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1 ‘ LIBRARY
3 _) o a Michigan State
University

This is to certify that the
thesis entitled

THE ROLE OF POLOXAMER 188 IN THE PREVENTION OF
NECROTIC CELL DEATH FOLLOWING A SEVERE BLUNT
IMPACT

presented by

Daniel Michael Phillips

has been accepted towards fulﬁllment
of the requirements for the

MS. degree in Mechanical EnﬁeerinL

 

 

 

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2/05 p:lClRC/Date0ue.indd-p.1

 

THE ROLE OF POLOXAMER 188 IN THE PREVENTION OF NECROTIC
CELL DEATH FOLLOWING A SEVERE BLUNT IMPACT

By

Daniel Michael Phillips

A THESIS

Submitted to
Michigan State University
In partial fulﬁllment of the requirements
For the degree of

MASTER OF SCIENCE
Department of Mechanical Engineering

2005

ABSTRACT

THE ROLE OF POLOXAMER 188 IN THE PREVENTION OF NECROTIC
CELL DEATH FOLLOWING A SEVERE BLUNT IMPACT

By
Daniel Michael Phillips
Clinical studies of traumatic joint injury often document early pain and
development of chronic diseases, such as osteoarthritis (OA). Various studies from the
literature suggest that OA can be initiated by cell death that occurs in articular cartilage
during mechanical trauma to the joint. The current study investigates chondrocyte cell
death following blunt trauma and the roles of apoptosis and necrosis. TUNEL analysis
was used to determine that a small degree of apoptotic cell death was observed in bovine
chondral explants 48 hours following blunt impact. In previous studies on chondral
explants from our laboratory it was observed that 30 — 4O % of the cells die in the ﬁrst 24
hours following this same mechanical insult, therefore it was concluded that necrosis,
rather than apoptosis, must be the primary mechanism of cell death. A nonionic
surfactant, Poloxamer 188 (P188), was explored as a potential tool for early intervention
into the disease process, as this surfactant has been shown to repair damaged cell
membranes in other cell types. Bovine cartilage explants were treated with P188
following trauma and showed a signiﬁcant decrease in the amount of cell death versus
explants not treated with P188. When explants pre-treated with P188 were compared
with those pre-treated and post—treated following trauma, it was determined that the post-
treatment of cartilage with P188 had a greater effect than pre-treatment. In these studies,
P188 was found to have a greater effect at lower levels of impact stress than at higher

levels of trauma. Finally, the effectiveness of P1 88 was investigated in the in vivo

ACKNOWLEDGMENTS

I would ﬁrst like to thank my mentor and professor Dr. Roger Haut for giving me
the chance to work in his laboratory. Without his help, I never would have made it this
far.

I would also like to thank the other members of my thesis committee Dr. Michael
Orth and Dr. Neil Wright.

I would like to thank Cliff Beckett for all of his technical expertise and Jean
Atkinson for her help with the care of the animals.

I would like to acknowledge my fellow students in the Lab for all their help and
support: Eric Clack, Eric Meyer, Vijay Jayaraman, Steve Rundell, Joanne Ewen, and
Derek Baars.

I would like to thank my parents for all their love and support. They have always
believed that I could make it here and never gave up on me when I began to slip up in the
past.

I would like to thank Traci, who has always been there for me and will continue
to push me to do great things. Without your help I never would have ﬁnished this thesis.

And lastly, to Rigby and Autumn who are always ready with a slobbery kiss just

when I really need it.

iii

TABLE OF CONTENTS

LIST OF FIGURES ........................................................................................................... vi
INTRODUCTION ............................................................................................................... 1
CHAPTER 1

THE ROLE OF APOPTOTIC CELL DEATH FOLLOWING BLUNT TRAUMA IN
CARTILAGE EXPLAN TS

Introduction .................................................................................................................. 14

Methods ........................................................................................................................ l 7

Results .......................................................................................................................... 21

Discussion .................................................................................................................... 24

References .................................................................................................................... 27
CHAPTER 2

THE USE OF A NON-IONIC SURFACTANT (P188) TO SAVE CHONDROCYTES
FROM NECROSIS FOLLOWING IMPACT LOADING TO CHONDRAL EXPLANTS

Introduction .................................................................................................................. 29

Methods ........................................................................................................................ 32

Results .......................................................................................................................... 36

Discussion .................................................................................................................... 40

References .................................................................................................................... 42
CHAPTER 3

THE ROLE OF POLOXAMER 188 IN SAVING CHONDROCYTES FROM
NECROTIC CELL DEATH FOLLOWING VARIOUS MECHANICAL LOADS

Introduction .................................................................................................................. 44

Methods ........................................................................................................................ 46

Results .......................................................................................................................... 49

Discussion .................................................................................................................... 5 1

References .................................................................................................................... 53
CHAPTER 4

THE EFFECT OF PRE-TREATMENT VERSUS POST-TREATMENT OF
POLOXAMER 188 IN TREATING CHONDROCYTES DAMAGED BY
MECHANICAL LOADS

Introduction .................................................................................................................. 55
Methods ........................................................................................................................ 57
Results .......................................................................................................................... 61
Discussion .................................................................................................................... 63
References .................................................................................................................... 66

iv

CHAPTER 5

THE USE OF A RABBIT ANIMAL MODEL TO DETERMINE THE IN VIVO
EFFECTIVENESS OF POLOXAMER 188 IN SAVING CHONDROCYTES
DAMAGED BY MECHANICAL LOADS

Introduction .................................................................................................................. 67

Methods ........................................................................................................................ 69

Results .......................................................................................................................... 76

Discussion .................................................................................................................... 79

References .................................................................................................................... 84
APPENDIX A

Standard Operating Procedures .................................................................................... 86

LIST OF FIGURES

Note: Images in this thesis are presented in color.

