HUMAN BONE GROWTH IN VITRO

Elaine Parker

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

Graduate Department of Dentistry, in the University of Toronto

O Copyright by Elaine Parker 1999

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Human Bone Growth In Vitro

Master of Science, 1999 Elaine Parker

Faculty of Dentistry. University of Toronto

Abstract

The successful development of methods for culturing human osteogenic cells is of

considerable interest and importance to the bone biology. biornaterials and tissue

engineering fields. This is the First study that has described the development of a

morphologically identifiable bone matrix made by human bone marrow stromal cells, in

both primary and first passage cultures. Using scanning and transmission electron

microscopy this bone matrix was s h o w to comprise of an interfacial afibrillar matrix

which? in the rat has been identified as the in vitro equivalent of the cernent line matrix

found at bone remodeling sites. Above this cernent line matrix a collagenous bone matrix

was assembled. in ahich osteocalcin. a key marker for mature osteogenic cells, was

incorporated. Finally the developrnent of osteocytic cells and the normal mineralization

of the collagen matris illustrated the maturation of this bone tissue. Having developed a

system for culturing expanded human stromal ce11 populations it is our hope to utilize this

system for the examination of 3-dimensional scaffolds for bone tissue engineering

applications.

Acknowledgmen ts

1 would like to express my sincerest appreciation to my supervisor Dr. J.E. Davies

for his professional guidance, enduring support and kindness throughout the course of my

graduate studies. My time in the group has been a rewarding experience that has

provided me with a foundation ont0 which 1 can build my future endeavours.

1 would also like to thank Dr. J.E. Aubin. Dr. S.N.M. Heersche and Dr. T.M. Murray for

senring on my graduate cornmittee. 1 have appreciated their advice. critical assessment of

my work and overall support, which helped to keep my project focused.

I extend my most sincere thanks to al1 memben of JED's group. especially X. Shen and

A. Shiga. for their help. advice. encouragement. kindness and friendship.

1 have also very much appreciated the cooperation of Dr. J. Wedge, at the Hospital for

Sick Children, who provided the marrow cells for my work.

Finally to my husband William. whose support. encouragement and loving

companionship enabled me to me to attain the necessary determination to complete this

project. To my parents for their love. concem and attention which has always been

unwavering in al1 of my endeavours.

Contents

Title

Abstract

Acknowledgcments

1 General Introduction

Tissue Engineering 1 .A. 1 General 1 .A.2 Bonr Tissue Engineering

Microstructure & Composition of Bone 1 .B. 1 In vivo Bone Formation and Osteogenesis 1 .B.? Bone Cells

1 .B.2.a Osteoblastic lineage 1 .B.2.b Osteoclasts

1 .B.3 Bone Matrix Composition 1 .B.3.aCollagen 1 .B.3 .b Major non-collagenous proteins 1 .B.3 .c Mineral phase

1 .BA The Cernent Line

Human Bone Growth In Vitro 1 .C. I Bone Explant Cultures 1 C . 2 Bone Marrow Cultures

1 .C.2.a In vivo and in vitro animal rnodels 1 .C.2.b Human marrow

1 C 2 . b . l In vivo 1 .C.2.b.2 In vitro

Modeling Bone Formation In Vitro

2 Hypothesis

3 Objectives

4 Materiais and Methods

4.A Ccll Culture 4.A. 1 Ce11 lsolation and Primary Culture 4.A.2 Subculture

iii

1.B Histological Studies 4.B. 1 Alkaline Phosphatase 4.B.2 Haematoxylin and Eosin 4.8.3 Tetracycline Labeling 4.B.4 Embedding and Sectioning Protocols

4.B.4.a LR white 4.E3.4.b Paraffin embedding and sectioning

4.B. 5 Iinmunolabeling 4. B.5 .a Perosidase labeling of paraffin sections

4.C Scanning Electron Microscopy (SEM), Energy dispersive X-ray Microanalysis (EDX) & Backscattcring Electron Imaging (BSEI) 4.C. 1 Scanning Electron Microscopy 4.C.2 Energy dispersive X-ray Microanalysis 4.C.3 Backscattering Electron lrnaging (BSEI)

4.D Transmission Electron Microscopy

5 Results

Light Microscopy of Standard Cultures 5 .A. 1 Tetracycline labeling 5 . A 2 Alkaline phosphatase 5.A.3 Immunoperoxidase labeling

Scanning Electron Microscopy of Standard Cultures

Transmission Electron Microscopy of Standard Cultures

Control Cultures

Dystrophic Mineralization S.E. 1 Electron Microscopy 5.E.2 Effects of B-Glycerophosphate on mineralization

Subcultures 5.F. 1 Subculture 5 .F.2 Light microscopy 5.F.3 Scanning electron microscopy

6 Discussion

6.A Light Microscopy

6.B Ultrastructure

6.C Dystrophic Mineralization

6.D Normal vs. Dystrophic Mineralization the Role of PGP

6.E Bone Matrix Formation in First Passage Cells

6.F Applications for Human Bone Marrow Cultures

7 Conclusions

8 Future Work

9 References

1. Introduction

1. A Tissue Engineeri~tg

I.A. 1 Ceneral

The increasing demand for organ and tissue transplants has driven the emergence

of innovative technologies such as tissue engineering. Tissue engineering was fint

defined in 1987. at the US National Science Foundation in Washington D.C.. as the

application of the principles of both engineering and life sciences towards the

development of biological substitutes to restore. maintain or improve the function of an

organ or tissue (Narem. 1991). This definition encapsulates the current approach in tissue

engineering. which has been to place cells in association with 3-dimensional matrices to

regenerate or augment the function of a tissue. Advances in ce11 biology and its recent

union with materials science have led to the development of unique biological/material

composites that can act as viable replacement tissues. The earliest and most significant

advances in tissue engineering have occurred in the area of artificial skin development. a

product which has found a much needed application in the treatment of bum victims.

Advances in cell culture technoloa have been key to the establishment of the

tissue engineering field (Narem, 1992)? marked specifically by the development of

artificial skin. The work of Rheinwald and Green (1 975) was pivotal to the tissue

engineering field. Here for the first time normal human cells. epidermal fibroblasts. were

propagated successfully in serial culture. Modifications to this technique enabled

investigators to isolate autologous keratinocytes. from srnaIl skin biopsies. which couid

be g r o w and harvested as sheets of cells suitable for grafting (Green et al., 1979). Tliese

original grafis however were difficult to handle and lacked a dermal component

(Eaglstein and Falanga, 1997). To solve these problems biocompatible materials have

been rmployed. One exarnple of this has been the use of collagen-glycoaminoglycan

matrices onto which epidermal cells can be grown (Doillon et al.. 1988. Yannas et al..

1989). These matrices act as a support or delivery system for the cells to a site. In

addition the material provides an artificial dermal layer into which fibrovascular ingrowth

can occur. More recently, in the skin tissue engineering field. materials such as

bioresorbable polymers have been used to create biologically engineered dermal tissue

(Hansbrough et al.. 1992. reviewed by Eaglstein and Falanga. 1997). This procedure

involves the isolation and expansion of nronatal foreskin fibroblasts in vitro. which are

then seeded ont0 a polyglycolic mesh. The mesh acts as a framework and support on

which these cells can easily proliferate and develop a tissue which is very similar to a

normal dermal matrix. Il lustrated in the continuing developrnent of mi ficial skin

technologies is the increasing dependency on both ce11 biologylculture and material

science. For tissue engineering in general Minuth et al. (1998) has cited two key

principles. First. the establishment of novel culture techniques for the isolation,

proliferation and manipulation of ceils in vitro; second. the creation of appropriate

carriers for ce11 delivery into a patient. where the biomatrix or scaffold can adequately

support ceil differentiation ancilor rnaintain phenotype. Adherence to these two principles

is central to the advancement of the field including the new challenge of bone tissue

engineering.

1.A.2 Bone Tissue Engineering Currently bone tissue is the second most transplanted tissue in the USA and

Europe (Martin et al., 1997). where it is used to regenerate bone to repair large bone

defects. resulting from non-union fractures or the removal of tumors from osseous sites.

or in orthopedic procedures. such as spinal fusion. Autologous bone, specifically

trabecular bone. is considered the "gold standard" for grafting (Yaszemski et al.. 1996).

However there is only a limited supply of autologous bone and its retrievai is associated

with high donor site morbidity (Burschardt. 1987). An alternative source of graft material

is allograft bone, and although more readily available its use is associated with slower

patient healing and the risk of disease transfer (Burschardt. 1987). Given these concems

there is a need to create other bone substitutes. The application of tissue engineering

principies may provide a viable alternative.

The bulk of the work in bone tissue engineering has centered on creating suitable

ce11 carriers. The ideal carrier would possess many of the charactenstics of trabecular

bone grafis. These features. reviewed by Yaszemski et al. ( 1996) and Burchardt ( 1987).

include an open porous structure which can be repopulated with mesenchymal stem

cells/osteochondral progenitor cells and allows graft revascularization and therefore the

delivery of nutrients and local cueing factors important to osteogenic differentiation.

Second. a graft should be able to support both osteoinduction. the differentiation and

maintenance of the osteogenic phenotype. and encourage osteoconduction. Finally

trabecular grafis. contrary to other bone grzfis, are ofien completely replaced by new bone

during the remodeling process. In terms of artificial substitutes. this requires that the

material should either undergo chernical degradation or cellular resorption so that it c m

eventually be replaced by bone. There are several materials that have been investigated

for this purpose. which have some if not potentially al1 of these properties.

Biodegradable polyrners are one class of material that may be suitable for bone

tissue applications (reviewed by Yaszemski et al.. 1996, Hollinger et ai., 1993). In

general. it is believed that the structure and composition of polymers are easily

manipulated to meet the specifics of an application such as the creation of foams with an

open porous structure resembling trabecular bone. These materials cm undergo either

ce11 ular or c hemical degradation and therefore can eventuall y be replaced by bone.

Idrally the degradation of the polyrner should be controllable and predictable. where the

products from this process can be easily metabolized and removed from the body.

Several different polymers and their 3D constructs have been investigated for their ability

to support osteogenesis and bone ingrowth. Of major interest are poly-hydroxyesters

such as polylactic acid. polyglycolic acid (Puelacher et al.. 1996) and poly

lactidelglycolide copolymers (Ishaug et al.. 1994. Ishaug-Riley et al.. 1997a. 1997b, Holy.

1998). Cerarnics such as tncalcium phosphate and hydroxyapatite are currently being

investigated for hard tissue regeneration applications (Hollinger et al., 1996). The

primary reason for their investigation is the similarity of the material to the minera1 phase

of bone, which is considered desirable for both osteoinduction and osteoconduction

(Yaszemski et al.. 1996). Finally, the development composites made from both

biodegradable polymers and cerarnics combines the benefits of both these classes of

materials. therefore providing a unique alternative for tissue engineering applications

(Laurencin et al.. 1996).

Considerable work remains in the development and characterization of materials

suitable for skeletal regeneration. The development of suitable ce11 carriers is but one

aspect of bone tissue engineering. Efforts rnust also focus on the development of

methodologies for the isolation and in vitro expansion of cellular populations with

osteogenic potential. where upon these cells can be delivered to an osseous defect and aid

in its regeneration (Caplan. 1994).

Current bone tissue engineering approaches have focused on utilizing

mesenchymal stem cells (MSCs) for the regeneration of skeletal tissue defects. MSCs are

defined as multipotential progenitors cells whose progeny give rise to skeletal tissues

such as cartilage. tendon. muscle and bone (Caplan. 1991 ). Bone tissue engineers have

focused their energies on this population of cells. because of their recognized role in the

development. maintenance and repair of bone (reviewed by Caplan. 1991. Bnider et al..

1994). These cells which are believed to be housed in the marrow and periosteum of

bone (Triffit. 1996), travel to remodeling sites where they will undergo osteogenesis or to

fracture si tes where osteochondral di fferentiation will occur. In fracture healing.

osteogenesis will be favoured provided that a vasculature is present at the site and that the

fracture site is immobilized. The local cueing factors involved in osteogenesis are as yet

poorly understood. they include cytokine/growth factors. which are provided by both

osteogenic cells and neighbouring cells such as vascular endothelial cells (Bruder et al..

1994). A better understanding of these events could be usehil when developing strategies

which involve MSCs.

Although many tissue engineering strategies rely upon fully differentiated cells.

the re-implantation of MSCs or early osteoprogenitors rnay be the most practical

approach to bone tissue regeneration (Bruder. 1997). MSCs by definition are not limited

in number of mitotic divisions they can undergo (Caplan, 1991). which is in contrast with

mature osteogenic cells. This feature of primitive osteogenic precursors improves the

feasibility of in vitro expansion. It is predicted that only moderate volumes of mmow

aspirates will be needed to rapidly obtain MSC populations in sufficient numbers for

skeletal regeneration procedures (Caplan and Bruder. 1 997).

Attempts to isolate and study human MSC populations or early progenitors, have

focused on the stromal or adherent cells derived tiom the mononuclear cells population of

the marrow and periosteum. Human marrow stromal or periosteal fibroblastic cells have

a demonstrated capacity to differentiate into several mesenchymal cell types including

adipocytes. chondroblasts and osteoblasts (Nahahara et al., 1991, Haynesworth et al..

1992. Rickard et al.. 1996). This population of cells c m be defined as multipontential.

but clona1 analysis of human marrow stromals would indicate that it is not homogeneous

(Kuznetsov et al., 1997). A definitive MSC remains to be identified.

Atternpts to assess the capacity of multipontential marrow stromal (MMS) cells

for skeletal tissue regeneration have been perfonned in both rabbit and rat models.

Cultured autologous MMS cells injected into defects of the rabbit were found to slightly

accelerate healing vs. the controls (Niedzwiedzki et al., 1993). Kadiyala et al. (1 997a)

used H U C P carriers to implant MMS cells into non-union defects of the rat femur.

Bone formation was found to be significantly higher in defects where cultured stromal

cells had been delivered in comparison to control sites where either no cells or whole

marrow preparations were used. The camer used in this instance was not ideal for use in

tissue engineering strategies. because it lacked macroporosity needed for tissue ingrowth.

Furthenore the experiment was not long enough to assess in vivo scaffold degradation.

