they are

positive for STRO-1 and CD-146/MUC-18. After transplantation into immunocompro-

mised mice, SHED were found to induce bone formation, generate dentin, survive in the

mouse brain, and express neural tissue markers. It has been suggested that SHED represent

a population of multipotent stem cells that are more immature in the cell hierarchy than

previously examined postnatal stem cell populations.

Subsequent research using frozen pulp and bone marrow sections has indicated that

MSC are located in the perivascular regions and constitutively express STRO-1, CD 146,

and α smooth muscle actin on the cell surface 90. The perivascular localization of these

cells is consistent with previous reports of localization of progenitor cells in the periodontal

ligament.

- 19 - 19

In light of these findings, the identification of stem cells within the periodontal

ligament will be a significant development towards a tissue engineering approach to obtain

periodontal regeneration. Furthermore, the ready availability of periodontal ligament tissue

from redundant teeth, such as third molars, may provide a supply of stem cells that may be

utilized for regenerating, not only the masticatory apparatus, but also other body tissues.

Therefore, the objectives of this study are to isolate, identify and characterize PDL

stem cells (PDLSC) as well as demonstrate their ability to differentiate into adipose, chon-

drogenic, and mineralized type tissues. This research is based on the hypothesis that undif-

ferentiated progenitor cells exist within the PDL and demonstrate capabilities to differenti-

ate into chondrogenic, adipogenic and bone like tissues.

- 20 - 20

- 21 - 21

ISOLATION AND CHARACTERIZATION OF MULTIPOTENT HUMAN PERIO-

DONTAL LIGAMENT STEM CELLS.

by

ISABEL C. GAY, SHUO CHEN1, MARY MACDOUGALL

Institute of Oral Health Research, School of Dentistry, University of Alabama at

Birmingham, Birmingham, AL

1Department of Pediatric Dentistry, Dental School, University of Texas Health Sci-

ence Center at San Antonio, San Antonio, TX

Submitted to Journal of Orthodontics and Craniofacial Research

Format adapted for thesis

ABSTRACT:

Periodontal ligament (PDL) repair is thought to involve mesenchymal progenitor

cells capable of forming fibroblasts, osteoblasts and cementoblasts. However, full charac-

terization of PDL stem cell (SC) populations has not been achieved. Here we isolate and

characterize PDLSC and assess their capability to differentiate into bone, cartilage and

adipose tissue. Human PDL cells were stained for STRO-1, FACS sorted and expanded

in culture. Human bone marrow SC (BMSC) served as a positive control. PDLSC and

BMSC were cultured using standard conditions conducive for osteogenic, chondrogenic

and adipogenic differentiation. Osteogenic induction was assayed using alizarine red S

staining and expression of alkaline phosphatase (ALP) and bone sialoprotein (BSP). Adi-

pogenic induction was assayed using Oil Red O staining and the expression of PPARγ 2

(early) and LPL (late) adipogenic markers. Chondrogenic induction was assayed by col-

lagen type II expression and toluidine blue staining. Human PDL tissue contains about

27% STRO-1 positive cells with 3% strongly positive. In osteogenic cultures ALP was

observed by day-7 in BMSC and day-14 in PDLSC. BSP expression was detectable by

day-7; with more intense staining in PDLSC cultures. In adipogenic cultures both cell

populations showed positive Oil Red O staining by day-25 with PPARγ 2 and LPL ex-

pression. By day-21, both BMSC and PDLSC chondrogenic induced cultures expressed

collagen type II and glycosaminoglycans. The PDL contains SC that have the potential to

- 22 - 22

differentiate into osteoblasts, chondrocytes and adipocytes, comparable to previously

characterized BMSC. This adult PDLSC population can be utilized for potential thera-

peutic procedures related to PDL regeneration.

- 23 - 23

INTRODUCTION

The dental attachment apparatus consists of two mineralized tissues; cementum and

alveolar bone, with an interposed fibrous, cellular and vascular soft connective tissue

termed the periodontal ligament (PDL). The PDL provides anchorage and support to the

functional tooth, dispersing the mechanical forces associated with mastication. The PDL

cell population is heterogeneous, consisting of two major mesenchymal lineages, fibroblas-

tic and mineralizing tissues, further divided into osteoblastic and cementoblastic subsets (1-

4). In the adult PDL, the progenitors for both cell lineages originate in the paravascular

zones (5). This progenitor stem cell (SC) population is enriched by precursors derived

from the endosteal spaces of the alveolar bone (6). Cell kinetic studies suggest that some of

these progenitors migrate toward alveolar bone and cementum where they terminally dif-

ferentiate into osteoblasts, cementoblasts and fibroblasts (5,7). Cell migration and differen-

tiation pathways are the basis for remodeling and healing in periodontal tissues, and it is

likely that some form of signaling has evolved to orchestrate, in a temporally and spatially

appropriate manner, these repopulation and differentiation responses (8). However, a de-

tailed understanding of the biochemical and molecular events associated with the remodel-

ing process in the periodontum has not been determined due to the diversity of cells and

tissue types. Methods that can identify specific PDL cell populations and PDL stem cells

(PDLSC) are of central importance for our understanding of remodeling and healing in

these tissues.

- 24 - 24

By definition, a stem cell has the ability to proliferate in culture for indefinite peri-

ods and give rise to specialized cells. Embryonic stem cells derived from the inner cell

mass of the embryo are termed pluripotent because they can give rise to many cell types

but not all the cells necessary for fetal development. In addition, multipotent SC have been

identified in young and adult tissues, and are capable of further differentiation. Examples

are blood and skin SC that play a key role in homeostasis and tissue repair by continually

replenishing our blood cells and skin layers throughout life.

Adult SC, once thought able to develop only the same type of tissue, have recently

been shown to be able to develop into many types of specialized cells (9). These data sug-

gest that adult SC have outstanding potential for the development of novel therapies such

as tissue transplantation and regeneration. It is well documented that adult bone marrow

(BM) contain a population of SC that retain the capacity to differentiate along different

lineages including reticular, adipogenic, fibroblastic, osteogenic, and chondrogenic (10-13).

In addition, BMSC can interact with other cell phenotypes in bone marrow microenviron-

ments to influence and regulate a variety of physiological pathways including bone remod-

eling (14,15), hematopoiesis (16-18) and angiogenesis (19). Effective techniques have

been developed to isolate BMSC using specific antibodies to cell surface markers with

fluorescence activated cell (FACS) sorting to enrich cell populations.

Stem cell differentiation processes have been successfully demonstrated in vitro by

exposing undifferentiated stem cells to a variety of microenvironments and tissue culture

conditions (20). Adipocyte differentiation is accompanied not only by gross changes in cel-

lular morphology, but also by the transcriptional activation of many genes. A member of

the nuclear receptor superfamily of ligand-activated transcription factors identified as per-

oxisome proliferator-activated receptor gamma 2 (PPARγ2) has been shown to be ex-

pressed early in the adipocyte differentiation program. It acts synergistically with

- 25 - 25

CCAATT enhancer-binding protein α to coordinate the adipocyte differentiation cascade.