INTRODUCTION
Figure l .......................................................................................................................... 1
Figure 2 .......................................................................................................................... 2
Figure 3 .......................................................................................................................... 3
Figure 4 .......................................................................................................................... 4
Figure 5 .......................................................................................................................... 6
Figure 6 ......................................................................................................................... 7
Figure 7 ......................................................................................................................... 8
Figure 8 ......................................................................................................................... 9
CHAPTER ONE
Figure l ........................................................................................................................ 14
Figure 2 ........................................................................................................................ 15
Figure 3 ........................................................................................................................ 18
Figure 4 ........................................................................................................................ 20
Figure 5 ........................................................................................................................ 22
Figure 6 ....................................................................................................................... 23
CHAPTER TWO
Figure 1 ........................................................................................................................ 30
Figure 2 ........................................................................................................................ 30
Figure 3 ........................................................................................................................ 31
Figure 4 ........................................................................................................................ 33
Figure 5 ........................................................................................................................ 36
Figure 6 ....................................................................................................................... 38
Figure 7 ....................................................................................................................... 39
CHAPTER THREE
Figure 1 ........................................................................................................................ 47
Figure 2 ........................................................................................................................ 49
Figure 3 ........................................................................................................................ 50
CHAPTER FOUR
Figure l ........................................................................................................................ 58
Figure 2 ........................................................................................................................ 61

vi

CHAPTER FIVE

Figure l ........................................................................................................................ 68
Figure 2 ........................................................................................................................ 69
Figure 3 ........................................................................................................................ 70
Figure 4 ........................................................................................................................ 71
Figure 5 .................................... 71
Figure 6 ....................................................................................................................... 72
Figure 7 ....................................................................................................................... 73
Figure 8 ....................................................................................................................... 74
Figure 9 ....................................................................................................................... 76
Figure 10 ..................................................................................................................... 77
Figure 11 ..................................................................................................................... 78

vii

Introduction
The annual cost of automobile accidents is estimated to be $47.8 billion (Rice et
al., 1989). Lower extremity injuries (Figure 1) account for approximately $21.5 billion

dollars annually for treatment, rehabilitation, and lost workdays (Miller et al., 1995).

 

Figure l. Lowereimemity injtuiesamountfor morethan$20 billiondollarsanmnlly,
withalargepercentageoftheseinjuriesoccurﬁngtotheknee.

Studies show that the number of deaths from automobile accidents is declining, but a
high rate of lower extremity injuries still occur (Dischinger et al., 1992). The most recent
analysis of the National Accident Sampling System database shows that approximately
10% of total injuries per year are to the knee (Atkinson and Atkinson, 2000). The
analysis also shows that greater that 50% of knee injuries per year result in contusions,
while less than 5% result in bone fracture. Several studies have shown the presence of
chronic joint disease with the absence of bone ﬁacture (States et al., 1970; Upadhyay et
al., 1983). The most common chronic disease affecting joints is osteoarthritis.
Osteoarthritis (0A) is a chronic disease of the articular cartilage in diarthrodial
joints. Treatment of 0A is both difﬁcult and expensive, and rarely results in relief of
conditions without completely replacing injured joints. Therefore, it is critical that new

techniques for the treatment and prevention of 0A are developed. Speciﬁcally, this

thesis investigates the possible prevention of CA by cellular rescue through the use of a
surfactant, poloxamer 188.

The human body is a complex system composed of many tissues, each with their
own composition and function. The skeletal system provides a framework for organs and
structures that allows movement through interaction with the muscular system. Joints
must transfer forces and absorb high loads with very little friction to permit this
movement. This task is accomplished by articular cartilage (Figure 2) found within

diarthrodial joints.

 

Figurez. 'I'hebonesofthekneejointareooveredwithcartilage, whichiscapableof
distributingtheloadspr‘oduoedaaossthejoint. Thecartilagprovidesthejointwithanear
frictionless anfaoe for movement and minimal wear.

Articular cartilage, along with the synovial ﬂuid, provides the joint with a near
frictionless surface for movement and minimal wear. Cartilage is made up of two phases,
a solid phase and a ﬂuid phase. The solid phase is made up of collagen, proteoglycans
and chondrocytes, while the ﬂuid phase is made up of water and electrolytes. The
macromolecules of the solid phase assemble in the extracellular space to form an
electromechanically and biochemically functional extracellular matrix which is essential
to the ﬁmction of cartilage as a load-bearing tissue (Quinn et al., 1998). Collagen is an

elastic protein that provides the cartilage with its tensile properties. Proteoglycans are

proteins with one or more glycosaminoglycan (GAG) chains attached. The GAG chains

are negatively charged molecules that are closely associated with positive ions that attract
water and impart many of the physical properties to cartilage. This provides an osmotic
pressure to the cartilage matrix, causing tension in the collagen, and allowmg the
cartilage to support compressive loads. The integrity of articular cartilage is highly
dependent on the matrix being intact. Damage to this structure can produce irreversible
changes that accumulate over time because of the low metabolic rate of cells in this

structure.