Despite this. these experiments do illustrate an important role for MMS cells in bone

regeneration strategies since bone growth was irnproved by enriching this cet 1 population

at the defect site. It has also been suggested that cells be directed ex vivo towards

osteogenesis prior to implantation (Caplan, 1994) and thus possibly improving the bone

healing process. Both dexamethasone (dex) and basic-fibroblast growth factor (bFGF or

FGF-2) are being examined for this purpose (Krebsbach et al. 1997. Martin et al. 1997).

The development or improvement of cellular expansion strategies requires

suitable assays to quanti* the osteogenic potential of the cells afier their manipulation.

The defining characteristic of mature osteoblastic cells. according to Beresford et al.

( 1993). is the ability to produce an identifiable bone matrix. Marrow stromal cells

espanded in vitro. derived from animal species such as rat. dog and chicken have a

demonstrated capacity for bone formation both in vivo and in vitro. This is not the case

with human stromal cells, where in vivo assays. such as the diffusion charnber. remain the

only reliable system for evaluating the osteogenic capacity of the cells. Human cells have

s h o w a varied capacity for bone formation in vitro. which may depend on the culture

conditions used. The development of a reliable system for examining bone matrix

elaboration by marrow stromal cells has yet to be described and characterized.

A human marrow culture system. where the events of bone matrix elaboration

could be identified and studied. would provide a usehl tool for the tissue engineering

field. Materials of interest could be evaluated for their ability to support osteoblast

differentiation and function within a controlled environment. It could be used to assess

the potential value of factors such as BMPs, which are of current interest in skeletal

regeneration strategies (Reddi. 1994. 1995). Finally such a system would provide a more

convenient rnethod to evaluate the osteogenic potential of marrow stroma1 cells following

in vitro manipulation.

1.8 Microstructure and Composition of Bone

1.B.I In vivo Bone Formation and Osteogenesis Embryonic skeletal formation occurs via two distinct process. endochondral or

intramenibraneous ossification (reviewed in Bloom and Fawcett. ( 1 986)). Endochondral

ossification is responsible for the formation of the long bones in the appendicular

skeleton. the vertebral column and the pelvis. Endochondral bone formation is an

indirect process which occurs upon a cartilage framework. In this process mesenchymal

cells di fferentiate down a chondrogenic pathway to form a cartilagenous model.

Eventually the chondrocytes will become hypertrophic. the cartilage matrix undergoes

calcification and this is followed bp the vascularization of the tissue. Vascularization

allows for the delivery of osteognic precursors, in the perivascular tissue. which will

differentiate and deposit bone on the remnants of calcified cartilage. In contrast during

intramernbraneous ossification bone formation occurs in a more direct fashion. In this

process, rnesenchymal cells aggregate, proliferate and differentiate into mature

osteoblasts to form the matrix of the flat bones of the cranial vault and facial skeleton. In

both processes. endochondral or intramembraneous ossification. the resultinç bone matrix

is referred to as primary or woven bone. Primary bone is characterized by a randomly

distributed and loosely packed collagenous matrix. which is rich in osteocytes. Primary

bone. for the most part. is replaced during the remodeling process by secondary bone.

which is lamellar bone in humans. Secondary bone is distinguished by the cernent line

which contours a resorption site (Hattner et al., 1965). creating an interface between the

old bone and the newly formed matrix. The collagen fibres. in human secondary bone.

are laid down in parallel to fonn larnellae of 5-7 mm in thickness. The orientation of

these fibres changes direction by 90" in every subsequent layer. These lamellae give

secondary bone a more orderly appearance in cornparison to woven bone matrix.

The formation of both primary and secondary bone results from the synthetic

activities of mature osteoblasts. Several criteria are used to identify osteoblastic cells

these include (Aubin and Liu, 1996): ( 1 ) the ability to elaborate a tissue that cm be

recognized as bone. (2) cuboidal cells which are found on the surfaces of actively forming

osteoid. (3) post-proliferative alkaline positive cells which can be recognized by their

expression of biochemical markers specific to the phenotype. These include bone matrix

proteins, certain cytokines and expression of the appropriate hormonelcytokine receptors.

Osteoblasts are believed to originate from mesenchymal stem cells contained in

the marrow and periosteum. however MSC rernain to be formaily characterized (Triffit,

1996). The stages marking the differentiation pathway from stem cells to the

differentiated phenotype are il1 defined. In the series of differentiation steps proposed by

Aubin and Lui (1996). the highly proliferate primitive precursors. such as MSC or

osteoprogenitors. progress through to the pre-osteoblast, which has a limited proliferative

capacity and finally to post mitotic cells such as the osteoblast and terminally

differentiated cells. like the osteocyte. Four stages of the osteoblast lineage have been

identified. These inciude preosteoblasts. mature osteoblasts. bone lining cells and

osteocytes. Here again the specific events occumng during maturation are only

beginning to be defined (Aubin and Tursken. 1996). Several different tools are used. or

are being developed. to identiQ cells during osteoblast differentiation and maturation

(reviewed by Aubin and Tursken. 1996). These include morphological criteria.

biochemical markers and monocional antibodies directed against surface antigens.

specific to cells at a particular stage in the process. Given that the present study is mainly

a histological examination of mature hurnan osteogenic cells. and the matrix they

elaborate in culture. this discussion will focus on morphological features which can be

used to classify cells of the osteoblast lineage (reviewed by Kelly et al.. 1983. Bloom and

Fawcett. 1986).

1.B.t Bone Cells The cells nomially associated with bone are those of the osteoblastic lineage and

osteoc lasts. These cells are essential for the production. maintenance and remodeling of

the bone matrix.

1. B. 2. a Osteo blust lit1 eage

The osteoblast lineage includes the mature osteoblasts, preosteoblasts, bone lining

cells and osteocytes. The mature osteoblast was defined by Aubin and Lui (1996). (see

above section 1 .B. 1 ) Ultrastnicturally these cells are characterized by a large

eccentrically placed nucleus, a large Golgi region. numerous mitochondria and an

extensive endoplasmic reticulum al1 of which are indicative of a highly synthetically

active cell.

Some osteoblastic cells will eventually discontinue matrix production to become

bone lining cells. Bonr lining or resting cells are inactive elongated cells. Thcy are

found covering a bone surface. where the matrix is no longer being actively formed. It

has been postulated that bone lining cells may be reactivated and resume synthetic

activity (Dobnig and Turner. 1995). They are also thought to play an important role in

the initiation of the remodeling process (Parfitt. 1 986).

Osteoblasts may also differentiate into osteocytic cells. Osteocytes gradualiy

become entrapped in their owvn matris. As an osteocyte matures the organelles associated

with high synthetic activity begin to vanish and large of amounts glycogen are seen to

accuniulate (Scott & Gliniicher. 1979). These cells, housed in their lacunae. remain in

contact with other osteocytes. bone lining cells and osteoblasts through both intercellular

and extracellular communication mechanisms. Osteocytes extend their narrow

cytoplasmic processes through the canaliculi of bone. communicating with other cells

through gap junctions (Doty. 198 1. Palumbo et al. 1990). Signaling molecules are also

believed to diffuse extracellularly through the lacuno-canaliculi network providing a

means of extracellular communication (reviewed by Nijweide et al.. 1996). Osteocytes

are generally believed to have a major role in the maintenance of bone. where they act as

mechanosensors (reviewed Duncan & Turner. 1995. Cowin, 199 1 ). Young osteocytes

may also be involved in the maturation (Baylink & Wergedal, 197 1 ) and mineralization

of the osteoid (Mikuni-Takagaki et al. 1995).

Preosteoblasts are considered to be the immediate precursors to the osteoblast. In

vivo they are found directly behind mature osteoblastic cells actively depositing matrix.

They. like the mature osteoblast, express al kaline phosphatase. but the synthetic capacity

of these cells is more limited. Other early precunors or the osteoprogenitors appear as

spindle shaped cells. with a high nucleoplasmic ratio. They have very few organelles and

ofien have large accumulations of cytoplasmic glycogen (Scott & Glimicher. 197 1 ).

Osteoclasts are responsible for the resorption of bone. They are classically

identified as large. rnutlinucleated. acid phosphatase tartrate resistant positive (+TRAP)

cells directly associated wi th the bone matrix. Ultrastructurally they are found to have

numerous niitochondria. several well developed Golgi regions. an extensive endoplasmic

reticulum and an abundance of vacuoles and lysosomes. Two other morphological

features are used to distinguish osteoclasts. these are the ruffled border and the sealing

zone. The ruffled border is a region which interdigitates with the bone matrix. this is the

zone of active resorption. The clear zone or sealing zone surrounds the ruffled border

creating a barrier between it and the extracellular space.

1.B.3 Bone Matrix Composition

1. B. 3. a Cdagtw

Collagen is about 90% of the total protein found in bone. where it provides both

the framework and strength to the tissue. Collagen type I predominates but small

amounts of type III and V are also found. Al1 of the collagen in bone forms intu "quarter

s taggered" fi brils and c m be identi fied ultrastructurally by their 67nm banding pattern

(Van der Rest & Gamone. 199 1 ).

Collagen type 1 molecules consist of 3 al and la2 polypeptides. which f o m into a

triple helis motif. Features of collagen synthesis that are critical to the establishment of

this motif and the subsequent formation of functional collagen fibres have been reviewed

extensively by Prockop (1979). Firstly the amino acid sequence is critical to the

formation of the triple helix motif. This sequence consists of Gly-X-Y repeats where

about a third of the X and Y residues are proline and hydroxyproline respectively. The

glycine residues. which are small. can be contained on the inside of the triple helix. The

stabilization of this conformation however is very dependent on the hydrosylation of

proline. The enzymatic hydroxylation of both proline and lysine residues require oxygen

(O2) . ~ e ' + and ascorbic acid. If this process is in anyway impaired. triple helix formation

at physiological temperatures is seriousl y compromised. Disulfide bridging between the

a chains at their globular carboxy terminal ends, is another post-translational

modification important to helis formation. This process in essence helps nucleate triple

helix formation (Prokop, 1990. Kuivaniemi et al.. 199 1 ).

Following procollagen assembly the molecule is secreted into the extracellular

space where specific arnino and carboxy peptidases remove the propeptides to form the

mature collagen molecules. The mature collagen molecules quickly self assemble into

fibres. Propeptide cleavage therefore is the last stage at which collagen assembly can be

controlled (Kuivaniemi et al., 1 99 1 ). During fibrillogenesis interchain bridging between

molecules further improves the strength of the fibres (Rossert & Crombrugghe. 1996).

These intermolecular bridges forrn between lysine and hydrosylysine residues.

I . B.3.b Major non-collagenous proteins

There are many proteins associated with bone other than collagen. ln general they

serve to facilitate matris remodeling and maturation. The following is a bnef summary of

both the distribution and proposed functions of the major non-collageneous constituents

of bone.

Osteocalcin (bone gla protein) is a y carboxyglutamic acid protein which accounts

for about 10-20% of the non-collageneous protein in bone (Gallop et al.. 1980).

Osteocalcin is normally found distributed diffusely throughout the matrix of bone

(McKee et al.. 1993). It is nearly csclusively rxpressed in mature osteoblasts and

osteocytes. it is therefore used as a key marker of the mature phenotypr (Hauschka et al..

1989).

Osteocalcin may have several functions in bone. It is reportedly espressed either

at or afier the onset of mineralization possibly implicating it in the rnineralization process

(reviewed by Hauschka et al.. 1989). Biochemical data would support this assertion.

This protein contains several gla residues which impart osteocalcin with a high affinity

for free calcium and calcium containing minerals (Hauschka & C m . 1982). Osteocalcin

has also been shown to inhibit crystal growth in vitro, therefore implicating it in the

regulation of matnx mineralization (Roomberg et al.. 1986. Van de loo et al.. 1987).

Osteocalcin may also be important to the recruitment and differentiation of

osteoclasts. Briefly osteocalcin acts as an chemoattractant for osteoclasts (Chenu et al.,

1994). Secondly bone particles which are deficient in osteocalcin are both resistant to

resorption (Lian et al.. 1984. Defranco et al., 1991) and have a decreased ability to direct

osteoclast differentiation (Defranco et al., 1992. Glowacki and Lian 1987).

Osteopontin (OPN) is an acidic phosphorylated glycoprotein. It is expressed at

most stages of osteoblast maturation including preosteoblasts. mature osteoblasts and

osteocytes (Mark et al.. 1987, Sodek et al. 1995). However. osteopontin is expressed in

many other ce11 types including monocytelmacrophages. kidney cells and lining epithelial

cells (reviewed by Denhart & Guo. 1993). In bone. osteopontin is heavily localized in

certain morphologically distinct regions.

Osteopontin is found at the mineralizing front of bone where it is believed to be

involved in the control of crystal growth. This protein has been shown to inhibit growth

of hydrosyapatite crystals (Hunter et al.. 1994. 1996). where osteopontin is thought to

bind the calcium of hydroxyapatite through its polyaspartic sequence.

Osteopontin is also closely associated with both cell/matrix interfaces. lamina

limitans. and interfaces such as the cernent line (Shen et al.. 1993. McKee et al.. 1 993.

McKee and Nanci. 1993. 1996 a. b). In the lamina lirnitans OPN probably facilitates

matris ce11 adhesion through its RGD sequence. The nature of the adhesion is probably

dependent on the location OPN in the matrix (reviewed by McKee and Nanci. 1996a.

1996b). At a bone surface OPN possibly facilitates osteoclastic adhesion (Reinholt et al..

1990), where as OPN at a resorption site may direct early matrix events and

preosteoblastic adhesion (Mckee and Nanci, 1996). On the lacuno-cannicular walls OPN

ma) act as a mechanotransduction molecule for osteocytes (Butler et al.. 1996).

Bone sialoprotein (BSP) is an acid phosphorylated glycoprotein, which contains

an RGD sequence. It is expressed and closely associated with the initiation of

mineralization in bone (Bianco et ai., 1 99 1 ). Bone sialoprotein has been shown to

nucleate hydroxyapatite formation. in a steady state agrose gel system (Hunter &

Goldberg. 1994 Hunter et al., 1996). therefore implicating this protein in the initiation of

bone matrix mineralization in vivo.