Importantly, the peroxisome proliferator response element has been identified in the pro-

moter regions of several enzymes associated with triglyceride metabolism (21). LPL en-

codes lipoprotein lipase, which is expressed in heart, muscle, and adipose tissue. LPL func-

tions as a homodimer and has the dual functions of triglyceride hydrolase and

ligand/bridging factor for receptor-mediated lipoprotein uptake (22).

Cells isolated from postnatal mammalian BM have also the potential for differentia-

tion into cartilage when implanted in vivo (23). However, attempts to develop in vitro con-

ditions in which mesenchymal stem cells, derived from postnatal mammalian bone marrow,

will progress down the chondrogenic lineage has been less successful. A system that cul-

tures cells in aggregates has been described (24, 25) and utilized in studies of terminal dif-

ferentiation of growth plate chondrocytes (26). This culture system allows cell-cell interac-

tions analogous to the ones that occur in pre-cartilage condensation during embryonic de-

velopment. However, this cell configuration is not sufficient for the induction of chondro-

genesis; it requires defined medium and bioactive factors such as TGF-β (27). Evidence of

chondrogenic differentiation includes the appearance of toluidine blue metachromasia and

immunohistochemical detection of type II collagen (28).

Recent findings show that mesechymal progenitors can differentiate into osteoblast-

like cells when challenged with dexamethasone, β-glycerophosphate and ascorbic acid in

vitro. These cells express terminal phenotype identification markers such as Runx2 pro-

moter activity, alkaline phosphatase (ALP), bone sialoprotein (BSP), osteopontin (OP), os-

teocalcin (OC), and collagen type I, as well as mineral deposits in the extracellular matrix

(29). In this study, we report that PDL tissue contains undifferentiated progenitor cells,

adult SC, that are capable of differentiation into bone, cartilage and adipose-like tissues

comparable with the well-defined BMSC. Also, we have demonstrated PDLSC capability

- 26 - 26

to express gene products compatible with differentiated cell types. This, therefore, clearly

demonstrates the existence of progenitor undifferentiated STRO-1 positive cells that could

potentially be used for PDL regenerative therapies as well as the possible formation of a

true PDL apparatus associated with a titanium implant surfaces.

MATERIALS AND METHODS

Cell Isolation, FACS Sorting and Cell Culture

Disease free impacted third molars were collected from 10 patients, 18 to 26 years

old, at the Oral and Maxillofacial Surgery Department at the University of Texas Health

Science Center at San Antonio (UTHSCSA). Individuals were selected on the basis of good

general health and elective surgery procedures, following the approved guidelines of the

UTHSCSA Institutional Review Board.

PDL cells were scraped from 3rd

molars, enzymatically digested for 1 hour at 37°C

in a solution of 3 mg/ml collagenase type I (Worthington Biochem, Freehold NJ) and 4

mg/ml of dispase (Worthington Biochem, Freehold NJ). PDL cells from different individu-

als were pooled and single cell suspensions were obtained through a 70 μm cell strainer

(Falcon BD, Franklin Lakes NJ). Samples were centrifuged at 400 g for 10 minutes and

resuspended in blocking buffer (HBSS, 20 mM HEPES, 1% normal human serum, 1% bo-

vine serum albumin, 5% FCS) for 20 min. on ice and stained with STRO-1 IgM antibody

(Developmental Studies Hybridoma Bank, Iowa City IW) (2 μg/ml) for 1 hour on ice. A

goat anti-mouse IgM FITC secondary antibody (Molecular Probes, OR) (2 μg/ml) was in-

cubated for 1 hour. Cell surface-marker STRO-1 positive cells were analyzed and sorted by

flow cytometry. This procedure was performed on a FACScan with automated cell deposi-

tion unit (Becton Dickinson, Parsippany, NJ) equipped with an argon laser and data ana-

- 27 - 27

lyzed with CellQuest software (Becton Dickinson, Parsippany, NJ) at the Institutional Flow

Cytometer Core Facility (UTHSCSA, San Antonio, TX).

Cells were expanded with Dulbecco's modified Eagle's medium containing 10%

FCS and 1% antibiotics. The cultures were incubated at 37oC in a 5% CO2 atmosphere.

Human BMSC kindly provided by Dr. A Caplan (Case Western Reserve University, Cleve-

land OH) were grown under the same conditions as PDLSC and used as a positive control.

Cultures at 2nd

or 3rd

passage were utilized for all experiments, unless otherwise stated.

Cell Growth Rate Assay

To compare cell growth capabilities we determined the growth rates on both cell

populations (BMSC and PDLSC). Cells were plated at a density of 5 X 103 cells per cm

2 in

12 well plates and cultured with DMEM containing 20% FCS and 1% antibiotics for 0, 48

and 96 hours. At harvest, cells were washed twice with PBS and released from the culture

surface by the addition of 200 μl of trypsin-EDTA 1X for 10 min at 37°C. The cell suspen-

sion was transferred to a vial containing 9.8 ml of 0.9% NaCl. Cell number was determined

with a Coulter Counter (Coulter Electronics Inc, Hialeah, FL). Cells harvested in this man-

ner exhibited greater than 95% viability on the basis of Trypan blue exclusion.

Clonogenic Assays

Colony forming units were evaluated for the BMSC and PDLSC using DMEM

(GIBCO/BRL) with 20% FBS (Equitech-Bio, Kerrville, TX) under standard culture condi-

tions at 37°C. At day 7 and 14, cultures were fixed with 10% formalin (Fischer Scientific,

Fair Lawn, NJ), stained with 0.1% toluidine blue (Sigma-Aldrich, Milwaukee, WI) and ob-

- 28 - 28

served under an inverted microscopy. Aggregates of 50 cells or more were scored as colo-

nies, results were recorded and compared between PDLSC and BMSC cultures.

Differentiation Culture Conditions

Osteogenic Induction: PDLSC and BMSC were plated at 5x103 cm

2 in 24 well plates. At

24 hours the media was changed to an osteogenic inducing media (Cambrex Bio Science

Walkersville, MD). Control groups were treated with DMEM containing 10% FBS and 1%

antibiotics only. Cells were assayed at 7, 14, 21, 28, 35, and 42 days for ALP expression

and calcium deposits using alizarine red S staining.

Chondrogenic Induction: BMSC and PDLSC populations (2.5 X105 cells) were centrifuged

in 15 ml polypropylene tubes (Corning Incorporated Life Sciences, Acton, MA). Cell pel-

lets were treated with chondrogenic differentiation media (Cambrex Bio Science Walkers-

ville, MD) supplemented with 10 ng/ml of TGF-β3 (Cambrex Bio Science Walkersville,

MD). Pellets were left free floating for 14 and 21 days and fixed with 10% phosphate buff-

ered formalin.