 

Figure 3. Osteoanhtiﬁsisachmnicdiseaseofﬂlearﬁcnlareartilagefwndinload
bearingjointa Dmingﬂieprogressionofmediseasethecarﬁlageisdegraded,leavingbone
onboneoomact. 'l‘hisconditioncausesextremepainandlossofmobility.

In 0A patients, articular cartilage degrades (Figure 3), resulting in the bones of
the joint coming into contact. This condition can cause extreme pain and loss of
mobility. Acute trauma to the joint is known to increase the risk of 0A. Destabilization
of the knee joint due to anterior cruciate ligament rupture or meniscal damage is also
known to cause radiographic signs of 0A in patients (Kerin et al., 2002). Other
mechanical inﬂuences that cause abnormal forces, such as obesity and muscle weakness,
are also linked to the progression of 0A. Degenerated tissue also has decreased stiffness
and a thickening of the subchondral plate, which have been hypothesized to contribute to

the eroding of the cartilage and the onset of 0A (Ewers et al., 2000).

“.4

L.

Chondrocytes, the resident cells of cartilage, are sensitive to mechanical stimuli,
such as shear strains that develop as a result of blunt trauma. Cell death correlates with
the degree of mechanical disruption on the surface of the cartilage (Oyen-Tiesma et al.,
1999). These researchers also put forth the theory that the majority of cell death occurs
near surface ﬁssures, and with the absence of surface disruptions, minimal cell death
would be observed. Morel et a1 made a similar observation that cell death appeared to be
associated with superﬁcial cracks for relatively high strain rates, but that cell death also
occurred in the absence of cracks, suggesting diﬁ‘erent sets of contributing
micromechanical factors (Morel et al., 2002). Early 0A changes are often observed as

ﬁssures on the articular cartilage surface (Krueger et al., 2003).

Amoulai surtace

Zones

S ADP! ma tangential (10- 2000) {7/

Middle (40-6000)

Deep (30%) ‘X, jv‘y‘,‘ h '1‘,"

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Tide mark

Figure 4. Cartilage is made up of three zones: the superﬁcial, the middle and
the deep. The zones are delineated by the orientation of the collagen ﬁbers. In the deep
zone the collagen ﬁbers are ananged' m a vertical orientation, in the superﬁcial zone the
ﬁbers are arranged parallel to the cartilage surface. Chondrocytes are arranged along
the collagen ﬁbers m the matrix

Chondrocytes are responsible for maintaining homeostasis within the matrix of
cartilage. These cells mediate the synthesis, secretion and degradation of matrix

proteoglycans, glycoproteins and collagen (Figure 4). Shear strains in the matrix of the

cartilage damage chondrocyte cell membranes and interrupt ion gradients leading to
swelling and eventually death. The strain rate, peak stress and peak strain developed in
cartilage during mechanical loads are important parameters for the determination of the
extent of mechanical injury. Cell viability and matrix damage may be aﬂ‘ected by the
speciﬁc applications of strain, strain rate or peak stress (Kurz et al., 2000). Following
injury a reduction in cell density may occur affecting the progression of lesions and their
subsequent repair (D’Lima et al., 2001). This study also hypothesized that further matrix
degeneration and the development of ﬁrll thickness cartilage lesions may result from cell
death and the absence of tissue homeostasis provided by cells.

It is therefore important to understand how mechanical forces affect cartilage. A
theoretical model of cartilage as a transversely isotropic biphasic material under high
rates of loading suggests high shear stresses on the surface of the explant (Ewers et al.,
2001). For a low rate of loading shear stresses may be more uniformly distributed
throughout the thickness of the explant. Due to high rates of loading, brittle material
behavior of the cartilage may cause a smaller zone of high shear stresses coinciding with
cell death adjacent to lesions (Ewers et al., 2000). In contrast, low rates of loading will
cause a more ductile response in cartilage and a larger zone of high shear stress at the tip
of the propagating cracks.

Chondrocytes die by one of two modes, necrosis or apoptosis (Blanco et al., 1998;
Loening et al., 2000; Lucchinetti et al., 2002). Necrosis is the mode of cell death that

occurs when a cell’s membrane is damaged (Figure 5).

VI

.5'
v. I.“

 

Figures. Necrosisischaracterizedbyadisruptionofthecellmembrane. An
omoﬁcimbalanceisaeawdbymelossofmembmmmwgrhy,meceﬂsweﬂswimwater
andevenhnllyhnstsmleasingﬁsceﬂuhrmntentmitssunoundingmvimnmm

The cell swells with excess water and eventually bursts. Necrosis has been referred to as
‘cell murder’ because otherwise healthy cells die due to an external inﬂuence. One study
notes, following blunt impact, most of the observed cell death is necrotic, but after day 1,
a small number of cells show characteristic ultrastructural features of apoptosis (Tew et
al., 2000).

Necrotic cell death can be detected in tissues by using membrane exclusion dyes,
which cannot penetrate intact cell membranes. Because necrotic cells have compromised
cell membranes, ethidium homodimer can enter cells and become incorporated into the
DNA of the cell and cause it to ﬂuoresce. Viable cells are counterstained using a
compound called Calcien AM that is metabolized in the cytoplasm resulting in a

ﬂuorescent byproduct. Tissue sections can then be viewed under a ﬂuorescence

microscope or assayed using ﬂow cytometry.

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Figureé. Dmingapoptosisthenucleusandqtoplasmofthecell
condenmthemrclearnmtaialiscleavedmmmandtheceﬂular
membraneblebstopackageﬂleceﬂularmatenalimaapoptoticbodiesforeasy
removalbyphagocytes.