Osteonectin (SPARC. secreted protein. acidic rich 1 cysteine) was first descnbed

by Termine et al. (1 98 1) in bone. but has since been found in many other tissues. The

structure and function of the protein have been reviewed by Lain and Sage ( 1 994). In

vitro studies using endothelial cells have implicated osteonectin in the control of cell

proliferation. cell rnorphology. growth factor binding and enzyme modulation. The

specific rolc for osteonectin in the osteoblast is yet unresolved.

The major proteoglycans of bone are decorin and biglycan. Both contain

chondroitin sulfate side chains and a leucine rich core protein. Decorin is normally

expressed in both preosteoblasts and mature osteoblasts. Biglycan is produced later in the

maturation of both osteoblast and maturing osteocytes. The role of biglycan and decorin

in bone is not clear but has been briefly reviewed by Robe? et al. (1 996). These

proteoglycans may modulate ce11 matrix interaction, regulate or inhibit mineralization and

help in the transduction of sheer forces in the case of biglycan.

1. B.3. c Mirierat Phase

Bone is about 65% inorganic. mostly in the form of hydroxyapatite

Ca10(P04)6(OH)2. Constituents such as carbonate and elements like Mg and Na are also

found to be incorporated into the crystal lattice of the hydrosyapatite. The exact chernical

composition however is variable and is influenced by factors such as the age of the tissue,

source of tissue and mineral age (Glimcher, 1984). In mineralized bone tissue the apatite

appears as needle-like crystals with an average thickness of 3 nrn and length of 40 nm,

which are arranged aiong the axis of the collagen tibre (Bloom and Fawcet. 1986).

1.B.1 The Cernent line

Bone is continually being remodeled throughout life. The process serves many

purposes these include (Ott. 1 996): ( I ) mineral homeostasis in the body. (2) the repair of

tissue damage. (3) a response to mechanical strain. There are several stages in the

remodeling process these include the activation. resorption. reversal and formation phases

( Eriksen. 1986. Vaananen. 1993. Parfitt. 1994). During activation bone lining cells

prepare the bone surface to be remodeled. Following tliis. mononuclear osteoclastic

precursors are recruited to the new remodeling site where they wiil undergo

differentiation. Here on the bone surface they will fuse to form mutlinucleated

osteoclasts which are responsible for the degradation of bone during the resorption phase.

The reversal phase. which follows. is considered to be the period between active

resorption where osteoclasts are clearly present and the formation phase where new

fibrillar bone matrix is formed by mature osteoblasts. During the reversa1 phase light

microscopy reveals that the newly resorbed surface is populated by mononuclear cells

(Tran Van. 1982). whose identity has not been formally established. Parfitt (1994) has

placed cernent line formation in the reversal phase.

Scanning electron microscopy of surface remodeling sites in vivo has provided

funher insight into the formation of the cernent line. Following the resorption of bone by

osteoclasts a globular material is deposited on the demineralized collagen surface. These

deposits fuse to form a continuous layer of afibrillar matrix. which is believed to be the

cernent line (Zhou et al.. 1994). This phenornenon has also been modeled in cultures of

rat stroma1 cells undergoing osteognesis. Here globular accretions are laid d o m ont0

the surface of the culture vesse1 eventually forming a confluent layer (Davies et al.. 199 1 .

Hosseini et al.. 1996). Immunogold labeling of this globular material revealed the

presence of osteopontin (Shen et al.. 1993). which has also be shown to be heavily

localized in the cernent line in vivo. This suggests that a differentiating osteogenic ce11

population is responsibie for cernent line creation at remodeling sites in vivo.

The cernent line lias been described as a collagen free or poor structure. whicli

does not stain with silver saits (Weidenreich. 1930). Scanning and transmission electron

microscopy of this structure would also support this assertion (Frasca. 198 1 Schaffler et

al.. 1987). X-ray microanalysis has suggested that the levels of both calcium and

phosphorus are lower in the cernent line than lamallar bone. In addition the Ca/P ratio of

the mineral phase of this structure is higher then the mineralized collagen matrix

(Schaffer et al.. 1987. Burr et al.. 1988). From this. Burr et al. (1988) suggested the

mineral of the cernent line may differ from that of collageneous bone matrix. Howriver

BSEI constantly shows cernent lines of an electron density greater than that of the

surrounding matrix which may indicate a relative hypermineralization, therefore

contradicting the EDX findings.

1 .C Human Bone Growth In Vitro

1.C.1 Bone Explant Cultures

Human osteogenic cells were first successfully cultured by Beresford et al. ( 1 984).

The cellular population. isolated from the outgrowths of trabecular bone. espressed niany

of the typicai markers of the osteoblast phenotype (Beresford et al.. 1984.1985). These

included collagen type 1 synthesis. high levels of alkaline phosphatase espression and

osteocalcin production which was responsive to 1, 25(OH)2 vitamin D3 administration.

These "bone-derived" cells have since been shown to produce many other non-

collageneous proteins associated with bone matrix including ostropontin. osteonectin.

biglycan and decorin (Beresford et al.. 198% Robey. 1989).

However it was the work of Robey and Termine ( 1985) that first described human

bone rnatris formation in vitro. Subcultures of trabecular bone outgrowths produced

mineralized nodular structures, in the presence of ascorbate and P-glycerophosphate. The

biochemical analysis of the whole culture was also consistent witli previous work.

Ultrastructural analysis of the nodular structures, found in bone derived cultures.

was provided by Gotoh et al. (1 990) and Kassem et al. (1 992). Their findings confirmed

the presence of a highly synthetically active ceil population, characteristic of osteogenic

cells. as well as an extensive woven estracellular collagenous matrix. which seemed to

mineralize nornially. Here the needle-like apatitic crystals aligned along the avis of the

fibres. The afibrillar mineralized matris of a cernent-line structure was not described in

either of these studies which may reflect the state of differentiation of the cells harvested

h r these cultures.

Despite the use of this culture system for over a decade. the human bone matris

developed in vitro remains to be exarnined in equivalent detail to that of animal species

such as rat (Beresford et al. 1993). Biochemical characterization of the matrix itself

includes the work of Slater et al. ( 1 994a.b) where colloidal gold irnmunolabeling was

used. Here cellular outgrowths from ernbryonic bone. not adult bone. were used. The

matrix produced in this system forms in sheets across the culture vessel. rather than

nodules. but the ultrastructure of the tissue. seen by both scanning electron microscopy

and transmission electron microscopy. is similar to that found in nodule forming cultures.

Osteocalcin. which is expressed in both mature osteoblasts and young osteocytes. was

found to be distributed diffusely throughout the matrix. Growth factorslcytokines.

commonly found in normal bone. such as TGF-bl . IGF-1. IGF-II and bFGF were

clustered in focal groups surrounded by electron dense regions in close association with

the collagen. These sarne electron dense regions. often near sites of mineralization, also

positively labeled for chondroitin sulfate. Chondroitin sulfate being the common

glycosaminoglycan associated with bone proteoglycans such as biglyan and deconn.

Human bone esplant cultures have provided a valuable tool for addressing

questions related to hurnan bone ce11 behaviour. It has eliminated concems regarding the

use of non-human systerns. where species variation can be seen (Bereslord, 1993). As a

result human cells are increasingly relied upon to study the etiology of bone metabolic

disease (Marie. 1994). to assess the biocompatibility of endosseous implant materials

(Gegoire et al.. 1990. Serre et al.. 1993.- where it is believed that in vitro assays should

be perfonned with ce11 populations which match those at the implant site (Oliva et al.?

1996). and to study the regulation of bone ce11 behaviour by factors that include hormones

like. 1.25 vitamin Dl and glucocorticoids (Beresford et al., 1984. 1986. Kasperk et al..

1995). fluoride (Kopp and Robey. 1 WOa 1 WOb. Kassem et al.. 1994) and age

(Pfeilschifier et al.. 1993. Fedarko et al.. 1992).

More recently. whether to increase the number of harvested primitive

multipotential progenitor cells in comparison to that available from bone fragments

(Beresford et al.. 1993). to facilitate study of the earliest stages of osteogenic ceIl

differentiation (Hayneswonh et al.: 1996. Rickard et al.. 1996). or to adopt a more

practical strategy for the harvesting of a patient's MSC for gene therapy (Prockop. 1997)

and bard tissue engineering applications (Caplan & Bruder. 1997. Bruder et al.. 1994). an

increasing number of groups now focus on the culture of harvested marrow stromal ceIl

populations.

1.C.2 Bone Marrow Cultures

1. C. 2. a Irr vivo a d in vitro animal modds The osteogenic capacity of bone marrow as demonstrated by bone matrix

production. has been illustrated in several animal species. Both whole marrow and

cultured marrow stromal cells delivered into an in vivo environment. either in diffusion

chambers or calcium phosphate carriers. undergo osteogenic differentiation and produce

bone (Fnedenstein et al.. 198% Ashton et al.. 1984. Mardon et al., 1987. Kayiyala et al..

1997. Goshima et al.. 1991). Cultured marrow stromal cells from chicken (Kamalia et al..

1992). rabbit (Fredenstein et al.. 1987. Johnson et al.. 1988). dog (Kadiyala et al.. 1997).

rat (Maniatopoulos et al.. 1989) and ferret (Graziano. 1998) can also be induced in vitro

to prodiice rnineralized nodules. The rnatnx of nodules grown from cultures of rat or

ferret stromal cells is similar to woven bone in vivo. Agents used to induce osteogenesis

in these in vitro studies include dexarnethasone. and 1,25 (OH)2 vitamin D3. Given the

information from these animal studies attempts to evaluate the osteogenic capacity of

human bone marrow using both in vivo and in vitro assays has been made.

1 .C.2.b, 1 In vivo Whole marrow derived from humans is limited in its osteogenic capacity in

cornparison to animal derived specimens. Davies (1 987) inoculated diffusion chambers

with human adult marrow and implanted them in the peritoneal cavity of rat. These cells

synthesized a collagenous matrix. but mineralized bone formation was not observed. Bab

et al. (1988) was able to demonstrate bone formation in chambers containing whole

marrow from children. but not adult donors. These results most likely reflect the limited

number of stromal cells that are contained in the marrow, which may be as low as 1 in 10'

to 1 o6 cells in adults (Caplan. 1994 Lazarus et al., 1995) and the probable drcreased

osteogenic capacity of these cells with increasing donor age (Quarto et al.. 1995). Human

stromal cells which are first isolated in culture prior to re-implantation do demonstrate

osteognic capacity across a wide range of donor ages (Haynesworth et al., 1992, Gundle

et al.. 1995. Krebsbach et al.. 1997). These cells c m be expanded in culture over several

passages and still maintain their osteogenic potential. However the vehicle used for re-

implantation and the conditions of the population expansion have a bearing on the level

of matris production. Ceramics such as HNTCP can easily support osteogenesis. this

contrats poly(1actic acid) or demineralized bone matrices which, generally. do not

(Krebsbach et al.. 1997). The in vivo osteogenic capacity of the cells can also be greatly

enhanced by in vitro expansion with factors such as dexamethasone or FGF-2 (Krebsbach

et al.. 1997. Martin et al.. 1997).

1 .C.2. b.2 In Vitro

Isolated human marrow stroma1 cells can be in induced to espress many of the

characteristics associated with mature osteoblastic cells. such as alkaline phosphatase

( ALP), PTH responsiveness. collagen type 1 and osteocalcin synthesis (Vi lamitiana-

Amedee et al.. 1993. Beresford et al.. 1994. Cheng et al.. 1994. Rickard et al.. 1996).

These markers continue to be expressed even in cells which have undergone several

population expansions. However, as reported by Jaiswal et al. ( 1997) subcuItures of

rnarrow derived cells do not t o m bone nodules as seen in either rodent marrow cultures

(Maniatopoulos et al.. 1988) or those derived from human bone fragments. Indeed.

instead of nodule formation. these human marrow cultures produce a patchwork of

apatite-like minerai across the culture (Cheng et al.. 1994. Kassem et al.. 199 1. Jaiwsal et

al.. 1997. Bruder et al.. 1997). This pattern of mineralization is similar to that reported in

human periosteal cultures by Koshihara et al. (1 987) and Nohutcu et al. ( 1997). While it

has been suggested that this fonn of mineralization models intermembranous ossification

(Jaiswal et al.. 1997). morphological evidence to support this has not been reported.

Primary cultures of human marrow derived cells have yielded discrete areas of

mineralization more reminiscent of bone nodule formation (Sel1 et al.. 1998). The

ultrastructural evidence of the rnineralized collagenous matrix, although limited. would

indicate nom~al mineralization (Gronthos et al.. 1994). The sequence of events that

comprise the formation of hurnan bone matrix by the differentiating osteogenic ce11

populations has not yet been explored.

1. D Modeling Bone Formation ln vitro The rat marrow culture system first described by Maniatopoulos et al. (1988) has

provided a valuable tool for modeling early matris events in vitro. Here dexamethasone

is used to induce osteogenesis in cultures of rat stromal cells. Desamethasone has been

shown to positively influence nodule formation in cultures of rat calvarial cells. but its

presence is absolutely required for nodule formation to occur with rat marrow stromal ceil

populations. Ascorbic acid. a necessary CO factor for collagen synthesis and P-

glycerophosphate. which provides an organic form of phosphate for mineralization

(Tenenbaurn & Hrersclie. 198 1). are both added to the system. The great utility of this

system is that it provides a larger number of early osteogenic precunors. than that

available from bone derived ce11 populations and therefore provides a good system to

evaluate early events in matrix formation.

The differentiating osteogenic cells. which colonize a culture vesse1 eventually

form multilayered ce11 sheets. have been shown to elaborate an afibrillar interfacial

matrix. Early on this matrix appears. as seen in transmission electron microscopy. as an

amorphous electron dense material. Osteopontin and chondroitin sulfate (CS-56) have

been localized in tliis interfacial layer. using immunolabeling (Davies, 1996). which is

consistent with the biochemistry of cernent lines seen in vivo. Bone sialoprotein has also

been associated with early matris deposition as seen by irnmunofluorescent labeling at the

ce11 culture substrata (Peel. 1995). During the elaboration of this matrix collagen can

only be localized intercellularly. In fact mineralization of this matrix occurs independent

or in the complete absence of collagen assembly (Hosseini et al.. 1996).

The mineralized matrix appears as globular accretions. which eventually fuse to

form a confluent afibrillar mineralized sheet. These structures only form underneath

developing bone nodules and their morphological appearance in the SEM is similar to the

cement line deposits seen in vivo at surface remodeling sites (Zhou et al.. 1994).

