the ferret: in vitro animal for of … · ilms (~steolo~ic" discs), resorption events and...
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THE FERRET: A POTENTIAL IN VITRO SMALL ANIMAL MODEL FOR THE STUDY OF OSTEOGENESIS AND
OSTEOCLASIS.
Victor M. A. Graziano
A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Dentistry
University of Toronto
O Copyright by Victor M. A. Graziano, 1998.
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MODEL FOR THE STUDY OF OSTEOGENESIS AND
M. Sc, thesis, 1998 Victor M. A. Graziano
Graduate Department of Dentistry, Centre for Biomatenals, University of Toronto.
ABSTRACT
It has recently been shown that the ferret may be employed as an m vivo mode1 for
the study of metabolic bone diseases m k e y et al., 1995). Unlike the rat, the ferret
exhibits BMU-based remodehg that paraUeIs the in vivo remodehg of human bone and
m e r displays siniilar physiological responses to PTH administration and estmgen
depletion. Induction of bone loss by estrogen depletion can occur either via ovariectomy
or, uniquely, by reducing lit photopenods. However, the ability of ferret marrow explants
to bction as an in vitro osteogenic and osteoclastic source is an essential step in
demonstrating the overail utility of this species for fiiture metabolic bone studies.
Modifying a strornal ceU culturing technique developed for the rat (Maniatopoulos et al.,
l988), ferret marrow explants h m 1 1- 13 week old young males were cultured in a-
minimal essential medium contaiaing 15% fetal bovine senun, antibiotics, and
combinations of: ascorbic acid, Na-B glycerophosphate andor dexamethasone.
Fully supplemented cultures containing dexamethasone demonstrated the abiiity to
elaborate mineralized interfacial matrices on culture substrates with which mineralizing
collagen fibers were intimately associated. Osteoblasts became entrapped in this
mineralizing rnatrix to become osteocytes. Furthemore, these cultures stained positively
for the alkaline phosphatase, von Kossa and tetracycline reaction. Energy dispersive X-ray
and X-ray difnaction analysis verifid a signincatlt calcium and phosphorus component of
the elaborated matrix and that the crystal nature of such rnineralized nodules was tbat of
embryonic type or poorly crystalline hydroxyapatite.
Stromal œ U plating density assays of fhst passage œil populations. demonstratecl a
linear relatiomhip between cell plating density and nahie count. Plating density however
had no significatlce on nodule size. Furthermore, if d tures contiiining dexaraethasone
were plated on resorbable substrates, either bovine bone slices or calcium phosphate thin
Ilms (~steolo~ic" discs), resorption events and tartrate resistant acid phosphatase (TRAP)
putative osteoclasts were obsewed.
Ferret IMKUW explants cultured in the absence of dexamethasone similarly
displayed the ability of TRAP positive mdtinuclear putative osteoclasts to resorb
commercially available substrates and bovine bone marrow slices. Resorption events were
puantitated and found to be more abundant in such culture conditions. However, under
dexamethasone absent conditions c d cultures did not elaborate mineralized bone nodules.
The work reporteci herein demonstrates the feasibility of employing ferret marrow
culture systems for bone cell assays and provides a new in vitro mode1 to complement the
in vivo use of the species to elucidate the etiologies of metabolic bone diseases such as
osteoporosis.
iii
CONTENTS ....................................................................... ABSTRACT I
.............................................. ACKNO WLEDGEMENTS rn ........................................................................ CONTENTS v
....................................................... FIGURES & TABLES oc ABBREVIATIONS ................................. ..... ............... XI
1 . GENERAL INTRODUCTION ............................................. i
..................... ...... ......*. 1 .A. METABOLIC BONE DISEASES .. .. 1
.......................... .......................... 1 .A. 1 . Osteoprosis .. 1
........................ 1 .B. OSTEOPOROTIC ANIMAL MODELS .... .... 4
1.B.1. In Vivo ............................... .. .. 4 ...... ................ 1.B.l.a. TheFerret ................................ .....,... 4 1.B.l.b. OthersAnimalModells .......................... .... 6
......................... ..................... 1 . B . 2. In Vitro .. ....... .. 8
............. 1 . C. IN VITRO OSTEOGENIC & OSTEOCLASTIC MODELS 10
............................ 1 . D. STRUCTURE & COMPOS~ON OF BONE 11
....................................... 1 . D. 1 . Macro & Microarchitecture 11 ......................................... . 1 . D. 1 .a Woven Bone 13 ....................................... . 1 . D. 1 b. LameIIar Bone 13
1.D.2. BoneCeh .................... ..., ................................ 14 .......................... 1 . D. 2 .a. Osteoblasts & Osteocytes 14
.................... ............. 1 . D . 2. b . Osteoclasts ..... .. .. 16 ................................................ 1 . D. 3 . Bone Composition 18 ............................... . 1 . D . 3. a CoUagen .. ........... 18
................................. 1 . D . 3 . b . Ground Substances 19 ................................ . 1 . D. 3 c . Mineral Component 20
1.E. BONEMODELING & REMODELING ................................. 20
....................... ................. 1 . E. 1 . Surface Remodeling ... 23 .................. 1 . E . 2. Intracortical Remodeling/Haversian Systerns 23
.................. 1 . F . THE REMODELING INTERFACE/CEMENT LINE 25
.......................... 1 . F. 1 . Structlrre, Development & Fuuction 25
1.G. THEFERRET .................................. .. .......................... 27
.............................. 4 . MATERIALS AND METHODS ... ....... 31
4.B. HISTOLOGICAL STUDES (LM) .................................... 33
4 . B . 1. Alkaline Phosphatase Ac tivity .................... ... ......... 33
4.B . 2. Tartrate Resistant Acid Phosphtase Activity .................. 33
4.B . 3. Tetracycline Labelhg ............................................ 34
4.B.4. Von Kossa ..................................... 34 ................ 4 . B . 5 . Embedding Protocols ......................... .. ........ .... . 35
4.C. SCANNING ELECTRON MICROSCOPY (SEM) & ENERGY
DISPERSIVE X-RAY MICRO ANALY SIS ( D X ) .................... 37
...................... 4 . D . X-RAY DIFFRACTION (XRD) ... ................ 38
4.E. TRANSMISSION ELECTRON MICROSCOPY (TEM) ............... 38
4.F. O ~ T E ~ ~ L A ~ ~ ~ R E ~ ~ R P ~ ~ N A S S A Y ............................... 39
4 . F . 1 . Osteologicm Calcium-Phosphate Thin F i ................... 39
............................ 4.F.2. Bovine Bone Slices ... ............. 40
4 . G . D-SONE (+) SERIAL DILUTION AS SAY ............... 41
................................................ S.A. ZN VWO ARCH~~ECTURE 42
...................................... 5 .A. 1 . TrabecularEndosteal Bone 43
5.A.2. CorticalBone .................................. i ................ 45
................... ........... 5.B. DEXAEIIETHASONE (+) CULTURES ... 49
................... ......................... S.B.1. Cellcultures ., ...,... 49
5 .B. 1 .a. Primary Cultures ................... .... ......... 49
5.B.l.b. FirstPassageCultures .............................. 51
................................ ...*........... . 5.B 2. Histochemisny ... 52
................... ......... 5 . B .2. a. Alkaline Phosphatase .. 52
................... 5.B.2.b. Tetracycline ... .............. 52
5.B.2.c. vonKossa ............................................ 52
.............................................. . . 5 B 3. Electmn Microscopy 55
........... 5 .B. 3 .a. Scanning Electron Microscopy (SEM) 55
. . ....... 5 .B. 3 b Energy Dispersive X-ray Analysis (EDX) 58
. ...... 5 .B. 3 c . Transmission Electron Microscopy 60
...................... . 5 . B 4. X-ray Diffr;u:tion (XRD) .. ....... ... ... 63
................................... . 5 C. DEXAMETHASONE ( 0 ) CULTURES 66
.......................... . .......**... 5 . C. 1 Histochemistry .. .... .. 66
5 . C . 1 .a. Tartrate Resistant Acid Phosphatase (TRAP) .... 66
................... 5 . C . 2. osteologic" Calcium-Phosphate Thin ~i lms 66
............................................... . 5 . C . 3 Bovine Bone Slices 68
.............. 5.D. DEXAMETHASONE (+) SERIAL DILUTION ASSAY 73
. 6 DISCUSSION ........................................................................ 80
................................ 6.A.1. TheRat .................... ,.,. 81
6.A.2. TheFerret ......................................................... 83
6.B. OSTEOGENIC ASSAYS ................................................... 8s
6 . B . 1 . Bone Nodule Chamterization .................................... 85
6 . c. OSTEOCLASTIC RESORPI~ON ASS AYS .............................. 87
6.D. D-SONE (+) SERIAL DILU~ONASSAYS ............ 88
7 . SUMMARY ......................................................................... 91
............................. ............................... 8. CONCLUSION ......... 92
9 . REFERENCES .................................................................. 93
Figure Figure Table Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure
1.A.l.: l.A.2,: 1: 1.D.l.: 1.D.2.: 1.D.3.: 1.E.l.: 1.G.l.: 4.F.l .: S.A.: 5.A*l*A.: 5.A .l. B.. 5.A.l.C.: S.A.2.A.: 5.A.2.B.: 5.A.2.C.: 5.A.2.D.: 5.Ae2+E.: 5.B.l.A.: 5.B.l.B.: 5.B-2.A.: 5.B.2.B.: 5.Be2.C+: S.B.2.D+: S.B.3.A.: 5.B.3.B.: 5.B-3.C.: 5.B.3.D.: S.B.3.E.: S.B.3.F.: S.B.3.G.: 5.B.3.H.: 5.B-3.1.: 5.B.3.J.:
.................... ...... . HeaIthy Vs Osteoporotic Bone ... 2
Minerai Density vs . Menopause ................... .. .......... 2 Resorption & Formation-deficient modek .................... 7
............................... Anterior view of a human femur 12
............................................... Osteoblast iineage 15 ...................... .. ...... Osteoclast Morphology .. .... 17
................... Basic multicehlar or Bone Metabolic Unit 24
.................. Mustela puroriusfuro (The domestic ferret) 28
Bovine Bone & Osteof ogicTY Disc .............................. 40 ............................................. Ferret Femoral Bone 42
.......................................... Ferret Trabecular Bone 43 .......................... Low Magnincation Trabecular Bone 44
High Mag . Trabecular Bone (Howship's Lacunae) .......... 44 ........................... Ferret Cortical Bone (Cross Section) 46
................................ Haversian Secondary Osteons 46 ............ .............................. Bone Metabolic Unit .. 47
Head of Cutting Cone (Osteoclast LM) ...................... 47
Head of Cutting Cone (Osteoclast TEM. Montage) .......... 48
......................... 13 Day Rimary Cell Culture @ex+) 50
........................ 16 Day Primary Cell Culture @ex +) 50 ................................. Alkaline Phosphatase S taining 53
...................................... . Tetracycline Labeding .. 53 Re von Kossa Montage .................................... ... . 54
....................................... Post von Kossa Montage 54 Globular Accretions (Low Magmfication) .................... 56
Cell Process & Globular Accretion .......................... 56
Globular Accretions (High Magnification) .................... 56 ................................ Inteifacial Matrix with Collagen 56
.................................................. Accretion Front 57 Mineralized Matrix (SEM Cross section) ..................... 57 Energy Dispersive X-Ray Analysis ............................ 58
............................. EDX Dot Map Analysis (Calcium) 59
EDX Dot Map Anaiysis (Phosphorus) ........................ 59 EDX Dot Map Analysis (Uverlay) ........................ ... . 59
Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Fipre Figure Figure
.................... ................. SEM (Secondmy Image) .. 59 TEM Cross Section through a 28 Day Old Ferret Nodule . . 61
TEM of a Ceil and the Interfacial Matrix ........................ 61
TEM of Collagai Mineralization ................................ 62 XRD - Bone h m Various Species ........................... 64
XRD . Ferret Noduies Vs . Embryonic Chick Bone ......... 65 ûsuoclastic resorption cornparison ............................ 67
TRAP positive Cek on Osteologic" subsaata .............. 69
TRAP Positive Cells on Bovine Bone S k e s ................. 69 Resorption of OsteologicN CaP Layer (LM) ................. 70 Resorption of ~steologic" Ca/P Layer (SEM) ............... 70
3D Osteoclastic Resorption of Bovine Bone @ex -) ........ 71
Osteoclastic Resorption of Bovine Bone @ex +) ............. 71 (Montage) Resorption of Bovine Bone @ex -) .............. 72 CeU Plating Dilution Assay (T-25s) ........................... 75 Plating Density Vs . Nodule Count (Weeks 1. 2 and 3) ..... 76
Plating Density Vs . Nodule Count (Week 3) ................. 77
Plating Density Vs . Nodule Area (Weeks 1. 2 and 3) ....... 78
Plathg Density Vs . Nodule Area (Week 3) ................... 79
ABBREVIATIONS
l,2S DHVD avB3 a-MEM BGP AA
ALP BMU BSP-Ii Ca CS PG 1
CSPGII CS PG m Dex DEXA
Docs EDTA
EDX FACS
FBS FESEM FRP FSM
G ~ Y GnRH
HA HC1 IFM IGF- 1
IL- 1
IL-6 LM MMP NCP
1 3 dihydmxyvitamin D Vitronech mtegrin Alpha minniiai essential medium Beta glycerophosphate Ascorbic acid (Vitamin C) Alkaline phosphaîase Bone metabo1ic unit or basic mdticeildar unit Bone Sialoprotein II Calcium
Chondroitan Sulfate Roteoglycan 1 (Biglycan) Chondroitan Sulfate Pmteoglycan II (Decorin) Chondroitan Sulfate Proteoglycan III Dexamethasone Dual energy X-ray absortiometry Differentiating osteogeuic ceh Ethylenediaminetetracetic acid Energy dispersive X-ray analysis J3uorescence activated ceil sorting Fetal bovine semm Field emission xanning electron microscopelmicroscopy Fmal reaction product FuUy supplemented medium Glycine
Gonadotropin-releasing hormone Hydroxy apatite Hydrochlonc acid Interfacial mstrix Insulin-like growth factor 1 Interleukin- 1
Interleukin-6 Light mimscope/microscopy Matrix metaUoendoproteinase Non-collagenous proteins
OC-2
Ovx Pc-L'M P PBS PG PRP PTH RCB R U RGD SD SEM SM SSP-1 TC TCP TEM TGF-6 TRAP WHO
XRD
Osteoclast speafic cystein prote-
Ovariectomy Phase contrast light mimscopy
Phosphorous Phosphate b a e r salllie
Proteoglycan Rimary reaction product Parathyroid hormone Clonally deriveci rat bone cell line Clonal chondrogenic c d h e Arginine-LysineAspartic Acid Standard deviation S d g electron mimscopelmicroscopy Supplernented medium Secreted phosphoprotein 1 Tetracycline Tissue cuiîure polysîyrene Transmission elecîron rnicroscoPe/microscopy Transforming gmwth factor Beta Tartrate resistant acid phosphatase World health organization X-ray ciifkction
- XII -
1. GENERAL INTRODUCTION
Metabolic bone diseases are diseases which compromise the balance of bone
turnover and replacement These diseases fall m&r two categories: 1) osteoporosis and 2)
osteodacia (which are both catabolic or destructive in nature). Although many other bone
disorders exist (e.g. se--mineral deficiencies, genetic, acquired or developmental
disorders), the following sub-section will focus briefiy on the rnetabolic bone disease;
osteoporosis, as m vivo research previoudy perfonned by Maclcey et al. (1995), on the
ferret, provides pertinent information for this partidar animal model.
1.A.l. OSTEOPOROSIS
Osteoporosis is best demi as a metabolic bone disease in which there is a
decrease in bone mass and structural deterioration of the skeleton. Bones become porous
which increases bone fiagiiity and the propensity for hcture (World Health ûrganization).
A distinguishing characteristic of osteoporosis is the maintenance of a normal collagen to
mineral ratio, which separates it fkom osteomaiacia, a disease characterized by relative
deficiency of mineral content in relation to collagen. Osteoporosis is M e r described as a
syndrome with many etiologies and clinicd forms. Primaiy or secondary f o m exist, the
latter depending on the presence of specifc conditions, in particular, certain diseases,
previous surgery, or the use of resorption accelerating medications (Khosla et al., 1995).
Primary osteoporosis is divided M e r into Type 1 (postmenopausal) and Type II (senile)
osteoporosis. The disease begins its deleterious effect by reducing bone mas, usuaily
after peak bone mass has b e n acquired, akhough juvenile f o m exist. In mild scenarios,
with minimal bone loss, the disease is known as osteopenia and may provide early
wamings for potentially susceptible individuals. Osteopenia (Latin = small bones) is a
condition in which decreased bone mass is f o n d without a clear-cut association with
fractures. Individuals are considered osteopenic if their bone mineral density falls 1
standard deviation (SD) beiow the popdation mean value for an individual of that sex, race
and age. As the deterioration advances, and bone Ioss increases, the disease is termed
osteoporosis and represents a much pater risk for bone nactme (F~gure 1.A.1.).
Osteoporosis û defined as a condition in any patient who has a bone mineral density value
measured by dual energy X-ray absorptiomeâry (DEXA) that is 2.5 SD uni& below the
population mean for their age, race and sex (Figure 1 .A.2.). These dennitions have been
defbed by the World Health 0rgani;rlition (WHO).
Figure 1.A.1. & 1.A.2. The images on the left represent a h d i h y sample of üabedar bone (top) and osteoporotic bone (bottom). The right band illustration depicts the trend in bone density of postmenopausal women 6th age. Both figures were reproduced fiom the Merck Pharmaceutical osteoporosis web page.
A common misconception is that osteoporosis pertains either to post-menopaual
women or astronauts, the latter have been recorded to lose up to 19% of their skeletai mass
during Long duration missions (Hughes-Fulford et al.. 1996). Less common, although
quite as serious, are juvenile and senile forms of the disease, which are more likely brought
on by hyperparathyroidism or hyperthyroidism rather than estrogen deficiency (Rodan, et
al., 1995).
Many factors are now recognized to contn'bute to osteoporotic f k tu re risk, Some
depend on the status of the individual's well being or somatotype. Traits such as the
tendency to f a lack of adquate reflexes, or inadaqnate soft tissue mass to cushion h m
insdt, can ail be viewed as extraosseous factors. Direct osseous factors, Uce previously
accumdated fatigue damage h m eariier fds , decreased bone mass or loss of trabecular
C O M ~ C ~ X ~ S S , &O play crucial roks in assessing the fhcture potential of individual
patients (Heany, et al., 1995) and may provide targets for management. However, the
single most cornmon etiology of osteoporosis remains the estrogen depleted state most
women encormter after ovariectomy or menopause.
A reduction of circulating estrogen is thought to have a wide spread systemic
effect. The level of estradiol, the ~ecreted and biolopically active hormone synthesized in
the ovaries, drops from -100-1000 pmol/L to approximately 20-50 pmoÿL after
menopause and in tum is thought to lower circulahg PTH and 1,25 dihydroxyvitamin 4
(125 DHVD) s e m levels. Cellular sensitivity to PTH however, is heightened (Cosman,
et al., 1993), causing an increase in osteoclastic recniitment and remodehg activities.
Once remodehg activities are accelerated, net bone deposition lags b e b d net bone
resorption, as osteoclasts resorb bone much faster than it can be replaced (Rodan, et al.,
1995). With a decrease in 1.25 DHVD levels, intestinai absorption of calcium drops and
calcium homeostasis beguis to rely more and more on the released calcium stores fiom the
skeletal reservoir. Moreover, estrogen plays other important roles at the cellular level on
the regdation of local messmgers. It is known that estrogen can stimulate the secretion of
insulin-me growth factor4 (IGF-1) and its binding proteins, and may be responsible for
the activation of transforming growth factor-beta (TGF-B), a factor known to slow bone
resorption. Furthemore, it has been suggested that estrogen may affect osteoclasts directly
(Ousler, et al., 1991), but also suppresses the synthesis of bone resorbing factors such as
IL-1 and IL-6 (Girasole, et al., 1992).