Apoptosis or ‘programmed cell death’ is a normal physiological process that
maintains tissue homeostasis by eliminating cells that may be harmful to the organism.
During apoptosis, there is internucleosomal cleavage of DNA, the cytoplasm and genetic
material condense, and the cell membrane ‘blebs’ to package ﬁ'agments of cellular
material into apoptotic bodies to facilitate removal by phagocytosis (Figure 6). Internal
organelles retain their structure, but the nucleus, which is unaltered during necrosis,
invariably changes dramatically during apoptosis (Duke et al., 1996). Chen et al found
that apoptosis was a factor in the loss of cellularity in cartilage and that the percentage of
apoptotic cells increased with the duration of loading following repeated 5 MPa impacts
(Chen et al., 2001). Chen et al also make the suggestion that necrosis, rather than
apoptosis, is the earlier event in chondrocyte cell death (Chen et al., 2001). The acutely
traumatized joint is an ideal microenvironment for the occurrence of chondrocyte
apoptosis due to the convergence of several known mediators of chondrocyte
programmed cell death, including matrix injury, the inﬂux of inﬂammatory cells, the
release of proinﬂammatory cytokines, the generation of reactive oxygen species and

nitric oxide, and the potential for continued joint injury from mechanical overload (Kim

et al., 2002). In the long term, apoptotic bodies are retained because cartilage lacks a
blood supply and a source of tissue macrophages.

Apoptosis can be detected in cells by a method referred to as TUNEL: TdT—
mediated dUTP Nick End labeling. The phenomenon of nuclear disintegration during
apoptosis can be used as a marker to detect cells undergoing this process. Cleavage of
genomic DNA during apoptosis is detected labeling free (3 ’_-OH) termini with
ﬂuorescein labeled nucleotides. The reaction is catalyzed by terminal deoxynucleotidyl
transferase in a template independent fashion. The ﬂuorescent DNA is then detected on a
single cell level by ﬂuorescence microscopy or ﬂow cytometry. Although the most
efﬁcient way of identifying apoptosis is by the detection of DNA with strand breaks by
labeling them with biotin or ﬂuorescein-conjugated nucleotides, there may be situations
with extensive DNA degeneration, even selective to internucleosomal DNA, that may
accompany necrosis (Blanco et al., 1998; Chen et al., 2000).

Several non-ionic surfactants have been shown to repair damaged cell membranes
alter injury from physical stress, electrical trauma, and chemical and thermal stresses
(Hannig et al., 2000, Lee et al., 1992, Maskarinec et al., 2002, 2003). Poloxamer 188
(P188) repairs cells by incorporating itself into the damaged portion of the cell membrane
and therefore sealing the defect preventing necrotic cell death (Maskarinec et al., 2002,

Figure 7).

as
H «(001, CH2 9—{0 CH CH2 9—60 CH2 CH2 9-OH
x y z

Figural 'I‘hechemicalstructureofPoloxamer188. P188ismadeupof

twohydmphilicsubgrmpssmmmdingacennalhydrophobicsubgrmp. Duetoits
structure, P188 integrates rtselfrntobtologrcal membranes.

P188 is a triblock copolymer of the form poly(ethylene oxide)-poly(propylene oxide)-
poly(ethylene oxide) with a molecular weight of 8400 Daltons. P188 is a highly surface
active copolymer, which probably aids in its absorption and facilitates its insertion into
lipid monolayers (Maskarinec et al., 2002, 2003). As recent injection experiments have
shown when surface pressure of an artiﬁcial lipid monolayer is decreased below a
threshold level, therefore representing damage, P188 is easily inserted into the
monolayer. On the other hand, at high surface pressures P188 does not insert itself into
the artiﬁcial monolayer. Therefore P188 is incorporated only into the damaged portions

of membranes (Figure 8).

 
  

restoration of
membrane Hilﬁgrtly‘

 

feeradical ~a‘tJTcmkzad-ie 3f 51.2.3
sasver qmg S perox .1731“- m
b.

mitochondrion

Flgnrcll. DiagramshowingapossiblemechanismfortheinsertionofPlss
intothedamagedportionofacellmembrancNaskarinec,2002,2003). The
hydrophobicporﬁmofPlﬂmmahsonthehsideofﬂremembmepmtectedbytbe

P188 has been used for a range of purposes including as a mild detergent to cleanse
wounds, as an emulsiﬁer for non-polar lipids, and as a laxative. It is also used to coat the

surface of membrane oxygenators in order to reduce erythrocyte lysis during

cardiopulmonary bypass (Lee et al., 1992). Because cells dying by apoptosis maintain
intact membranes, P188 has no affect on these cells.

Chapter 1 of this thesis will investigate the role of apoptosis in cartilage explants
following a single blunt trauma. Chapter 2 will investigate the ability of P1 88 to restore
the integrity of damaged membranes, and ‘save’ these cells ﬁom necrosis. Chapter 3 will
examine the role of P1 88 pre-treatrnent only, versus both pre- and post-treatment in
preventing the loss of cell membrane integrity. Chapter 4 will document the
effectiveness of P188 at varying levels of stress. Chapter 5 will deal with the potential
action of P188 in a live animal joint following acute blunt trauma.

The potential for early intervention of a traumatized joint with P188 may
represent an important advancement in the treatment of this trauma patient. The research

presented in this thesis represents a ﬁrst step in this process.