The collagenous scaffold is assembled on this cernent line structure. Only after

the assembly and maturation of the collagen scaffold does the fibrous matris begin to

mineralize. The stages of niatrix elaboration which have been modrled in vitro and can

be identified morphologically are: the creation of the afibrillar collagen's matrix.

mineralization of this interfacial matrix. assembly and maturation of the collagenous

matrix and final1 y mineralization of the collagen scaffold. The morphological hallmarks

which are consistent with this sequence of events have most recently been modeled using

ferret marrow stroma1 cells (Graziano. 1998). but yet remain to be fully identified in

cultures of human marrow stroma1 cells.

2. Hypothesis

That the osteogenic ce11 population derived from the human marrow can be expanded

in p r i m q culture, harvested and remain capable of eiaborating a morphologically

distinct bone matris in vitro.

3. Objectives

1. Establish the primary culture conditions necessary to support bone formation by

human marrow stromal cells in vitro.

2. Characterize the matris developed in these primary cultures by identiSing the

histological hallmarks of bone.

3. Establish subculture conditions to support bone formation by human marrow stromal

cells in vitro.

4. Characterize the matrix developed in subculture by identi&ing the histological

hallmarks of bone.

4. Materials and Methods

4.A Cell culture

4.A.I Cell isolation and Primary culturc Femoral or iliac marrow containing trabecular bone fragments. isolated from donors ( a g s

1 %-18 years) undergoing elective surgery. were used as a source of marrow cells. These

fragments were flushed several times with 10 ml volumes of phosphate buffered saline

( P B S - ~ g ~ a ) to remove the marrow cells from the fragments. The ce11 suspension was

passed through a 100 pm nylon ce11 strained (Falcon) and subsequently centrifuged at 500

x g for 10 minutes at room temperature. The fragments were then discarded. The cells

were resuspended in 6 ml PBS and fractionated on a ficoll-plaque (Pharmicia) density

gradient run at 1 100 s g for 45 minutes at room temperature. The cells were isolated

frorn the gradient interface. counted on a Coulter counter and seeded at 2x 10' cells/cm2.

Cells were maintained in a M E M containing 15% fetal bovine serum (Gibco). 10%

antibiotic solution ( 100 mdml penicillin Ci, Sigma. 50 mglm1 gentamicin sulpliate.

Sigma, 0.3 mgml amphotericin B. Sigma). 50 m g h l L-ascorbic acid (AA. Sigma) and

1 0-8 M dexamethasone (Dex. Sigma) at 37'C in a hurnidified atmosphere of 95% air with

5% CO? for periods of 6 to 13 weeks. On day 4, cells were washed 2-3 times with PBS

to remove any non-adherent cells from the culture vesse1 and refed with fresh medium.

At this time three vessels were used to determine the number of adherent celIs. The cells

were quantified. by manually counting 4 different regions in the culture vessel. The

regions were randomly selected and had a set area, from this an estimate of the ce11

density was made. Refeeding occurred every 3 to 4 days. P-Glycerophosphate (PGP.

Standard conditions (3.5mM). 5mM. lOmM Sigma culture grade) was added to the

culture medium at either day I or following the appearance of ce11 multilayering at

approximately day 16. Negative controls included cultures where either Dex. AA or BGP

was absent from the culture medium.

I.A.2 Subculture

Three of the standard primary cultures listed in table 1 were subcultured on day 16.

primaries were kept as positive controls. The cells. first washed with PBS to remove

nonadherent cells and debris. were trypsinized in 0.005 % trypsidPBS for either 5

minutes or 15 minutes at 37'C in a humidified atmosphere of 95% air with 5% C02. The

cell suspension was passed through a 100 pm nylon cell strainer (Falcon) and

subsequently centrifuged at 500x g for 1 fl minutes ai room temperature. The pelleted

cells were resuspended in a M E M containing 15% fetal bovine serum (Gibco). 10%

antibiotic solution (100 mg/ml penicillin G.Sigma. 50 mg/ml gentarnicin sulphate. Sigma.

0.3rng/ml amphotericin B. Sigma). 50 rng/ml L-ascorbic acid (AA, Sigma). 1 0 - ~ M

desamethasone (Des. S ipma) and P-Glycerophosphate (PGP. 3.5 m M Sigma culture

grade). The isolated cells were then seeded at 5x 1 o3 cells/cm2 and maintained under the

same conditions as the primary cultures until mineralization or nodule formation

occurred.

1.A.3 Reseeding Non-adherent cells from primary cultures

Occasional non-adherent cells removed from prirnary cultures on day 4 were collected

and pelleied by centrifugation at 500 x g for 10 minutes at room temperature. The cells

were then resuspended in culture media. plated at 2 x 1 o5 cellslcm'. if possible. and

maintained under the same conditions as the primary cultures.

4.B Histological Studies

3.B. I Alkaline Phosphatasr Fast blue RR salt (10 mg. Fisher) was added to 0.5 ml of Naphthol AS-MX phosphate

alkaline solution. 0.25% (Fisher 85-5). made up to a total volume of 10 ml with DH20.

The solution was filtered (Whatman paper. 1 ) and used immediately. Whole culture

dishes were rinsed with PBS three times and stained for 5 min. They were then rinsed

several timcs in PBS. fixed in phosphate buffered fonnalin (pH 7.4) and mounted in

glycerol jelly.

4.B.2 Haematosylin and Eosin Staining was perfoned on paraffin sections of cultures grown on flesible bottom well

inserts (0.4 pm pore size. Falcon). Sections were deparaffinized in sylrne and rehydrated

through a graded ethanol series. The specimens were stained with Harris' modified

Haematosylin (Fisher) for 5 minutes. then washed with water to remove escess stain.

The slides were differentiated in O.Joh HCL/9j0h ethanol. washed in water. then quickIy

dipped in 1% ammonia solution and washed once again. Counter staining in Eosin

Yellowish solution 1% w/v (Fisher SO-E23) was done for approximately 20 sec. afler

which the sections were rinsed in 95% ethanol followed by absolute ethanol. three 2 min.

rinses were used. The slides were cleared in sylene and rnounted with DPX mountant

(Biochemika).

4.B.3 Tetracycline labeling Tetracycline was added to the cultures. at a concentration of 9pg/mI in t lie medium. 24 hr

prior to the termination of a culture. The whole cultures were photographed under UV

light. Some samples were rinsed in 70% ethanol. fixed in absolute ethanol ovemight and

air dried. Samples were either analyzed directly or embedded in LR White (London

Resin Co.) resin. Both the whole cultures and 30 pn undecalcified sections (see LR

white ernbedding) were viewed by UV-escited fluorescence rnicroscopy.

d.B.4 Em bedding and Sectioning Protocols

4. B.4.a L R White (Lotjdon Resin)

Samples were brought back into 70% ethanol and the tissue was infiltratcd with a 2: 1

resin to 70% ethanol mixture under vacuum for 30 minutes. This was followed by

infiltration wi th two changes of pure LR white resin held under vacuum for 1 hour each.

LR white resin was polymerized under anaerobic conditions and held under 50°C. The

ernbedded samples were sectioned to a 300 Fm thickness through both the culture and

polystyrene dish. The sections. fixed on glass slides. were polished to a final thickness of

approximately 30 pm.

4. B. 4.6 Para ffitiri embeddittg arzd sectioriing

Cultures grown on flexible bottom well inserts (.4 mm pore size. Falcon) were fixed in

2% Paraforrnaldehyde/PBS for 45 minutes. The inserts were then rinsed in PBS and

dehydrated through a graded ethanol series. Samples were kept in a 1 : 1 mix of absolute

ethanol and methylbenzoate overnight. The samples were then infiltrated first in

methy lbenzoate and then with a I : I methy lbenzoatefparaffin was ( 6 0 ' ~ ) under vacuum.

Final embedding was performed where the wax was kept at 60°C

4.8.5 l mmunolabeling

4. B. 5.a Peroxidme labeliirg of Para ffin Sectiom

Sections were deparaffinized in xylene. rehydrated through a graded ethanol series and

brought to PBS. Sections were incubated with blocking serurn. from the Vestastain

Universal Quick Kit (Vector lab. PK-8800). for 10 minutes. The sections were blotted.

rinsed with PBS aiid reblotted again to remove excess blocking serum. This was

followed by an incubation of hour with the respective primary antibody. The rabbit anti-

human osteocalcin IgG (BTI. BT-593) was used in a 1 : 100 ratio made in PBS, the mouse

anti-rat osteopontin IgG1 (DSHB. MPIIIBIO (1)) was used in a 150 ratio. Rabbit serurn

and mouse serum w r e the respective controls for each of these incubations. Following

the incubation with primary antibody the slides were rinsed with agitation in 3 changes of

PBS for total of 6 minutes. Incubation with the secondary antibody was done following

the protocol prescri bed in the Vestastain Universal Quick Kit with the following changes:

first. the slides were rinsed with agitation between steps in three changes of PBS for a

total of 6 minutes. second. a incubation period of 1 minute was used wirh the DAB

substrate (Peroxidase Siibstrate kit DAB Vector lab SK-4100). Sections were then rinsed

in tap water and counterstained with hematoxylin (see above protocol). The slides were

then cleared in xylene and mounted with DPX mountant (Biochemika).

4.C Scanning Electron Microscopy (SEM), Energy dispersive X-ray

Microanalysis(EDX) & Backscattering Electron Imaging (BSEI)

4.C.1 Scanning Electron iMicroscopy Cultures were washed 3 times with 0.1 M sodium cacodylate buffer (pH 7.1-7.4 at 2 5 " ~ )

were fixed ovemight in 2% paraformaldehyde. 2.5% glutaraldehyde in 0.1 M sodium

cacodyiate buffer (pH 7.2-7.4 at 4 OC). The samples were then dehydrated through a

graded ethanol series and critical point dried. Some of the cell layers were removed from

the nodule structures usine fine tweezers. These sarnples were either then gold coated or

carbon coated. Al1 samples were anaiyzed on a scanning electron microscope (Hitachi S-

570).

1.C.2 Energy dispersive X-ray Microanalysis

For some carbon coated samples the minera1 was analyzed on a Hitachi S-570 equipped

with an x-ray detector (Link Analytical 5929).

4.C.3 Backscattering Electron Imaging (BSEI)

LR white 30 Fm undecalcitïed sections previously viewed by UV-excited fluorescence

microscopy (see Tetracycline labeling) were carbon coated and observed in the scanning

electron microscope (Hitachi S-570) equipped with a Robinson back-scattering detector

for analysis.

4.0 Transmission electron microscopy

Cultures were thoroughly washed in O. 1 M Na cacodylate buffer (pH 7.2-7.4 at 25 O C )

and fixed for 4 hours in 2% paraformaldehyde. 2.5% glutaraldehyde in O. 1 M sodium

cacodylate buffer (pH 7.3). The samples were then post fixed in 1% osmium tetraoxide

in cacodylate buffer for 1 hr at roorn temperature. followed Dy en bloc staining with 2 %

uranyl acetate in 50% ethanol for 2 hr at room temperature. The sarnples were then

dehydrated througli a graded ethanol series before tissue infiltration first with 50% epon

resin/ethanol. followed by several changes of pure epon resin. The final epon change was

polymerized at 40 'C for 2 days and then 60 OC for 2 days. The polystyrene dish were

removed from the epon block. areas of interest were cut from the block and re-embedded

in epon. polymerization was done ovemight at 60 OC. Thick sections were cut and

examined. Blocks of interest were then trirnmed before sectioning on the Reichert

Ultramicrotome. Sections were mounted on fomvar coated copper gnds. Post

sectioning staining included 3% magnesium uranyl acetate in 70 % ethanol, followed by

lead citrate (Reynold's). The final sections were examined on a Phillips 400 T

transmission electron microscope. Some of the final sections were analyzed on a Hitachi

H-600 transmission electron microscope equipped with an x-ray detector (Link Analflical

5929).

5. Resutts

5.A Light Microscopy of Standard Cultures Human marrow stroma1 cells from a total of 22 donors were maintained in

primary culture under standard conditions (aMEM, 15% FBS. 100 mgml penicillin G .

50 mg/ml gentamicin sulphate 0.3 rng/ml arnphotericin B. 50 m g h l AA. 1 0 ' ~ M Dex

and 3.5 mM PGP). Donor ages ranged from 1 8 months to 1 8 years of age. this has been

summarized in table 1 .

The number of adherent cells per 10' cells seeded was determined on day 4 of

culture in 8 of these primaries (summarized in table 2). The number of adherent cells that

could be derivcd from a bone marrow preparation was found to be highly variable. as

reflected by the hipli standard deviation. even within the narrow range of donor ages that

were examined. A specific pattern between the number of adherent cells obtained and

donor age could not be formally established given the limited samples that were analyzed.

Based partly on these quantitative results and on the more general qualitative

observations of these cultures it could be said that teenage donors gave consistently lower

ce11 counts than infants. The influence of sampling location was not taken into account.

The adherent ce11 population generally reached confluence at approximately day

10. followed by evident ce11 multilayering by day 16 which was concomitant with a

change in ce11 shape to a more polygonal form. Bone nodule formation, as seen in figure

1A. normally occurred between 3 and 5 weeks. but was not seen in one culture. where the

donor was a 16 year old female. until day 60. Cells in the nodular area were first seen

packing tightly together. where they appeared to have a tessellated morphology. It was in

these nodular areas where mineralization was seen to occur (fie 1 C). Minemlization was

identified as opaque or dense areas. The mature minrralized matrix had an obvious three

dimensional appearance. It should also be noted that morphological evidence of

adipocyte formation was seen in these cultures generally after the first signs of

mineralization.

Though most of the cultures were capable of producing distinct nodular structures.

dystrophic mineral deposition was seen in some of the cultures grown under the standard

conditions. In these cases the minera1 formed in patches randomly across the culture (fig

1 B). This type of mineralization was found within the first 3 weeks of culture. Nodule

formation was not seen and the dystrophic mineral deposits were often associated with

cells that had a fi broblastic morphology. In cultures. which normally were confluent and

had evident cell multilayering prior to dystrophic mineral deposition. the cells were found

to detach and contract away from the culture dish (fig 1 D). Thinning of the ce11 sheet was

common. especially near large dystrophic mineral deposits where only a sparse number of

fibroblastic cells could be found. Given these observations it would appear that ceIl

viability was found to be severely compromised following dystrophic mineral formation.

sincr the) were not able to fully differentiate. The viability of these cells was not

fonally tested. Adipocytes which were normally seen in nodule forming cultures were

not observed in cultures where dystrophic mineralization was seen.