1.B. O S T E O P O ~ C ANIMAL MODELS
L B , ~ . IN vrvo A rational approach to developing osteopomtic models is to use what is known
about the human disease as the template for recreating a similar condition in animals. In the
case of osteoporosis, since the human pathophysiology is not M y understood and is
undoubtedly not caused by a single factor, most of the models in use today [mouse (Weiss
et al., 199 1), rat (Jee et al., 199 1 ; Matsumoto et al., 1985; Vailas et al., 1 !EU), rabbit (Wu
et al., 1990), pig (Spencer et al., 1979; Mosekilde et al., 1993), sheep (Newman et al.,
1995), dog (Geusens et al., 1991; Kbmel et al., 1991), and monkey (Mann et al., 1990;
Pope et al., 1989)] recreate perturbations in animais reflective of the key risk factors for the
disease in humans. Unfortunately the aforementioned species, particularly the rat which
has been well documente& displays physiologicat (Wronski, T.J. et al. 1991), and
dtrastnictural differences (Bagi, C.M. et al., 1997) when compared to the human skeletal
systems. Furthemore, ethical or hancial restrictions (see Newman, E. et al., 1995;
Geddes, A.D. 1996. for a review of the requirements for an ideal animal model) may also
deem larger animal models of littie utility. It is for this reason, that new models. including
the ferret (see below), are considered as potential species in elucidating the complexities of
metabolic bone diseases.
1.B.l.a. The Ferret
Although the ferret has been previously utilized in scientSc investigations,
particuiarly in the fields of virology, toxicology and immunology (Fox, J.G. 1988), a
ment study performed by Mackey et al. (1995) provides pdcuiar relevance to the study
of metabolic bone disease. The authors stress that there still exists a need for a small
animal model with basic multice1lula.r (or bone metabohc) unit @MU1)-based Haversian
type remodeling to rnimic the bone loss associated in postmenopausal osteoporosis, which
-
l A term coined by Frost, HM. (1973)
the rat, the most widely used mode1 (current goid standard) does not display. Tbe study
&O assesses the potential use of this species by examiniag the ferret's response to PTH
administration, estmgen depletion-induced bone loss (either via ovariectomy or reduced
thne spent in lit conditions) and maturaton of the p i e s skeleton. Their resuits
demonstrate that the ferret achieves s k e l d matLuity between 4 and 7 months of age, as
noted by closure of the epiphyseai growth plate, and mahiration of trabeculae h m thin
rods to thick rods and plates. This maturation proces is only noted very late in rat skeletal
developrnent (Jee, W.S.S. et al., 1991) and has led to the misconception that growth plates
do not fuse. PuIsatile injections of 0.02 mg/kg (1-34) WTH on a 24 hour basis for 12
weeks, versus a vehicle injection, resulted in a significant bone minerai density increase as
weil as trabecular tunnehg, a phenornenon only observed in larger animal models with
BMU-based Haversian bone remodeling (Boyce, R.W. et al., 1996). Estrogen depletion
studies document a senun estradio1 level drop as well as a uterine weight decrease, and
bone mineral density reduction associated, with bilateral ovariectornized and intact
photoperiod reduced (short') animals. These resdts were reportecl in comparison to
control intact groups2 and intact Iight cycled (lon2) ferrets. The mechaukm underlying
estrogen depletion-induced osteoporosis via photoperiod cycling remains rather
contmversial and is based on the secretion of melatonin h m the pineal gland. Altemi
photoperiods change melatonin release duration (darkness stimulating melatonin release and
daylight abolishing it). The melatonin pulse is read by a neural system (unknown at this
the ) and translateci into GnRH pulses from the hypothalamus, which then stimulates the
pituitary to release LWFSH. The systemic secretion of LH/FSH m e r regulates estrogen
secretion (direct communication with Dr. J Herbert, investigator into ferret photoperiod
dterations) .
' Intact light cycled (short) - - 8 hrs lighti16 hrs dark. Conte01 intact group - - 12 tus ligWl2 hrs da& Intact light cycled (iong) - - 16 Light/8 hrs dark
It is for these rwwns that the ferret may provide utility as a new üz vivo, rdatively
inexpensive and readily available, purposebred animal mode1 with potential use for skeletal
research. This inc1udes investigations hto the mechani,miic of estrogen depletion-induced
bone loss and mechanisms-of-action of bone therapeutic agents. Furthemore, altered
photoperiods may provide a repiOducib1e altemaîive to surgicd induction of estrogen
depletion via ovariectomy.
l,B.l.b, Other Animal Models
When attempting to understand the nahue of osteoporosis in humans, a disease not
yet M y understood and with many key factors, the need arises to recreate individual or
combinations of key risk factors, such as reduced estrogen levels or increase PTH levels.
to simulate the h a s e in healthy models. Recreating the osteoporotic state in animal
models, by inducing individual or combined alterations, results in the development of two
animal mode1 types. The h t mode1 represents a system with accelerated bone loss and
high rates of bone turnover, and is characterized as 'resorption dominant'. This mode1
investigates the nature of the osteoclast and osteoclastic resorption kinetics. These studies
potentiate the discovery of resorption suppressing drugs. The second mode1 termed
'formation deficient', investigates osteoblastic function and examines the role of anabolic
agents. These systems are characterized by low bone mass and demonstrate low bone
turnover. Both models have been examineci and utilized extensively in various animal
species. The following table provides a list of some of the aforementioned models and was
reproduced fkom The Rinciples of Bone Biology (pg. 1348).
ResorptionResorption-predominanCpred~~f Formation deficit-predominantb
Mode1 Source Mode1 Source
Ca-rest&ed ovx rat Lactahg, Ca-restricted ovx rat hctatblg C a - d c t e d
Druglachicéd Retinoid E=TH
Heparinued rat Acidifiecl rat Thyroid excess rat
Immobilized Sciatic denervectomy Tenotomized Taif suspendeci Bandageci hindlimb Space fiight
Immobiiized, ovx rat hobi f i zed , Ca- restricted rat
Lactating pig
Ovx dog Ca-cestricteci ovx dog
Ovx ferret Ca-restncted rabbit
Ovx monkey GnRH monkey Ovx baboon
Shen et crl. (1995) Stauffer et al. (1973) Vancierschma et al. (1993)
Matsumoto et al. (1985) Anderson et al. ( 1990)
Garner et aL (1987)
Trechsel et al. (1987) Russel et al. (1 970)
Monreai et al. (1990) Barzel(1976) Ongphiphadhanakul et al, ( 1992)
Turner and Bell (1986) Zeng et al. (1993) GIobus et al. (1986) Jee et al. (199 1) Vailas et al. (1992) Okumura et al. ( 1987) Weinreb et al. (199 1)
Aged normal mouse Aged nonnal rat -acceIerated m Aged ovx rat Smke-proue rat
Glucocorticoid treated rat Diabetic rat h o b i l i z e d rat I n n e o n - m M rat
Spencer ( 1979) Aged Ca-restricted ovx pig
Kimmel(1991) Aged dog Geusens et al. (199 1) Immobilized dog
Mackey et al. (1995) Glucocorticoid dog Wu et al. (1990) Glucocorticoid sheep
Jerome et al. (1994) Aged sheep Mann et al. (1990) Immobilized sheep Jerome et al. (1986); Aged monkey Thompson et al. ( 1992)
immobilized monkey
Weiss et al. (1991) Jee (1991) Matsushita et al. (1986) Ibbotson et al. (1992) Yamori et al. (199 1)
Simmons and Kunin ( 1967) Sasaki et al. (199 1) Jee et al. (1991) Lempert et al. (1991)
Mosekilde et al. (1993)
Jee et al. (1970) Waters et al. ( 199 1); Uhthoff and Jaworski (1978) -1s ( 1992) Chavassieu et al. (1993) Newman et al. (1995) Rubin et al. (1988) Pope et al. (1989)
' Accelerated boue 10s- Osteopenic.
l.B.2. IN VITRO
Current danring techniques evolved from previous prutocols, the first dating
back almost a hundred years to Carrel and Burrows' dtivation of amilt tissues outside of
the body (19 10). In vitro bone assays originated as organ culture systems that utilized
tissue explants, usually calvariae or long bone, and aimed to maintain the tissue's metabolic
integrity m vitro. Nefussi et al. (1982) demonstrated a short osteogenic capacity for such
organ long bone cultures providing a useful, however temporary, mode1 for the study of
osteogenesis in vitro. Furthemore, preparation of organ explants must be executed with
great care to maintah tissue vital@.
Cellular cdhnlng protocols soon foIlowed and were first developed by Fell in . 1932 to study the nature of bone cell populations devoid of bone fragments. Two noted
advances resuited h m Feu's work: 1) enymatic digestion of bone to release bone ce11
populations and, 2) mechanical isolation which was based on the migratory capabilities of
bone cells, from bone, onto tissue culturing substrata. The former was further modifiecl to
employ a coclaail of dissociative agents (trypsin, ElDTA, collagenases) which would assist
in the release of ceU populations (Wong and Cohn, 1974 & 1975). Further isolation of
these primary heterogeneous cultures have employed elatmphoretic separation (Puzas and
Jensen, 1982), differential adhesion techniques (Wong and Cohn, 1975) and fluorescence
activateci ceil sorthg (FACS) (Herbertson, A. et al., 1997) to try and obtain single cell
populations or monocultures. Other investigators have produced clonal ce11 Iines including
the RCB and RCJ lines fiom rat calvaria (Aubin et al., 1982) and the MC3T3-El ceII h e
nom mouse calvaria (Sudo et al., 1983). However, heterogeneity is still an issue amng
such populations and may aise due to partial transformation of such immortal clone lines
(Aubin et al., 1982).
One of the most =nt cell culturing protocoIs, developed by Maniatopoulos et al.
1988, which amse h m Friedenstein's (1970) eariier concept of marrow explantation,
employs phase-contrast light microscopy (pc-LM), histochemistry and
immunohistochemistry. scanning and transmission electm microscopy (SEM & TEM),
energy dispersive X-ray mimanalysis (EDX) and X-ray dispersive microanalysis (XRD)
as a means of characterihg the elaborated bone-like matrix. The observations fkom this
study show that bone-Iike tissue can be synthesized in vitro by cells cultured h m young-
adult rat bone marrow, provided that the medium contains ascorbic acid' (AA), Na beta-
glycerophosphate2 (6GP) and, particularly, dexamethasone3 @ex).
In light of the variety of culhumg protocols available, the use of in vitro cell
culturing assays have increased- For example, estrogen agonists have been tested in mouse
(Rogers, M.J. et al., 1996), rat (Grese, T.A. et al., 1997) and human (Somjen, D. et al.,
1996; Petilli, M. et al., 1995) culture systems. Glucocorticoid induced osteoclastic
resorption has been investigated in many species including rat (Gronowicz, G.A. et al..
1995) and human explanted cultures (Kasperk, C. et al., 1995). Ovariectomized rat
marrow has been explanted to study the effects of estrogen loss at the osteoclast progenitor
coiony forming unit-granulocyte macrophage (CFU-GM) level (S hevde, N. K. et al.,
1996), while in vitro effects of growth factors have been studied on rat marrow stroma1 cell
systems (Locklin, R-M. et al.. 1 995). Our intention herein is to characteriz the osteogenic
and osteoclastic potential of ferret marrow explants (utilizing many of the aforementioned
citeria associated with Maniatopoulos' work) to provide a new, characterized, mode1 for m
vitro metabolic bone research.
' Co-factor for lysyl & prolyl hydroxylase (coilagen synthesis). An orgaaic phosphate source. Synthetic glucocorticoid which stimulates osteoblasac differentiation.
1.C. IN VITRO OSTEOGENIC & OSTEOCLASTIC MODELS
In vitro osteogenic and osteoclastic models, in parti& the rat (BelIows, C.G. et al.,
1986; Grigonadis, A I et al., 1988; Maniatopoulos, C. et al., 1988; Bellows, C.G. et al.,
1990; Lang, H. et al., 1990: Davies. JE et al., 1991; Kidder, L.S. et al., 1993;
Herbertson, A et al., 1995; Ohgushi, H. et al., 1996), have been studied and documented
exhaustively in the fiterature. While the rat represents the current gold standard, mal1
animal, model for preIiminary investigation into the aforementioned research, other in vitro
modeIs have ken examined including the mouse (Hattersley, G. et ai-, 1989), rabbit
(Ahrengart, L. et al., 1986). cat (Pharoah, et al. 1985), porcine (Thomson, B.M. et al.,
1993), avian (Tenenbaum, H.C. et al., 1985; Teti, A. et al., 1990), bovine (Puelacher,
W.C. et al, 1996), and human (Beresford et al., 1984; Parker, E. et aL, 1997). These
animals represent potential modeIs which may provide new means to m e r investigate
osteogenic or osteoclastic mechanisms and actions.
However, it must be recognized that each model has its own advantages and
disadvantages, whether it be cost, size, or culture dynamics. FUTtherrnore, we must
combine the documented in vitro potential of each species with what we know of its
physiology in vivo . For example, it may be determinecl that the sheep represents a useful
animai model for in vitro osteoclastic study. However, sheep are niminids and regurgitate
their food. This may alter kinetics of oral drug absorption (Geddes, A.D. 1996) making
this animai a poor choice for orally dmhistered drug treatment assays. We must then
realize the limitation of each model and use the species that may best fulnU the mandate of
the study. Suitable guidelines for choosing such a mode1 have been outlined by Rogers et
al. (1993) and are defhed as 1) convenience, 2) devance (comparable to the human
condition) and 3) appropriateness. Appropnateness is M e r defïned as: use as an
analogue, transferability of information, background knowledge of physiological
properties, cost and avadability, adaptability to experimental manipulations, and ethicd and
social implications (ibid).
1.D. STRUCTURE & COMPOS~MON OF BONE
loDol0 MACRO & MICROARCEDTECTURE
Bone is an organized tissue composed of mineral, proteinacious and cellular
components. It consists of two tissue arrangements, and in long bone, is divided into three
anatomically distinct regions.
Trabecdar (or cancellous) bone is found principally in the metaphyseal and
epiphyseai regions of long bone (Figure 1.D. 1 .). The trabecular network is encaseci in a
layer of cortical bone and the two become confluent at the endosteal sudace. In the ad&
trabecuiar bone is generaily of hme11ar architecture (see below), the thickest trabeculae
containing Haversian osteons and neurovascular ingrowth. The metaphyd and
epiphyseal regioris are divided, durhg growth of the organism, by a region of continuously
proliferating cartilage tenned the epiphysed growth plate. Later in Me, around puberty,
this growth plate calcifies, fusing the metaphysis and epiphysis marking the cessation of
axial growth in that bone. Cortical or compact bone, the second fom of bone, represents
the entire cortex of long bone. It, too, is composed generally of lamella. bone in its mature
state, and contains bone marrow which is confluent with the marrow of the trabecuiar
cornpartment.
AU long bones are covered on their outer surface by layers of co~ect ive tissue
caiied the periosteum with the exclusion of points of articulation, ligament or tendon
attachment, and at points of nutrient artery supply. The periosteum is M e r divided into
two layers: 1) an outer fibrous layer and 2) a . inner layer of soft co~ective tissue. This
inner layer, at quiescent times, comprises potentially osteogenic cells referred to as resting
or lining cells. On the endosteal aspect of long bones is an homologous layer of ceils d e d
the endosteum. They &-te the medullarylendosteal surface h m the interna1 marrow
cavity. It is fiom these lining ceIl layers (endosteurn/jmer penosteum) that differentiating
osteogenic cells (DûCs) are recruited for bone synthesis.
I Human Femur Anterior View
- Greater trochanter-
Epiphysis
Metaphysis - Dlaphysis
Lateral epicond
Lateral condyle * Patellar Surface Medial condyle
Figure 1.D.1. A diagram of the antecior view of a human femur. Surface landmatks are labeled and a 'cut away' of the femur is provideci in the lower right corner. The cut away displays the relationship of the rnarrow cavity of the diaphysd region to the trabecdated marrow cavity found more comrnody in the metaphyseal and epiphyseal regions. (Image modified from Netter's Human Anatomy)
1.D.l.a. Woven Bone
The fht bone that appears in embryonic development and continues into the f h t
few months of neonatal Me is termed woven bone. Woven or prirnary bone can also be
found in the adult at sites of bone fractures where it bridges bone bgments and produces
the caüus, or new bone produced in some bone tumors. Woven, or primary bone has more
cells per unit volume than does larnellar bone, its mineral content varies which may cause it
to stain unevenly, and its cek are more randomly rirranged than lamellar bone. Its
extracellular substance is characterized by bundles of collagen fibers which are deposited in
an irreguiar, or interlachg and random manor. Despite the fact that the majority of woven
bone is resorbed and reqlaced by lamellar bone in humaos, some primary bone may persist
in combination with the latter near cranial sutures, in tooth sockets and near tendon and
iigarnent attachrnents (Pritchard, 1952).
1.Dmlob. Lamellar Bone
Lameilar bone begins to form approximately one month after birth in humans. By
four years of age, most woven bone is completely remodeled and replaced (Kaplan, F.S. et
al.. 1994). Ail bone, including the woven form, is resorbed by cek known as osteoclasts
(see below). Lamekir bone architecture results from modehg events associated with the
deposition of circumferential lamellae which serves to increase cortical diameter, or in the
remodehg events of woven or lamellar bone via the deposition of new rnature bone in
discrete units terrned secondary osteons. Each osteon is demarcated fiom previously
deposited bone by a morphologicaIly distinct structure known as the cernent ihe. Osteons
expressing concentrically arranged lamellae of lamellar bone, situated around neurovascular
bundles, are termed Haversian osteons after the Haversian canals they surround. In
humans, each larnella is approximately 5 - 7 p thick and consists of fine collagenic fibers
and hydroxyapatite crystals oriented in pamllel to one another within a single lamella, but
up to 90' ciifference between successive lamellae. Furthemore, each 1-Ila is separateci
nom the next by an interlamellar cementing zone through which no collagen fibers are
believed to extend (Gray's Anatomy).
Ali modehg activities responsibIe for the circumferential IamelIae of corticai bone
arise by direct synthesis of pre-bone or osteoid on pviously elaborated bone at the
periobteal or endosteal envelope. ûsteoblasts derived h m the periosteum and endosteum
are responsible for such events. Remodeling activities on the other han& are carried out by
basic multicellular or bone rnetabolic uni& (BMUs) which were first descriid by Frost and
d e s c r i i in detail under the subsection C'Modeling and Remodehg Events".
l.D.2. BONE CELLS
1.D.2.a. Osteoblasts & Osteocytes
Osteoblasts and osteoprogenitors arise h m undifferentiated plUnputent
mesenchymal cells found in the marrow of long bones and the comective tissue of the
periosteum and endosteum. Osteoblasts are M y differentiated to carry out the function of
bone formation and are characterized by means of morphological and biochemicd analyses,
osteocdcin immunohistochemistry perhaps the most specific. B y definition, osteoblasts
mut satiss certain criteria: 1) by their ability to elaboraie tissue recognizable as bone 2) are
documentecl on the surfaces of actively forming bone separatecl h m it by a thin layer of
non-mineralized osteoid 3) are considered pst-proliferative cuboidal ceus, which stain
positively for alkaline phosphatase at the cell membrane and may also be recognized by
their ability to synthesize a number of associated molecules, bone matrix proteins, certain
hormone receptors and cytokines (Aubh & Liu, 1996). Further morpho1ogical analysis
utiliang the LM and TEM displays a c e U with an extensive rough endoplasmic reticulum
(indicative of cells which manufacture protein, also responsible for the basophilie nature of
the cytoplasm) and eccentric nuclei distal to the bone surface. On completion of their bone
forming activity most osteoblasts revert to quiescent inactive cek and reside in the resting
cell population of the periosteum, endosteum or Lining cells of Haversian canals.