10

References

l.

Atkinson T, Atkinson P. (2000) Knee injuries in motor vehicle collisions: a study of
the National Accident Sampling System database for the years 1979-1995. Accid Anal
Prev. 32(6):779-86

Blanco FJ, Guitian R, Vazquez-Marni] E, de Toro FJ, Galdo F. (1998) Osteoarthritis

chondrocytes die by apoptosis. A possible pathway for osteoarthritis pathology.
Arthritis Rheum. 41(2):284—9.

Borrelli Jr. J, Torzilli PA, Grigiene R, Helfet DL. (1997) Effect of impact load on
articular cartilage: development of an innit-articular fracture model. J Orth Trauma.
11(5):319-326.

Chen CY, Burton-Wurster N, Lust G, Borden C, Bloom S. (2000) Cell death in
articular cartilage alter repeated impacts. 46* Annual Meeting Orthopaedic Resew'ch
Society.

Chen C, Burton-Wurster N, Borden C, Hueﬂ‘er K, Bloom SE, Lust G. (2001)
Chondrocyte necrosis and apoptosis in impact damaged articular cartilage. J Orth
Res. 19:703-711.

Dischinger PC, Cushing BM, Kerns TJ. (1992) Lower extremity fractures in motor
vehicle collisions: Inﬂuence of direction of impact and seatbelt use. 38th Annual
Proceedings, Association for the Advancement of A utomotive Medicine. pp. 319-325.

D’Lima DD, Hashimoto S, Chen PC, Colwell CW, Lotz MK. (2001) Impact of
mechanical trauma on matrix and cells. Clinical Orth Related Res. 391S2S90-S99.

Duke RC, Ojcius DM, Young JD. (1996) Cell suicide in health and disease. Sci Am.
275(6):80-7.

Ewers BJ, Haut, RC. (2000) Polysulphated glycosaminoglycan Treatments can
mitigate decreases in stiffness of articular cartilage in a traumatized animal joint. J
OrthRes. 18:756-761.

10. Ewers B, Dvoracek-Driksna D, Orth M, Altiero N, Haut R (2000) Matrix damage

and chondrocyte death in articular cartilage depends upon loading rate. 46“ Annual

Meeting Orthopaedic Research Society.

11. Ewers B, Krueger J, Dvoracek-Driksna D, Orth M, Haut R (2001) Chondrocyte

viability decreases over 24 hours post-impact in a mechanically traumatized cartilage
explant. 4 7“ Annual Meeting Orthopaedics Research Society.

11

12. Hannig J, Zhang D, Canaday DJ, Beckett MA, Astumian RD, Weichselbaum RR Lee
RC. (2000) Surfactant sealing of membranes permeabilized by ionizing radiation.
RadRes. 154(2):171-177.

13. Kim HT, Lo MY, Pillarisetty R (2002) Chondrocyte apoptosis following
intraarticular fracture in humans. Osteo Cart. 10(9):?47-9.

14. Kerin A, Patwari P, Kuettner IQ Cole A, Grodzinsky A (2002) Molecular basis of
osteoarthritis: biomechanical aspects. Cell Mol Life Sci. 59:27-35.

15. Krueger JA, Thisse P, Ewers BJ, Dvoracek-Driksna D, Orth MW, Haut RC. (2003)
The extent and distribution of cell death and matrix damage in impacted chondral
explants varies with the presence of underlying bone. J Biomech Eng. 125: 1-6.

16. Kurz B, Jin M, Patwari P, Lark MW, Grodzinsky AJ. (2000) Biosynthetic response
and mechanical propeties of articular cartilage aﬁer injurious compression: the
importance of strain rate. 46'” Annual Meeting Orthopaedic Research Society.

17. Lee RC, River LP, Pan F, Ji L, Wollmann RL. (1992) Surfactant-induced sealing of
electropermeabilized skeletal muscle membranes in viva. Proc Natl Acad Sci.
89:4524—4528.

18. Loening AM, James IE, Levenston ME, Badger AM, Frank EH, Kurtz B, Nuttall ME,
Hung H, Blake SM, Grodzinsky AJ, Lark MW. (2000) Injurious mechanical
compression of bovine articular cartilage induces chondrocyte apoptosis. Arch
Biochem and Biophys. 381(2):205-212.

19. Lucchinetti E, Adams CS, Horton Jr WE, Torzilli, PA (2002) Cartilage viability alter
repetitive loading: a preliminary report. Osteo and Cart. 10:71-81.

20. Maskarinec SA, Hannig J, Lee RC, Lee KC. (2002) Direct Observation of Poloxamer
188 insertion into lipid monolayers. Biophysical J. 82: 1453-1459.

21. Maskarinec SA, Lee, KC. (2003) Comparative study of poloxamer insertion into lipid
monolayers. Langmuir. 19:1809-1815.

22. Miller TR, Levy DT. (1995) The effect of regional trauma care systems on costs.
Arch Surg. 130(2):]88-93.

23. Morel V, Quinn TM. (2002) Micromechanical factors in cartilage injury under ramp
compression of explants for deformations applied near the “gel diffusion” rate. 48‘”
Annual Meeting Orthopaedic Research Society.

24. Oyen-Tiesma M, Deloria LB, Meglitsch T, Oegema TR, Lewis JL. (1999) Cell death

aﬁer cartilage impact depends on matrix damage. 45‘” Annual Meeting Orthopaedic
Research Society.

