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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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