Extenuhm proMeration Uning Cell
1 Pmoebobbst Plurfpotent
Stem Cell
Mature
Progenüurs form other mesenctiymal œlls Iiiducnng Mature
O adpocytes. fibrobiasîs and OSteobbt
Figure 1D.2. Postdated steps in the osteoblast üneage implying recognizable stages of differentiation. (Modined fiom Aubin et al. 1996).
Osteoblasts becorne entrapped in the matrix they secrete to become osteocytes. As
such, they mide in bony facunae and are responsible for the maintenance of their
surrounding matrix (Buckwalter et ai., 1995). Osteocytes remain in contact with other
osteocytes and osteoblasts (on the bone surface or near vascular supplies) through
inûmmix charnels d e d canaliculi. These channels permit the extension of cellular
processes which abut to other processes of neighboring cells. Gap junctions,
trammembrane pores formed by connexin proteins, d o w for the transport of ions and
metabolites from cell to cell, allowing communication and nutritional supply from the
vascular supply of Haversian canais (Doty, S.B. et al., 198 1). Therefore, lüce the
osteoblast, osteocytes also represent post-proMerative cells of the osteogenic heage which
are smaller in volume than their ostwblast progenitors. have lost many of their cytoplasmic
organelies, and have decreased alkaline phosphatase activity when compared to mature
osteoblasts (Nijweide et al., 1996). It has been proposed that two phenotypes for the
osteocyte might exist due to a transition in its functional state. This m i t i o n would
provide the capacity not only to synthesize, but also resorb maîrk to a limited extent
(Marks et al., 1996). The former represents a young osteocyte with an organelle repertoire
chacteristic of the osteoblastic phenotype, whereas the latter, the 'osteolytic osteocyte'
contains lysosomal vacuoles common to phagocytic cells and is devoid of many
c ytoplasmic organelles.
1-D.2-b. Osteoclasts
Osteoclasts are bone resorbing multinuclear giant cek which differentiate fkom
stem cell precursors of the hemopoetic lineage by the fusion of blood-borne monocytes
(Suda et al., 1996). These progenitors are recmited to target sites such as bone through
circulating blood. Omx differentiated, osteoclasts may reside in resorption/Howship's
lacunae which have been resorbed fkom bone, on bone surfaces. However, for f d l
osteoclastogenesis to occur, direct interactions with osteobiastic stroma1 cells are essential,
as has been displayed by the physicd separation of mesenchymal osteoblastic cells from
hemopoetic precursor populations by means of a membrane filter (Takahashi et al., 1988).
Osteoclasts are utilized in resorption of calcifieci cartilage and remodehg of living bone.
Furthemore, osteoclasts assist in maintainhg calcium homeostasis by releasing calcium
stores and elevating systemic calcium blood levels.
Osteoclastic cells commonly display a multinuclear morphology with phenotypic
expression of tartrate resistant acid phosphatase (TRAP) and, in most species, calcitonin
receptors on the cell surface. These phenotypic markers, dong with many others [avB3
(vitronecth) & a2B 1 (collagen) intergrins, mat& metalloendoproteinase-9 (MMP-9) and
the osteoclast specific cystein proteinase, OC-2) are also expressed in mononuclear TRAP-
positive ceil mRNA, which when plated on bone were also capable of forming resorption
lacunae (Wesolowski et aL, 1995). These in vitro hdings indicate that osteoclast-
associated phenotypes are expressed in mononuclear precunors and that the presence of
calcined tissue is not required to induce oste0~1ast~associated phenotypic expression.
OsteocIastic resorption initiates with cell attachent to bone via intergrin
interactions. The exact signahg events which regdates this attachment sequence have not
been elucidated. It is postulated that the cd33 intergrin, found on the osteoclast cell
membrane* is also the mediator for sealing zone attachment/formation. However, others
like V%Winen and Horton (1995) suggest that the sealing zone is equipped with a unique
cd-tematrix interaction which may provide an ideal target for resorption inhibition. As
osteoclasts prepare to resorb, they becorne highly polarized and &velop four distinct
membrane domains F~gure 19.3.). The sealhg zone, responsible for providing a tightly
Sealeci barrier around the acidic compartment of the niMeci border, also separates the
aforementioned domain h m the basolateral dornain. The nifned border becomes the
resorbing organ and the pen-membrane cornpontment (bone facing aspect) becomes tée
functional equivalent of a secondary lysosome (Akamine et al., 1993). A fourth membrane
domain has recently been discovered which may represent the accumulation of transcytotic
transport vesicles for exocytosis (Salo et al., 1996).
Figure l.D.3. The non-resorbing osteoclast is poIarized (1) but after attachment, shows three di£ferent membrane domains (2): d e d border (a), sealing zone (b), anri basal membrane (c). Once matrix degradation has starteci (3) a fourth membrane domain appears in the basal membrane (d). (Viihiinen - Priaciples of Bone Biology pp. 105)
Resorption of m i n e mahyr occm as protons are pumped h m the cell mto
the sealed extmcelluiar cornpartment. V-type ATPase pumps located in the d e d border
membrane of this compartment provide the dnving force for compartment acidif?cafion and
matrix solubilization. Functiod studies revealed that bone resorption in vitro could be
effectively blocked by inh i ion of vacuolar proton pumping with bafilomycin Al, a
specinc inhibitor of V-type ATPase activity (Mattsson et al., 1991). Once the mineral
phase of bone has been solubilized, an organic component remains. MMP-1, an
osteoblastic metalloendoproteioase, plays a major role in the degradation of bone covering
osteoid However, the literature conceming the production of MMP-I by osteoclasts is
currently rather controversial. Research in osteoclastic organic maeix resorption has
focused mainly on two major classes of proteolytic enqmes, namely lysosomal cystein
proteinases and MMPs, in particular MMP-9. Recent evidence indicates that cwrdinated
action of both of the aforementioned proteinases are crucial for the solubilization of nbrillar
type 1 collagen and other bone maîrix proteins.
l.D.3. BONE COMPOSITION
l.D.3.a. Collagen
Type I collagen is the most abundant extraceliular protein of vertebrates and is
essential for bone strength. 18 other collagen types exist ( V d o and de Crombrugghe,
1990), however type I is the main constirnent in bone. The mature collage type I rnolecule
is a triple helix composed of two al chains and one a2 c h a h These molecules M e r self
assemble into coilagen fibrils. The amino acid sequence of each a chah consists of Gly-
X-Y repeats which produce a left handed helïcal conformation. The 3 polypeptide chains
further assemble into a nght handed triple helix, where the Gly residue of each chain
resides in the centre of the helix to ailow for the tightest conformation possible. The X and
Y residues occupy the periphery of the molecule and 33% of the the, represent the amino
acids proline and hydroxyproline respectively. Hydroxyproline provides helicai stability to
the molecule and is an unique characteristic of coliagen.
The procollagen peptide is synthesized (following pre-mRNA transcription and
modification) in the endoplamiic reticulum and is fuaher glycosyIated in the Golgi
apparatus. The modifiecl polypeptide chains ine ~ecreted as propeptides and are modified
m e r by the cleavage of the N and C teIopeptides into the mature collagen molede.
These molecules then rapidly assemble into collagen fibrils. C m n t theory holds that
collagenesis must occur for mineralization of bone matrix to proceed (Aronow, et al., 1990;
Owen et al., 1990) although work by Davies et al. (1991) and Hosseini (1996) have shown
otherwise. The collagen's presence dong with a mineral nucleating factor(s), perhaps of
the noncollagenous protein family, or removd of a nucIeating inhibitor (perhaps
proteoglycan), provides the driving force for rnineralization initiation.
l.D.3.b. Gronnd Substance
Ground substance is the term coined for the amorphou matrix in which coUagen
fibers and hydroxyapatite (HA) crystals of bone rnatrix are embedded. The constituents of
this substance, through the mectianisms of dissociative extraction procedures, have been
isolated and characterized and comprises a mixture of non-collagenous proteins (NCP) and
proteogiycans (PG). The major proteoglycans of bone (CS PG 1, II and III), are isolateci
foUowing demineraikation with EDTA during extraction procedures, demonstrating their
strong association with the mineral crystals of bone (Sodek et al., 1991). The isolation of
two bone sidoproteins of the NCP famiIy [secreted phosphoprotein 1 (SSP-1) and bone
siaioprotein II (BSP-II)] at specific points in rnatrix elabration, have provided researchers
with a tool for postulating their proposed rde in osteogenesis. Both proteins have been
detected in association with and collagenase digestion extracts, implementing their
rolls as possible binders of Ca. Furthemore, both proteins express RGD cell attachent
sequences. SPP-1 (Shen et al. 1993) and BSP-II (Sodek et. al. loc. cit.) have also been
detected in pre-mineralized collagen extracts, which may indicate a role as a mineral
nucleator, or fuahermore in the regulation of HA crystal growth.
1.D.3.c. Mineral Component
Neariy two-thirds of bone matrk is inorganic. This inorganic matRx is typicdy
composed of rod-me crystals which range in size from 30 to 50 A in width and up to 600
A in length (Femandez-Moran, 1957). TEM analysis has documenteci an mtbaîe
association of such md-like crystals with the collagen network of mineralizing osteoid.
The majority of the ciystal's consti~ients are the elements calcium, phosphorous and
oxygen, however other elements like magnesiun and sodium have been detected (Skinner,
1979). When examined using X-ray diffraction m) analysis. a pattern similar to that of
hydroxyapatite [Ca,,(POJ,(OH)J is produced. However due to the aforementioned
incorporation of non-HA elements. the previous stoichiometric formula is inaccurate.
Some confusion between the distinct processes of modeling and remodehg exist.
Modehg is &&cd as the process characterimi by a change in bone shape or location of a
bone structure in space such as occm during growth, fracture repair, or response to altered
biomechanid stress. Remodeling is the process of turnover or replacement of discrete
packets of bone tissue by basic metabolic or bone multicellular d t s (BMU) (Geddes,
1996). It was Frost in 1973 whom first coined the phrase BMU and defined the unit as a
cutting cone of osteoclasts Ieading a nIling cone of osteoblasts (see intracortical
remodeling).
There are two major modes of bone modeiing. and both involve the replacement
of a pre-existing co~ective tissue by mineraiized bone. The development of primitive
connective tissue into bone is temed intramembmous ossification while the replacement
of a cartilage mode1 is termed endochondral ossification. Intramembranous ossification is
the characteristic way in which the flat bones of the cranial vault are formed. Mesenchpal
ceils derived h m the neural crest interact with the extraceUular matrix of the head epithelial
ceils to form bone (Hall, 1988).
During intramembranous ossification, the mesenchymd cells proHetate and
condense. Some cells change their morphology to becorne osteoblasts, cells capable of
seaeting bone maîrix. The seæted collagen-glycOSammogIycan maûix is able to bind
calcium salts which allows the matrix to become c a l M d In most cases, osteoblasts are
separaîed from the region of dcification by a layer of the prebone matrix (osteoid) they
have secreted however, some becorne trappeci in this matrix to become osteocytes. As
calcification proceeds, bony spicules radiate out from the center where ossincation began.
Furthemore, the entire region of calcifiecl spicules becomes swrounded by compact
mesenchymal cells that form the penosteum. The cells on the inner surf'e of the
periosteum also diffemntiate into osteoblasts and deposit bone matrix on existing spicules.
In this mamer, many layers of bone are formed.
Endochondral ossification involves the formation of cartilage tissue firom
aggregated mesenchymal cek and the subsequent replacement of this cartilage tissue by
bone (Horton, 1990). The cartilage tissue provides a modei for the bone that follows.
Bones such as those in the vertebral column, the pelvis, and the exaemities are initiated as
cartilage models and are later converted hto bone. This process coordinates
chondrogenesis (cartilage production) with osteogenesis (bone formation and growth),
while these skeletal elements are simultaneous1y bearing a load, growing in width, and
responding to local stresses. Soon after the cartilaginous "model" is formed, the cells in
the ceneal part of the model become dramatically larger and begin secreting a different type
of matrix, one that contains collagen X, more fibronectin, and less protease inhibitor.
These cells are the hypertrophie chondrocytes. Their matrix is more susceptible to invasion
by blood vessels from the perichondrium. Soon thereafter, capillaries from the
perichondnum invades the centre of the previously avascular cartilage shaft. As the
Carmage ma& is degraded, the hypertmphic cartilage ce& die, and osteoblasts begin to
secrete bone matcix on the partially degraded cartilage. Eventually, ai i the cartilage is
replaced by calcified cartilage, and fhaily bone.
As portions of the cartilage model are converted to calcified cartiiage and
mbsequently bone, an ossification b t is fomied between the newly synthesized
mineralized matrix and the remaining cartilage. This h n t spreads outward in both
directions h m the ossification center, and more cartilage is converted to bone. If this were
all, however, there would be no axial growth and our bones would remain the size of the
original cartilaginous model. As the ossification fbnt nears the ends of the cartilage model,
the chondrocytes near this fiont pliferate prior to undergohg hypertrophy. This pushes
out the cartilaginous ends of the bone, providing a source of new cartilage. These
cartilaginous regions at the end of the long bones are called epiphyseal growth plates. As
this cartilage hypertrophies and the ossification h n t extends M e r outward, the
remsining cartilage in the epiphyseal plate proMerates. This region forms the growth area
of the bone. Thus, bones continue to grow due to the pmliferation of cartilage cells which
subsequently hypertrophy, and die. As a result, new bony maeix is deposited dong the
cartilage matrix, by osteoblasts. As long as the epiphyseal growth plates are able to
produce chondrocytes, the bone continues to grow in length. However, ceIls of the growth
plate are very responsive to hormones, and th& proliferation is stimulated by growth
hormone and insulin-like growth factors. Other hormones are also responsible for the
cessation of growth. At the end of puberty, hi@ levels of estrogen or testosterone may
cause the remahhg epiphyseal plate carmage to hypertrophy. These cartilage cells grow,
die, and are replaced by bone. Without any m e r chondrocyte proliferation, growth of
these bones cease.
1.E.l. SURFACE REMODELING
SiIrface remodeling events are documented on periosteal, endosteal and trabecular
bone envelopes. However. the temporal and spatial relationships of these remodehg
events are not cleady understood. Uniike the tunneling phenornenon associated with BMU
based remodeling, d a c e remodehg is believed to be independent of the cwrdinated
efforts as seen in the BMU but stül consists of resorption, interfacial matrix elaboration,
and osteoid depositionai stages. Parfitt, A.M. (1983) describes the initiation of such
remodehg event by the digestion of mineral from bone surfaces by osteoclasts. leaving
scalloped resorption characteristics. Once resorption is completed, ostwgenic precursors
fiom the periosteum or endosteum differentiate into matrix secrethg osteoblasts and reverre
the resorptive events. Such a pmcess typically involves the deposition of a cernent liw
(described below), foiiowed by osteoid elaùoration and mineralization.
1.E.2. INTRACORTICAL REMODELING / HAVERSIAN SYSTEW
Trabecular and cortical bone is constantly tumed over from the earliest years in Me
until death. Investigations into the initiation of this remodeling process are under
investigation and are focused on the cellular and molecular mechanisms initiated by
mechanical stress (or lack thereof as in the case of microgravity). The machinery necessary
for the remodeling of bone is found in discrcte packages termed BMUs. Bone modeling
(or metabolic) units consist of a group of cells that participate in the coordinated event of
resorption and re-deposition of new bone (Figure I .E. 1 .).
Figure 1.E.1. Profile of a BMU. Position f is occupied by resorbing osteoclasts, 2) differentiating osteogenic precursors, 3) pre-osteoblasts, 4 & 5) osteoid synthesizing osteoblasts, 6) quiescent lining cells. The yellow matrix represents non-mineralited osteoid while the ssatified maîrk represents mineralized tissue. The position of the cernent line is not documented and osteocytes are not present. (Image source - Bone Histomorphometry, 1994).
The osteoclast, a hemopoetic denved cell, is responsible for the resorption of
previously existing bone. These cek bore through bone, excavaihg a cylindrical channel
with scaiioped characteristics, usually in p d e I with the long axis of the bone. This part
of the BMU machinery is temied the-cutting cone, as narned for its appearance in
Iongitudinally cut sections. Following this destructive unit is neurovascular invasion and
the second haif of the BMU machinery responsible for bone deposition. Osteoblasts
denved h m DOCs, which are responsible for the deposition of the cernent line, comprise
the Fillllig cone as termed for their anabolic nature. These mesenchymally derived ceUs
deposit osteoid which subsequently mineralizes, entraps osteoblasts making them
osteocytes, and fils in the previously bored-out channel. The completely remodeled packet
of bone is termed a Haversian secondary osteon afkr the cm& tirst documented by Sir
John Havers, but is anatomicaily recognized as a lamellated secondary osteon.
19.1. Structure, Development & Fonction
The term 'cernent line' or KittIinim (German), as £kt describecl by von Ebner in
1875, can b a t be d e s c r i i as an atïbdar mineralized matrix which serves as a bone to
bone, or bone to endosseous implant interface. The ceils postulated to provide this priming
mechanism were described by Parfitt (1983) to be of a pst-osteoclast, mononuclear
phenotype. These cek were also proposed to be capable of resorbing colIagen,
undegraded by previous osteoclast attacks, to M e r modify the nbrilar surface into a site
more suitable for cement line deposition. Following surface modification these ceils could
commence to elaborate the cernent lines at reversal sites. Studies conducted by Davies et al.
(1 99 1) however, have provided contrary morphological evidence which &monstrated that
cells of the mesenchymal lineage, in particular of osteogenic phenotype, are responsible for
the deposition of such cement lines. DOCs which initiate the elaboration of the cernent h e
then go on to elaborate unmineralized osteoid which would subsequently mineralize into the
coiiagenous matrix of bone.
The study of this morphologically distinct matrix provides particular devance h
the study of implant osseointegration, as it is the h t biologidy derived matrix to come
into contact with the implant surface. If an implant is subjeaed to micro-motion during the
interfacial matrix elabordtion and boue ingrowth period, a fibrous encapsulation forms
preventing the direct apposition of mineraiized matrix to the implant surface (piiiiar et al.,
1986). Such an interface would render dental implants useless as ngidness is an essential
quality expected. The cement h e is also the & which primes the reversal line of
resorption canals prior to the coupled event of bone synthesis. Such events have been
documented in vivo (Zhou et al. 1993) and Ur vitro (Davies, J.E., 1996 review), Davies
describes the initiation of cernent line elaboration as:
" starting with secretion and adsorption to the substratum of organic
cornponents, of which the major proteins are osteopontin and bone sialoprotein. Mineralization of this matrix occurs by the seeding of nanocrystalline calcium phosphate, which precedes the appeamnce of morphologically identifiable collagen fibers. Although cdagen is
synthesized by the differentiiating osteogenic cells that eIaboraîe the cernent line interface, it is not adsorbed to the rmderlying solid surface. Following the elaboration of the cement line matrix, coilagen fiber assembly occurs and is then mineralized to produce morphologically identifiable bone matrix".