12

25. Quinn TM, Grodzinsky AJ, Hunzinker EB, Sandy JD. ( 1998) Effects of injurious
compression on matrix turnover anround individual cells in calf articular cartilage
explants. J Orth Res. 16:490-499.

26. Rice DP, MacKenzie EJ. Cost of Injury in the United States ——A Report to Congress,
National Highway T raﬁic SqfetyAcbninistration, US. Department of T rartmortation
and Centers for Disease Control, US. Department of Health and Human Services.
1989

27. States JD. (1970) Traumatic arthritis: a medical dilemma. In: Proceedings of the 14'”
Annual Conference of the American Association for Automotive Medicine. 14:21-28

28. Tew SR, Kwan AP, Hann A, Thomson BM, Archer CW. (2000) The reactions of
articular cartilage to experimental wounding: role of apoptosis. Arthritis Rheum.
43(1):215-25.

29. Upadhyay S, Moulton A, Srikrishnamurthy K. (1983) An analysis of the late eﬂ‘ects

of traumatic posterior dislocation of the hip without fractures. J Bone Joint Surgery
65-B(2): 150-152.

13

Chapter One

The Role of Apoptotic Cell Death Following Blunt Trauma
In Cartilage Explants

Introduction

Osteoarthritis (0A) affects more than 21 million Americans, and is the leading
cause of disability in the US. each year (US census bureau, 2000). Clinically the disease
is characterized by joint pain and narrowing of the joint, as diagnosed by radiological
examination (Flores et al., 1998). Pathologically, the disease exhibits a loss of cartilage
and sclerosis of underlying subchondral bone. Histologically there is fragmentation of
the cartilage surface in the diseased joint, cloning of chondrocytes and eventually
violation of the tideka by blood vessels. Studies have associated the physiopathology
of articular cartilage and the time course of 0A with the existence of dead Chondrocytes
(Figure 1) (Blanco et al., 1998, Hashimoto et al., 1998, Hashimoto et al., 1998, Heraud et
al., 2000, Kim et al., 2000).

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cellularcontentsintoapoptoticbodies.

In recent times several new theories for the onset and development of OA have

suggested that chondrocyte apoptosis might be involved as the mode of cell death

14

(Blanco et al., 1998, Loening et al., 2000). In these studies chondrocytes in the
superﬁcial tangential and middle zones of human OA cartilage show the characteristics of
apoptotic cell death, such as nuclear chromatin condensation (Lucchinetti et al., 2002).
Apoptosis, also known as programmed cell death or ‘cell suicide’, is a normal
physiological process that maintains tissue homeostasis and is characterized by a
condensation of cytoplasm and by intemucleosomal cleavage of DNA. In its ﬁnal stages
of cellular death there is blebbing of the cell membrane in order to package fragments of

cellular contents into structures known as apoptotic bodies (Figure 2) (Chen et al., 2001).

 

Figurez. Pictrne showing cellularmembraneblebbingthatocarrsdrning apoptosis.
TheblebbingofthemembraneoccrnstopackageﬂreceﬂularcontentsiMoapoptoticbodiesfor
easyremovalbymacrophages.

In the long term apoptotic bodies are retained because cartilage lacks a blood supply and
a source of tissue macrophages (D’Lima et al., 1994). The acutely traumatized joint is an
ideal atmosphere for the appearance of cellular apoptosis due to several mediators of
programmed cell death including matrix injury, the inﬂux of inﬂammatory cells, the
release of proinﬂammatory cytokines, the generation of reactive oxygen species, and the
potential for continued joint injury ﬁ'om mechanical overload (Kim et al., 2002).
Cartilage wounding with a trephine shows cell death near the lesion due to a gradual

increase in apoptosis, and eventually resulting in a secondary necrosis (Costouros et al.,

15

2003). In contrast, canine cartilage explants subjected to low levels of cyclic
compression exhibit the development of cellular necrosis progressively increasing in
time, whereas apoptosis (TUNEL-positive cells) is not seen until 48 hours or more post
loading (Chen et al., 2001). Tew et al also documented that initially following trauma
most of the observed cell death is necrotic, but after 24 hours post impact a small number
of cells show the characteristic ultrastructural features of apoptosis (Tew et al., 2000).
Chen et al also noted that necrosis rather than apoptosis appeared to be the earlier mode
of cell death following impact loads, but that apoptosis was also involved, suggesting that
cell loss in injured cartilage may involve both apoptosis and necrosis (Chen et al., 2001).
The hypothesis of the current study was that a single, severe blunt insult on
cartilage would induce a small amount of apoptosis, but this type of cellular death would
not be signiﬁcant until 48 -— 96 hours post impact, based on the above literature. In the
current study bovine cartilage explants were subjected to a 30 MPa impact load. A
TUNELassaywasusedtoassessapoptoticcell deathatseveraltimepointswithinthe
ﬁrst96hours. Theimpactloadwasappliedatbothahigh(50 msto peak)andalow(1 s
to peak) rate of loading. These rates and levels of loading were chosen based on previous
studies in the laboratory (Ewers et al., 2000, Krueger et al., 2003), and based on other
studies in the literature (Atkinson and Haut, 1995, Atkinson et a1, 1998, Borrelli et al.,

1997).