Adipogenic Induction: PDLSC and BMSC were plated at 5x103 cm

2 in 6 well plates. At

100% confluence, three alternated cycles of induction/maintenance were performed utiliz-

ing Cambrex adipogenic induction medium and Cambrex maintenance medium (Cambrex

Bio Science Walkersville, MD). Control cultures were treated with Cambrex maintenance

medium. At day 25, after induction, cultures were fixed with 10% formalin (Fischer Scien-

tific, Fair Lawn, NJ) and stained with Oil Red O.

Culture Histochemical Staining

Osteogenic cultures were stained with 2% alizarine red S (Sigma-Aldrich, Milwau-

kee, WI), (pH 4.2) according to the manufactures instructions. Chondrogenic culture pel-

- 29 - 29

lets were embedded in 2% agarose followed by paraffin. Serial histological sections were

stained with 0.2% toluidine blue (Sigma-Aldrich, Milwaukee, WI) according to suppliers’

instructions. Adipogenic cultures were stained with 3% Oil Red O (Sigma-Aldrich, Mil-

wakee, WI) for 5 minutes and counterstained with Harris hematoxylin (Sigma-Aldrich,

Milwaukee, WI) for 1 min suing standard techniques.

In situ ALP Staining:

For localization of ALP activity, cultures were washed twice with phosphate-

buffered saline (PBS), fixed with 70% ethanol for 5 min, and washed for 5 min. in buffer

(100mml/l Tris-HCL, ph 9.5; 100 mmol/l NaCl; 50 mmol/l MgCl2). In situ ALP staining

was performed according to the supplier’s instructions (Bio-Rad, Hercules, CA)

Immunohistochemistry:

Chondrogenic culture samples were immunostained for collagen type II expression

using a commercial collagen type II antibody (Santa Cruz Biotechnology, Santa Cruz CA)

at a 1:100 dilution for 1 hour incubation according to the supplier’s instructions. The sec-

ondary antibody used was a rabbit anti goat IgG FITC (Molecular Probes, OR) (2 μg/ml)

incubated for 1 hour. Images were obtained at the optical facility, UTHSCSA under the

same parameters using an Olympus Wide Field microscope.

RT-PCR Analysis:

Total RNA was isolated from osteogenic and chondrogenic cultures (PDLSA and

BMSC) using RNA STAT-60 (TEL-TEST, Inc. Friendswood, TX.). First-strand cDNA

- 30 - 30

was produced from 500 ng of total RNA with random hexamers using MultiScribe reverse

transcriptase according to manufacturer’s instructions (ABI TaqMan Kit, Applied Biosys-

tems, Foster City, CA). After mRNA conversion to cDNA, 500 ng of target cDNA was

amplified with 1 Unit of Thermus aquaticus (Taq) polymerase (Perkin-Elmer Cetus, Bos-

ton, MA) using various specific primer sets.

Osteogenic Marker Primer Sets and PCR Conditions

Specific primer sets (10 pmol) included ALP (accession number NM_000478: an-

tisense 5’-atgcaggctgcaatacgccat-3’, sense 5”-atctttggtctggcccccatg-3”) and BSP (accession

number AH_006462: antisense 5’-ttcctcctc ctcttctgaactg-3’, sense 5’-atggcctgtgctttctaat-

3’). PCR amplification for each primer included denaturation at 95o C for 1', annealing at

58-60o C for 1' and an elongation step of 72

o C for 1' for a total of 40 cycles. GAPDH (ac-

cession number AL_136219: antisense 5'-gcaaagttgtcatgg atgacc-3', sense 5'-

ccatggagaaggctgggg-3') was used as an internal control for cDNA integrity. PCR products

were separated by agarose gel electrophoresis (2-4%) in 1 X Tris-borate buffer (0.01M

Tris-base, 0.1 M boric acid, and 00.2 M EDTA, pH 8.3).

Adipogenic Marker Primer Sets and Real-Time PCR Conditions

To verify gene expression in the adipogenic induction cultures amplification reac-

tions were analyzed in real-time on an ABI 7500 (Applied Biosystems Foster City, CA)

using SYBR Green chemistry and the threshold values calculated using SDS2 software

(Applied Biosystems, Foster City, CA) according to the supplier’s instructions. Thermal

- 31 - 31

cycling parameters were 95°C for 30 s and 60°C for 1 min for 40 cycles. Reactions were

performed in quadruplicate and threshold cycle numbers were averaged. A single melt

curve peak was observed for each sample used in data analysis, confirming the purity and

specificity of all amplified products. Expression fold was calculated according to the for-

mula 2 (Rt-Et)

/2 (Rn-En)

where Rt is the threshold cycle number for the housekeeping gene

(18S) from non-induced PDLSC and Et is the threshold number for the experimental gene

observed in non-induced PDLSC. Rn is the threshold value for the housekeeping gene in

induced PDLSC (18S) and En is the threshold cycle number for the experimental gene in

induced PDLSC (30). Real Time PCR primers were designed to the following human

genes: PPARγ2 (accession number D83233: antisense 5’-caggaaa gacaacagacaaatca-3’,

sense 5-’ggggtgatgtgtttgaacttg-3’); LPL (accession number NM_000237: antisense 5’-

gtggccgagagtgagaacat-3’, sense 5’-gaaggagtaggtcttatttgtggaa-3’), and 18S (accession num-

ber NM_022551: antisense 5’-agacctggagcgactgaaga-3’, sense 5’agaagtgacgcagccctcta-3’).

Statistical Analysis:

Each individual value represents the mean ± SEM of 6 individual sample measure-

ments, performed in triplicate. The data were analyzed by use of Student’s t-test. Signifi-

cance level was set at p< 0.05 for all tests.

RESULTS

To identify the existence of potential stem cells within the PDL cell population sin-

gle cell suspension of the PDL was plated on 4 chamber slides and cultured for 24 hours to

- 32 - 32

promote cell attach. As a positive control, purified BMSC were plated in the same manner.

Immunohistochemistry was performed using an antibody directed against a known stem

cell marker STRO-1. Experiments were performed in parallel on the PDL and BMSC cul-

tures to avoid possible time-related biological fluctuation. The BMSC, an enriched popula-

tion, served as a positive control due to their well-characterized stem cell properties. Fig-

ure 1 shows the immunohistochemistry of 3rd

passage human BMSC from a 31 year old

male and primary PDL cells obtained from a pool of patients (18 to 26 year olds). The

BMSC stained positive for STRO-1 confirming their stromal stem cell status (Fig. 1, B &

C). The PDL cell heterogeneity was confirmed showing a low number of STRO-1 positive

cells. Figure 1 panel H shows a single STRO-1 positive cell observed in the field of the

PDL mixed cell population, The negative controls lacking the primary antibody for both

cell populations shown no staining (Fig. 1, E, F, K & L).

Next, PDL single cell suspensions obtained from the 38 molars were stained with

STRO-1 antibody. Surface-marker STRO-1 positive cells were analyzed and sorted by flow

cytometry. A total of 27% of the PDL cell population was positive for STRO-1 and of that

population 3% were strongly positive. This enriched population had several intensity ratios

to the mesenchymal stem cell antibody as demonstrated by various peaks within the M1

region after sorting was performed a second time and the population re-analyzed (Fig. 2,

C). The PDL STRO-1 negative cell population analysis demonstrated 99.8% purity as seen

in Figure 2, panel D.