Documenting the succession of interfacial matrix elaboration utilizing the SEM,
one observes the deposition of hemispherical globular accretions (approximate1y 1 pm in
diameter) directly apposed to the sudface of the substratum. Cells, projecting processes
which directly abut such giobular accretiom, are often localized to such events. If such an
elaborating interfacial matrix was viewed at a Iaîer &te, the globular accretions would no
longer appear individual. As more matrix is synthesized, the accretions fuse, produchg the
confluent morphological feature homologous to the cernent line of bone. Recent SEM
examinations (Hosseini et al., 1996) in which rat osteogenic cultures were deprived of
ascorbic acid in otherwise fdly supplemented culhue conditions @ex and BGP), elaborated
such matrices in the absence of subsequently mineralizing osteoid. Cernent lines viewed h
such AA (-) assays, as well as fully supplemented culture conditions (FSM) [Dex, BGP,
AA] in TEM cross sections, displayed a matrix thickness of approximately 1 - 1.5 p.
1.G. THE FERRET
The European ferret (Figure l.G.l.), M'ela putonus furo, has been
domesticated for over 2000 years, although confusion exists as to its exact origin and early
use as a domesticated animal (Thompson. 1951). Ferrets in Empe and the British Mes
were used for rabbit hunting and rodent control, and even today remain popular for hunting
in some parts of the world. It wasn't until the 1900s, however, that the ferret was Eirst
formerly introduced as an animal model for biomedical research. Because the ferret was
seen so infhquently as a laboratory animal, and even less so in the mutine small anmial
veterinary practice, easily accessible sources of information on its physiology and diseases
were not &y available until recently.
The ferret's increasing popularity in research and as a pet is rnaidy the result of
large-scale commercial breeding. For exampie, Marshall Farms in New York state, has
been raising ferrets commercially for neariy 50 years and scientists can request a specific
sex, weight, and age of the animal for individual experiments. Although the ferret is
considered non-standardized in regard to an exact genotype and pedigree, its routine
availability in a clinically healthy state has aided its acceptance as a research animal.
Figure 1.G.1. Male 1 1 - 13 weeks old f e n u employed in this m d y weighed approximately 1ûûû - 1200 grams ami measured 40 - 45 cm in lengtti (kaci to tip of tail). Image pvided by Marshail Famis (N.Y .).
The ferret has k n proposeci as a new small animai mode1 for metabolic bone
research due to its bz vivo ability to paraUd human physiologid and skeletal systems and
responses (Mackey et al., 1995). However, the characterization of a ferret h vin0 bone
m m w culture system is an essential step in the overall assessrnent of the species utility for
future bone metabolic study. The hypothesis underlying the work reportecl herein is that
ferret bone marrow wiU, dependant upon d t u r e condition, produce either matrix
identifiable as bone as an end redt of osteoblastic fûnction, or differentiate resorptively
functional osteoclasts.
3. OBJECTIVES The primary goal of this snuly is to develop and characte& a ferret marrow
stromal system to assess the ùz vitro utility of ferret manow as an ostengenic and
osteoc1astic source for funue bone rnetabolic shidy. Femt femoral macro and
microarchitecture will be examined to detemine if BMU-based remodeling. an event not
observed in the rat skeletal systems. is present in this species. Furthemore, we will test
the ability of expIanted ferret marrow. modifying a previously established rat marrow
culturing technique (Maniatopoulos et al., 1988). to synthesize bone nodules and
demonstrate hctional osteoclastic resorption of commercially availab1e substrates or pre-
fashioned bovine bone slices.
4. MATERIALS AND METHODS
4.A. FERRET C m , BONE MARROW HARVESTING & CULTURE
The results presented herein were based on primary and first passage explants
derived firom ferret marrow stroma. The basic protocol employed was h t descnbed by
Maniatopoulos et d. (1988) and used extensively, on the rat, in our laboratory to elucidate
the events of cernent line and bone matex elaboration (Davies. et al., 199 1; Davies, 1996)
and osteoclast resorption (Davies, et al., 1991, 1993). The pmtocol designed by
Maniatopoulos was modifieci slightly to account for 2 factors: 1) the greater marrow
volume found in ferret femora and 2) a reduction in l3-glycerophosphate nom 10 mM to 5
rnM, to prevent ectopic rnineralization (as observed in human and rat cultures at this
concentration).
Marrow cells were harvested h m the femora of young adult male, 1 1 - 13 week old
ferrets (Marshal Farms, N.Y.). Males were chosen for this study to avoid fluctuating
estrogen levels associated with the f e d e estrus state and to parallel, as closely as possible,
the protocol initially established by Maniatopoulos et al. (1988). Ferrets were housed
individually in stainless steel dog transfer cages, fed Pinina cat chow ad lib and maintained
on a 12 hour on112 hour off light and dark cycle in a rwm temperature of 2 1-22'C. In
total. 7 ferrets were utilized for this study and were ktlled by CO2 inhalation. The femora
were excised aseptically at the Department of Comparative Medicine (University of
Toronto), cleared of soft connactive tissue and transported back to the Centre for
Biomaterials, on ice, in 10x the concentration of antibiotics [O. 1 gram of penicifi G (167
units/ml), 1 mi of gentamycin (500 pg/ml) and 0.3 ml of Fungizone (1 mgM) in 100 mls
a-Minimal Essential Medium (a-MEM) ] for m e r processing. They were then washed 3
times for 10 minutes each, in the same antibiotic solution, and rinsed in a-MEM,
The epiphyses were removed h m each femur and the marrow was expelled using
a 30 ml syringe fitted with a 20 gauge needle. The syringe contained 30 rnls of
supplemented medium [(SM) - described below], 15 d s of which was flushed through
each end of a single femora while the needle was rnoved up and down within the marrow
cavity. The expelled ceU solution was collec@d and 15 mls of additional SM was added to
the œil suspension, for a total of 45 mWfemora In the mt protocol, each femora was
flushed with a total of 15 mls SM. The c d suspensions were then passed through a 20
gauge n e d e to break up œil clumps, passed over a 100 pm aic con@ cen strainer (Becton
Dickinson Labware, Franklin, NJ) and pipetted several times to assure adequate
distribution of cells within the suspension. Supplementation of the cell suspension and
seeding on tissue culture polystyrene (TCP) substrats folIowed. Seeded cultures were
rnaintained in a humidifiecl aûnosphere of 95% air and 5% CO, at 37'C. The SM containeci
75% a-MEM, 15% Foetal Bovine Senun (FBS) and 10% of antibiotic concentration listeci
above. Further supplementation depended on the nature of the study. Assays prepared for
the examination of osteogenic activities contained combinations of 5 mM 8
glycerophosphate (BGP), 50 pghl ascorbic acid (AA) and 104 M dexamethasone @ex),
all supplied by Sigma. For tetracycline labeled culnires, both gentamycin and
picillin were replaceci by TC in the final culture refeeding (details to follow). ûsteoc1astic
assays were generally CUItured in Dex (-) conditions and supplemented with the same
concentrations of AA and BGP. However, other studies were prepared to examine
osteoclastic potentiai in the presence of 104 M Dex.
In our sub-culturing protocol, primary explant cell cultures were trypsinized
(0.05% trypsin and 0.53 EDTA - Sigma) on day 10 for 15 minutes, and collected in
centrifuging e s . Following centrifugation. medium and trypsin were aspirated nom the
tube and fresh, fdly supplernented medium (FSM), was added. Again, the pellet was
pipetted several times and passed through a 20 gauge needle and n al con@ flter prior to re-
plating at desired seeding densities, generally 10%ells/cm2. AU refeedings, prirnary or first
passage explants, o c c d the day after seeding to remove non-adherent ceus and
subsequently every Monday, Wednesday and Friday for the duration of the experiment.
43. HISTOLOGICAL STUDIES (LM)
4,B,l, ALKALINE PHOSPHATASE ACTIVITY
Localization of alkaiine phosphatase (ALP), was performed utilizing the m&ed
Azo dye method. Briefly, ferret marrow explants were grown on tissue culture polystyrene
in the presence of Dex until signs of mineraluation were apparent (minimum 11 days).
Whole samples were h e d at 4'C in 2.5% gluteraldehyde folIowed by incubation at pH 10
for 15 minutes with Na-a-naphthyl phosphate (organic phosphate) in tris buffer, in the
presence of Fast Violet Red TR salt. This technique provided a system in which the
primary reaction product (PRP), a naphthol is very stable and insoluble. This PRP is
coupled to a diazonium salt, Fast Red TR, producing the h a 1 reaction product (FRP)
which was deleted using a Leitz Diavert light microscope.
4.B.2. TARTRATE RESISTANT ACID PHOSPHATASE ACTIWTY
Samples fkom both Dex (+) and Dex (-) cultures were stained using the naphthol
AS-BI phosphate substituted naphthol method for tartrate resistant acid phosphatase
(TRAP). The hydrolysis of the naphthol AS-BI phosphate produces a-naphthol. This
PRP is also very stable and insoluble. The PRP is coupled to a diazonium salt, in this case
pararosanilin hydrochloride, which produces a red insoluble product at the site of enzyme
activity. The advantage of using this rnethod of acid phosphatase staining over other
methods (metal precipitation or unsubstituted Am dye coupling) is the fact that there is
better localization of the FRP and that this detectable product is stable in alcohol and
xylene, allowing the preparations to be dehydrated to xylene and mounted in resinous
mediums (Bankcroft, J. et al., 1990).
4.B.3. TETaACYCLINE LABELING
0.01 gm of tetracycline HCl powder (Sigma) was dissolved in 10 ml of deionized
water (999 m) for the preparation of [100x] tetracycline CTC) soiution. This solution
was filtered through a Millex-W 0.22 p pore size filter (Mülipore) and M e r diluted in
a-MEM to a [10x] solution (90 pglinl) to which 0.3 p&ml Fungizone (Sigma) was added.
The [lOx] TC solution was wrapped in foil and ~fkigeraîed at 4'C. When desired, it
substituted the standard [lh] anti'biotic in our SM, and chelated to matrix calcium m
culture over the course of 48 hours. Upon termination, cultures were briefly washed twice
in rmm temperature d i n e solution and twice in 70% ethanol. Fuially, the samples were
fked in 100% ethanol over night, rinsed a final time with 100% ethanol, and left to dry in
the absence of light
Observation of fluorescent mineralized nodules was carrieci out using a custom
made unit housing 4 ultraviolet (W -365 nm) lamps (Microfites ScientSc) positioned in
the shape of a square. The lamps were mounted on the b e r and upper aspect of the
housing unit to allow equal illumination of the sarnple nom all sides. A viewing port was
fashioned in the centre of the housing to allow for visual observation and photography
(Nikon F-601 camera equipped with Al? Micro Nikkor 60 mm lem and colour slide
Ecktachrorne - 400 ASA Kodak film). Nodule size and quantity was then quantitated by
converhg photographic images to digital TIFF files using a Photolook colour scanner
(Agfa). Once digitized, images were imported into NM Image shareware data acquisition
software (http:/lwww .nih. govl) previously instailed on a Po wer Macintosh (Apple)
personal cornputer. Nodule quantity and surface area was measured by detemiining a
standard image threshold wbich best ~flected nodule shape as initiaiiy observed on nIm.
The data was then exported and converted to an Excel (Microsoft) file and andyzed.
4.B.4. VON KOSSA
The von Kossa d o n is an assay by which the presence of phosphate can be
&terminai and localized UnIike the TC method by which fluorescent molecules chelate to
calcium, detection of phosphate is made possibIe by a substitution of calcium with süver
ions. Samples &y for the von Kossa method were incubateci in 1% Siiver Nitrate and
exposed to UV light for 1 hour. Following the incubation, sampfes were washed 3 tinaes
with distilled water. Now samples were treated with 2.51 Sodium Thiosulphate for 5
minutes and washed again 3 times with distiüed water. Fiially, samples were counter-
stained with 1% Safarin Orange, for 10 seconds and washed 3 tinses again in distilleci
water. Cultured cells were fixed and rinsed with non-phosphate buffered saline before
staining to d o w for maximal reaction.
4.B.5. EMBEDDING PROTOCOLS
Following the expulsion of the marrow, femoral diaphyses were fixed in Kamovski's
fixative (2% paraforrnddehyde and 2.5% giuteraldehyde in 0.1 M sodium cacodylate
buffer, pH 7.2-7.4, at 4OC) and subjected to mild decalcification for par* embedding
(below) or prepared nondecalcifîed, for embedding in Osteo-Bed (Polyscience Inc.). The
following is a brief description of the embedding protocols for each.
Femora cleared of comective tissue and marrow were placed in 1.125% fomic
acid/sodium citrate (pH 5.0) and demineralized for a minimum of 3 weeks. The solution
was changed every second day and the decdcifjmg bone was kept on a Red Rotor mtating
table (Hoefer Scientific Instruments, San Francisco) to assure solution mixing. Afkr 3
weeks, samples were radiographed to determine if decalcification was complete. ûnce
having achieved adequate demineralization, deterrnined by the level or radio opacity, the
femora were left in ninnllig water overnight prior to dehyàration to neutralize the
decal-g action of the formic acid
The dehydration pmess consisted of a &d series of submersions (repeated
twice for each value) in 50%. 7096, 8046, 95% and 100% ethanol solutions. The femora
were dehydrated for a minimum of 8 hours in each solution. Samples were cleared in 1: 1
ratios of 100% methylbenzoate/lOo% ethanol and nnally, 10046 methylbenzoate alone mtil
the bone appeared transparent. Infiltration of chloroform for 4 hours and
chlorofodparaffin for 2 hour, followed by pure paraffin infiltraiion (under vacuum at
60°C) foliowed clearing. Samples were ready for embedding in paraffin (~urgipath.). A
Histostat (American Optical) tissue embedder was used for this procedure.
Once the paranin had hardened, 4 pm thin longitudinal or cross sections were cut
using an American Opticai microtome (model820) with a cutting angle set to 5'. The cut
paraffin was floated in a heated bath of water (48 - 50°C). The ribbons of par& were
carefuliy mounted on glas siides (WWR CanLab, 1" x 3") then deparaffinized 3 times for 5
minutes in xylene, and rehydrated in decreasing concenirations of alcohol to water prior to
stiiining. Slides were bien stained in Harris' Hematoxylin (~urgipatkf") for 5 minutes and
counter-stained in Aqueous Eosin. A second series of ethanol washes occurred, this time
reversing the solution concentrations back to 10096, finishing with a wash in xylene.
Coverslips were added to the skies and mounted with Permount (Fisher).
Osteo-Bed EmOedding
Undecalcified portions of ferret femora were fixed using I O 8 neutrai formalin and
dehydrated through tsvo, 24 hour washes in 7096, 95% and 100% etbanol concentrations,
and finaily twice for 24 hours in xylene. The femora were then placed in Osteo-Bed I for 2
days at 4'C. Infiltration of Osteo-Bed II commenced after Osteo-Bed 1, in vacuum for 4
hours and moved again to
followed for Osteo-Bed
the 4'C refiigerator for two more days. The same procedure
III [Osteo-Bed II contains lg/lûû ml benyle peroxide
(polymerization activator) wMe Osteo-Bed III contains 2.5g1100 ml]. Afkr 2 days, the
Osteo-Bed Wfemur cornplex was moved to a 35 'C hot bath and Ieft to polymerize
compieteiy over night.
The S U r f k e of the fblly polymerized Osteo-Bed block was polished to a fine W h
(with 180 to 4000 grit wet and dry paper) at which point the block was glued to a
microscope slide with 5 minute epoxy. A sample of the embedded bone was then cut h m
the mounted block using an Isomet precision saw with diamond w a f e ~ g blade (Buehler
Ltd., IL.), and polished d o m to 20 - 50 pn using wet and dry paper, aforementioned Lf
desired, the exposed bone surface, prior to mounting was stained for 15 minutes with
toluidine blue foilowed by a 70% alcohol rime. Counter staining followed with a 7 minute
Van Gieson's stain, and one last rinse with 70% alcohol. SampIes were allowed to air dry
and were M y viewed under the iight microscope (LM). If staining was adequate, the
block was glued to the g las slide with 5 minute epoxy. The remaining block was cut away
h m the slide using the precision saw and polished again for M e r restaining and
remounting.
4.C. SCANNING ELECTRON MICROSCOPY (SEM) & ENERGY DISPERSIVE X-RAY MICROANALYSIS (EDX)
Osteogenic and osteoclastic cultures, as well as femoral bone hgments prepared
fiom freshly excised ferret femora, were observed by SEM. In order to view endosteal and
trabecular surfaces of long bones, femora from ferrets were cleared of soft connective
tissue, rnanually, via the expulsion of marrow with a syringe containing SM and m e r by
5 minute immersion in NaOCI. Karnovski fixation for 4 days, sonkation and keze
fracniring employing liquid nitrogen, mbsequently followed. The fhgments were
examineci under a dissecting microscope in order to retrieve portions of bone that displayed
both trabeculae and endosteal envelopes. These portions were then dehydrated through a
series of ethan01 washes (as above), critically point dried fiom CO2 (Autosamdn 810,
Tousimis Corp., R o c M e , MaryIand, USA), mounted on duminum stubs and gold coated
(Poiaron ES00 2 cool sputter coater, England) prior to examination.
Cell cultrnes were fixed for a 2 hour minimum in Karnovski's fixative. FolIowing
fixation, areas for examination were cut away h m the various tissue cuIture polystyrene
containers, dehydrated through graded ethanol series comprising 5096, 70%, 80%, 95%
and 100% alcohol washes, and finally critical point dned fiom CO,. Nodules were
observed in one of two subsets. The f b t comprised an untouched version of the nodules,
while the second consisted of 'peeled' samp1es which had a majority of their ceIlular and
fTbrilar content removed by gentle dissection with forceps. Specimens were then mounted
on alirminum stubs and gold coated to a thickness of - 12 - 15 m prior to SEM
examination.
Cultures displaying mineralized nodules were analyzed in an Hitachi S-570 SEM
(Hitachi, Tokyo, Japan). For analysis utilizing energy dispersive X-ray microanalysis
(EDX), samples were prepared as above, mounted on aluminurn stubs, and carbon coated
(Edwards E 12E4 Vacuum Coater, England) prior to examination. Al1 EDX analyses were
performed on an Hitachi S-4500 T;ESEM (Hitachi, Tokyo, Japan), fitted with an ISIS
(Link) EDX analyzer. Images were capbired on Quartz PCI based image acquisition
software (Quartz Imaging Corp., Vancouver, Canada) which could record and store
imaged data on removable media
4.D. X-RAY DIFFRACTION (XRD)
Ferret femora were excised, cleared of remaining soft tissue and rinsed in distillai
water. Both elaborated minedzed nodules and femora were M e r dehydrated, cleared in
anhydrous ether then pulverized. SampIes of bone powder from each source were
analyzed by X-ray difnaction using a Rigaku difhctometer using Cu K a radiation and a
highly crystalline mineral fluorapatite as a standard.
4.E. TRANSMISSION ELECTRON MICROSCOPY (TEM) Cultures ready for TEM anaiysis were rinsed with a-MEM 2-3 times, fouowed by
0.1 M Cacodylate buf5er. then nxed in Kamovski's fixative for 2 hours prior to p s t
fixation in 1% osmium tetroxie/l.S% sodium ferrocyanide (to preserve c e U membrane
morphology). AAer 3 repeated washes in 0.1 M Cacodylate buffer and 70% ethanol,
enbloc staining with 7.5% magnesium uranyl acetate (protein sstaining) in 70% ethanol
commenced for 90 minutes in the dark, at room temperature. A series of alcohol gradecl
dehydrations follow& 70%, 80% and 95%. twice for 5 minutes each. Then 100%
bonded EtOH 3 times for 10 minutes each. SampIes were infiltratexi with a series of
alcohol-epon mixtures (2: 1.1 : 1, 1 :2) and finally with 3 changes, 1 hour each, of pure epon
(epox 8 12) under vacuum. The epon polymerization initiated ovemight at 40°C in vacuum
and continued at 60°C for 3 more &YS. nie embedded mineralized tissue was separated
fkom the tissue culture polystyrene, and areas of interest were re-embedded in beam
capsules. Silver to pale gold thin sections were cut with a ultra-microtome (Isomet) and
mounted on copper grids. The sections were restained with 7.5% magnesium w y 1
acetate in 70% ethanol and double stained with Reynold's lead citrate (lipid content).