Dystrophic mineralization was more common in cultures where PGP was added

early to the culture (see table 1). It should however be noted that delaying PGP addition

in 5 of the primary cultures increased the risk of ce11 detachment. This was most

pronounced in cultures from pre-pubescent donors. The success of the cultures may have

also been influenced by the semm lot used. A summary of the cultures. conducted under

standard conditions, can be found in table 1 .

Table 1 Summary of cultures done under standard conditions*. - - -

Sex PCP ~ddi t io t Ï Serum (yr) 1 Age Dav 1 Late Lot

-Su bculture 1-subculture

8 * Standard Conditions is culture medium supplernented with 50 m d m l L-ascorbic acid. 10- M dexamethasone and 3.5 mM P-Glycerophosphate.

Table 2 Effects of age on the number of adherent cells.

Bone

Ye* y es yes YeS YeS yes no no

Age (~ears)

11!2 2 4 5 7 7 11 18 .

Ave 381 STD 41 1

*please note n=3

Sex

M F F F F M M F

# Adherent per 1 0' cells seeded' (sTo)

1316 (83) 555 (65) 56 (13)

612 (100)

31 (8) 218 (8) 217 (16)

45 (12)

Figure I Primary human bone marrow cultures grown under standard conditions, where

3.5 mM of PGP was used. A) Dark Field micrograph of a mature bone nodule at

day 42 of culture. The mineralization (light area) was heaviest at the central

region of the nodule (F.W. 2.7 mm). B) Dark field micrograph of dystrophic

minera1 formation (F. W. 1.1 mm). Here the minerai had a random and speckled

appearance. which was not isolated to a single discrete region in the field of

view. C) Phase contrast micrograph of the edge of a mature nodule at day 42.

The mineralized region of the nodule. the opaque area (star). was bordrred by

cells which were densely packed together and had a polygonal morphology

(arrow) (F.W. 2.7 mm). D) Dystrophie mineral was not coincident with cell

aggregates as seen in nodule forming cultures. lnstead sparse mineral patches

were associated with a thin layer of elongated fibroblastic cells (arrow) (F.W.

1 - 1 mm).

S.A. 1 Tetracycline labeling Tetracycline, which chelates with the ~ a " , is often used to label the mineralizing

front of forming bone. The tetracycline labeling in the cultures coincided with the

mineralized nodular areas which had first been visualized in phase optics (fig 2A ). The

binding of tetracycline to these discrete areas in the nodule forming cultures was in

contrast to the random labeling of small minera1 patches across the culture dish found in

the dystrophie samples (fig 2B).

Using the tetracycline labeling the mineralizing fronts in the nodular tissue could

bc visualizrd in cross-sections of mature nodules (fig 3A). The distribution of tetracycline

labeling in these cross-sections wris shown to be coincident with the eIectron dense

regions seen in the backscattered micrographs (fig 3B). These electron dense regions

were assumed to be rich in calcium.

5.A.2 Alkaline phosphatase Alkaline phosphatase activity could be detected, in many of the ceIl colonies. very

early in the culture penod (fig 4A). The majority of cells reaching confluence displayed

sonie lrvel of alkaline piiosphatase (fig JB). No perceivable changes in the staining

pattern or in its intensity could be visually noted until the commencement of nodule

formation. The most intense alkaline phosphatase staining was observed highlighting the

cuboidal cells at the center of incipient nodules or at the borders of more mature

rnineralized matnx (fig 4 C&D)' while the less differentiated cells at the periphery of

these areas stained more weakly. In control cultures. where Dex was absent. alkaline

phosphatase staining was hardly perceivable (data not shown).

Figure 2 Cultures grown under standard ce11 culture conditions. labeled with tetracycline

24 hr prior to termination. A) Labeling in the normal nodule forming cultures,

was localized to discrete regions in the culture (insert). Each region

corresponded with a single nodule. where intense labeling was greatest at the

center of the nodule (F. W. 2.7 mm). B) Labeling of the dystrophic

mineralization was ubiquitous (insert). This type of minera1 was not isolated to

discrete regions. which could be identified as nodules. Labeling was random

and diffuse. highlighting very small clusters (F. W. 2.7 mm).

Figure 3 Tetracycline labeled bone nodules in cross-section seen in A) Scanning electron

microscopy B) Backscattered electron imaging C) Fluorescent microscopy. The

mineralizing fronts. labeled in the last 24 hr of culture. appeared as bands in the

fluorescent micrograph. Many of these regions were coincident with electron

dense regions. which are thought to represent the calcium rich regioiis. in the

backscattered image. (F. W 450 pm).

Figure 4 Alkaline phosphatase staining of cultured stromal cells fiorn a 7 year old

female donor. A) At day 7 of culture many alkaline phosphatase positive

colonies were noted. The staining was seen on the fibroblastic-like cells at the

center of this colony (F.W. 900 pm). b) At day 13 of culture the cells were fully

confluent. Variable levels of staining could be noted on the majority of the

fibroblastic cells in the culture (F. W. 900 pm). C) An increase in the intensity of

alkaline phosphatase staining on the cells immediately bordering the mineralized

area of the nodule could be noted (arrow) (F. W. 3.6 mm). D) Intense alkaline

phosphatase staining highlighted the very compacted rounded or polygonal cells

(arrow) bordering mature nodular structures (F.W. 900 pm). In cornparison

weak staining was seen on the less differentiated, fibroblastic cells. at the outer

most prriphery of these nodules. Alkaline phosphatase activity was also lcss

apparent on cells overlaying heavily mineralized areas (star).

5.A.3 lmmunoperoxidase Iabeling Maturing nodules were used for the immunoperoxidase labeling. At both the

tissue/membrane interface and overlying the matrix of these nodules. there was a

monolayer of cells in close association with one another. The interfazial matris of these

nodules however was often disrupted when the membrane. ont0 which the cells were

seeded. detached during processing. The extensive extracellular matrix. of about a

maximum depth of 100 Fm. separated these cell layers. The nodules also had several

randomly distributed cells embedded in the matrix. The beginning of matrix

mineralization was identified as dark opaque regions seen in the tissue sections (fig 5A).

These regions were separated from the surface cells by a thin layer of osteoid. Osteocalcin

(OC) staining was intense and randomly distributed throughout the matrix of the tissue

(fig 5B). The controls. where non-immune rabbit serum (fig SC) or seconda- antibodies

were used. showed either no appreciable or low levels of background staining. In both

OC and the non-immune serum controls staining was noted to be more intense on the ce11

layers at the interface and surface of the nodule.

Attempts to stain for osteopontin (OPN) yielded only weak results. Although

labeling was limited to the peripheries of the tissue, specifically at the tissue/substrate

interface, the levels of label was no higher than the background labeling found in the

control cultures. Therefore these OPN results were negative.

Figure 5 Light micrographs of paraffin cross-sections of nodules at day 40 of culture. A)

The haematoxylin and eosin of the bone nodule highlights an extensive

acidophilic extracellular matrix. Contained within this matris was numerous

embedded cells (arrow heads) There was also evidence of rnatris mineralization

(star). A tliin layer of osteoid could been seen separating the overlying ceil layer

from the mineral (arrow) (F.W. 900 pm). B) Rabbit anti-human osteocalcin

staining was seen to be distributed diffusely throughout the osteoid tissue (F.W.

900 pm). C ) The control. where rabbit non-immune serum was used instead of

the prirnary antibody, had only a very faint background staining (F.W. 900 pm).

5.B Scanning electron microscopy of Standard Cultures Globular accretions were deposited by the differentiating osteogenic celis ont0 the

surface of the polystyrene culture dish (fig 6A). The developing accretions accurnulated

on the substrate surface forming a nearly confluent layer of afibrillar interfacial matrix

(fig 6B). The fibrous matrix. forming the bulk of the nodular structure. was assernbled on

top of this cernent line matrix. In figure 6c the collagen cornpartment was seen to have

formed in alternate lasers of highly calcified collagen and less mineralized collagen. This

matrix appears to be very heavily calcified making the individual collagen fibres hardly

distinguishable (fig 6C). Removing the overlying osteoid layer ofien revealed the

presence of srnal! cells. 4 5 pm in diameter. whicli had a morphology typical of

osteocytes (fig 6D). These cells were seen nested in lacuna-like cavities in the matris.

The matrix immediately surrounding these cells was ofien times heavily mineralized.

5.C Transmksion Eiectron Microscopy of Sfatidard Cultures At the surface of the nodule there was a preponderance of cells with large

arnounts of glycogen. in a clusters (fis 7 9r 8). These cells had a flattened appearancr

and were somrwhat removed from the more active cells and the bulk of the developing

matrix. The level of glycogen was substantially reduced in the more mature cells. which

were characterized by the presence of an extensive and well developed collection of

prorein synthetic organelles (fip 7 & 9). The cells at the tissue substrate interface differed

only in their more flattened morphology.

At the tissue substrate interface, electron dense globular structures could be seen

(fig 9A). The interfacial globular accretions occupied a hali micron space betrveen the

culture substrate and the bulk of the overlying matris. Ver). few collagen fibres could be

seen occupying tliis space. The collagen fibres. identified by the characteristic cross

banding pattern. were laid d o m in parallel to one another forming large groupings in the

tissue (fig 9B). The fibrils were found in these clusters were either laid down

perpendicularly or in the horizontally plain of the section. The orientation of fibres in one

grouping were found to be orthogonal to those in another group. In the lower regions of

such nodules several cells were observed whose morphology was reminiscent of early

osteocytic cells. embedded in a mineralizing collagenous scaffold (fig 7 & 1 0). Although

these cells still possessed synthetic organelles. the processes of the entnpped cells were

seen estended though rudimentary cannicitla-like channels in the matris tlirough which

cell/cell contact was noted (fig 10C). Glycogen was seen to accumulate in these cells.

The collagen scaffold adjacent to these cells demonstrated morphological evidence of

rnineralization. which could be noted on bot11 single or small aggregates of the collagen

fibres (tig 9C).

Extending from the periphery of thesr discrete rosette appearances or aligned

along the collagen fibres one could see dark needle-like crystals. typical of hyciroxyapatite

(Fig 11A). Surrounding the heavily rnineralized regions. were electron dense or grey

zones. Morphologicai evidence of apatitic crystal formation was not noted in these grey

regions. The levels of calcium or phosphorous seen, using energy dispersion X-ray

analysis of the gray regions and the surrounding unrnineralized collagenous matrix, were

hardly perceivable (fis 1 1 B a&@. The EDX spectra of the very dark electron dense

regions. where mineralization had been previously identified. had a strong calcium

(ka1.2) prak and although masked by arsenic the phosphorous peak could also be clearly

detected (fig 11B c).

Figure 6 Scanning electron micrographs of a standard culture containing nodules A) a

differentiating osteogenic ce11 (arrow) in association with newly deposited

globular accretions (arrow heads) at the base of a nodule. These plobular

accretions have a diameter of about 1-2 pm. (F.W. 45 pm). B) Removal of the

overlying cell sheet revealed the extensive collagen scaffold of a large nodule.

At the interface. between this matrix and the culture dish. was an almost

confluent mat of globular accretions. forming the cernent line. (F. W. 90 pm). C)

The heavily calcified matrix. seemed to form in layers (arrows) (F. W. 23.1 pm).

D) Osteocytic cell with extended processes ernbedded in its lacuna. The fibrous

matrix immediately surrounding the cell was found to be calcified. (F.W. 45

Pm).

Figure 7 Transmission electron niicrograph depicting a vertical cross-section of a nodule

grown under standard culture conditions. With the exception of the large rotund

ce11 (arrow head) at the surface of the structure, the majority of the cells (small

arrows) in the upper region of the nodule had an elongated shape. a high

nucleo/cytoplasmic ratio and evidence of glycogen deposits. The matris in this

portion of the nodule was much sparser in cornparison to the lower region.

Embedded in the dense extracellular niatrix in the lower portion of the scaffold

was several cells (arrow). which have taken on a osteocytic morphology. At the

base of the nodule was a group of more flattened cells. which overlay the

globular accretions at the surface (F. W. 88 pm).

Figure 8 Transmission electron micrograph of the surface of a nodule. where the cells

had large accumulations of glycogen. in a-clusters (arrow). These elongated flat

cells had a paucity of synthetic organelles and were surrounded by a sparse

fibrous extracellular niatrix (F.W. 9.3 pm).

Figure 9 Transmission electron micrographs of a nodule grown under standard conditions

A) At the tissue substrate interface there were srveral eleciron dense globular

masses (small arrows) which been deposited. begiming the formation of an

afibrillar matrix at the culture substrata. An elongated cell. whose collection of

synthetic organelles was notable, could be seen above (F. W. 20 pm). The

collagenous matrix seen in (B) could be identified by the fibril banding pattern.

as seen on the group of fibres parallel to the section (small arrows). The

majority of the densely packed fibres seen here had been laid d o m in large

groupings in which the fibres in one group lay at right angle to those in the other

group. Extended through into the matrix was the cytoplasrnic process of an

osteocytic cell. The ceIl immediately overlying this dense region of collagen

niatris had an extensive endoplasmic reticulum (F. W. 20 pn). C)

Mineralization of this collagen matris could be morphological identitird. on

small discrete bundles of collagen fibres. as extremely electron dense regions

with a rosette appearance (small anows). (F.W. 20 pm).

Figure 10 Many of the typical features of osteocytic cells could be seen in this

transmission electron micrograph of a human bone nodule A) Two young

osteocytic cells in close contact to each other (small arrows). Still present in

these cells was a fairly prominent endoplasmic reticulum and several

mitochondria. The processes of these cells were seen to be extended through the

dense collagenous matris of this tissue (F. W. 52 pm). B) As the osteocytes

matured glycogen was seen to accumulate in their cytoplasm. as the number of

synthetic organelles decreased (F.W. 28 pm). C) The glycogen forrned in alpha

clusters (arrow). These cells were often be seen in contact with other cells in the

tissue (srnall arrow) (F.W. 10 pm).