To investigate the potential of the enriched PDLSC to undergo mitosis compared

with the BMSC, cell suspensions from both cell types were plated and counted at 0, 48, and

96 hours using a Coulter Counter. At 96 hours, PDLSC demonstrated a statistically signifi-

- 33 - 33

cant (p<.05) nearly double number of cells as compared to the BMSC, whereas at 48 hours

there was no significant difference between the two cell populations (Fig. 3).

The ability of PDLSC to form characteristic stem cell adherent clonogenic cell clus-

ters of fibroblast-like cells was compared with BMSC. Both PDLSC and BMSC showed

the ability to form multi-cell clusters (Fig. 4, A & B). PDLSC showed the formation of

about 50 colonies (Fig. 4, C) generated from single cells cultured at low density (5x103

cm2) for 7 days; whereas BMSC generated 35 colonies. These results showed statistically

significant difference with PDLSC having an increased colony forming unit behavior as

compared to BMSC. To investigate the potential of PDLSC to differentiate into mineraliz-

ing osteoblast or cementoblast-like lineages, we induced the formation of mineralized ma-

trix containing calcium deposits by the addition of culture media containing L-ascorbate-2-

phosphate, dexamethasone and β-glycerophosphate (osteogenic media). After 48 days in

culture, PDLSC showed calcium deposits, whereas at day 35, BMSC demonstrated calcium

deposits as visualized by alizarine red stain (Fig. 5). Staining of the PDLSC cultures was

associated with cell clusters whereas staining within the BMSC cultures was more diffuse

seen throughout the culture. To confirm the differentiation of PDLSC towards an os-

teoblast/cementoblast-like tissue, we investigated the presence of ALP involved in the early

mineralization events and BSP a gene expression product known as a late mineralized tis-

sue marker (Fig. 6). Immunohistochemical evaluation (Fig. 6, A-C) and RT-PCR analysis

(Fig. 6, D) showed that osteogenic induced PDLSC cultures express known mineralized

tissue markers: ALP and BSP. The positive control samples BMSC cultures were positive

for both proteins. In situ staining for ALP activity was detectable by 14 days of culture in

the PDLSC as compared to 7 days for the BMSC cultures. However, by 21 days no detect-

- 34 - 34

able difference in level of ALP activity was seen between the two cell populations. ALP

expression was confirmed by RT-PCR analysis was well as the expression of a late miner-

alized tissue marker BSP. Expected amplification products (ALP 288 bp and BSP 204 bp)

were detectable in the PDLSC and BMSC cultures as early as 7 days.

GAPDH, a housekeeping gene, served as a positive control for the PCR reaction.

Control samples were negative for all tested markers at all time points.

We next assessed whether PDLSC, like BMSC, had the potential to differentiate

into other cell lineages such as adipocytes. After 25 days of culture with an adipogenic in-

ducing media, PDLSC as well as BMSC developed oil red O positive lipid-laden droplets

(Fig. 7, A & C) whereas control cultures grown in control media failed to produce similar

results (Fig. 7, B & D). These finding clearly correlate with an up-regulation in the expres-

sion of two adipocyte specific early/late differentiation transcripts LPL and PPARγ2, re-

spectively. As shown in Figure 8 panel E quantitative RT-PCR normalized against the

pseudogene 18 S (31) showed increase in the two adipocyte specific gene markers in both

cell populations. While the two transcripts increased in the PDLSC cultures, the change in

the later marker PPARγ2 was greater in the BMSC culture showing a nearly 10 fold in-

crease.

Another well-characterized feature of undifferentiated mesenchymal stem cells such

as BMSC is their capability to differentiate into chondrocytes, the process of chondrogene-

sis. After 14 days of exposure to chondrogenic differentiation media containing 10 ng/ml of

TGF-β3, BMSC express metachromasia with toluidine blue stain as well as positive immu-

nohistochemistry for collagen type II a gene marker for chondrocyctes. PDLSC failed to

express metachromasia at day 14, although they were positive for collagen type II expres-

sion. At 21 days both cell populations show chondrocyte-like cell morphology as well as

the expression of collagen type II and toluidine blue metachromasia. Staining with

- 35 - 35

touidine blue demonstrates the accumulation of glycosaminoglycans in the extracellular

matrix (Figs 8, 9). Negative controls exposed to control media were not possible to obtain

due to the fact that stem cells fail to form aggregates unless they are exposed to differentia-

tion media.

DISCUSSION

Our findings clearly demonstrate the successful isolation and characterization of

undifferentiated progenitor cells contained in human PDL that can be expanded in vitro,

providing a unique reservoir of stem cells obtained with minimally invasive procedures.

Therefore, the PDL apparatus represents a viable alternative to obtain potentially high

numbers of cells for regenerative procedures without the necessity of bone marrow aspira-

tion or more invasive procedures that will result in increased morbidity.

Our experiments show PDLSC multipotential capability to differentiate in vitro to-

wards osteogenic, adipogenic and chondrogenic tissues. These results positively correlate

with the well characterized BMSC when exposed to appropriate culture conditions. In this

study we found that PDLSC are similar to other mesenchymal stem cells such as BMSC

(32-34) and dental pulp derived stem cells (35-38) with respect to their expression of the

STRO-1 marker. The development of a series of monoclonal antibodies raised towards

BMSC surface antigens, along with other antibodies developed to characterize BM stromal

cells has been crucial for the immunophenotyping of these cells. Results have shown that

the antigenic profile of BMSC is not unique, presenting features of mesenchymal, endothe-

lial, epithelial and muscle cells (39). Several research laboratories have joined efforts to

find the potential “gold standard” antibody for stem cell identification. From these, STRO-

1 IgM antibody has shown consistent, promising results (40-42). In our laboratory, other

- 36 - 36

surface antibodies, such as SH2, SH3, SH4, endoglin (CD105) and CD44 (43-46) yielded

100% positive results which would seem to indicate that all PDL cells are multipotent (data

not shown). These results are not compatible with heterogeneous PDL cell population that

contains fibroblasts, osteoblasts, cementoblasts and progenitor cells. After exhaustive sur-

face marker testing we were able to isolate and purify a PDLSC population derived from

the PDL using the STRO-1 antibody.

PDLSC demonstrated by 48 hours the same growth rate as BMSC. At 96 hours

PDLSC cell numbers were almost double those of BMSC cells. Similar findings have been

reported by other authors for other dental tissue derived stem cells such as those isolated

from permanent tooth pulp (35-38). The exact mechanism for this increased cell growth

remains obscure. The nature of this difference may arise from several determinants, includ-

ing the procedure used to harvest the cells, the age difference of the PDLSC donors (18-26

years) versus the BM donor (37 years). Another possibility is that dental tissue derived

stem cells exhibit higher mitotic properties that cells derived from BM aspirations, specifi-

cally BMSC. As a consequence of faster cell growth, the clonogenic capabilities of PDLSC

were superior as compared with BMSC.