Samples were then viewed using a Philips EM 400T TEM.
4.F. OSTEOCLASTIC RESORPTION ASSAYS
4.F.1. OSTEOLOGIC" CALCIUM-PHOSPHATE TEtIN FILMS
Sub-micron calcium phosphate ceramic thin nIms known as 0steologic" discs
(Milleniun Biologix Inc. Canada) were generously provided by the manufacturer (see
below for image). These thin films have displayed the ability to be resorbed by
multinucleate, TRAP positive putative osteoclasts (Davies et al., 1993) and provide a
means for a quick and reproducible methoci to cddate resorption percentages per sample.
Furthemore, Nakamura et al. (1996) displayed F-actin dots (actin ring) formation of
seeded osteoclasts on such substrates homologous to those seen on caicified dentine slices
but not demineralized dentine or type I coUagen gel ma&. The nIms are composed of a
triple crystal layer of calcium phosphate which is deposited on a quartz support The discs
arrive? pre-sterilized by ethylene oxide, in 24 well trays. Since the depth of the t h film
averages 5 pm and the quartz support is non resorbable. these discs pmvide a means of
compuhg and quantitahg xesorption activities in a 2 dimensional piane without the
dimension of depth as a vande. The transparent nature of the cpr& support also dlows
for transmitted LM examination of fesorption activities in vitro once the overlying CaP
layer is resorbed. This feature is not possible with opaque substrates like bone. Discs can
as be prepared for SEM examination following the aforementioned SEM protocols.
4.F.2. BO- B O m SLICES
Bovine long bone was purchase from a local abattoir and cleared of marrow and comective
tissue. Using a drill press fitted with a trephine, longitudinal cyhders of bone
appruximately 12 mm in dianieter, were cut and prepared for sectioning (See image below).
Using an h m e t precision saw fumished with a diamond wafering blade, bone slices in the
shape of discs approximatefy 1 mm thick were fashioned These discs were prepared to
resemble the dimensions of the ~steologic" discs provided by Mi1Ienium Biologix Inc. (see
above). Foliowing sectioning, the discs were immersed in NaOCl for 5 minutes to remove
di organic matter, riased thoroughly in water and stored in phosphate buner sahe (PBS).
When needed, bone &CS were given a fksh rime in PBS and placed in 24 celi weU culture
trays (Coming G l a s Wodrs, NY) pnor to inoculation with b h l y expelled femt marmw.
43.1. Bovine Bone Vs. Osteologic" Substratum. Above are two importeci images, one of a pre-fashioned bovine bone slice (left) and a commercially avaiiable CaP Osteologic" disc (right). The bovine bone slices were f&oned with an identical thickness and approximate diameter. Resorption events are visible to the naked eye on the commercial substraîa (right) while resorption events on bone must be viewed by SEM.
Two M y confluent, T-75 Dex (+) flash (Sarstedt Inc. No, were trypsinized after
10 days of primary culture. Celk were counted ushg a Coulter comte? (Couiter
Electronics LtcL) and a final concentration of 4 x 104 cellslml was prepared in a volume of
30 mis of FSM. Senal dilutions followed in which 15 mls of this initial suspension was
used to seed 3 T-25 flasks (5 mliflask) providing a c d plating density of 2 x 105 ceWT-
25. The remaining 15 mls of nrst passage c e k was combined with 15 mls of freshly
prepared FSM, to bring the volume back up to 30 mls, thus halving the nnal concentration
of cells to 2 x 10" c e W d . This ceil suspension was pipetted many times to assure proper
rnixing of the celUFSM soiution. At this pink 15 m l s of the cell solution was transferred
to 3 new T-25 flasks with a final cell population of 1 x 1@ celldï-25. Serial dilutions
were repeated 3 more times to prepare c d populations ranghg h m 2 x 105 ceM-25 to
1.25 x IO4 cefls/T-25. This assay was undertaken to determine if the expression of
mineralized bone nodules (reflection of initial osteoprogenitor population), or nodule size,
was related to the number of ceils plated.
5.A. IN Vwo ARCHITECTURE
kfi and right femm from 7 different male, 11 - 13 week old, ferrets were
m e a d h m proximal to distal ends (including articular cartilage surfaces) and found to
range from 4.3 cm to 5.2 cm h length with a mean of 4.79 f 0.33 cm (Figure 5 .A.).
Cross sections through the diaphyseal portion of fenet femora pvided cortical and
marrow cavity measurements of 1 f 0.08 mm in thickness and 2.13 f 0.15 mm in diameter
respectively. Cortical and medullary cavity measurements were coIIected h m 4 samples,
half way between each epiphyseal plate. In vivo bone architecture and remodehg activities
of femoral bone are discussed in sections 5.A.1 - 5.A.2, and display both light and
scanning electron microscopy images.
Figure S.A. Freshly excised ferret femur meastuhg approximately 5.2 cm. This femur was removed h m an 11 - 13 week old male ferret weighing approrimatefy 1100 grams, prior to mam>w expulsion.
Ferret endosteal and h-abecular macr~archiecaire was observed to comprise a
trabecular network of anastomosing rods and small plates which made numemus jmctions
with the endosteal wdl. However, no resorptive activity was visible as overlying cellular
layers including the endosteum and other cellular debris, prevented d a c e examination
(Figure S.A. 1 .A). Resorption bays known as Howship's lacunae (S.A. 1 .B), became
evident on both endosteal and trabecular surfaces once samples were immersed in NaOQ
for 5 minutes to remove d a c e organic constituents. These laamae were bounded by
characteristic scdloped resorption borders fomed by resorbing osteoclasts (5.A. l .C).
Figure S. A. 1 .A. Tmbeah mrfkes prior to NaOCl immersion. Cells of the endosteum and marrow obscure the underlying bone surfaces. Modehg and remodeling activities cannot be seen. Field widtll = 500 pl.
Figure S.A.1.B. & 5.A. 1.C. An endosteaVtrabecular junction. The image on the right is a magnincation of the boxed a n a in the left image. Howship's resorption lactmae with Scafloped borders (mm) indicative of osteoclastic resorption are apparent and abundant following NaOCi immersion. FieId widths 400 pm (B) and 80 pm (Cl-
S.A.2. CORTICAL BONE
Examination of 1 1- 13 week old cross and longitudinal ferret bone sections reveaied
BMU-based remodeling l a n h k s and events (Figures 5.A.2.A to 5.A.2E.). These
teniporally and spatially coorduiated activities display the resorptive nature of the cutting
cone and the osteogenic ability of the f i k g cone 5.A.2.C). MuItinucIear
osteoc1asts 5.A.2D.j were evident in cutting cones of remodehg unit5 and
displayed polarized morphologies with interdigitating projections; the ruMed border, wbich
were intimate1y associated with bone. Figure 5.A.2E documents the polarized
morphology of an actively resorbing osteoclast Intracellular vesicles were noted near the
mffled border while two visible nudei were located distally to the resorbing front. The
cytoplasm was aiso occupied by cellular organeiles, many golgi and RER. The arrows of
figure 5.A.2.D. indicate the direction of cutting cone advancement based on cell and
remodeling channel orientation.
The osteogenic unit, termed the nIling cone, followed the resorbing osteoc1astic
component of the BMü. Flattened putative osteogenic precursors adhered to the newly
resorbed reversal line. These DOCs which initiated the deposition of an interfacial matrix9
would M e r differentiated into the more mature osteoblastic phenotype. As such, the
synthesis of osteoM commenced and was observed as a yellowish-green colour by primary
toluidine blue and secondary Van Gieson's staining (Figure 5.A.2.C). As the osteoid
mineralized, some osteoblasts became entrapped in the matrix they secreted to become
osteocytes. The entire remodeling event gave rise to Haversian osteons (Figure 5.A.2.B)
characterized by the concenhic arrangement of embedded osteocytes in IameUar bone
around a central neurovascular canal. LameUae were not documented to exceed 5 rings.
Figure S.A.2.A. Low magnincation of a cross section tbrough ferret corticai bone. Apparent are the many secondary HaverSiacl osteons present in this 11 - 13 week old bone- Field width = 1.37 mm.
Figure S.A.2.B. A cross section through fefiet corticai bone. 2 fiversian osteons are visible, each comprising concenîric rings of embedded osteocytes and lamellar bone. Field width = 140 p.
Figure 5. A. 2. E . Transmission e l m n micrograph montage of the k a d of a cutting cone with cells displaying osteoclast morphology. Note the interdigitating microprojections of the osteoclastic müied border in direct association wiîh bone, intraceliular vesicles and a binuclear morphoIogy (Special rhankn to Dr. Okada). Field width = 45 p.
5.B. DEXAMETHASONE (+) CULTURES
S.B.1, CELL CULTURES
5.B.La. Primary
Femt bone marrow œlls were seeded on TCP in the presence of AA, BGP and
Dex. Cek initially displayed a spinde shape morphology while cell layers were non-
confiuent. Coduency of T-75 seeded flasks was achieved by the 6th &y of culture and
celi multilayering became apparent by &y 9. Upon achieving confiuency and
rnultilayering, foci of cells in certain areas of the fi& adopted a more cuboidd morphology
commonly associatecl with differentiating osteogenic cells. On the 1 lth &y of primary
culture and thereafter, mineralizing nodule eiaboration cornmenced as observed by visual
detection. Mineralizing mairices appeared white to the eye however, under phase-contrast
microscopy, as figures 53.1 .A and 5B. 1 B. The visible detection of the muieralizing cell
foci was not preceded by microscopic detection. Instead, nodule mineralization (visible or
microscopic) seemed to occur rapidly between successive viewings (usually 24 hours
apart). Figure 5.B. 1 .A. displays three, 2 day old m i n e m g nodules on day 13. The
nodules appear to have a reticular appearance, most likely due to uneven rnineralization,
with variable peripheral opacity. Figure 5 .B. 1 .B. displays the position of the previously
documented nodules, on &y 16. The individual nodules of figure 5 .B. 1 .A. have fused to
form a much larger single nodule spanning 2.5 mm across through its longest axis. More
obvious in figure 5.B. 1 .B. is the reticular nature of the nodule and the opaque periphery
surrounding it.
Figore 5.B.1.A. & S.B.1.B. Initial elaboration of 3 nodules (auows) in Dex (+) cultures on day 13 (A). By &y 16, the nodules have grown and fused into one large nodule (arrow) in Figure B. The rnineralized nodules in both images display a reticular-like appamme with varying peripheral opacity. The ce& of the culture display a confluent cuboidal morphology with rnultiiayering of cells residing at the nodule penphery. Field width = 2 mm.
5.B.l.b. First Passage
F i passage, subcultured ferret marrow populations similady displayed the ability
to elaborate nodules, however the f o d m of mineralized tissue was not obssrved until
the third week of subculture (first seen on &y 22). A h gentle trypsktion, primary
plated celis were re-plated at a concentration of 104 ceWm2 unless otherwise stated mx (+) Dilution Assay]. Early cultures dispIayed an initial spindle cell shape morphology
while in non-confluent States (as observed in primary culture). By the 7th day of sub-
culture, cell codiuency was achieved and the earliest signs of c d multilaye~g were
evident on the 16th &y.
5.B.2. HISTOCHEMISTRY
5.B.2.a- Alkaline Phosphatase
The histochemical staining technique for ALP was employed on our Dex (+)
cultures seeded on TCP for osteogenic assay. Cek at the irnmediate periphery of
elaborating nodules displayed multilayering and were bighly ALP positive (Figure
5.B.2.A.) as is typical for the pre-osteoblastic and osteobIastic phenotype. At higher
magnincations, the localization of the stain was found to associate with the ceIl plasma
membrane, as the ALP enzyme! is an ectoenqme, and proviàed a means of disceming cell
plasma membrane boundanes.
5.B.2.b. Tetracycline
48 hours of cell culture in the presence of TC provided adequaie time for the
antibiotic to chelate to calcifiecl n0dUJ.e~. The tetracycline Iabeling methd was employed to
detect the presence of calcium in mineralizing matrices and to attempt to reproduce the
results observed by the chelation of tetracycline to calcium in the rat marrow culturing
system. AU mineralized nodules in primary culture or first passage culture P x (+)
Diiution Assay J displayed the ability to fluoresce (Figure 5.B.2.B.) and provided means
for nodule analysis utilizing NIH Image acquisition software.
5.B.2.c- Von Kossa
The von Kossa staining rnethod was performed early in this study's inception, for
the detection of calcifieci maeix. Nodules from severai samples, elaborated in primary or
subculture conditions, were able to react positively for the staining technique as concluded
by the colour change of unstained mineralized nodules (Figure 5.B.2.C.) to solid black
(Figure 5.B.2.D.).
Figure 5.B.2.A. Intense Iocalization of ALP is found at the periphery of deveIoping bone nodules (center of field). As DOCs display high leveis of this ectoenyme, it ïs postulateci that this position is occupied by Merentiatiug pre-osteoblastic ceiis assisting in mineralization. FieId width = 1.4 mm.
Figure S.B.2.B. Fluorescent detection of mineralized tissue is made possible by tetracyciine chelation to calcium. Nodules fluoresced providing a method to obsenre nodule distribution, quantity and size. Field width = 4.43 cm,
Figure 5.B.2.C & 5.B.2.D. Montages of pre (C) and p s t @) von Kossa stained mineraüzed nodules
5.B.3. ELECTRON MICROSCOPY
5.B.3.a. Scanning Electron Microscopy (SEM)
Scanning electron mimgraphs of elaborating bone-like rnatrix exhiiited a mdtistep
procedure resulting in hlly mineraiized nodules. Individual mineralized anbrilar globular
accretions, were evident in culture as early as &y 10. DOCs were fomd in the hmedbte
proximity of globular accretions and extended cell processes which were observed to abut
dkctly to the accretions @gures 5.B.3.A. & 5.B -3.B .). The acætions were also found
in direct apposition to the substrate (TCPI and ~easured on average between 0.8 pxn to 1.3
~indiameterandapproximately500-750miaheight (Figures 5.B.3.B. & 5.B.3.C.).
As culture time was extendeci and elaboration of nodules advanced, the individual globuiar
accretions fused to form a confluent afibrilar interfaciai maîrix (Figure 5.B.3.D.)
homologous to the cemnt line of remodehg bone, or that seen in direct contact with
endosseous implants surfaces. As the interfacial matrix manired and fused, the
initiation of collagenesis commenced. Collagen fibers became intimateiy associated with
the interfacial matrix (Figure 5.B .3 .D.) and subsequently mineraiized. As nodules
mineralized, they continued to grow in diameter by the M e r deposition of globuiar
accretions at the nodule periphery (Figure 5B.3E.). These globuiar accretions also fused
and became coduent with the previoudy depsited interfacial rnatrix. Osteoid was
subsequently deposited above the new IFM. Nodules also grew in thickness by the
continwd proliferation and differentiation of osteogenic cells. Osteoblasts synthesizing
osteoid periodicdy became entrapped in their to becorne osteocytes. In this mamer,
ma& elaboration continued, and nodules grew in thickness (Figure 5.B.3.F.) as weil as
maintaining a full cornplement of osteogenic celis.
Figure 5.B.3.A. & S. B.3.B. Initial deposition of globdar acctetioas (arrow heads). Osteogenic c e k (arrows) extend ceU process which abut directly with the accretions (Figure B, arrow). Field width = 40 p (A) and 2 m.
Figure S.B.3.C. & 5. B .3. D . Giobular accretions measuring approximately t - 1.2 p in diameter (C). Figure D displays a confluent interfacial matrix with intimately associated collagen fibers (arrows). Field width = 3.20 prn (C) and 16 pm (D).
Figure S.B.3.E. & 5.B.3.F. The micrograph on the left displays the continuous elaboration of globular accretions ( m w s ) outward from a mineraiizing nodule. Figure F provides a 45 ' tilt thtough a fnctured nodule displaying the mineralized profile of a mature nodule at 23 days. Field width = 80 pm (E) and 62 pm (F).
S.B.3.b. Energy Dispersive X-ray Analysis (EDX)
28 day old Dex (+) cultures, with many eiaborated mineraiized nodules. were
prepared for EDX analysis. Samples displayed prominent peaks for calcium and
phosphorous (Figure 5B.3.G.). EDX dot map analysis localized the sipals to a source.
The calcium source 5B.3.HJ and phosphorous source (Figure 5B.3.1.). when
superimposeci (Figure 53.3 J.). displayed a co-localization which were congruent with the
position of the globular accretions seen in the secondary SEM image (Figure 5.B.3.K).
1 2 3 4 6 Energy (keV)
Figure 5.B3.G. Energy dispersive X-ray miroanalysis spectra displaying prominent calcium and phosphorous spikes for globuiar accretions.
Figures 5.B.3.H. - 5.B.3.K Energy dispersive X-ray dot map analysis of globular accretions (K) provided the signal location of calcium (H) and phosphornus (0. These superimposed signals (J) were congruent with the location of g1obuIa.r accretions in the secondary SEM image. Field widtfis for H through K = 12 p.
S.B.3.c Transmission Electron Microscopy (TEM)
Mmeralized nodules, 28 days into culture, were mmhated and prepared for 'IEM
analysis. Samples were processed h m various areas of many nodules which provideci
evidence of nodule elaboration at many ciifferent stages. Figure 53.3-L. displays a cross
section through a matare nodule. The interfacial mahYr was seen in direct contact
with the substrate (TCP). M y associated with the IFM, was both minefalized (MM)
and non-minedized matru or osteoid (NMO). Four entrapped osteocytes are seen in this
rnimgraph displayhg various levels of entrapment in mineralized ma&. In the bottom
left corner of the micrograph, an osteocyte (OC) is fidiy entrapped in mineralized matrk
At the upper right comer of the micrograph, a . OC is completely surrounded by NMO.
This micrograph portrays a typical profile of elaborating bone matru in vitro. Figure
5B.3.M. documents an earlier event in the eelaboration of a developing nodule. Globular
ametions (GA) were stiU at low concentrations and had not yet fused to form the interfacial
rnatrix or cernent line. Found directiy above the GAs was a cell containing rough
endoplasmic reticuium associated with protein synthesis. Figure 5.B.3.N. displays the
initiation of mineral nucleation centres. The mineralization of the rnatrix commenceci on
coIlagen fibers which at higher mapfication displayed h e rod or pin like structures
typical of individual hydroxyapatite cxystals.
Figure S.B,3,L. TEM mimgraph of part of a mineraking boue nodule. A confluent interfacial matnx 0 is seen in direct contact with a tissue culture polystyrene (TCP) substraie, Also evideat is mineraüzing matrix (MM) and osteoid (NMO) which have en- several osteocytes (OC). Field width = 30 m.
Figure 5.B3.M. A rough endoplasmic reticulum (RER) nch ceU, cornmonly associateci with protein synthesizing ceils and the osteoblast phenotype, is seen directly above two globular accretions (GA). Field width = 3.8 p.
Figure 5.B,3.N. TEM micrograpb displayhg non-mineralized coUagen (NMC), made evident by its banding pattern, and mineralized coUagen (MC). Fieid width = 3 p.