16

A . ‘ IA ’ .
if?
a J 1 \. l ' a - I I ’ 3) 1.
I ‘ I l ' ’ .‘

Methods:

Four bovine forelegs from 2 mature animals were obtained from a local abattoir
within six hours of slaughter. The legs were skinned and rinsed with water prior to
exposing the metacarpal joint in a laminar ﬂow hood. A biopsy punch (Miltex
Instrument Company, Bethpage, NY) was used to make forty-four 6mm diameter
chondral explants ﬁom the lower metacarpal surface of the limbs. The explants were
separated from the underlying bone using a scalpel. The specimens were washed three
times in Dulbecco’s Modiﬁed Eagle Medium: F12 (DMEM, Invitrogen Co., Grand
Island, NY, #12500-062). The explants were then equilibrated for 48 hours in
DMEM:F 12 (2 explants per well, approximately 1 mg of cartilage per 1 ml of media)
supplemented with 10% fetal bovine serum (Invitrogen Co), 21.9 mg/ml glutamine,
additional amino acids and antibiotics in a humidity controlled incubator (37° C, 5% C02,
95% humidity).

The explants were randomly assigned to three treatment groups, unimpacted

controls, a high rate of loading group (50ms to peak load), and a low rate of loading

group (Is to peak) (Figure 3).

17

1 Hour
8 Hours

48 Hours

96 Hours

1 Hour

11:44 8Hours

48 Hours

96 Hours
9 Control

(00000000

 

F'ignre3. Diagramshowingtheexperimentaldesign Explantswerefrozenat 1,8,
480r96hourspostimpactandstainedforTUNEL.

All loaded explants were taken to 848 N (~30 MPa), following a 5 N preload, in
unconﬁned compression between two highly polished stainless steel plates using a
haversine loading protocol with a servo-controlled hydraulic testing machine (Instron,
model 1331, Canton, MA). Three biomechanical parameters, peak load, time to peak,
and maximum explant compression were documented in each experiment. The explants
were then re-incubated for 1, 8, 48 or 96 hours in ﬂash DMEM:F12 supplemented
medium. Following incubation, the explants were irnbedded in Optimal Cutting
Temperature media (OCT, Tissue-Tek O.C.T. compound, Sakura Finetek USA Inc,
Torrance, CA), frozen, and stored at -80°C. 8pm thick slices were cut through the entire
thickness of the explant using a cryotome (HistoSTAT rnicrotome, AO Scientiﬁc
Instruments, Buffalo, NY). The slices from the central area of the explant were
immobilized on glass slides, air dried and stored at —80°C. A control explant was treated
with DNase I (Promega, Madison WI) for 10 minutes at room temperature for use as a

positive control. The explant slices were stained for apoptotic nuclei by the terminal

18

deoxynucleotide transferase mediated UTP nick end labeling method (TUNEL, Promega
DeadEnd Fluorometric TUNEL System, Promega, Madison WI). Brieﬂy, the explant
slices were ﬁxed in 4% formaldehyde and then permeabilized in 0.2% Triton X-100. The
slices were then incubated in a 20 ug/ml proteinase K solution (Promega, Madison WI)
for 8 minutes at room temperature. Next, the slices were incubated in an equilibrium
buffer containing a nucleotide mix and TdT enzyme at 37°C for 1 hour to perform the
reaction. Finally the slices were counterstained with a 1 jig/ml propidium iodide solution
for 15 minutes at room temperature to highlight non-TUNEL positive cells. The
specimens were then viewed under a ﬂuorescence microscope (Leica DM LB (frequency:
50-60 Hz), Leica Mikroskopie und Systeme GmgI-I, Wetlzar, Germany) and
photographed using a digital camera (Spot Digital Camera, Diagnostic Instruments Inc).
Each explant slice was divided into three zones: superﬁcial (top 20%), middle (middle
50%), and deep (bottom 30%). Two parallel lines were drawn approximately 2 mm apart
and perpendicular to the explant to outline an area for determination of viable and non-
viable cells. Each explant was divided into its upper 20%, middle 50%, and lower 30%

using each line as a guide (Figure 4).

19

JUpCliiCIJI Lune

 

Figure 4. Using image software, the explants were divided into three zones, the
superﬁcial (top 20%), the middle (50%), and the deep (bottom 30%). Lines were drawn on
the photo to represent the zones.

Another line was drawn centrally on the photograph to help construct the horizontal lines
that demarcated each zone of the explant. This photographic processing was performed
using a commercial program (Corel Draw, Corel Inc., Dallas, TX). A blinded observer,
using image analysis software (Sigma Scan, SPSS INC., Chicago, IL), quantiﬁed cell
viability in each zone of the explant.

The percentage of TUNEL-positive cells was quantiﬁed in each zone. The
maximum applied load, time to peak load and maximum explant compression were
determined from each experiment. A two-factor ANOVA was performed on these
biomechanical parameters and the percentage of TUNEL-positive cells in each group of
the explants. Time (1, 8, 48, 96 hours) and treatment (high rate, low rate or control) were
the factors. A Student-Newman-Keuls (SNK) post-hoe test was used to determine

diﬁ‘erences between groups.

20

Results:

The experiment was performed to determine the percentage of TUNEL-positive
cells following unconﬁned compression of chondral explants following a low (ls to peak)
and a high (Sms to peak) rate of loading. Analysis of the high rate of loading data for the
various groups (1, 8, 48, and 96 hours post trauma) indicated no signiﬁcant diﬂ‘erences
(p>0.05) in any biomechanical parameter. The maximum applied load, maximum
explant compression and time to peak load were (Mean: SD) 832.51: 7.5 N, 0.296:b 0.068
mm and 0.056d: 0.003 ms, respectively. In the low rate of loading experiments the
biomechanical parameters also did not vary signiﬁcamly (p>0.05) between groups, but
themaximumloadandtimetopeakwerediﬁ‘erentthanthoseinthehighrateofloading
experiment. The corresponding values of maximum load, maximum compression and
time to peak load were 851.7:1: 1.1 N, 0.3343: 0.041 mm and 1.0% 0.02 sec, respectively.