The osteogenic potential of the heterogeneous population of PDL cells has been

demonstrated earlier with in vitro procedures as well as the ability of such cultures to form

a mineralized matrix (47). In our series of experiments PDLSC fail to produce such results

at all time points tested. Mineralization was induced after 14 days of exposure to differenti-

ating media. Our data and other published data (35-38,48) shows the potential of PDLSC to

form mineralized deposits in vitro as previously has been shown with other mesenchymal

cell populations (49). However, PDLSC formed sparse calcified focal nodules that took

longer to form as compared with their BMSC counterparts. These results are supported by

- 37 - 37

the gene expression profiles and protein expression patterns as determined by RT-PCR

analyses, in situ expression, and immunohistochemistry.

A possible explanation for the delay in differentiation seen in the PDLSC cultures is

the presence of terminally differentiated cell types in the enriched population. By FACS

sorting techniques, we were able to obtain a 73.3% enrichment of STRO-1 positive cells,

thus it is possible that differentiated cell types such as fibroblasts interfered with the miner-

alization process. In other words, it is possible that PDLSC used in our experiments repre-

sent a more heterogeneous SC enriched population mixed with a greater subpopulation of

terminally differentiated cell types. Another possible explanation for this finding is that our

PDLSC population is at a different stage of commitment as compared to the BMSC.

Recent in vivo data has shown that SC differentiation may be due to cell fusion with

other differentiated cell types (50,51). Although it is known that several various cell types

reside in PDL tissue, adipocytes have not been reported as a native component. In our se-

ries of experiments, we were able to demonstrate that an inductive adipogenic tissue culture

media can induce PDLSC and BMSC to form characteristic oil red O positive lipid laden

droplets that are comparable to those found in adipocyte cells. This phenotypic conversion

was also correlated with the expression of the early adipogenic transcription factor PPARγ2

and the later marker LPL. Similar results for dental pulp SCs and PDLSC have been pub-

lished (35-38, 48).

A differentiation pathway that has not been reported for PDLSC is their ability to

form chondrocyte like tissues when exposed to chondrogenic media with the addition of

TGF-β3. We successfully induced not only a shift in cell morphology towards a chondro-

cyte cell morphology, but also the expression of collagen type II and proteoglycans as indi-

cated by metachromasia with toluidine blue stain seen at 21 days. The resulting differenti-

ated tissue shows characteristics representing a prechondroyd differentiation stage. Carti-

- 38 - 38

lage is not a normal tissue type associated with the periodontal apparatus or surrounding

areas, which further demonstrates the multipotent potential of PDLSC to differentiate into

multiple cell types.

The specific cues to initiate the proliferation and differentiation of PDLSC in vivo

are not known. The ability to isolate, expand in culture and direct the differentiation of

PDLSC in vitro to particular lineages provides the opportunity to study events associated

with commitment and differentiation. From the various unique culture conditions required

for terminal differentiation, we conclude that basal nutrients, cell density, spatial organiza-

tion, growth factors and cytokines present in the culture media have a profound influence

on PDLSC differentiation. PDLSC cultured in each of the differentiation conditions may

also produce autocrine and paracrine factors that are essential for specific lineage progres-

sion. The PDLSC described here have the ability to proliferate extensively and maintain

their capability to differentiate into multiple cell types in vitro, establishing their SC char-

acteristics. Their cultivation and selective differentiation should provide further under-

standing of this important progenitor of multiple tissue types.

We are currently evaluating the behavior of these cells in vivo on an osteoconduc-

tive scaffold environment as well as their interaction with SLA titanium surfaces for the

possible formation of a natural PDL on the surface of dental implants. Therefore, PDLSC

can potentially be isolated and expanded in vitro for their use in PDL regenerative thera-

pies, with minimal discomfort due to invasive procedures commonly performed.

- 39 - 39

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ology and pathology, J Periodontal Res 1991; 26:144-54. 2. Pitaru S, McCulloch CAG, Narayanan SA. Cellular origin and differentiation

control mechanisms during periodontal development and wound healing, J Perio-dontal Res 1994; 19: 81-94.

3. Liu WH, Yacobi R, Savion N, Narayanan AS, Pitaru S. A collagenous cementum-derived attachment protein is a marker for progenitors of the mineralized tissue forming cell lineage of the periodontal ligament, J Bone Miner Res 1997; 12: 1691-99.

4. Gottlow J, Nyman S, Karring T, Linde J. New attachment formation as the result of controlled tissue regeneration, J Clin Periodontol 1984; 11: 494-503.

5. McCulloch CAG, Melcher AH. Cell density and cell generation in the periodontal ligament of mice, Am J Anat 1983; 167: 43-58.

6. McCulloch CAG, Nemeth E, Lowenberg B, Melcher AH. Paravascular cells in en-dosteal spaces of alveolar bone contribute to periodontal ligament cell populations, Anat Rec 1987; 217: 2233-42.

7. Roberts WE, Chase DC. Kinetics of cell proliferation and migration with orthodon-tically induced osteogenesis, J Dent Res 1981; 60: 174-81.

8. Melcher AH. On the repair potential of periodontal tissues, J Periodontol 1976; 47256-60.

9. Mann LM, Lennon DP, Caplan AI. Cultured rat pulp cells have the potential to form bone, cartilage, and dentin in vivo. In: Davidovitch Z, Norton LA, editors. Biological Mechanisms of Tooth Movement & Craniofacial Adaptation. Harvard Society for the Advancement of Orthodontics, Birmingham, AL: EBSCO Media: 1996, p. 7-16.

10. Caplan, AI. Mesenchymal stem cells, J Ortho. Res 1991; 9:641-50. 11. Owen, M. Lineage of osteogenic cells and their relationship to the stromal system.

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12. Dennis, JE, Haynesworth, SE, Young, RG, Caplan, AI. Osteogenesis in Marrow-Derived Mesenchymal Cell Porous Ceramic Composites Transplanted Subcutane-ously: Effect of Fibronectin and Laminin on Cell Retention and Rate of Osteogenic Expression, Cell Transpl 1992;1:23-32.

13. Jaiswal, N, Haynesworth, SE, Caplan, AI, Bruder, SP. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro, J Cell Biochem 1997; 64:295-312.

14. Bruder, SP, Fink, DJ, Caplan, AI. Mesenchymal Stem Cells in Bone Development, Bone Repair, and Skeletal Regeneration. J Cell Biochem 1994; 56:283-294.

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15. Mbalaviele, G, Jaiswal, N, Meng, A, Cheng, L, Van Den Bos, C, Theide, M, Hu-man Mesenchymal Stem Cells Promote Human Osteoclast Differentiation from CD34+ Bone Marrow Hematopoietic Progenitors. Endocrin 1999; 140: 3736-43.