5.B.4. X-RAY DIFFRACTION m) Both pulverized cortical bone samples and mineralized bme nodules, eiaborated m
vitro, were analyzed iuüizing an X-ray diflktonieter. Ferret bone disp1ayed a crystalline
hydroxyapatite-like specûum associated with mid to long rauge crystal order of HA,
homologous to those produced by p u l v e d rat and dog bone (Figure 5.B.4.A.).
Prominent pe&s were noted at 26,32 and 40 2 0 angles.
Ferret bone nodules grown for 21 days on TCP (Figure 5.B.4.B.) produced an
XRD spectra not unlike that representative of embryonic developing chi& bone however,
displayed a more prominent peak at the 40 2 0 angle. Fetal ferret bones were not avadable
for analysis.
Bone XRD Cornparison
CPS
26 32 Angle (2 Theta) 40
Figure 5.3.4.A. XRD diffraction of ferret, dog and rat bone. Al1 t h e species display a pattern similar to that of highly crystalline mineral hydroxyapatite.
In Vitro Vs. Embryonic Bone
26 " Angle (2 Theta) 40
Figure S.B.4.B. XRD patterns cornparhg 21 &y old in vitro femt bone nodules to 12 &y old embryonic chick bone. Both specaa display values consistent with amorphous substances compnsing short range hydroxyapaiite c r y d order.
S.C. DEXAMETEASONE (-) CULTURES 5.C.l. HISTOCHEnaSTRY
Cultures maintained in the absence of Dex, for 13 days, did not display signs of
osteogenic activity in the form of mineralized nodule elaboration. This observation is
consistent with previous =ports on cuItured rat marrow systems which concluded the
necessity of Dex in culture medium for osteoprogenitor differentiation and mahix
synthesis. ALP staining assays were not attempted for this reason.
5.C.l.a. TRAP
Ferret marrow plated for 1 1 days on ~steologic" discs (Figrire 5 .C. 1 .A.) and bovine bone
slices (Figure S.C.l.B.), in the absence of Dex or preence, to assay the effects of Dex on
resorption percentage, displayed populations of TRAP positive cells which displayed the
multinuclear phenotype of osteoclasts. Attempts to quantitate TRAP positive populations
fded, however, resorption percentages were measured between Dex (+) and Dex (-)
Osteologic"' discs (see below).
5.C.2. OSTEOLOGIC" CALCIUM-PHOSPHATE THLN FILMS
Femt primary marrow plated on osteologic discs in both Dex (+/-) culture
conditions was able to display functiod osteoclastic resorption. Rior to culture
termination, LM examination in situ, documented the presence of large ceils with round
morphologies, occupying resorption iacunae. Morphologies were made distinguishable by
phase LM however, overlying heterogeneous cell populations made the detection of
multinuclearity inconclusive. Cd layers were removed by NaOCl immersion in order to
expose the thin film surface for resorption v i s ~ t i o n and quantitation. Evident by both
LM (Figure 5.C.2.A) and SEM (Figure 5.C.2.B.) observation was the resorption of
portions of the calcium phosphate thin film. Scalloped resorption pit borders seen bz vivo,
indicative of osteoclast resorption, were noted in vitro. Partial resorption of CaP layers
was also documented in both LM and SEM images, in and aear resorption iacunae
boundanes.
Resorption quantitatim in Dex (-) culture conditions of 5 oste~logic" discs, using
an automaîed ~ i c m s t ~ device, pmvided a h a 1 rewrptive niean of 1.88 f 0.4% of the
measured surface area In cornparison, resorption quantitation of Dex (+) samples yield a
significantly lower (@.O resorption value of 0.7 1 0.14%.
Osteoclastic Resorption Comparison
signtficant difference (pe0.025)
Figure 5. C.2. Percent of calcium phosphate thin film resorbed by osteoclasts in Dex absent (1.8846, blue bar) and Dex containing (0.7% burgundy bar) culture conditions. A significant diffmnce is noted between the two sampIe popdations with a confidence interval of p4I.025.
S.C.3. BOVINE BONE SLICES
Cultures plated on bovine bone slices, whether in the presence (Figure 5.C.3.A)
5.C.3.C.) or absence (Figure 5.C.3.B.) of Dex, were able to resorb portions of the bovine
bone surface. Resorption pits known as Howship's iacunae were evident in both
conditions however more abundant in Dex (-) cultures. A 3D image of a resorption event is
seen in figure 5.C.3.A. and illustrates the depth associaîed with some resorption lacunae.
Quantitation of resorption pit volumes or total resorption per sample was not an objective of
this preiiminary investigation and therefore not undertaken. Resorption events were
usually observed as having pitted characteristics with scalloped margins (Figures 5.C.3.A
and 5.C.3.B.) thought to be due to several osteoclasts at one site. or as trough-like
resorption events (Figure 5.C.3.C.) indicative of individual osteodasts resorbing while
migrating. Ali attempts to observe nXed osteoclasts in resorption lacunae by removd of the
overlying cell layers failed Cells responsible for such resorption events may have been
removed in the dissection, or possibly migrated to other sample locations.
Figure S.C.1.A. & 5.C.l.B. The ~IIOWS in (A) display the position of TRAP positive, putative osteoclasts. Cultures seeded on both Ostedogic" discs (A, with methyl green counterstaïn) and bovine bone slices (B), b t h Dex (+/-)] displayed the presence of TRAP positive ce&. Most TRAP (+) ceils displayed a vacuolar morphology and under higher magnification, multinuclearity. Field widths = 1-13 mm (A & B).
Figure 5.C.2.A. & 5. C.2.B. Osteoclastic resorption events noted on Osteologic" thin films by both LM (A) and SEM (B) observation. Common to both samples are the scalloped resorptive borders noted in in vivo resorptive events. Field widths = 90 pm (A) and 150 pm (B).
Figure 5.C.3.A. & 5.C.3.B. 3D reconstruction of a bovine bone slice d a c e a . a 13 day Dex (-) culture (A). Resorption pits are seen boring deep into the b u e matrix (green lem on left eye). A similar bone slice, this time cultured in a Dex (+) conditions (B). Evidence of resorption was okrved, but was less common than ihaî seen in Den (-) conditions. Field widths = 100 pm (A & B).
Figure 5.C.3.C. SEM montage image of an osteoclastic resorption 'trough'. This resorption event may be the result of two osteoclasts as a resorption lacuna ridge sepamk~ the upper thîrd of the event fiom the Iower two thirds, Field widtù = 72 pm for black data bar.
S.D. DEXAMETHASONE (+) SERIAL DILUTION ASSAYS
Serial dilution assays were prepared to determine if a direct relationship existed
between plating density and bone nodule count, or plating density and bone nodule size.
C d plating densities were senally diluted from 4 x 104 ceiis/rnl(2 x 10' ceUs/T-25) to
2.5 x lo3 cells/d (1.25 x ld cells/T-25). Nodule count (a direct reflection of initial
osteoprogenitor population) was found to increase with both time and plating density.
These trends were evident by visual assessrnent (Figure 5.D. 1.A.) of the plated T-25s-
however a software assisted nodule count was employed using NIH Image data
acquisition shareware.
Results illustrated in figure 5.D. 1 B. displayed that as plating density increased,
the number of nodules increased (pd.025). The number of nodules formed in vitro for
each of the 5 seeding densities were analyzed by t h e period and regression analysis was
applied. All three periods (day 7, 14, and 21 termination points) displayed strong
correlation between nodule count and plating density with regression values of R~ =
0.941 1 (week l), 0.7754 (week 2) and 0.9873 (week 3 shown in Figure 5.D.l.C.).
Furthemore, for each seeding density, tirne was found to have a profound effect on
nodule formation as the number of nodules increased significantly (pc0.025) within
identical plating density from week to week.
No trend was observed between nodule size (mm2) and plating density in a 3
week comparison (Figure 5.D.l.D). Figure 5.D.l.E. shows the lack of correlation
between mean nodule size and plating density (R2 = 0.25) at 21 day post nodule
detection.
It is important to note that the serial dilution assay resuits reported herein were
calculated h m the data generated fkom only one expiment. Due to time restrictions,
the opportwiity to reproduce these rrsults was not avaitable. However, the relationships
between plating density and nodule size or nodule count matched those reported by
BeUows et al. (1990) in a sunilar subculhued study on rat rnarrow explants.
2 x 1 0 5 Cel ls/T-25
Week 1
Week 2
Week 3 1 '
Figure 5.D.I.A. T-25 flasks representing ceii plating densities of 1.25, 5 and 20 x 10' ceiidï-25 for weeks one, two and three. The trend displays an increase in nodule count from week 1 to week 3 and from low concentrations to high. However, no significance is reported when nodule size is compared to plating density (p4.025).
Nodule Number Vs. Plating Density
Plating Density x 104
ce1 l s /TX
Week significant difference (p<0.025)
Figure 5.D.I.B. Results obtained from a senal dilution assay. Bone nodule count is compared to ce11 plating density when densities are doubled once, and cwice above and below 5 x 10' cells/T-25- The trend displays an increase in nodule count fiom Iow concentration to the next, correlating to an increase of initiai differentiating osteogenic stem cells. The bars represent the mean number of nodules elaborated in 3 T-25s at I 95% confidence limits.
Nodule Count Vs. Plating Density
Figure 5.D.l.C. Relationship of nodule numkr to pIating density. Densities started at 1-25 x IO' cellsiT-25 tïask and doubled in concentration to a final density of 2 x IV ceIldT-25 fiask. Fint passage cultures were maintained in vitro for 21 days after mineralkation commenced. A direct correlation exists between the ce11 seeding density and the number of nodules formed. The points plotted represent the mean number of nodules elaborated in 3 T-î5s at f 95% confidence limits.
Nodule Area Vs. Plating Density
wesk * significant difference ( ~ ~ 0 . 0 2 5 )
Plating
Density
Figure 5.D.l.D. Nodule sizes present in 5 different plaring densities as recorded over a 3 week period. No trend is evident.
Nodule Area Vs. Piating Density
. . . . . . . . . . _ - . < - - - . . _ - . _ . - . - c - - . . - " . . ,. ,* . .; .. -. . . . . . . . . - ..-i L : .. . 7 - - . .:
. . . . . . * , . . - . & . . - . .:, :-. .-,7;->** . -- . . . - '- - . - . *
. . ' : ..:: . - . . . : - - . . . . . : * ...... : . . . - - - . * . -. - . . - - , . , . : . . - . < - . . . . . . . . . . . . . . . . . . . - . -. ,
_ L / . - , . .L- Y.?: . - - - - : * a . - : -. . ' - . . . . . .<a. . . - . : -.,. - - . . .. . . . . . . . . . - . - . - . . - .: . . - - : - . : . . , - - - -:' - * ", -*:y Y.;;?:: ' . . , , . : . . . . . . a . . .... . . . r - ..- . , . . . . . . - . . - . . . ' . . +y<:! .. , . . - , - . : -
O 5 t O 15 20 25
Plating Density x 10'1 T 2 5 FI2 = 0.2503
Figure 5.D.l.E. Relationship between nodute area and plating density at week 3. The area of the nodules formed at different piating densities were measured by MH Image analysis. No significant relationship existed between nodule size and plating density.
6. DISCUSSION
The study reporteci herein was initiatecl to examine the osteogenic and osteoclastic
potentid of ferret m m w ceils in the presence or absence of specific supplements @ex.
AA, BGP). While the rat model represents the most commonly studied and "compulsory
model for pre-clinical and chical evaluation of agents used in the treatment or
prevention of postmenopausal osteoporosis" (FDA guidelines, 1994), other studies
document irrefutable evidence, reporting dissirnilatities in rat skeletogenic and
physiologie processes when compared to humans (see below). Nevertheles, explanted
rat bone marrow cultures have provided a powerful tool in the elucidation of in vitro
osteogeniclosteoclastic mechanisms.
The inability of the rat model to parallel human physiology has led to Proctor and
Garnble's investigation into the discovery of a new, s m d sized, animal mode1 (ferret) for
the study of metabolic bone disease (Mackey et al., 1995). Arhüe a pilot study performed
on the ferret displayed utility of this species in the elucidation of etiologies and possible
treatments for osteoporosis, it's use as a complementing in vitro mode1 was uncertain
More the present investigation was undertaken. The remainder of the discussion wiil
focus on the advantages and disadvantages of the rat and ferret model for the study of
osteoporotic research, and the in vitro results reported herein.
6.A. ANIMAL MODELS OF OSTEOPOROSIS
6A.1. TEE RAT
The rat model has been utilized in numerous in vivo and in vitro bone rdated
studies (Jee et al., 1991; Matsumoto et al., 1985; Vailas et al., 1992; Bellows, C.G. et al.,
1986; Grigonadis, A E et al., 1988; Maniatopoulos, C. et al., 1988; Lang, H. et al., 1990;
Davies, J.E et al., 1991; Kidder, L.S. et al., 1993; Zhou et al. 1994) and has provided
scientists with a srnall, flordable and standardized (in regard to genotype and pedigree)
animal model. However, certain characteristics of rat skeletal development Vary nom
those reported in the human. While surface remodeling (Frost, 1969; Tran Van et al.,
1982) endosteal resorption (Jones and Boyde, 1977) and trabecular modeling (Ke et al.,
1992) have been documented, it has long been considered that rat cortical bone does not
undergo intracortical remodeling (Foote, 19 16; Enlow, 1958; Jowsey, 1968). A study
perfonned by Ruth et al. (1953) was able to display secondary rat cortical remodeling by
restricting calcium from the animal's diet, rendering the animal hypocdcemic. This
subsequently lead to the resorption of intracortical bone. With the r e m of calcium to
the animal's diet, Ruth was able to initiate re-deposition of mineralized tissue on
previously resorbed revend sites. Although this 'induced' remodeling event mimicked
bone remodeling with resorption preceding deposition, the events were independent of
one another unlike those observed in tme BMU-based remodeling, documented in other
species (Cooper et al., 1966; Pd&, 1983; Gray, 1980; Mackey et al., 1995). Zhou et al.
(1993) have since documented intracortical rernodeling of rat bone in young male Wistar
rats, but describe the secondary osteonai architecture of rat bone to be "better organized
bone of stratifïed structure, and distinct fkom surrounding primary bone, although not of
l a d a . architectureTT.
Moreover, rats are considered continuous growers. Such species depend on the
grinding of their teeth to prevent excessive tooth emption. Similarly, epiphyseal growth
plates of such animais are commonly considered not to cal-, allowing for continuous
axial growth throughout Me. Jee et al. (1991) has describecl this as the partial truth. Rat
epiphyseal growth plates in tibia have been documented to close, but only in very old
animais (16-18 months). It is likely that many researchers use younger, skeletdy
immature rats, which have led to the misconception, or that the animal dies before such
an age. Such a model, unless utilwd at a late stage in its skeletal development would not
mirnic the mature skeletal state of post-menopausal women. Bagi et al. (1997) compared
the histoanatomical and structural characteristics of the femoral neck between human and
rat models. A signincant clifference in mcro- and microanatomy of the proximal femur
was found, a site quite susceptible to osteoporotic fracture in humans. The percent of
cortical bone component is much higher in rats measuring 72.5 % relative to humans at
12.5%. Furthemore, cortical bone at the femoral neck in rats is evenly distributed,
whereas in humans there is a signincant difference in the amount of bone found on the
superior aspect compared to the inferior half of the femoral neck. Considerable
differences also exist in the amount and distribution pattern of the trabecular network.
Other studies performed on the rat, in which pulsatile administrations of PTH
were administered, failed to display the trabecular remodeling phenornenon documented
in larger mammalian species such as the dog and human (Boyce, kW et al., 1996). Rat
bone having smaller trabecular diameters on average however, may represent the
underlying m o n . Wronski et al. (1991) further provide a List of factors, displayhg the
disadvantages associated with the use of the rat model for human bone studies. ûther
than the aforementioned fxtors, Wronski states that the young rat skeleton is not only in
a state of axial growth but modeling events dominate as a means of skeletal adaptation to
load or hcture repair in the absence of subsequent remodeling turnover. Furthemore,
rats lack the impaired osteoblastic function seen in late stages of human estrogen
deficiency-induced osteoporosis. Wronski speculates that "perhaps the rat is too short
lived for thc development of osteoblastic insufficiency after ovarïectomy and is not
predictive of the late estrogen deficient state in women".
6.A.2. THE FERRET
Although the femt represents a new candidate animal model in the bone
metabolic field, it menu strong consideration for M e r investigation into the elucidation
of new therapies for reversal or prevention of postmenopausal bone loss. Mackey and
coworker's 1995 paper entitled 'The ferret as a small animai model with BMU-based
remodeling for skeletal research' has provided an impressive ni vivo pilot study. Of
importance is the documented early cIosure of growth plates between 4 - 7 months post-
natally. These animals unlike rats can live up to 1 1 years of age (Fox, 1988) in captivity,
and hence provide a much younger, skeletally mature, osteoporotic model should Ovx be
induced at such a t h e . PTH has an anabolic effect when administered in a pulsatile
fashion reflecting a physiologie response to the hormone much like larger mammalian
animal models (Boyce et al. 1996). Furthemore, BMU-base Haversian remodeling is
observed in this species noted as early as week 1 1 pst-natally. Finally, reduced systemic
estrogen levels induced via Ovx or exposure to reduced photoperiods (short1), lead to a
senun estradiol level &op, an uterine weight decrease and bone mineral density
reduction. Reduction of tirne spent in lit conditions provides a reversible alternative to
Ovx which may in tum provide a modd for the study of the anabolïcally induced revend
of osteoporosis.
The ferret as an in vitro animal mode1 also deserves M e r attention. Explanted
cultures were easily prepared and cultured for the study of bone nodule elabration and
characterization as well as the study of osteoclastic resorption. The ferret provided an
easily maaaged animal which required little attention and relatively low maintenance
costs. This species also houses a larger marrow supply and possesses a skeletal anatomy
which remodels more closely to our own. Characterization of elaborated bone-Iike tissue
paralleled that documented in the rat. Furthemore osteoclastic resorption of resorbable
substrates demonstrated the utiLity of ferret marrow for osteoclastic assay as weU.
Of particular importance is the difference in estrus patterns noted between
humans and ferrets. Human femdes g e n e d y cycle every 28 days prior to menopause.
Rats are similarly polyestms and cycle every 5 - 6 days. The ferret however is a
monestrus induced ovulator. Estms cycles start in late March and continue through to
August. Ovulation is induced by copulation at any time during estrus and fernale ferrets
wiIl remain in such a state until bred. However, if left in an estrus state females may be
susceptible to aplastic anemia; the depletion of hemopoetic stem cells which may lead to
death. Intact female ferrets can be cycled out of estrus with GnRH injections as
displayed by Mackey et al. (1995). For our studies, male ferrets were utilized to avoid
l intact light cycled (short) - - 8 hrs light/l6 brs dark.
- 84 -
fluctuating hormonal levels associated with intact estrous fernales, and to paralle1 as
closely as possible the maww culhiring protocol introduced by Maniatopoulos et al.
(1988).
While the evidence supporting the use of the ferret for rnetabolic bone research is
growing. we must exercise caution as the ferret itself represents a newly investigated
animal model for scientific research. It has taken many y e m for the rat model to become
so widely accepted and characterized. Further investigations into ferret skeletogenic and
metabolic bone mechanisms are strongly recommended.
63. O S T E O G ~ C ASSAYS
6.B.l. BONE NODULE CHARACTERIZATION
This study has shown that ferret rnarrow stroma, much Wre the rat, has the
potential to elaborate a mineralize matrix which is bone-like in nature provided
dexamethasone, ascorbic acid and beta-glycerophosphate are added to culture medium.