Figure 1 I A) The mineralized matrix appears as very electron dense regions. Needle-

like crystals (arrows) can be seen extending from these rosette appearances and

along a few of the fibres. The banding pattern of collagen cm be seen in the

fibres at the bottom of the micrograph (F.W. 3.5 pm). B) Highly mineralized

regions (a) are often surrounded by grey electron dense areas (b) that form a

border between sites of active mineralization and the naked collagen fibres (c)

(F.W. 2.5 pm).

a Range (keV) Net Integral Calcium L a, b 26-.42 29 Calcium K a 1,2 3.58-3.80 1 09 Calcium K b l 3.90-4.12 88 PhosphorusKal 1.90-2.10 54

Range (keV) Net lntegnl Calcium L a, b -26-.42 23 Calcium K a 1,2 3.58-3.80 89 Calcium K b l 3.90-4.1 2 114 Phosphorus K a l 1.90-2.10 155

c Range(keV) Netlntegral Calcium L a, b -260.42 56 Calcium K a 1,2 3.58-3.80 201 7 Calcium K b l 3.90-4.12 468 PhosphorusKal 1.90-2.10 1264

I

O t 1 I l I I 1 I I t 1 1 t 1

F;-an 12 EDX of zone a,b and c as seen in fig 1 IB. (Y axis, kcounts X axis keV)

5.C. 1 Control Cultures Cultures where dexamethasone was not present failed to show any signs of

osteogenic diffeïentiation. The cells maintained a flattened and fi broblastic morpho logy.

The cells rapidly reached confluence eventually developing into multilayered sheets.

which often detached from the culture vesse1 after severai weeks in culture. There was no

evidence of either normal nodule Formation or dystrophic mineralization. SEM of

cultures before cell detachment did not demonstnte either the presence of an interfacial

afibrillar matrix or a collagenous matrix.

In the absence of P-glycerophosphate (BGP) the cultures were very prone to ceIl

sheet detachment. In cultures. which could be maintained. non-mineralized nodular

structures were noted. Sporadically these nodules were mineralized. however the level of

mineral indicated by the tetracycline labeling appeared to be reduced in comparison to the

standard cultures.

Cultures which w r r not supplemented with ascorbic acid normally s w e vey

prone to dystrophic mineral formation. Tlir confluent ce11 sheet would begin thinning

and ce11 detachment would occur in these cultures following dystrophic mineral

deposition. In fact very few cells remained in these cultures. When the cultures were

viewed in the SEM a sheet of mineral spheres. similar to those seen in the dystrophic

cultures. were found. The ce11 sheet which normally would be seen overlying the rnatrix

was no longer visible having been covered with these dystrophic deposits (fig 13).

Figure 13 Scanning electron micrograph of cultures in the absence of ascorbic acid A)

The ce11 sheet was obscured by the mineral deposits (F.W. 300 pm). B) Beneath

the clusters of these small minerai spheres there was a little evidence of cells or

extracellular matris (F.W. 45 pm).

S.D.1 Electron Microscopy In standard cultures. which failed to produce discrete bone nodules. SEM analysis

reveaied a random deposition of mineral over the cells and sparse collagen matris (fig

14). These sphrres with diameters of around 1 Fm. formed large aggregates. Energy

dispersive X-ray microanalysis in the SEM confirmed that these structures contained

signifiant levels of calcium and phosphorus (fig 15).

5.D.2 Effects of P-Glycerophosphate on mineralization Cultures of stroma1 cells from 4 donors were used to examine the effects of

increased PGP on mineralization. Phase niicroscopy andior ultrastructural data was used

to assess or coniïrm the nature of the minera1 in culture. The findings of this study have

been summarized in table 3.

Illustrated in figure 16A is the typical appearance of normal mineralization or

nodule formation. These structures were typical of cultures containing 3.5 m M PGP. In

contrast whcn the levels of PGP rench 10 mM a random and diffuse pattern of

mineralization. identical to that previously identified as dystrophic mineralization. was

consistently seen (fig 16C). PGP supplemented at 5 m M levels gave variable rrsults.

While dystrophic rnineral was often noted in these cultures (fig 16B). nodule formation

was also seen to occur. The severity or levcl of dystrophic minera1 was in general lower

with decreasing levels of PGP (fig 168 insert). The appearance of dystrophic rnineral in

general inhibited the normal development of the tissue. In figure 16 it was seen that the

cells. in cuitures supplemented with 10 mM of PGP, would only form a very thin

multilayer and a tissue which was very sparse in comparison to nodule foming cultures

where 3.5 m M of PGP was used.

SEM examination of cultures (fig 17). first identified in phase microscopy to

display dystrophic mineralization. revealed that minera1 deposits were very sirnilar to

those which have been previously described as dystrophic mineral. Transmission electron

microscopy of the most severe dystrophic rnineralization. in cultures containing 10 mM

PGP. revealed the presence of large electron dense masses occupying areas where there

was an absence of any obvious collagenous matrix (fig 18). The cells in these cultures

were thin with a paucity of synthrtic organelles. The collagenous extracellular matris. if

present. was normally very sparsç in comparison to normal cultures. However a

confluent afibrillar matrix was O fien noted. In general however dystrophic mineralization

inhibited the normal developrnent of the tissue, this however was highly variable being

dependent mainly on the level of PGP used. For instance although ectopic minera1 had

been prcviously identified. usine SEM. in cultures containing 5 niM PGP there were

regions of the tissue in these samr cultures in which mineralization. that appeared

normal, could be seen (data not shown).

Table 3 The effects of B-Glycerophosphate on mineralization

1 Donor Age 1 Sex 1 p-Glycerophosphate (mM) 1

*Nodule formation was not seen, but areas of both dystrophic and normal mineralization were seen when viewed ultrastructurnlly.

, (yeaW 14 4 8 5

M F M F

3.5 5 10 nodule nodule nodule nodule - dystrop hic

dystrophie Ibone* dystrophic

dystrophic /nodule

d ystrophic dystrophie dystrophie

Figure 14 Scanning electron micrographs of dystrophic mineralization in cultures grown

under standard conditions A) The sparse layer collagen matrix and very

elongated cells were covered by large clusters of randomly distributed minera1

(arrow) (F.W. 180 pm). B) These clusters were formed from individual mineral

spheres. The clusters were found to rest directly on the cells (F.W. 45 pm).

Range (keV) Net lntegral Calcium L a, b .26--42 -32 Calcium K a 1,2 3.58-3.80 40143 Calcium K b l 3.904.12 5029 Phosphorus K a l 1.90-2.10 21392

Figure 16 Phase contrast micrographs of cultures supplemented with varying

concentrations of P-GP. Ail of the cultures were of cells derived from a 5 year

old male donor (inserts are photographs of tetracycline labeled cultures taken

under UV light) A) A culture containing 3.5 m M P-GP produced discrete

nodules (insert). These nodules had al1 the morphological hallmarks of normal

mineralized nodule formation (F. W. 1.1 mm). B) & C) No nodules were noted

in cultures containing either 5 m M or 10 m M concentrations of 0-GP. The

mineral was diffuse and more heavily localized to the edges of the culture dishes

(insert). Ectopic rnineralization was associated with a sparse monolayer of

fibroblastic cells (F.W. 1.1 mm).

Figure 17 Scanning electron micrographs of cultures suppiemented with either A) 5 m M

PGP (F. W. 225 pm) or B) 10 m M PGP F. W. (300 pm). In both sets of culture

conditions mineral deposits were seen to overlay the culture. obscuring the

sparse cell sheet.

Figure 18 Transmission electron micrograph of cultures supplemented with 1 O m M of

BGP. There was only a very sparse ce11 sheet and no visible evidence of

collagen matris. The mineral deposits. seen here as electron dense clusters, were

found on the ceIl sheet or at the surface of the culture substrata (F.W. 90 pm).

5. E Subcultures

5. E. La Su bculture Three attempts to subculture with the prescribed methods were made. in the first

experiment only cells at the surface of colonies were retrieved. with a short digestion

period. This contrasted the long digest subcultures where the majority of cells were

collected. In the other two attempts. al1 subculture conditions resulted in the detachment

of the entire. but intact. cell shert. Mechanical dismption was needed to retrieve the

cells.

S. E. 1. b L igli t Microscopy In the first expenment rnorphologically identifiable nodular structures were found

in cultures of cells. retrievrd from primaries where a short digestion period was used.

The mineralization of these structures was first detected afier 6 weeks of subculture (fig

19). These early nodules were similar to those noted in the primaries. However they

appeared to be more numerous fusing together and covering the majority of the culture

dish. Large numbers of adipocytes were present prior to mineralization. This differs

from the primaries where the apprarance of adipocytes was either coincident with. or

followed. the initiation of mineralization.

In contrast. subcultures where a longer digestion period was used to harvest the

cells failed to provide a normal mineralized matrix. Evidence of dystrophic

mineralization was noted at the end of three weeks in these subcultures (fig 20).

The following two attempts to replicate these results in two subsequent

subcultures failed? although it should be noted that the primary cultures. which wrre

maintainrd as positive controls. also failed to produce normal bone matris.

5.E.2 Scanning Electron Microscopy Scanning electron microscopy of the nodules elaborated in the successful

subcultures revealed the presence of an extensive fibrous matrix (fig 2 1 A). Interfacing

between the collagen and polystyrene surface was a sparse collection of globular

accretions (fig 21 B). However they did not appear to have formed a confluent layer at the

time these cultures were examined. The fibrous matrix was densely rnineralized and

there was evidence of pre-osteocytic cells embedded in the collagen scaffold (fig 3 1 ).

5.F Non-adherentcells Non-adherent cells. which are normally removed and discarded at day 4 remain

viable. in some cases capable of osteogenic activity. Crlls retrirwd either form the

primary or from the subsequent cultures of reseeded cells were able to form colonies of

adherent cells. More importantiy nodule formation was noted in some. but not al1 of

these cultures. Although the leveis of osteogenic activity were not quantified. in

comparison to the primary the amount of matrix formed appeared to be less extensive.

Figure 19 Nodules formed in subculture (short digest) A) Dark field micrograph of an

incipient nodule. seen in the center of the field. ai day 60 of culture. Adipocytes

(small arrows) were clearly evident immediately surrounding the nodule (F. W.

2.7 mm). B) Phase contrast micrograph of this new nodule. where the polygonal

cells have begun to condense and aggregate. Adipocytes were also seen in these

same regions (arrow). The opaque areas indicate the first morphological

rvidence of mineralizaiion (F. W. 1.1 mm). C) Numerous adipocytes were seen

amongst or near the mineralizing tissue (arrow). The morphology of the cells in

association with mineralizing areas (star) was similar to those in the incipient

nodule (arrow head). Individual nodules are seen to cover the culture vesse1

(insert) (F. W. 1.1 mm).

Figure 20 Dystrophic minerai was predominate in subcultures where cells were

harvested using a long digestion period. A) Dark field micrograph depicting

minera1 (light areas) which was haphazardly distributed over the culture (F.W.

2.7 mm). B) Phase contrast micrograph of diffuse patches of mineral (mow

head) that were seen across the sparse ce11 sheet. The cells had an elongated

fibroblastic morphology (F. W. 1 . 1 mm). Evidence of nodule formation was not

seen.

Figure 2 1 Scanning electron micrograph of nodule forming subcultures. A) Subcultured

cells produced an extensive fibrous extracellular matrix. as seen in the right hand

corner of the micrograph (F.W. 260 pm). At the base of this nodule (arrow) the

globular accretions of the cement line could be seen beneath the collagen mat at

the outer edge of the nodule. seen at higher magnification in (B) (F. W. 60 pm).

C) The fibrous matrix was seen to be very heavily calcified in regions. The

individual fibres were almost indistinguishable. as they were encased in minera1

(F. W. 1 2 pm). D) Cells resembling osteocytes were also noted in the matris.

The cytoplasmic extensions radiate outward to the walls of the lacunae housing

these cells (F.W. 45 pm).

6. Discussion The results reported have unequivocally demonstrated that morphologicaily

identifiable bone matrix can be elaborated in vitro in cultures of human stroma1 cells

harvested from femoral and iliac bone marrow. This bone matrix comprised an

interfacial afibrillar globular matrix which, in the rat has been identified as the in vitro

equivalent of the cement line matrix found at bone remodeling sites (Davies. 1996). This

matrix was laid down on the culture dish surface in both primary and first passage

cultures and separated the culture substrata from the collagenous bone matrix above. The

latter mineralized and contained osteocytes which themselves displayed ce11 processes

which radiated through the surrounding bone matris. The most superficial layer of such

nodules were not rnineralized and were putatively identified as an osteoid layer which

separated the mineralized collagen from the overlying layer of tessellated cells. In

transmission electron microscopy these cells and others associated with the nodule. were

rich in endoplasmic reticulum and other cytoplasmic organelles typical of synthetically

active cells. while those cells becoming surrounded by rnineralized matrix accurnulated

glycogen, a recognized hallmark of osteocytes (Scott & Glimcher, 1971). These

morphological markers of bone matrix perrnitted a clear cornparison with another form of

biornineralization which appeared in some cultures. The latter exhibited a randoin.

punctate distribution. which has been described by others as biornineralization

representative of intramembranous ossification (Jaiswal et al.. 1997). By clearly

dernonstrating the elaboration of bone matnx (vide supra) we were able to define the

latter "biomineralization'.. while dependent on the presence of cells, as a form of

dystrophic mineralization rather than bone forniation. The term dystrophic

mineralùation is used, in the context of this study, to specifically describe

mineralùation occurring in the cultures which bears no morphotogical resemblance

to bone nodule tissue, grown in cultures of animal derived osteogenic cells, as

reported by other investigators (Nefussi et al.. 1985, Luria et al.. 1987. Bhargava et al..