16. Majumdar, MK, Thiede, MA, Haynesworth, SE, Bruder, SP, Gerson, SL. Human Marrow-Derived Mesenchymal Stem Cells (MSCs) Express Hematopoietic Cyto-kines and Support Long-Term Hematopoiesis When Differentiated Toward Stromal and Osteogenic Lineages, J Hematotherapy and Stem Cell Res 2000; 9: 841-8.

17. Reese, JS, Koc, ON, Gerson, SL. Human Mesenchymal Stem Cells Provide Stro-mal Support for Efficient CD34+Transduction, J Hematotherapy 1999; 8:515-23.

18. Majumdar, M, Thiede, M, Mosca, J, Moorman, M, Gerson, S. Phenotypic and Functional Comparison of Cultures of Marrow-derived Mesenchymal Stem Cells (MSCs) and Stromal Cells, J Cell Phys 1998; 176: 57-66.

19. Egginton,S. The Role of Pericytes in Controlling Angiogenesis In Vivo, Adv Exp Med Biol 2000; 476:81-99.

20. Filip S, English D, Mokry J. .Issues in stem cell plasticity. J Cell Mol Med 2004; 8:572-7.

21. Nuttall ME, Patton AJ, Olivera DL, Nadeau DP, Gowen M. Human trabecular bone cells are able to express both osteoblastic and adipocytic phenotype: implications for osteopenic disorders. J Bone Miner Res 1998; 13 :371-82.

22. Sparkes, RS, et al. Human genes involved in lipolysis of plasma lipoproteins: map-ping of loci for lipoprotein lipase to 8p22 and hepatic lipase to 15q21. Genomics 1987; 1: 138-44.

23. Meinel L, et al. Engineering cartilage-like tissue using human mesenchymal stem cells and silk protein scaffolds Biotechnol Bioeng 2004; 5:379-91.

24. Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo J. In vitro chondrogene-sis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 1998; 10:265-72.

25. Sekiya I, Vuoristo JT, Larson BL, Prockop DJ. In vitro cartilage formation by hu-man adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis. Proc Natl Acad Sci 2002; 2:4397-402.

26. Ballock RT, Reddi AH. Thyroxine is the serum factor that regulates morphogenesis of columnar cartilage from isolated chondrocytes in chemically defined medium. J Cell Biol 1994; 126: 1311-18.

27. Kim, MS et al. Musculoskeletal Differentiation of Cells Derived from Human Em-bryonic Germ Cells, Stem Cells 2005; 23:113-123.

28. Yates KE. Demineralized bone alters expression of Wnt network components during chondroinduction of post-natal fibroblasts. Osteoarthritis Cartilage 2004; 12:497-505.

29. Minguell JJ, Erices A, Conget P. Mesenchymal stem cells. Exp Biol Med 2001; 226:507-20.

30. Beckler A, et al. Overexpression of the 32-kilodalton dopamine and cyclic adeno-sine 3’,5’Monophosphate-regulated phosphoprotein in common adenocarcinomas, Cancer 2003; 98:1547-51.

31. Zhang Z, Harrison P, Liu Y, Gerstein M. Millions of years of evolution preserved: A comprehensive catalog of the processed pseudogenes in the human genome, Ge-nome Res 2003; 13:2541-58.

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32. Caplan, AI . Mesenchymal stem cells, J. Ortho. Res, 1991; 9:641-50. 33. Owen, M. Lineage of osteogenic cells and their relationship to the stromal system.

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34. Bruder, SP., Fink, DJ., Caplan, AI. Mesenchymal Stem Cells in Bone Development, Bone Repair, and Skeletal Regeneration. J. Cell Biochem 1994; 56:283-94.

35. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo, Proc Natl Acad Sci 2000; 97: 13625-30.

36. Shi, S, Robey, PG, Gronthos, S. Comparison of human dental pulp and bone mar-row stromal stem cells by cDNA microarray analysis, Bone 2001; 29:532-39.

37. Gronthos S, et al. Stem cell properties of human dental pulp stem cells, J Dent Res, 2002; 81: 531-35.

38. Miura M, et al. SHED: Stem cells from human exfoliated deciduous teeth, PNAS 2003; 100: 5807-12.

39. Minguell JJ, Erices A, Conget P. Mesenchymal stem cells. Exp Biol Med 2001; 226: 507-20.

40. Dennis JE, Carbillet, JP, Caplan AI, Charbord P, (2002), The STRO-1+ Marrow Cell population is multipotential, Cell Tissues Organs 2002; 170: 73-82.

41. Simmons PJ, Torok-Storb B.( 1991) Identification of stromal cell precursors in hu-man bone marrow by a novel monoclonal antibody, STRO-1. Blood 1991; 1: 55-62.

42. Stewart K, Monk P, Walsh S, Jefferiss CM, Letchford J, Beresford JN. (2003) STRO-1, HOP26 (CD63), CD49a and SB-10 (CD166) as markers of primitive hu-man marrow stromal cells and their more differentiated progeny: a comparative in-vestigation in vitro. Cell Tissue Res 2003; 313: 281-90

43. Haynesworth, SE, Baber, MA, Caplan, AI. Cell Surface Antigens on Human Mar-row-Derived Mesenchymal Cells are Detected by Monoclonal Antibodies, Bone 1992; 13: 69-80.

44. Xu W, et al. Mesenchymal stem cells from adult human bone marrow differentiate into a cardiomyocyte phenotype in vitro. Exp Biol Med 2004; 229:623-31.

45. Zhang CC, Lodish HF. Murine hematopoietic stem cells change their surface phe-notype during ex vivo expansion Blood 2005; 105(11):4314-20.

46. Boiret N, et al. Characterization of nonexpanded mesenchymal progenitor cells from normal adult human bone marrow. Exp Hematol 2005; 33:219-25.

47. Mouri Y, Shiba H, Mizuno N, Noguchi T, Ogawa T, Kurihara H. Differential gene expression of bone-related proteins in epithelial and fibroblastic cells derived from human periodontal ligament. Cell Biol Int 2003; 27:519-24.

48. Seo BM et al. Investigation of multipotent postnatal stem cells from human perio-dontal ligament. Lancet 2004; 10:149-55.

49. Pittenger M et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284:143-47.

50. Ying QL, Nichols J, Evans EP, Smith AG. Changing potency by spontaneous fu-sion. Nature 2002; 416, 545–48.

51. Terada, N. et al. Bone marrow cells adopt the phenotype of other cells by sponta-neous cell fusion. Nature 2002; 416, 542–45.

- 42 - 42

FIGURE LEGENDS

Figure 1. Immunohistochemistry of human BMSC and PDL cells with mesen-

chymal stem cell marker STRO 1. Panels A-F represent images from BMSC cultures

bright field (A & D), fluorescence (B & E) and overlay of both images (C & F). Panel B

and C show positive cell staining for STRO-1 of the BMSC population (20X). Panels E,

and F represent the negative control results lacking the primary antibody (20X). Panel H

and I show the STRO-1 staining with a single positive cell observed in the visible field

(10X), in contrast to the negative controls with no staining shown in panels K and L. Im-

ages were obtained with an Olympus Wide Field Microscope at the Imaging Core Facility

University of Texas Health Science Center at San Antonio (UTHSCSA). Details of the ex-

periments are described under Materials and Methods section.