Characterization of the elaborated m a t e included morphological and ultrastructural
evidence generated from LM, SEM, TEM and X-ray diffkaction analysis as wel1 as
histochemical assay. This was in accordance with the methods employed in the mmow
explant study performed on the rat by Maniatopoulos et al. (1988).
Immunohistochemical assay was considered in our characterization protocol, however,
due to the Iack of species specifc antibodies for the non-collagenous proteins of ferret
extracellular matrix or type 1 collagen, this was not attempted. Otherwise,
ultrastnicturally and histochemically, our observations displayed strikingly similar results
to those documented by Maniatopoulos and coworkers (ibid).
The characterization of mineralizing ma& as well as the cells in the immediate
vicinity of the elaborated tissue is essential in determining the bone-like attributes of the
matrix itseif. Since no single dennitive marker exists for bone, a series of analyses were
employed to provide concrete evidence as to the composition and structure of in vitro
bone-like tissue. Many investigators in their characterization of what appears to be in
vitro mineralized ma&, consider these products to be bone-like merely by
microanalytical detection of mineral composition or by pure histochemical &ta (Cheng et
al., 1994; Bruder et al., 1997). Investigations into the arrangement of organic
constituents such as the presence of coliagen or non-collagenous proteins and their ability
to bind and nucleate calcium salts, opposed to ectopic or dystrophie forms (Howlett et al.
1984, 1986), is crucial in the overall characterization of the matrix. Furthemore, cell
morphologies and phenotypes provide supportive evidence as to the Iheage of such ceIis
and the tissue they support. Our mineralized tissues were determined to be bone-like by
employing many of the criteria established for rat bone-like tissue (Maniatopoulos, 1988,
thesis for review).
6.C. Osmocumc REsomo~ Ass~ys
Assays were conducted to examine the resorptive potential of osteoclast-like
ceiis explanted from ferret marrow. As marn>w sources comprise both hemopoetic and
mesenchyrnal cell populations (Owen, 1985) it would seem reasonable to employ a
marrow source for both osteogenic and osteoclastic assay. Cultures were seeded on both
resorbable Ca/P thin f W (Osteologic" discs) and pre-fashioned bovine bone slices.
Furthemore, cultures were plated in FSM conditions or FSM Dex (-) conditions which
have previously displayed the ability to differentiate muitinucleate osteoclasts (Davies, et
al., 1991 and 1993).
The effect of dexamethasone on the differentiation of osteoclasts from
hemopoetic sources and on isolated osteoclasts themselves have been rather
controversial. Dex has been documented as an in vitro stimulator of osteoclastogenesis
(Gronowicz, et al., 1990) by inhibiting the endogenous production of granulocyte
macrophage-colony stimulating factor (Shuto et al., 1994), which may function as a
regulator of osteoclast formation, or by stimulating osteoclastogenesis fiom hemopoetic
blast cells (Kaji, et al., 1997). Conversely, Dex has been implicated as an inhibitor of
osteoclastogenesis (Stern, 1969; Raisz et al., 1972; Liskova-Kiar, 1979) in a dose
dependant manner (Tobias et al., 1989) while displaying complete inhibition by
progesterone antagonism. As progesterone comptes for the glucocorticoid nuclear
receptor, this result suggests a receptor mediated mechanism. Glucocorticoids also
oppose many of the actions of IL- 1 (Thomson et al., 1986) which has profound
osteoclastogenic effects. Furthemore, Taylor et al. (1993) have shown in cocultures
that as chondroprogenitor cell lines are stimulated to differentiate to the more mature
chondrocyte phenotype in the presence of marrow stroma, the differentiation of
osteoclasts decreases. It appears as though Dex has a secondary effect on osteoclast
inhibition via the differentiation of chondroprogenitors whic h, w hen mature, cease to
release a soluble factor essential for osteoclastic differentiation.
The effects of Dex on our culture system appeared to agree with the latter notion
that Dex has an inhibitory effect on osteoclastogenesis. Whether the effect of
dexamethasone on osteoclasts was duect or secondary, acting via local factors which in
turn may alter the kinetic of osteoclastogenesis, or simply alter the viability of mature
osteoclasts in the ferret marrow system, has yet to be elucidated. The ferret osteoclastic
assay was simply a means to characterize the potential of ferret marrow further. It was
not employed to determine the role of dexarnethasone on the system.
6D. DEXAMETHA~ONE (+) PLATING DENSITY MAY
The undenaking of a plating density assay in which cell suspensions were seriaüy
diluted and plated at five different concentrations, halving the original concentration of 4
x 104 celldml (2 x 10' ceUs/T-25) each instance to a final concentration of 2.5 x 103
ceUs/d (1.25 x 104 cells/T-25), allowed us to determine the relationship between bone
nodule count and plating density or time. It is postulated that in vitro bone nodule
elaboration is the result of osteogenic precursor ceil transfer fiom the marrow stroma to
culture. If this concept is valid, we would expect that as ceU plating density increased,
inducable osteogenic colonies responsible for the development of mineralized nodules
would also increase in a directly proportionai manner to the number of cells plated.
These colonies would arise fom single osteoprogenitors which were transferred nom the
original explant. It is however possible that the limiting cell type is an inducer of
osteoblastic differentiation rather tha. the osteoprogenitor itself (Beilows et al., 1989). In
such a case, osteoprogenitors would be present in excess but would only differentiate in
the presence of an inducer cell or its secreted local factods. The results s-d in
section 5.D. show that ferret bone marmw cells, at mid to high plating densities
synthesize a predictable number of mineralized bone-like nodules in vitro which are
&pendant on both cell plating density and time. ThÛ would be in accordance with either
theory (inducer or actual osteoprogenitor as limiting cell). That the number of nodules
increased with t h e in plating densities of similar concentration suggests the possibility
that new ceus. perhaps derived from stromal stem cells, entered the osteoblastic pathway
with tirne. Whether each of the nodules fonned in this study were derived form a single
cell (clonal colony unit) or fiom multiple cells has not yet been determined. Limiting
dilution kinetic studies may provide insight into thk question.
Our ferret m m w seeding density assay appeared to behave in a manner simüar
to that observed in a serially diluted (Aubin et al., 1990) first passage rat stroma assay. In
both cases, bone nodules were elaborated in a linear manner with strong regression values
approaching 1 when cell plating densities were plotted against the number of bone
nodules elaborated. This ability may be contingent on a number of different factors and
are reviewed by Maniatopoulos (1988). However, stromal marrow systems prepared
from both rat and femt marrow explants display different relationships to those
documented in rat calvarid systems (Bellows et al., 1989). Both calvarial and stromal
assays document strong correlations between seeding density and nodules counted.
However, values for calvarial systems intersect the origin displayhg completely linear
relationships at all densities. Stroma1 systems (both rat and ferret) conversely appear to
display non-hem relationships at low platùig densities. Bellow. [oc. cit. postulated thaî
cooperativity between different categones of cells in rat calvarial cell populations was not
required for nodule elaboration and that only one cell necessary for bone nodule
formation was Iimiting. If however, hemopoetic cell populations are retunied to culture
in rat stromal serial dilutions, such results follow a linear relationship at low plating
densities which also pass through the origin. These hdings indicate that the expression
of osteogenesis by bone marrow osteoprogenitors may be under the regulation of other
cells in the bone rnarrow stroma
1. UnIike rats, the ferret displays BMU-based remodehg in cortical bone, which led to
the elaboration of Haversian type secondary osteons. Osteonal channels were cmted
by multinuclear gimt, putative osteoclasts, which displayed characteristic ninled border
morphologies. Osteoblasts subsequentiy deposited new osteoid which would later
rnineraiize ia lari.ie11ar fashion. Observed secondary Haversian osteons consisted of
central neurovamilar canals, embedded osteocytes, and up to 5 concentric rings of
lamehr type bone.
2. Both primary and f k t passage cell cultures, maintaineci in fully supp1emented medium
conditions (AA, BGP, and Dex) on tissue cul^ polystyrene, were able to elaborate
mineralized matrices which were preceded by the deposition of an interfacial matrix
(homologous to the cernent line of bone). Eiaborated mineralized matrices were
chamcterized ultrastnicRiraUy aad histochemidy and were found to display bone-like
qualities.
3. Serial dilution assays designed to asses the osteogenic capacity of explanted ferret
marrow in M y supplemented culture conditions, documented a hear relationship
between cell platkg deosity and nodule count. The relationship between nodule size
and plating density however, displayed no correlation.
4. Rimary marrow explants seeded on comrnercialiy avdable resorbable calcium
phosphate thin nIms (Osteologic" discs) and bovine bone slices, displayed the ability
of tartrate resistant acid phosphatase, putative osteoclasts to be resorptively active.
Explants seeded in M y supplemented culture conditions displayed a significantly
d e r (p4.025) resorption area value (%) when compared to cultures see&d on
other ~steologic" discs in the absence of the synthetic glucocorticoid, dexamethasone.
8. CONCLUSION The ferret in vitro mamw culhire system provides a new and ideal environment in
which the mechanisms of ostcogenic and osteoclastic function can be observed and
assessed. Dependent on dture medium supplementation, ferret marmw osteogenic or
osteoclastic ce& are capable of bone-like matrix elaboration and bone or calcium phosphate
thin nIm resorption respectively. Furthemiore, osteoclastic resorption is decreased
significantly in the presence of dexamethasone.
Ahrengart, L., Lindgren, U. (1986): Prevention of ectopic bone f o d o n by local
application of ethane- 1-hydmxy- 1.1-diphosphonate (EHDP): an expxhental
snidy in rabbits. J. Orrhop. Res. 1: 18-26.
Akamine, A., Tsukuba, T., Kimura, R., Maeda, K., Tanaka, Y., Kato, K, Yamamoto,
K. (1993): Increased synthesis and specific localizaton of a major lysosorna1
membrane sialoglycopmtein (LGP107) at the d e d border membrane of active
osteoclasts. Histochemistry. 100: 10 1-1 18.
Aronow, M.A., Gerstenfeld, L.C., Owen, T.A., Tassinari, M.S., Stein, GA, Lian, J.B.
(1990): Factors that promote progressive development of the osteoblast phenotype
in cultured fetal rat calvaria cells. J. Cell Phsiol. 143: 2 13-22 1.
Aubin, LE., Heersche J.N.M., Medes, ML, Sodek, J. (1982): Isolation of bone ceIl
clones with differences in growth, hormone responses and extraceilular matrix
production. J. Cell BioL 92:452-46 1.
Aubin, J.E., Fong, S. W., Georgis, W. (1990): The influence of non-osteogenic
hemopoetic cells on bone formation by bone marrow stroma1 populations. J. Bone
Min. Res. Absîracts (s81) #3 1.
Aubin, J.E., Liu, F. (1996): The Ostcoblast Lineage. In: Bilezikian, J.P., Raisz, L.G.,
Rodan, G.A. (eds). Principles of Bone Biology. Academic Press, San Diego, pp.
5 1-67,
Bagi, C.M., Wilkie, D., Georgelos, K., Williams, D., Bertolini, D. (1997):
Morphologhl and structural characteristics of the proximal femur in human and
rat. Bone. 2l:S6 1-267.
Bankcroft, J.D., Stevensons, A. (1990): Enyme Histochemistry. Io: Bankcroft, J.D.,
Stevensons, A. (eds). Theones mid Practice of Histological Techniques. Churchill
Livingstone, New York, pp. 387.
Bellows, CG., Aubin, JE., Heersche, JN.M., Antosz, ME. (1986): Mineralized bone
nodules formed ik Miro hm enymaticaUy rieleased rat calvaria œil populations.
Calci$ Tiss. In?. 38: 143- 154.
Bellows, C.G., Aubin, J.E. (1989): Detemiination of numbg of osteoprogenitors present
in isolated fetal rat caivaria ceUs in vitro. Devel. Biol. 133%-13.
Bellows, CG., Heersche, JN.M., Aubia, JE. (1990): Detemiinaton of the capacity for
proWeration and differentiation of osteoprogenitor cells in the presence and
absence of dexamethasone. Devel. Bio. 140: 132- 1 3 8.
Beresforci, J.N., Gallagher, J.A., Poser, LW. (1984): Roduction of osteocalcin by
human bone cek ih vitro. EffectS of 1,25(OH)P3, 24,25(OH)P3, parathymid
hormone and glucocorticoids. Metab. Bone Dis. Relat. Res. 5229-234.
Boyce, R.W., Paddock, C.L., Fraaks, A.F., Jankowsky, M.L., Eriksen, E.F. (1996):
Effects of intermittent m ( 1 - 3 4 ) alone and in combination with l,25(OH)@, or
risedronate on endosteal bone remodeling in canine cancellous and corticai bone.
J. Bone Miner. Res. 11:6ûû-613.
Jaiswal, N., Haynesworth, S.E., Caplan, A.I., Bruder, S.P. (1997): Osteogenic
differentiation of purined, culture-expanded human mesenchymal stem ceils m
vitro. J Cell Biochem. 64295-3 12.
Buckwalter, J.A., Glimcher, M.J., Cooper, R.R., Recker, R (1995): Bone Biology. Part
1. Structure, blood supply, ceUs, matrix and mineralization. J. Bone Joint Surg.
77-A: f 256-1275,
Carrel, A., Burrows, M.T. (1910): Cultivation of adult tissues and organs outside of the
body. Amer. Med. Assoc. J. 55: 1379- 138 1.
Canalis, E. (1983): Effects of g i ~ c ~ ~ o ~ c o i d s on type 1 collagen synthesis, aikaline
phosphatase activity, and deoxyninucleic acid content in cultured rat caivariae.
Endocrinology. 1 l2:93 1-939.
Chaisson, ILB. (1958): Laboratory anatomy of the white rat Brown, W.C. (ed).
hibuque, Iowa. pp. 1-36.
Cheng, SL., Yang, J.W., Rif', L., Zhang, S.F., Avioii, L.V. (1994): Diffmmtiaîion of
human bone marrow osteogenic stromal œlls m vitro: Induction of the osteoblast
phenotype by dexamethasone. Endocri1~12ogy -134277-286.
Consensus Deveiopment Conference V. (1993): Diagnosis, prophylaxis, and m e n t of
osteoporosis. Am J. Med 6546-650.
Cooper, R.R., Milgram, J.W., Robinson, RA. (1966): Morphology of the osteon. An
electron microscopie sîudy. J. Bone Joint Surg. 48: 123% 1270.
Cosman, F., Shen, V., Xie, F., Seibel, M., Ratcliff, A., Lindsay, R. (1993): A
mecbanism of estrogen action on the skeleton: protection against the resorbing
effects of (1-34) hPTH infusion as assessed by biochemical markers. Ann.
Untern. Med. 118:337-343.
Davies, JE., Chemecky, ELT Lowenberg, B., Shiga, A. (1991): Deposition and resorption
of calcined matrix in vitro by rat bone marmw cells. Ceil Mater. 1:3-15.
Davies, J.E., Shapiro, G., Lowenberg, B. (1 993): Osteoclastic resorption of calcium
phosphate ceramic thin nIms. Ce11 Mater. 3:245-256.
Davies, J.E. (1996): In vitro modehg of the bonelimplant interface. me Anat. Rec.
245:426-445.
Doty, S.B. (1981): Morphological evidence of Gap Junctions between bone cells. Calcjc
Tiss, Int. 33509-5 12,
Enlow, D.H. (1958): A comparative histological study of fossil and ment bone tissues.
Part III, Tex. J. Sci. 1 O: 187-230.
Eriksen, E R (1994): Bone Histomorphometry. In: Axehoci, D., Melser, F. (eds). Raven
Press, New York,
F.D.A. (1994): Guidelines for preclinical and clinical evaiuation of agents used in treaûmmt
or prevention of poaîmenopausal osteopomsis. Dbiiion of ntefabolh and
endocrine drug products, FDA. April
Feu, H.B. (1932): The osteogenic capacity in vitro of periosteum and endosteum isolaîed
h m the limb skeleton of fowl embryos and young chicks. Amtomy. 66:157-
180.
Fernandez-Moran, H. (1957). Electron microscopy and X-ray CWhction of bone.
Biochem Biophys. Acta. 23:260-264.
Foote, J.S. (19 16): A contriution to the comparative histology of the femur. Smithsonian
Contrib. To Knowledge. 3595.
Fox, J.G. ( 1988): Taxonomy, history and use. Biology und diseases of the ferret. Lea and
Febiger, USA. pp. 3-13.
Friedenstein, AJ. (1970): The development of fibroblast colonies in monolayer cultures of
guinea-pig bone marrow and spleen ceus. Cell Tissue Kinet. 3:393-403.
Frost, H.M. (1969): Tetracycline based histological analysis of bone remodeling. Caleif
Tiss. Res. 3: 2 1 1 -23 7.
Frost, H.M. (1973): Bone remodeling and its relationship to metabolic bone diseases.
Charles C. Thomas, Sprin&eld, Illinois.
Geddes, A.D. (1996): Animal Models of Bone Disease. In: Bilezikian, J.P., Raisz, L. G.,
Rodan, G.A. (eds). Principles of Bone Biology. Academic Ress, San Diego, pp.
1343-1352.
Geusens, P., Schot, L.P., Nijs, J., Dequeker, J. (1991): Calcium deficient diet in
ovariectomized dogs limits the effects of 17 beta-estradio1 and nandrolone
decanoate on bone. J. Bone Min. Res. 6:79 1-797.
Girasole, G., Jilka, R.L., Passeri, G. (1 992): 17 beta-estradiol inhibits interleukin-6
production by bone marrow-derived stroma1 ceus and osteoblasts in vitro. J. Clin.
ïnvest. 89:883-89 1.
Gray, H. (1980): Osteology. In: Williams, P L and Warwich, R. (eds). Gmy's Anatomy
36th e d Churchill Livingstone, New York. pp. 230-41 8.
Grigoriadis, AB., Heersche, J.N.M., Aubin, JE. (1988): DBefentiation of muscle, fat,
cartilage, and bone from progenitor ce& present in a bone-deriveci clonal œlI
population: effect of dexamethasone. J Cell Biol .106:2 139-5 1.
Grese, TA., Cho, S., Fidey, D.R., Godfkey, AG., Jones, C.D., Lugar, C.W., Martin,
M.J., Matsumoto, K. Pe~ington, L.D., Winter, M.A., Adnan, M.D., Cole,
H.W., Magee, DE., Phdlips, DL., Rowley, E.R., Short, L.L., Glasebrook,
A.L., Bryant, H.U. ( 1997): Structure-activity relatiomhips of selective estrogen
receptor modulators: modincations to the 2-arylknzothiophene core of raloxifene.
J. Med. Chem. 40: 146- 167.
Gronowicz, G.A., McCarthy, M.B ., Raisz, L.G. (1990): Glucocorticoids stimulate
resorption in fetal rat parietal bones. J. Bone Min. Res. 5: 1223- 1230.
Gronowicz, G.A., McCarthy, MB. (1995): Glucocorticoids inhibit the attachment of
osteoblasts to bone extraceUular matrix proteins and decrease beta Lintegrin
levels. Endocrinology. 136:598-608.
Hall, B.K. (1988): The embryonic development of bone. Am. Sci. 76:174-18 1.
Hattersley , G., Chambers. T.J. (1 989): Calcitonin receptors as markers for osteoclastic
differentiation. Endocrinology. 3: 1606-1 6 12.
Heany, R.P. Matkovic, V. (1995): Inadquate Peak Bone Mass. In: Riggs, B.L. &
Melton, L.J. (eds). Osteoporosk Etiology, Diagmsis mid Munagement, Second
Edition. Lippincott-Raven, Philadelphia. pp. 1 15- 13 1.
Herbert, J. (1989). Light as a multiple control system on reproduction in mustelids. In:
Seal, U.S., Thorne, E.T., Bogan, M.A., and Anderson. J.H. (eds).