1988. Satomura and Nagayama. 1 99 1 ).

In general. there are three methods to culture osteogenic cells which in tum are

capable of bone matrix production (for reviews see Davies. 1990. Beresford et al., 1993,

Majeska. 1996). First, the isolation of osteogenic cells from enzymatically digested rat or

mouse calvarial tissue. The cell population isolated is heterogeneous. representing

different stages of differentiation. However the pool of very early osteogenic precursors

is significantly lower than that found in ce11 cultures derived from the marrow. Second.

marrow esplants provide a source of stroma1 cells which under the appropriate

conditions. normally requiring the addition of dexamethasone, can result in differentiation

of osteogenic cells. Third, cells can also be isolated from populations which have

rnigrated frorn esplantrd bone fragments. The majority of researchers who have

described human bone growth in vitro have employed the latter method. ncrmally

utilizing trabecular outgrowths. The human trabecular bone cultures have been

successhilly employed ta demonstrate the growth of morphologically and biochemically

identifiabie bone in vitro. This capacity for bone formation was first, successfully

described by Robey and Termine (1 985). However more recently, several authors have

emphasized the benefits of employing human bone rnarrow to derive an osteogenic

population. There are several different reports. but they have signifiant contradictions

with respect to the type of mineralization seen in these cultures. Indeed there is very little

histological or ultrastructural data provided to adequately analyze these different types of

biomineralization.

Despite the considerable interest. and the associated importance, of having

reliable methods For cuituring osteopnic cells in bone ce11 biology. biomaterials and

tissue engineering fields, there have been no convincing reports of the development of

morphologically identifiable bone matrix by human marrow stromal cells in vitro.

Several researchers have attempted to examine the osteogenic differentiation of

human marrow stromal cell populations in vitro (Kassem et al.. 199 1. Viiamtiana-

Amedee et al.. 1993. Beresford et al.. 1994. Cheng et al ., 1994. Richard et al.. 1996.

Jaiswal et al.. 1997). Many of these studies have utilized dexarnethasone which is also

known to induce osteogenesis, as seen by bone nodule formation, in stromal cells of dog

(Kadiyala et al., 1997). chick (Kamalia et al.. 1992), rat (Maniatopoulos et al.. 1988) and

mouse (Luria et al., 1987) origin. These endeavours have provided consistent results with

respect to certain criteria indicative of osteogenic behaviour. These include one or more

of the following: an increase in alkaline phosphatase activity. which is seen to occur in

the vast majority of the cells, PTH responsiveness, the up-regulation of osteocalcin in

response to 1,25,(0H)2 vitamin D3 and the deposition or formation of hydroxyapatite.

However there are differing reports with respect to the nature of the biomineralization

elaborated in human stromal ce11 cultures.

Biomineralization in hurnan stromal cultures, derived from both marrow (Kassem

et al.. 199 1, Cheng et al., 1994, Jaiswal et al., 1997) and periosteurn (Koshihara et al.,

1987). many times has been described as ubiquitous, occumng in the absence of nodule

formation. The light microscopy of these cultures revealed that the variable sized clusters

of mineral were randomly distributed. The cells associated with the minera1 were often

fibroblastic in morphology. These mineralkell clusters referred to as "mineralized

cellular aggregates" (Jaiswal et al., 1997. Bruder et al.. 1997) were found to detach from

the culture substrata. The identification of the mineral has been limited to EDX and x-ray

diffraction analysis, where it was classified as hydroxyapatite. Unfortunaiely there are no

ultrastructural evaluations of the cells, their matrix or the minera1 given in any of these

studies. This is in contrast to findings from stromal and calvarial animal cell cultures

where nodules develop from foci of osteoprogcnitors. The elaboration of bone matrix.

which Later mineralizes. is localized to these regions. The absence of nodule formation in

human stromal ce11 cultures has been attributed to the homogeneity or uniformity in the

osteogenic response of this cellular population (Jaiswal et al.. 1997). Clearly however

both the present study and the work of several others (Shibano et al.. 1998. Sel1 et al..

1998) wouid indicate that these cells. as seen by phase microscopy. can indeed produce

structures resembling bone nodules and that osteogenesis occurs in a limited portion of

the cellular population. Interestingly, despite the proven capacity of cells in our culture

system to produce bone nodules there were discrepancies between cultures with respect to

the type of biomineralization. paralleling those differences seen in the Iiterature between

random vs. isolated sites of mineralized nodule formation. Comparing these two types of

biomineralization using morphological and uitrastructural criteria, ive were able to clarify

andior distinguish between mineralization associated with bone matrix maturation and

dystrophic biomineralization which c m be mistakenly associated with. or used as

evidrnce of. the mature osteoblast phenotype. In doing so we have. to our knowledge.

been able to provide the only thorough morphological/ultrastnictural characterization of

the human rnarrow stromal cells and the matrix they produce in vitro.

6.A Light Microscopy Many of our early observations of the human marrow stromal cells are very

similar to those described in the literature. First. only a small proportion of the seeded

cells were found to attach to the culture vessels. The number of cells which can be

retrieved from the marrow. for the age range examined. was generally in accordance with

Caplan (1 994). The variability in the number of cells is probably influenced by the donor

age. However a specific trend in the number of adherent cells could not be formally

established. given the limited data collected. Donor age has a documented influence on

the number of stromal cells retrieved (Caplan. 1994) and their osteogenic potential (Bab

et al.. 1988. Hanswonh et al.. 1997. Nakahara et al.. 199 1). Additional factors which may

have some influence. but were not exarnined, include donor sex, sampling location and

other mitigating influences specific to the donor. In addition the serum lot may play a

role. According to Lennon et al. (1996) serurn composition influences the selection and

at tachent of the stromal cells. the specific factors which contribute to this are yet poorly

defined.

The majority of the early adherent ce11 colonies in Our primary cultures were

alkaline phosphatase positive. which is in agreement with the observations of Beresford

et al. (1994). Several weeks into culture these cells proliferate to form a sheet of

multilayered cells whose morphology and levels of alkaline phosphatase staining

appeared fairly uniform. It is at this point where what we have described as dystrophic

mineralization can occur. The nature of this type of biomineralization, its effects on the

cells and the factors which contribute to its occurrence will be discussed separately.

However. in general the culture conditions employed in this study supported nodule

formation rather than dystrophic biomineralization.

The progression of osteogenesis could be morphologically identified both in phase

microscopy and in serial cross-sections of the early nodules (data not show). as an

alteration in ce11 morphology to a polygonal (cuboidal) form and an increase in ce11

density in discrete regions of the culture. It is speculatçd that the increase in ce11 density.

Iorming a three dimensional structure of multilayered cells. creates a microenvironnient

which is important to the promotion of osteogenic differentiation (Nijweide et al.. 1982.

Tenenbaum et al.. 1 986. Nefussi et al., 1989). In addition. there is an apparent increase in

alkaline phosphatase activity, which is coincident with the morphological changes just

outlined. Alkalinr phosphatase is recognized as an early marker of the osteoblastic

lineage (reviewed by Caplan? 199 1. Aubin & Turskin. 1996). However its expression is .

not limited to this ce11 type and is often associated with adipogenic cells, whose

development has been noted in our cultures. In fact the CO-expression of osteoblast and

adipogenic markers. including alkaline phosphatase are expressed in clonal stroma1 ce11

colonies (Rickard et al.. 1997). More importantly than the expression of this enzyme are

the morphological transformations accornpanying its expression' which according to

Nefùssi et al. (1 989) who modeled these events in rat calvarial cultures. is a key evrnt in

osteoblastic differentiation.

[t is here in the interior of the nodular regions where mineralization is first

localized. The tetracycline label ing. which is ofien used to identiQ the mineralizing

fronts of bone (reviewed by Ibsen, 1985), is detected in discrete regions throughout the

nodular matrix. These regions correlate very closely with electron dense regions in the

backscattered electron images, which could be indicative of the calcium of

hydrox yapati te.

Mineralization whether it be dystrophic or associated with nodule formation was

dependent on the presence of dexamethasone in culture. This contrasts with the human

explant cultures where mineralized nodule formation can occur in the absence of external

stimulation with drx. This situation parallels the effects of glucocorticoids on rat

calvarial cells vs. stromal cells. Whereas dexamethasone was found to increase the

number of nodules and the amount of mineralized matrix in calvarial cultures. it is not

essential for bone matnx formation (Bellows et al.. 1987. Bellows & Aubin. 1989).

However. Maniatopoulos et al. ( 1988) reported that dexamethasone was required for

nodule formation in their marrow stromal cultures. This reflects the less differentiated

phenotype of stromal cells populations. according to Beresford et al. (1993).

In general. the rnorphology of these rnarrow derived nodules resembles those

described in human adult trabecular outgrowth cultures (Robey & Termine. 1 985) and in

calvarial models (Bellows et al.. 1986). Overlaying the extensive acidophilic

extracellular matrix was a layer of flattened tessellated cells. Within the matrix there were

several embedded cells resernbling pre-osteocytes. Mineralization is contained to the

central portion of the structure. The overlaying ce11 sheei is separated from this area of

active rnineralization by a seam of osteoid. as would be expected in in vivo bone

formation. Immunolabeling of the matnx produced by these cells has revealed the

incorporation of osteocalcin. a bone matrix protein presumably made by mature

osteoblasts. Osteocalcin. produced at basal levels, has previously been localized

intracellularly and in the media of human stroma1 ce11 cultures (Vilarntiana-Amedee.

1993), but not in the tissue produced in these cultures. It should be noted that attempts to

localize the protein in the tissue of non-nodule forming cultures have been reported to

give negative results (Jaiswal et al.. 1997). This was attributed to the low basal levels of

osteocalcin resulting from the inhibitory effects of dexamrthasone.

6.8 Ultrastructure At the upper most surface of the developing nodules large bulbous cells could be

noted in the transmission electron micrographs. Examination of the cells overlying the

developing bone matrix. with scanning electron rnicroscopy. often times revealed a

collection of tessellated cells with nurnerous blebs and filopodia. which were

distinguished from the more flattened fibroblastic cells at the periphery of the nodules

(data not shown). Bhargava et al. (1 988) and Nanci et al. (1996) made similar

observations in cultures of rat calvarial cells. The membrane blebbing increases ce11

surface area which is thought to improve nutrient uptake in the cells overlying the

developing nodules. In addition in the upper region of the nodule there are several

elongated cells. with a paucity of synthetic organelles, features which are associated with

preosteoblasts or early osteogenic precursors. The large accumulation of glycogen seen in

these cells. has also been descnbed by Scott & Glimcher (1 97 1) in early osteogenic

precursors in the rat tibia.

The afibrillar matnx which was assembled at the substrate tissue interface, closely

resembles the cernent line descnbed in cultures of rat (Davies et al.. 1991) and ferret

(Graiano. 1998) stroma1 cells. The cells were often in close association with the

globular accretions of this matrix, which may imply a ceIl mediated process. In rat the

formation of this matrix is dependent on the presence of an osteogenic differentiating ce11

population. but independent of the presence of ascorbic acid (Hosseini et al., 1996). The

absence of both the cernent line and the mineralized collagenous matrix of bone in

cultures lacking dexamethasone would support this assertion. In addition the globular

accretions were limited to regions of nodule formation. This was confirmed in the SEM

examination where the concentrated collections of accretions were limited to specific

regions of the dish, often extending just beyond the fibrous matrix of the nodules.

Tetracycline labeling was used to conf in the distribution of these nodules. When

labeled cultures were exarnined either by light or confocal microscopy (data not sliown)

small. presumably mineralized, areas whose dimensions match those of the accretions.

could be seen at the base of the newly developing nodules and/or extending just beyond

the bulk of the mineralized matrix. It is unlikely that the creation of these structures are

the result of spontaneous mineral deposition since. in the latter, one would expect the

pattern of distribution to be more random and ubiquitous. as is seen in the examples of

dystrophie mineralkation reported herein.

The extensive densely packed fibrous matrix was identified as collagen by the

periodic banding pattern seen in the transmission electron micrographs. The type of

collagen however was not identified. Previous studies have demonstrated that pro-

collagen type 1 can be detected in hurnan marrow stroma1 ce11 cultures. where the

conditions promote osteogenesis as seen by nodule formation (Shibano et al.. 1998). The

matris was highly organized with the bundles of collagen fibres arranged onhogonally to

one another.

Minerai was found in association with collagen fibres deep in the matrix. The

feathery needle-like crystals that were seen extending from foci of collagen

mineralization or which were aligned dong the individual fibres. are rerniniscent of

hydroxyapatite crystals seen in the mineralization of bone in vivo. These crystal however

have not yet been confirmed by x-ray diffraction as the hydroxyapatite of bone. The

newly mineralizing regions of collagen were often surrounded by an electron dense

amorphous substance. Inspections of these areas either morphologicall y by TEM or

chemically using EDX analysis gave no detectahle indication of mineral. However very

early crystal formation may be beyond the detection limits of these methods. These

regions likely represent the accumulation of non-collagenous proteins important to the

initiation or control of the mineralization process. Irnmunogold labeiing of electron

dense regions, closely associated with mineralizing matrix, in developing bone both in

vivo (Chen et al., 1994) and in vitro (Slater et al.. 1994a), have demonstrated a localized

increase in chondroitin sulphate, osteopontin and bone sialoprotein. Irnrnunological

analysis of the regions detected in Our cultures would probably yield comparable results.

The maturation of the matrix is rnarked by not only by the progression of the

mineralization process. but also by the terminal differentiation of osteogenic cells. The

morphology of these osteocytic cells closely resembles the cells described in vivo

(Holtrop. 1975. Jande. 197 1 ). Young osteocytic cells. identified by their collection of

synthetic organelles and their extended ceIl processes. which presumably create a cellular

communication network within this bone matrix. The more mature osteocytic cells show

a gradua1 atrophy of their synthetic organelles and begin to accumulate large amounts of

glycogen. a phenornenon described by Scott & Glimcher (1971) in fetal rat tibia. Finally

a few of these small cells could be found embedded in the heavily calcified tissue which

they have created.

The ultrastructure of the matrix produced in human stromal cell cultures has al1

the morphological hallmark events in bone matrix elaboration. In summary these include

the establishment of an afibrillar matrix proteinacious layer which mineralizes. followed

by the assembly of a collagenous matrix containing osteocytes which in tum matures and

becomes mineralized.

6. C Dystrop hic Mineralka tion The thorough histological and ultrastructural examination of the tissue elaborated

in human rnarrow stromal ce11 cultures reveals that the nodular matrix produced in these

cultures is very similar to normal bone matrix. In cornparison the tissue produced in

cultures where dystrophie mineralization is seen bears little resemblance to bone tissue.

Dystrophie mineralization often appeared in SEM as spheres, of approximately one

micron in diarneter, which were seen to form large aggregated clusters on the ce11 sheet.