Figure 2. FACS Sorting of STRO-1 Positive PDL Cells. Primary digested pooled

adult human PDL cells were immunostained with STRO-1 antibody and cells analyzed on a

FACScan with automated cell deposition unit (Becton Dickinson, Parsippany, NJ)

equipped with an argon laser and data analyzed with CellQuest software (Becton Dickin-

son, Parsippany, NJ) at the UTHSCSA Institutional Flow Cytometry Core Facility. A) The

gated cell population. B) The percent of the STRO-1 positive cell population identified

(27%). C) A 73.3% enrichment of STRO-1 positive PDL cells at second cell sorting.

Strongly positive STRO-1 cells represent around 3% of this total cell population. D)

Analysis of the negative STRO-1 cell population with a 99.8% purity.

- 43 - 43

Figure 3. PDLSC and BMSC Cell Proliferation Rates. PDLSC and BMSC were

cultured for 0, 48 and 96 hours in 12 well plates with media changes every other day. The

cells were harvested by trypsinization and counted with a Coulter Counter. The data are

from 1 of 3 representative experiments each yielding similar results. Values are the mean ±

SEM for 6 cultures (P<0.05).

Figure 4. PDLSC and BMSC Colony Forming Units. At day 7 after plating,

PDLSC and BMSC were fixed with 10% formalin and stained with toluidine blue. Aggre-

gates of 50 or more cells were considered colonies and counted. A) PDLSC representative

colony (10X). B) BMSC representative colony (10X). C) Graph represents a statistically

significant difference in total colony number between PDLSC (n=50) and BMSC (n=35)

cultures. Values are the mean ± SEM for 6 cultures (P< 0.05).

Figure 5. Expression of mineral deposits within PDLSC and BMSC long term

cultures. At day 35 BMSC show mineral deposits as is evidenced by alizarine red stain.

Those deposits are distributed throughout the tissue culture well. In contrast, PDLSC cul-

tures demonstrate calcium deposits at day 48 and the distribution was seen as dense foci

associated with cell clusters (20X).

Figure 6. BSP and ALP gene expression profiles of osteogenic BMSC and

PDLSC cultures. BMSC and PDLSC were grown in osteogenic (experimental) or normal

(control) media for 7, 14 and 21 days. ALP staining increased temporally in the experimen-

tal induced groups whereas the control groups failed to express detectable enzyme activity

(A & B). Immunohistochemistry images reveal the presence of BSP positive cells after 7

days of culture (10X) in both cell populations. Staining was more intense in the PDLSC

and continued through day 21. Negative controls, lacking primary antibody showed no

- 44 - 44

staining at all days tested (C). RT-PCR image for ALP, BSP and the housekeeping gene

GAPDH on PDLSC cells. At day 7 there is no detectable ALP expression. Starting at day

14, ALP expression increases throughout whereas BSP remains as a constant expression

from day 7 through day 21 in culture (D). Lanes were loaded as follows: 1) PDLSC 7 days

induced; 2) PDLSC 7 days control; 3) PDLSC 14 days induced; 4) PDLSC 14 days control;

5) PDLSC 21 days induced; and 6) PDLSC 21 days control.

Figure 7. Characterization of adipogenic differentiation of PDLSC and BMSC

cultures. Panel A shows BMSC induced cultures at 25 days with visible positive oil red O

staining lipid droplets. This is in contrast to the control cultures grown for the same period

in normal media (B). Adipogenic Induced cultures of PDLSC also formed positive lipid

droplets after 25 days in culture (C), These droplets were not seen in the negative control

cultures (D). Magnification 20X. Panel E shows the RT-PCR analysis of adipogenic mark-

ers demonstrating statistical significant upregulation of PPARγ2 and LPL in BMSC. Direct

comparison between BMSC and PDLSC demonstrates increased production of both gene

expression markers and lipid accumulation in BMSC. Values are the mean ± SEM for 6

cultures (P< 0.05).

Figure 8. In vitro chrondogenic differentiation of PDLSC and BMSC. Panels A

and C show BMSC cultures after 14 and 21 days, respectively treated with chondrogenic

differentiation media (10X). Panels B and D show PDLSC cultures after 14 and 21 days,

respectively treated with chondrogenic differentiation media (10X). In both cell popula-

tions metachromasia and cell morphology correspond with embryonic stages of cartilage

formation.

- 45 - 45

Figure 9. Immunohistochemistry for collagen type II expression in PDLSC and

BMSC chrondrogenic induced cultures. BMSC and PDLSC were cultured as aggregates

in a differentiation media for 14 days (A & B) and 21 days (C & D), Top rows show im-

ages correspond to samples exposed to primary collagen type II antibody; whereas bottom

rows show negative control images lacking collagen type II antibody (10X). Both BMSC

and PDLSC induced cultures were strongly positive for type II collagen expression at the

time points tested.

- 46 - 46

Figure 1

J L K

G H I

D FF

A

E

B C

- 47 - 47

A B

27% M1

R1

73.3% 99.8 %

C D

M1M1

Figure 2

- 48 - 48

STEM CELL GROWTH

0

2

4

6

8

10

12

14

16

0 hour s 48 hour s 96 hour s

HOURS

PDLSC

BMSC

A

Figure 3

- 49 - 49

- 50 - 50

STEM CELL COLONY FORMING UNITS

0

10

20

30

40

50

PDLSC BMSC

7 DAYS

CO

LON

Y U

NIT

S60

A

A

B

C *

Figure 4

PDLSC

BMSC

Control Induced

Figure 5

- 51 - 51

21 days

14 days

21 days

BMSC PDLSC C D 7 days

14 days

7 days

Control 21 days

B Control Induced Control Induced

AL

BSP

GAPD

M 1 2 3 4 5 6 +

BMSC PDLSC A

Figure 6

- 52 - 52

A B

C D QRT-PCR ON BMSC

0

2

4

6

8

10

12

LPL PPAR gamma 2

GENE

FOLD

INC

REA

S

Figure 7

E

E

QRT-PCR ON PDLSC

0

20

40

60

80

100

L PPAR gamma2

GENE

PER

CEN

T IN

CR

EAS

LP

- 53 - 53

- 54 - 54

A B

BMSC PDLSC

14 days 21 days

Figure 8

C D

B PDLSC

Type II Control

Type II Control

A BMSC

Figure 9

- 55 - 55

FUTURE PROSPECTS FOR PERIODONTAL REGENERATION

Numerous clinical techniques, including bone grafts, root surface conditioning, bar-

rier membranes, and various growth factors, have been utilized over the years in an attempt

to achieve PDL regeneration. Unfortunately, current therapeutic measures are unable to

obtain predictable regeneration, thus underscoring the importance of restoring or providing

the cells and microenvironment capable of initiating and promoting new periodontal tissue

formation.