Conservation Biology and the Black-foted Feret. Yale Univ. Press. pp. 138-
159.
Herbertson, A., Aubin, J E (1995): Dexamethasone dten the sub-population make-up of
rat bone marrow stromat ceU cultures. Bone Min. Res. 10:285-294.
Herbertson, A., Aubin, J.E. (1997): Ceii sorthg enriches osteogenic populations in rat
bone mam>w stromal ceil cultures. Bone, in press.
Horton, W.A. (1990): The biology of bone growth. Growth Genet. Hom. 6(2): 1-3.
Horowitz, M.C. (1993): Cytokines and estrogen in bone: Anti-oskoporotic effects.
Science. 260:626-627.
Hosseini, M.M.. Peel, S.A.F., Davies, J.E. (1996): Collagen fibers are not required for
initial ma&k mineralization by bone cells. Cells & Mut. 6: 233-250.
Howlett, C.R., Owen, M., Cave, J., WiIliamson, M., Bab, I., Maybee, S., Trifitt, J.T.
(1 984). Zn vitro minerabation and aikaiine phosphatase activity in cultures of
rabbit bone marrow s t r o d cells. Calcf T h Int. 36(Supp. 2 ) S-67.
Howlett, C.R., Cave, J., Williamson, M., Farmer, J., Ali, S.Y., Bab, I., Owen, M.
(1986). Minerahaiion in m vitro cultures of rabbit marrow stromal cells. Clin.
Orthop. 213:25 1-263.
Hughes-Fulford, M., Lewis, ML. (1996): Effects of rnimgravity on osteoblast growth
activation. Exger. Cell Res. 224: 103- 109.
Jee, W.S.S. (199 1): The aged rat mode1 for bone biological shidies. Cells Mater. 1:75S-
84s.
Jones, S.J., Boyde, A. (1977): Some morphologicd observations on osteoclasts. Cell
Tiss. Res. 185387-397.
Jowsey, J. (1968): Age and species clifferences in bone. ConieZZ Vetergiarian.
58(supp):74-94.
Kaji, H., Sugimoto, T., Kanatani, M., Nishiyama, K, Chihara, K. (1997):
Dexamethasone stimulates osteociast-like ceII formation by directly acting on
hemopoetic blast cells and enhances osteoclast-like œll formation stimulated by
parathymid hormone and prostaglandin &. Bone Min Res. 12734-741.
Kaplan, F.S., Hayes, W.C., Keaveny, TM., Boskey, A., Einhorn, T.A., Iannotti, J.P.
(1994): Fom and Function of Bone. In: Simon, S.R. (ed). Orthopedic Busic
Science . Port City Press, pp. 127- 184.
Kasperk, C., Schneider, U., Sommer, U.. Niethard, F., Ziegler, R. (1995): Differential
effects of glucocorticoids on human osteoblastic ceil rnetabolism in vitro. Cal@
Tissue In?. 573120-126.
Ke, H.Z.. Jee, W.S.S., M o , S . Li, X I KimmeI, D.B. (1992): Enects of long term
daily admhktratXon of prostaglandin-E, on maintahhg elevated proximal h'bid
metaphyseal cancellous bone mass in male rats. Calcif Tiss. Int. 50245-252.
Khosla, S., Melton, 1. (1995): Secondary Osteoporosis. In: Riggs, B.L. & Melton, L. J.
(eds). Osteoporosis: Etiology. Diagnosis mtd Management. Second Edition.
Lippincott-Raven, Philadelphia. pp. 183-204.
Kidder, L.S., Klein. G.L., Gundburg, C.M., Seitz, P.K., Rubin, N.H., Simmons, D. J.
(1993): Effccts of alirminum on rat bone œil populations. Calcg Tissue Int.
5:357-36 1 .
Kimmel, D.B. (1991): The oophorectomized beagle as an experimental mode1 for estrogen
depletion bone loss in the aduit human. Cells Mater. k75S-84S.
Lang, H., Mertens, T., Gerlach, K.L. (1990): Bone regmeration following the
implantation of osteoblasts fiom ceil cultures. Dtsch Z Mund Kiefr Gesichtschir.
3:224-228.
Liskova-Kiar, M. (1979): Mode of action of cortisol on bone resorption in vitro. Am. J.
Anat. 156:63-68.
h ~ k h , RM., Williamson, M.C., Beresford, JN-, T s t t , J-T., Owen, ME. (1995): In
vitro effects of growth factors and dexamethasone on rat rnairow stroma1 cek.
CIà O h o p . 3:27-35.
Mackey, M.S., Stevens, ML., Eberts, D.C.. Tressler, DL.. Combs. K.S., Lowry,
C.K., Smith, P.N., McOsker, J E . (1995): The ferret as a smaii animal mode1
with BMU-based remodehg for skeletal research. Bone. 17: 19 1 S- 196s.
Maniatopoulos, C., Sodeck, J., Melcher, AH. (1988): Bone formation bz vitro by stroma1
cells obtained fiom bone marrow of young adult rats. Ce11 and Tiss. Res.
254:3 17-330.
Mann, D.R-, Gould, KG-, Collins, D.C., (1990) A potentiai primate mode1 for bone loss
d t i n g k m medical oophorectomy or menopause. "1. Clin. Endocrinol. Met&.
71: 105-1 10.
Marks, S.C. Jr., Hermey, D.C. (1996): The Structure and development of bone. In:
Bilezikian, J.P., Raisz. L.G., Rodan, G.A. (eds). Prkiples of B o ~ e Biology.
Acadexnic Press, San Diego, pp. 3-14.
Matsumoto. T e . Ezawa, L, Monta, IL, Kawanobe, Y.. Ogata, E. (1985): Effects of Vit D
metabolites on bone metabolism in a rat mode1 of postmenopausal osteoporosis. J.
Nu*. Sci. Vitminul. 31(supp):S6 1 -S65.
Mattsson, J.P., Vaananen, K., Wallmark, B., Lorentzon, P. (1991): Omeprazol and
banlomycin, two proton pump inhibitors: differentiation of their effects on gastric,
kidney and bone El(+)-transloeating ATPases. Biochem. Biophys. Acta.
lO6S:26 1-268.
McCarthy, T.L., CentreUa, M., Canalis, E. (1989): Regulatory effects of insulio-like
growth factor 1 and II on bone coUagen synthesis in rat calvarial cultures.
Endocrinology. W:3O 1-309.
Merck & Co.. Inc. Webpage: http://www.merck.com
Mosekilde, L., Weisbrode, S.E., et al, (1993): Evaluation of the skeletal effects of
combined mild dietary restriction and ovariectomy in Sinclair S- 1 rninipigs: A pilot
study . J. Bone Mher. Res. 8: 1 3 1 1 - 1 3 2 1.
Nakamura, I., Takahashi, N., Sasaki, T., Jimi, E., Kurokawa ,T., Suda, T. (1996):
Chemicai and physical properties of the extrace1lula.r ma&k are required for the
acth ring formation in osteoclasts. J Bune Miner Res. 12: 1873- 1879.
Nehissi, J-R., Vignery, A., Puzas, J.E., Baron, R (1982): Histomorphometry and
autoradiography of cultumi fetal rat long bones. Anat. Rec. 2û4: 105- 1 12.
Netter, FH. (1989): Atlas of human anatomy. Ciba-Geigy, USA. 4.459.
Newman, E., Turner, AS., Wark, J.D. (1995): The potentid of sheep for the study of
osteopenia Current status and cornparison with other animai models. Bone.
16277s-284s.
Nijweide, P.J., Burger, E.H., Klien Nuiend, J., Van der Plas, A. (1996): The Osteocyte.
In: Bilezikian, J.P., Raisz, L.G., Rodan, G.A. (eds). Phciples of Bone
Biology. Academic Press, San Diego, pp. 115-126.
Ohgushi, H., Dohi, Y., Katuda, T., Tamai, S., Tabata, S., Suwa, Y. (1996): In vitro
bone formation by rat mmow cell culture. J. Biomed. Mut. Res. 32:333-340.
Ousler, ML, Osdoby, P., Pyfferoen, I., Riggs, B.L.. SpeIsberg, T.C. (19%): Man
osteoclasts as estrogen target cells. Proc. Nat1 Acad. Sei USA. 88:6613-6617.
Owen, M. (1985). Lineage of osteogenic cells and their rdatiomhip to the stroma1
population system. In: Peck, W.A. (ed). Bone mid mineri research, vo1.3.
Elsevei~, -Amsterdam, pp. 1-25.
Owen, TA., Aronow, M., Shalhoub, V., Barone, LM., Wilming, L., Tassinari, M. S.,
Kennedt, M.B., Pockwinse, S., Lian, J.B., Stein, G.S. (1990): Progressive
developnent of the rat osteoblast phenotype in viîm. Reciprocal relationships in
expression of genes associated with osteoblast proMeration and merentiation. J.
Cell Physiol. 143:420-430.
Parfitt, AM. (1983): The physiologicd and cIinical significance of bone
histomorphometric &ta In: Recker RR (ed). Bone h i s t o m o r p h o ~ : techniques
mrd ùiterpretation. CRC Press, Boca Raton, Florida, pp. 143-223.
Parker, E., Hosseini, MoM., Davies, JE. (1997): Human bone tissue growth m vitro.
19th Ann Meeting of the Am. Soc. for Bone Min. Res. (Abstract F298)
Petilli, M., Fiorefi, GaT Benvenuti, S., Frediani, U., Gori, F., Brandi, ML. (1995):
Interactions between iprifiavone and the estrogen receptor. Calcg Tissue Znt.
56: 160-165.
Pfeilschifer, J., Seyedin, SOM., Mundy, G.R. (1988): Transforming growth factor beta
inhiits bone resorption in fetai rat long bone cultures. J. Clin. Invest. 82:680-
685.
Pharoah, M. J., Heersche, J.N.M. (1985): 1 ,î5-DihydroxyVitadn D3 causes an iacrease
in the number of osteoclast-iike ceIls in cat bone rnarrow cultures. Calcif: Tissue
Int. 37:3 276-8 1 . Pilliar, R.M., Lee, J.M., Maniatopoulos, C. (1986): Observations on the effects of
movement on bone ingrowth into porous-surfaced implants. C h Olthop.
208: 108-1 13.
Pope, N.S., Gould, K.G., Anderson, D.C., Mann, D.R. (1989): Effects of age and sex
on bone density in the rhesus monkey. Bone. 10: 109-1 12.
Pntcharcl, J.J. (1952): A cytologicd and histochemid study of bone and cartilage
formation in the rat. J. Anat. 86:259.
Puelacher, W.C., Vacanti, J.P, Fermo, N.F., Schloo, B., Vacanti, C.A. (1996): Femoral
shaft reconstruction using tissueengineered growth of bone. J. Oml Mdlofac.
Surg. 3:223-228.
Rizas, J.E., Jensen, LA. (1982): Ezectrophoretically separated bone tell types fiom the
fetal calvarium: a histociiemical and biochemical study. Himchem. J. 14561-
571.
Raisz, L.G., Tnrmmel, CL., Werner, J.A., Simmons, H. (1972): Effect of
glucocorticoids on bone resorption in tissue culture. Endocrinology. 90:961-967.
Rodan, G.A., Raisz, L-G., Bilezikian, J.P. (1996): Pathophysiology of Osteopomsis. In:
Bilepkian, J.P., Raisz, L.G., Rodan, G.A. (eds). PTinciples of Bone Biology.
Academic Press, S a . Diego. pp. 1343-1352.
Rogers, J.B., Monier-Faugere, M.C.. Mailuche, H. (1993) Animal modds for the study
of bone loss after cessation of ovarian function. Bone. 14:369-377.
Rogers, M.J., Chilton, KM., Coxon, F.P., Lawry, J., Smith, M.O*T Suri, S., Russell,
R.G. (1996): Bisphosphonates induce apoptosis in mouse macrophage-like cells
in vitro by a nihic oxide-independent mechanism. J Bone Miner. Res. 11: 1482-
1491.
Ruth, EB. (1953): Bone saidies, II. An experimental study of the Haversian-type vascula.
channels. Am. J. Anat. 93:429-455.
Salo, J., Metsikko, K., Paiokangas, H., Lehenkari, P., VWiinen, K. (1996): Bone-
resorbing osteoclasts reveal a dynamic division of basal membrane into two
different domains. J. Ce11 Sci. 106: 30 1-307.
Shen, X., Roberts, E., Peel, S.A.F., Davies, J.E. (1993): Organic extracellular matrix
components at the bone ceW substratum interface. Cells Mat. 3: 257-272.
Shevde, N.K, Pike, J.W. (1996): Estrogen modulates the recruitment of myelopoietic celi
progenitors in rat through a stroma1 ceil-independent mechanism. Blood.
87:2683-2692.
Shuto, T., Kukita, T., Hirata, M., Jimi, E-, Koga, T. (1994): Dexamethasone stimulates
osteoclast-like cell formation by inhibithg granulocyte-macrophage colony-
stimdating factor production in mouse bone marrow cultures. Endocrinology.
l34:112l-l126.
Skinner, H.C.W. (1979): Bone: Ce1Inlar and Molenilar 0rgani;ration. In: AIbright, LA.
and Brand, ILA. (eds). Scient@ Barns of Ortopaedics. Appieton-Century-Crofts,
New York, pp. 105- 134.
Sodek, J., Zhang, Q., Goldberg, H.A.. Domenicucci, C., Kasugai, S., Wrana, J.L.,
Shapiro, H., Chen, 1. (199 1): Non-collagenous bone proteins and their rok in
substrate-induced bioac tivity . In. Davies, LE. (ed). nie Bone-Biomaterial
Interface. University of Toronto Press, Toronto, pp. 97-1 10.
Somjen, D., Waisman, A., Kaye, AM. (1996): Tissue se1ective action of tamoxifen
methiodide, raloxifene and tamoxifen on d e kinase B activity in vitro and in
vivo. J Steroid Biochem. Mol. Biol. 59~389-396.
Spencer, G.R. (1979): Pregnancy and lactational osteoporosis. Animal model: Porcine
lactational osteoporosis. Am. Pathol. 95227-280.
Stem, P.H. (1969): Inhibition by steroids of parathyroid hormone- induced " ~ a release
fiom embryonic rat bone in vitro. J. Phannacol. Erp. mer. l68:2 1 1-2 17.
Suda, T., Nobuyuki, U., Nobuyuki, T. (1996): Cek of Bone: OsteocIast Generation. In:
Bilezikian, J.P., Raisz, L.G., Rodan, G.A. (eds). Princeles of Bone Biology.
Academic h s s , San Diego, pp. 87- 103.
Sudo, H., Kodama, H.A., Amagai, Y., Yamamoto, S., Kasai, S.C. (1983): In vitro
differentiation and caicification in a new clanal oseogenic ceil h e denved fiom
newborn mouse calvaria. J. Cell Biol. 96: 19 1- 198.
Takahahi, N., Akatsu, T., Udagawa, N., Sasaki, T., Yamaguchi, A., Moseley, J.M.,
Martin, T., Suda, T. (1988): OsteobIastic ce& are involved in osteoclast
formation. Endocrinology. 123:2600-2602.
Taylor, L.M., Turksen, K., Aubin, LE., Heersche, J.N.M. ( 1993): The osteoclast
dinerentiation in cocultures of a clonal chondrogenic ceiî line and mouse bone
marrow cells. Endocrinology. 133:2292-2300.
Tenenbaum, H.C., Heersche, J.N.M. (1985): Dexamethasone stimulates osteogenesis in
chick penosteum in vitro. EndocrU10logy. 85: 1 175-22 1 1.
Teti, A., Grano, M., Colucci, S., Argentho, L., Zmbonin =one, A. (1990):
OsteobIast-osteoclast interaction in bone resorption. Preliminary results. Bull Soc
Ital Biol Sper. 665 427-3 1.
Thompson, A S D . (195 1): A history of the ferret. J. Hist. Med. 6:47 1 .
Thomson, B.M., Saklatvala, J., Chambers, T J. (1986): Osteoclasts mediate hterleukin 1
stimulation of bone resorption by rat osteoclasts. J. Erp. Med. 164: 104-1 10.
Thomson, B.M., Bennett, J., Dean, V., Triffitt, J., Meikle, MC., Lovendge, N. (1993):
PreIiminary characterization of porcine bone marrow stromal cek: skeletogenic
potential, colony forming activity, and response to dexamethasone, transfonning
growth factor beta and basic fibroblast growth factor. J. Bone Miner. Res.
10: 1173-1 183.
Tobias, J., Chambers, TJ. (1989): Glucocorticoids impair bone resorptive activity and
viability of osteoclasts disaggregated from neonatal rat long bones.
Endocrînology. 125: 1290- 1295.
Tran Van, P., Vignery, A., Barror, R. (1982): An electron microscopie study of the bone
remodeling sequence in the rat. Ce11 Tiss. Res. 225283-292.
V m h e n , K., Horton, M. (1995): The osteoclast clear zone is a specialized œll-
extracellular matrix adhesioa structure. J. Cell Sci. 1 O8 : 2729-2732.
ViBnhen, K. ( 1996): Osteoclast Function: Biology and Mechanisms. In: Bilezikian, J . P.,
Raisz, L.G., Rodan, G.A. (eds). Principles of Bone Biology. Academic Press,
San Diego, pp. 103-1 13.
Vailas, A.C., Vanderby, R. Jr., et al. (1992): Adaptions of young adult rat cortical bone to
14 days of space fiight. J. Appl. Physiol. 73 (supp):4S-9s.
von Ebner, V. (1875): Ueber den feineren Bau der Knochensubstanz S A . Akad. Wiss.
Wien, math.-nat. KI. 72:49- 138.
Vuorïo, T., de Crombrugghe, B. (1990): The f d y of collagen genes. h a Rev.
Biochem. 59:837-872.
Weiss, A., Arbell, I., Steinhagen-Thessen, E., Si1berman.u. M. (199 1): Structural changes
in aging bone: Osteopenia in the proximal
172.
Wesolowski, G., DUO^^, L.T., Lakkakorpi, P.T.,
femurs of f e d e mice. Bone. 12: 165-
Nagy, R-M-, Tenika, K., Tanaka, H.,
Roda, G.A., Rodan, SB. (1995): Isolation and characterization of highly
enriched, perfusion mouse osteoclastic cells. Erp. Ce11 Res. 219:679-686.
Wong, G.L., Cohn, D.V. (1974): Separation of parathymid hormone and calcitonin
sensitive ce&, f h n non-responsive bone ceils. Natzwe. X2:7 13-7 15.
Wong, G.L., Cohn, D.V. (1975): Target cells for parathyroid and calcitonin are different:
enrichment for each cell type by sequential digestion of mouse calvaRa and
selective adhesion to polymeric surfaces. Proc. Natl. Acad Sci. USA. 72: 3 167-
3171.
World Health Organization (WHO) webpage: http:/lwww .w ho.org .
Wronski, TJ*, Yen, C.F. (1991): The ovariectornized rat as an animal mode1 for
postmenopausal bone loss. Cells Mater. 1:69S-74s.
Wu, D.D., Boyci, R.D., Fi, T.J., Burr, D.B. (1990): Regional patterns of bone loss and
altered bone remodehg in response to calcium deprivation in laboratory rabbits.
CaZcif. Tiss. Int. 47: 1 8-23.
Zhou, H. (1993): Cernent üne formation in rat fernord bone. M.Sc. Thesis, uni ver si^ of
Toronto, Depariment of Dentistry.
Zhou, H., Chemecky, R., Davies, J.E. (1994): Deposition of cernent at reversal lines in rat
femoral bone. J. Bone Min. Res. 9:367-374.
l MAGE EVALUATION TEST TARGET (QA-3)