The transmission electron micrographs demonstrated that these islands of minera1 debris

which were seen both above or just beneath the ce11 layer. These calciurn/phosphate

deposits remained to be unequivocally identified. but are probably hydroxyapatite.

Furthemore the appearance of these deposits had a pathological effect on the cultures.

The cells' viability and their capacity to produce an extracellular rnatrix was often

severely compromised. In the most extreme cases there was only a limited arnount of

collagen production and very little evidence of normal mineralization of the collagen

fibres. Encasernent of the limited fibrous matrix was probably accidental rather than a

ce11 directed phenomenon. but this was not the focus of our study. The degree to which

the matrix formation and ce11 viability were impaired seemed to be related to degree of

dystrophic mineralization, this in turn was influenced by increasing the level of PGP used

in the cultures. In general dystrophic mineralization appeared to impede the progression

of osteogenesis in this cellular population as refiected by their inability to produce a

matrix resembling bone.

6. D Normal vs. dystrophic mineralization the role of pGP There are varying opinions regarding the need for PGP in osteogenic cultures.

Mineralization of the extracellular matrix produced by either rodent stroma1 cells

(Maniatopoulos et al.. 1988) or calvarial cells (Bellow et al., 1986, Escort-Charrier et al.,

1983) in culture has been reported to be dependent on the administration of PGP or free

phosphate to the medium. While this may be true. some reports described the formation

of mineralized nodules in the absence of PGP. suggesting that PGP merely facilitates or

augments the level of mineralization (Owen et al., 1990, Slater et al., 1994). In the

present study PGP was generally needed for mineralization of the bone nodules and was

often required to maintain the cells in culture. since its absence resulted in ce11

detachment. However in cultures where the cell sheet integrity was maintained there

were rare occasions where a limited amount of mineralized nodule formation could be

noted. The number of nodules and level of minenlization in cornparison to standard

cultures was significantly lower. This occurrence rnay be related to the youth of the

donors used in this study. Where human embryonic bone explant cultures (Slater et al..

1994) rnineralize in the absence of an exogenous source of phosphate. beyond that what is

found in the media, this has not yet been reported to occur in cultures utilizing adult bone

tissue.

Inclusion of PGP provides an organic source of phosphate. The mechanism by

which PGP facilitates the initiation of mineralization has been linked to alkaline

phosphatase activity whicli is substantially increased near sites of nodule formation. The

enzyme is thought to hydrolyze the PGP to provide a localized increase in inorganic

phosphate. which may be important to the initiation of mineralization (Tenenbaum. 1987.

Bellows et al.. 1992). Blocking the hydrolytic activity of the enzyme. with levarnisole.

prevents mineral deposition from being initiated. Despite the regular use of PGP in

osteogenic cultures, rnany have speculated on its physiological relevance and the type of

rnineralization which will occur in its presence.

It has been demonstrated in several studies that biomineralization in the presence

PGP rnay reflect aikaline phosphatase activity rather than the osteogenic character of the

cells. If this is the case, spontaneous deposition of dystrophic minerai would not be seen

in cultiires where Dex is absent and alkaline phosphatase activity is iow. In fact

significant levels of calcium phosphate deposition have been reported to occur in cultures

of skin fibroblastic cells cultured with PGP and exogenous alkaline phosphatase. at levels

simulating those excepted in osteogenic models (Khouja et al.. 1990). Similarly

hydroxyapatite formation has been found to occur in cultures of non-osteogenic cells

transfected to express bone alkaline phosphatase. when their medium is supplemrnted

with PGP (Hui et al., 1997). In this second study the hydrolytic activity of the enzyme

enhanced. but was not absolutely required for minera1 deposition to occur. The mineral

which formed deposited randomly over the filters which the cells were seeded. TEM of

these minera1 deposits revealed clusters of minera1 spheres. similar in appearance to the

dystrophic mineral we see in Our cultures. These findings could in pan explain the

sporadic occurrence of dystrophic mineralization in Our cultures.

The morphological indications of nodule formation in our cultures are seen 3 to

10 weeks following the expression of alkaline phosphatase. This lag period potentiates

the formation of dystrophic mineralization. since the hydrolysis of BGP as suggested by

Chung et al. ( 1992) should result in an increase of inorganic phosphate (Pi) in the

medium. which would facilitate the nucleation of dystrophic mineral. As inferred from

our data. an increase in PGP would elevate the risk and possible arnount of dystrophic

mineral by increasing the level of Pi which would accumulate in the cultures. Chung et

al. (1992) suggest that the arnount of PGP should not exceed 2 mM. a number based on

the estimated level of physiologically available inorganic and hydrolyzable phosphate

level espected in vivo. Previous reports of nodule formation by human marrow stroma1

cells have limited the levels of Frer phosphate to approximately the suggested range.

Alkaline phosphatase itself may also nucleate dystrophic mineralization. The

phosphotidyl inositol glycolipid anchor of alkaline phosphatase was implicated in the

initiation of hydroxyapatite formation in the work of Harrison et al. (1995). The early

expression of this enzyme may result in an increased accumulation of insoluble alkaline

phosphatase (i.e. with the anchor in a vesicle or as a membrane fragment) in the media.

The risk of dystrophic mineralization therefore may not just reflect the quantity of PGP

added. but the level of aikaline phosphatase expression and the possible accumulation of

it in the medium prior to nodule formation. This may account for the variation in

biomineralization occuning at lower levels of PGP supplementation. If the progression

of osteogenesis is delayed. due to donor specific influences. or is inhibited by the

eiimination of ascorbic acid (Xiao et al., 1997) from the culture this could increase the

likelihood of dystrophic mineralization. More frequent media changes might reduce the

possible accumulation of factors which could initiate dystrophic mineral deposition.

Finally BGP has been shown to elevate the release of metalloproteinase into the

medium of both osteoblast (Dean et al., 1994) and chondroblast (Schellar et al.. 1995)

cultures. lncreasing levels of PGP may contribute to an elevation of degradative enzymes

in the medium of our human osteogenic cell cultures, which into tum may exacerbate ce11

sheet disaggregation and detachment as was seen in our system.

6.E Bone Mafrix formation in first passage cells Presently there has been only one recent study where subcultures of expanded

human marrow stroma1 cells demonstrated the capacity for nodule formation (Shibano et

al.. 1998). Illustrated in this report was a very limited arnount of nodule formation as

seen by phase microscopy, but there was no ultrastructural analysis of the tissue.

In our primary cultures we were able to define the rnorphological criteria of

normal bone rnatrix formation and a sequence ~f events comprising its formation. which

is similar to that seen in other systems. It therefore should be reasonable to assume that

harvested human bone marrow stroma1 cells first expanded in primary culture. would

maintain their capacity to make bone matrix in vitro. Our early attempts to harvest the

primary cells (data not presented here) utilized the harvesting protocol used for the rat

stroma1 culture system (Davies et al., 199 1 ). Using this procedure we found that

dystrophic biomineralization occurred in these subcultures, even when nodule formation

was seen in the primary control cultures. Therefore it would appear that the harvesting

procedures may affect the cells and their capacity to produce normal mineralized matrix.

The initial studies examined shortening the digestion to 5 from 15 minutes. in

addition to lowering the amount of trypsin used. Here we found in Our first experiment

that cells harvested using a shonened digestion period were able to produce bone nodules

whose ultrastructural morphology. as seen in SEM. was very similar to that seen in the

primaries. This included the afibrillar globular accretions of the cement line matrix. an

extensive collagenous matnx which seemed to calci@ normally. In addition terminally

differentiated osteocytic-like cells were clearly visibly embedded in the lacunae created in

that matrix. In contrat the cultures where the standard digestion time was used showed

evidence of dystrophic mineralization indicated by the random deposition of mineral in

the absence of nodule formation? the nature of this mineral being confirmed by SEM

analysis.

It would appear from these results that longer exposures to trypsin had a

deleterious effect on the cells and their ability to progress through osteogenesis or that

cells which rnay not have the potential to undergo osteogenic differentiation recover more

readily from long exposure to trypsin over those cells that do have that capacity. The

severity of the effects trypsin may have could depend on the celi type or stage of

differentiation. This may in turn select against cells which will undergo osteogenesis or

delay the process. while cells recover from the treatment. This however is speculative at

best. A repetition of the experimental results will be needed to confirm that a limited

exposure to trypsin is favourable for harvesting.

Unfortunately. we have bern unable to replicate the results in our subsequent two

experirnents. Dystrophic mineralization was seen in both Our subsequent attempts to

subculture the cells using both short and long digestion periods. However given that we

saw dystrophic mineralization in Our control prirnary cultures, it would seem likely that

donor specific influences may have been at play. This may also illustrate the need to

make additional modifications to our culture conditions in order to further minirnize the

prevalence of dystrophic mineralization in gneral.

The influence of the harvesting procedure remains to be more fùlly exarnined and

understood. it should to noted the culture conditions used to expand the ceIls may also

need to be re-exarnined making them more suitable for tissue engineering applications.

Bruder et al. (1997) examined the expansion of stroma1 ce11 populations in the absence of

any osteogenic inducers. such as dex. allowing for the proliferation of the cells without

differentiation. However when the osteogenic activity of tliese cells was assoyed in vitro.

after various numbers of passages. only random biomineralization was seen. This is

probably due to the high levels of PGP which were used in their cultures. Another

approach has been to expand the cell population with factors. such as FGF-2 (Martin et

al. (1997). This study found that FGF-3 was preferable to either dex or the absence of

exogenous factors. excluding those found in the serum. because it mainteined cells in

their more immature state and potentiated the expansion of osteo-progenitors as indicated

by the in vivo assay.

6. F Applications for Human Bone Marrow Cultures Multipotential bone marrow stromal cells. because of their known role in bone tissue

repair and remodeling. are ideally suited to bone tissue engineering stratepies. The

therapeutic potential of autologous transplantation of ex vivo expanded marrow stromal

cells for bone regeneration has been demonstrated in both rat (Kadiyala et al.. 1997) and

canine (Bruder et al.. 1998a) models. In addition ex vivo expanded human marrow

stromal cells loaded on ceramics and implanted into critical size femoral defects. in adult

athymic rats. were found to help in the generation bone in these sites (Bruder et al..

1998b). Given the potential for bone marrow stromal cells in this type of cellular ther&

requires the development of a "high standard and reproducible systems for the ex vivo

expansion" of these cells according to Martin et al. (1997). The development of

appropriate ex vivo expansion strategies requires an adequate method to assess the

osteogenic potential of the cells (Bruder et al., 1997). However until now the most viable

rnethod for testing this. for human bone rnarrow derived populations was using in vivo

assays of bone formation. Having established culture conditions which promote normal

bom matrix fonation we have a convenient in vitro assay for testing the osteogenic

potential of these cellular populations.

In addition human marrow stromal ce11 systems can be used to test in vitro the

ability of 3-dimensional material constructs to support osteogenesis and bone rnatris

formation. Since the materials of interest to the tissue engineering tield must be able to

do several things: deliver human marrow stromal cells to a site. allow for osteogenic

differentiation and ultimately support bone matrix fonation. Given this mandate i t

would seem appropriate to utiiize this same cellular population in an in vitro system

which will be used study the above parameters in the evaluation of a material construct of

interest for the tissue engineering field.

Bone biologists have utilized the bone nodule assay, using mainly animal derived

cells. to study osteogenesis and factors influencing the osteoprogenitor population.

Researchers have relied on this assay because to date there are no other unequivocal

markers of the osteoblast or their early precursors. The capacity to form tissue which is

morphologically and biochemically similar to in vivo bone remains the only, albeit

indirect. measure of osteogenesis (Aubin & Herbertson, 1998). In addition there has been

increasing interest in utilizing cells of human ongin to study both normal and

pathological bone ce11 differentiation and behaviour, thereby eliminating any concems

related to species differences (Beresford et al., 1984). The first bone nodule assay

utilizing human cells. relied on the outgrowths of bone explants. Although the system is

used with some frequency the isolated ce11 population is thought to provide a more

limited number of early osteogenic precursors, in cornparison to that available from the

marrow (Beresford et al.. 1993). In this study we have been able to demonstrate that

human marrow stroma1 cells under the appropriate culture conditions can producr bone

nodules and that the tissue they produce resembles bone. thereby providing a useful assay

not just to the tissue engineering field but also those in bone biology.

7. Conclusions From the results reported herein it can be concluded that human bone marrow

stroma1 cells can produce in vitro a mineralized tissue whose ultrastructural morphology

is very similar to bone seen in vivo. Al1 the histological hallmarks signifjing the events

of bone matris elaboration and maturation cm be noted in these cultures. This type of

mineralization. which is associated with nodule formation is limited to only a portion of

the ce11 population and greatly differs from the dystrophic "biomineralization" which has

been mistaken for bone mineral by others. Finally these cells c m be expanded in culture.

hmested and maintain their capacity to form normal bone matrix in vitro.

8. Future Work Current efforts in bone tissue engineering to design 3-dimensional scaffolds must

include the evaluation of these structures to support osteogenic differentiation and bone

matrix formation on their surfaces. Efforts in Our own laboratory have relied upon the rat

stromal ce11 systern for these types of studies. These constructs however will eventually

be used as carriers for human stroma1 cells and therefore it would be advantageous to

utilize these same cells in the analysis of materials of interest. It is Our intention to

develop a 3-D culture systern. These efforts will based on the culture conditions

rmployed for 2-D substrates described in this study, where normal bone matrix

production by human bone rnarrow stromal cells was supported. As was the case in the

2-D system bone matrix elaboration will be used as a measure of osteogenic activity.

Using the culture systern. outlined in this study. 3-D rnaterial constructs will be evaluated

for their ability to support osteogenic ce11 ingrowth, osteogenesis and bone matrix

maturation. The rnatrix will be evaluated first by its morphological and later biochemical

characteristics.

In addition to the development of the 3-D culture system efforts will be made to

optimize and standardize the ce11 expansion and harvesting procedures to be more

suitable to tissue engineering applications. In this work we will need to examine a wider

range of donor ages. Given the youth of the donoe used in this study we have esaminrd

a somewhat idealized population with respect to the osteogenic activity of cells retrieved

from the marrow. We need to determine the culture conditions to espand the osteogenic

population and establish whether in vitro bone growth can still be supported in 2-D and 3-

D cultures wl-iere the cells have been obtained from the marrow of adult donors.

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