From a biological perspective, in order for periodontal regeneration to occur, the

availability of appropriate cell types, together with a favorable local environment promot-

ing cell migration, adhesion, proliferation and differentiation, all need to be precisely coor-

dinated both temporally and spatially. Thus, a tissue engineering strategy for periodontal

regeneration that exploits the regenerative capacity of stem cells residing within the PDL,

grown in a three-dimensional construct and subsequently implanted into the defect may

help to overcome many limitations with current regeneration modalities 91. In doing so, the

need for recruitment of various different cells to the site is negated and the predictability of

the outcome may be enhanced.

Mesenchymal stem cells are already being utilized in regenerative medicine, most

notably in orthopedic medicine where they have been shown to facilitate repair of bone 92

and cartilage 93. Furthermore, MSC have also been shown to produce de novo myocardium

- 56 - 56

94 as well as assist in the functional recovery following spinal cord injury 95. The potential

of stem cell-based tissue engineering has also been harnessed in the reconstruction of mur-

ine teeth using cultured stem cells which when transferred into renal capsules, resulted in

the development of tooth structures and associated bone 96. These results provide signifi-

cant advances towards the creation of artificial embryonic tooth primordia from cultured

stem cells that can be used to replace missing teeth following transplantation into the adult

in order to fulfill the criteria of true biomimetic tissue regeneration.

The plausibility of a stem-cell based tissue engineering approach to achieve perio-

dontal regeneration and formation of a true periodontal ligament around dental implants is

supported by animal studies demonstrating that periodontal ligament cells cultured in vitro

can be successfully reimplanted into periodontal defects and or around titanium dental im-

plants in order to promote true regeneration 97,98.

In light of these findings, the identification of stem cells within the periodontal liga-

ment with the ability to achieve new attachment formation in vivo is a significant develop-

ment towards a tissue engineering approach to obtain periodontal regeneration. Further-

more, the ready availability of periodontal ligament tissue from redundant teeth, such as

third molars, may provide a supply of stem cells that may be utilized for regenerating, not

only the masticatory apparatus, but also other body tissues.

SUMMARY AND CONCLUSIONS

Our findings clearly demonstrate the successful isolation and characterization of un-

differentiated progenitor cells in the periodontal ligament that can be expanded in vitro,

providing a unique reservoir of stem cells obtained with minimally invasive procedures.

- 57 - 57

Therefore, the periodontal ligament apparatus represents a viable alternative to obtain poten-

tially high numbers of cells for regenerative procedures without the necessity of bone mar-

row aspiration or more invasive procedures that result in increased morbidity.

Our experiments show PDLSC multipotential capability to differentiate in vitro to-

wards osteogenic, adipogenic, and chondrogenic like tissues. These results positively corre-

late with the well characterized BMSC when exposed to appropriate culture conditions. We

have found that PDLSC are similar to other mesenchymal stem cells such as BMSC32-34 and

dental pulp derived stem cells35-38 with respect to their expression of the STRO-1 marker.

The specific cues to initiate the proliferation and differentiation of PDLSC in vivo are

not known. The ability to isolate, expand in culture, and direct the differentiation of PDLSC

in vitro to particular lineages provides the opportunity to study events associated with com-

mitment and differentiation. From the different assay conditions required for terminal differ-

entiation, we conclude that basal nutrients, cell density, spatial organization, growth factors,

and cytokines present in the culture media have profound influence on PDLSC differentia-

tion. PDLSC cultured in each of the differentiation conditions may also produce autocrine

and paracrine factors that are essential for lineage progression. The PDLSC described here

have the ability to proliferate extensively and maintain their capability to differentiate into

multiple cell types in vitro, establishing their irrefutable stem cell characteristics and nature.

Their cultivation and selective differentiation should provide further understanding of this

important progenitor of multiple tissue types.

Future challenges include the molecular engineering of PDLSC to provide potential

gene therapy in patients affected with genetic diseases. It would be beneficial to engineer a

patient’s own PDLSC to replace a defective gene and restore basic cell genotype and normal

- 58 - 58

function. To permanently modify PDLSC with retroviral vectors proven to be efficient and

neutral with respect to cell function represents a viable research option. In this respect, these

tools will provide not only long-term production of a desired protein, but also the opportunity

to design constructs able to modulate, or silence the expression of genes associated with

genetic diseases of the craniofacial skeleton.

Another possible application of multipotential PDLSC might be the percutaneous de-

livery of these cells to a localized lesion such as cysts or cleft defects in bony tissues. The

use of a semisolid carrier, via injection directly to the defect through the epithelial barrier,

represents an alternative approach that will be less invasive than a surgical procedure. A

number of injectable carriers are currently available, such as calcium phosphate and calcium

sulfate, polylactide and polyglicolide acids, which could be used to deliver PDLCS with

combinations of growth factors or cells alone. With further development, direct deliver of

cells without open surgery could provide a major clinical advantage.

Current dental implant treatment relies on the ability of bone to interface with the ti-

tanium surface in a process known as oseointegration. This interface may be improved by

the development of cementum on the implant surface, along with the generation of a true

PDL between the newly formed cementum and alveolar bone. This type of ideal connection

will provide the implant with loading dispersion capabilities better able to resist occlusal

forces.

The ultimate goal of our stem cell research will be to recreate a tooth bud that can be

surgically implanted in an edentulous area in order to develop a tooth like structure on its

own. A recent study 99 showed that cells isolated from un-erupted tooth buds have the ability

to reorganize into dental like structures when transplanted with a carrier in vivo. Further-

- 59 - 59

- 60 - 60

more, it can be envisioned that tooth buds can be formed by introducing mesenchyme from

dental or non-dental sources or using different types of stem cells in conjunction with oral

epithelium. One study using a similar approach generated tooth bud like structures in rodents

96.

In conclusion, current science clearly indicates that the use of stem cells for regenera-

tion, reconstruction, or bone repair is feasible. Translation of these advances into clinical

practice will occur. This endeavor is multidisciplinary in nature, with the periodontist and

the researcher playing a substantial role. However, there are still limitations in knowledge

that need to be solved before this “biomimetic dentition” becomes a clinical reality.

GENERAL REFERENCES

1. Jan Lindhe, Thorkild Karring, Mauricio Araujo, (2003), Anatomy of the Periodon-tum. Clinical Periodontology and Implant Dentistry. Davidovitch and L.A.Jan Lindhe, Thorkild Karring, Nikklaus P. Lang (ed), Blackwell Publishers, 4th ed 1-49.

2. McCulloch CAG, Bordin S, (1991), Role of fibroblast subpopulations in periodon-tal physiology and pathology, J Periodontal Res, 26:144-154.

3. Pitaru S, McCulloch CAG, Narayanan SA, (1994), Cellular origin and differentia-tion control mechanisms during periodontal development and wound healing, J Periodontal Res, 19: 81-94.

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INSTITUTIONAL REVIEW BOARD APPROVAL

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