TUNEL staining ofthe impacted explants showed only a minimal (<0.5% of all

cells) percentage of TUNEL-positive cells prior to 48 hours post trauma (Figure 5).

21

16-

O1Hou
Dal-bu ‘F
I48l-bu
12+ '98“!!!

14-

 

 

%Apoptotlc Gels
a: a:

 

 

 

, _rI—J.h_ ,r-Iu-L—L

Cortrol MRabLnadm

Flares. Histogramshowdngdretotalpercentagerrt‘TUNﬂpositivecells

foﬂowingbhmtinmactlmdmgatl(whhebam),8(diagmnlstﬁpedbms),48(gray

hars),and96hours(blaekbars). Exphntswererandomlyassignedtooneofthree

groupszconuoLlughmteofloading(50msmpeak),mxlbwrateofloarhng(lsto
peak).

Following a high rate of loading the chondral explants showed only a minimal (<0.5% of

 

all cells) number of TUNEL-positive cells for each time point to 96 hours. Conversely,
in the low rate of loading experiments the percentage of positively stained cells was 7.9:
5.9%and2.8i3.4%atthe48and96hours, respectively. ThepercentageofTUNEL
positive cells in controls at each time point was minimal (<0. 1%), while nearly all of the
cells within the explant were stained in the DNase positive controls. In those explants
showing TUNEL positive cells after loading, these cells were limited to the superﬁcial
zone in all groups (Figure 6). In the low rate of loading group at 48 hours and 96 hours
the percentage of TUNEL-positive cells in the superﬁcial zone was 38.1:1: 26.9 % (n=4)

and 33.4:1: 35.7 % (n=4), respectively (Figure 6).

22

 

 

 

 

 

 

 

 

 

 

 

70
1
m4
Imam
lulu.
50+ DDaapZone
2
34m
3
E...
(U
a?
20.
10«
0 __ q-T'— — _
‘III as “I ”I
l
Catch

 

ll‘lgrrre6. ’I‘lnpercentageof’lUNﬂpositivecellsinthethreezmesofthe
cartilage: superﬁcial(top20%), middle(50%)anddeep(bottom30%). Therewasa
signiﬁcamdiﬂ‘crencemﬂrepercemageofTUNELposiuvecdlsvmtheconﬂol
upmmﬂlemperﬁcialzonesmwand96hmuspoa4mpaafmthehwrateofhading
W

23

Discussion:

This study documented the role of apoptosis in chondrocytes following blunt
trauma to cartilage. Chondral explants were loaded to 30MPa in unconﬁned compression
at low (Is to peak) and high (50ms to peak) rates of loading. The explants were
examined at several time points following loading to determine the percentage of
TUNElrpositive cells. The hypothesis of this study was that the majority of cells injured
during compressive loading would not be TUNEL-positive.

In the study TUNEL-positive cells were observed after a low rate of loading at 48
and 96 hours, but only minimal (< 0.5%) staining was observed in the high rate of
loading. Since the applied load was only slightly (2 %) greater in the low versus the high
rate of loading experiments, we believe one possible factor responsible for the
appearance of apoptotic cells was the rate of loading. Yet, the mechanism of this
response is unknown. The percentage of TUNEL-positive cells following a low rate of
loadingwasapproximately8%at48 hoursandlessthan3 %at96hours. Incontrast, an
earlier study by this laboratory documented approximately 66 % of cells dead at 24 and
96 hours in chondral explants exposed to 30MPa of loading (Ewers, 2001). Thus, the
results of the current study suggest that the primary mechanism of cell death resulting
from blunt mechanical loading is via the necrotic pathway. This result compared well
with experimental data from adult canine chondral explants following low intensity
(5MPa) cyclic impacts (Chen, 2001). In that study, cartilage cyclically loaded for 2 hours
showed 32% of the chondrocytes had leaky cell membranes characteristic of necrosis.
While only 1% of these chondrocytes were TUNEL-positive 4 hours later (at 6 hours), at

48 hours the percentage of TUNEL-positive cells rose to 73%. In this study and in the

24

current investigation, TUNEL-positive cells were limited to the upper zones of the
explant. While TUNEL-positive cells have been thought to largely reﬂect cellular death
via apoptosis, Chen ct al (2000, 2001) and others have also documented TUNEL-positive
responses in necrotic cells (Grasl-Kraupp, 1995, Tew, 2000, Blanco, 1998). Without
verifying the existence of apoptotic bodies using transmission electron microscopy the
mechanism of cell death in the TUNEL-positive cells of the current study is yet
unknown. A possible scenario may be that these cells were less severely traumatized by
the mechanical insult. 0n the other hand, death in these cells may have been due to a
cell-mediated response produced by necrosis in adjacent cells (Levin, 2001).

A limitation of the current study was that osteochondral explants were not
utilized. The attachment of the explant to underlying bone helps limit the extent of cell
death in all zones for a given level of loading (Krueger, 2003). A reduction in the
percentage of cell death in osteochondral versus chondral explants at 30 MPa of
compressive loading is currently thought to be primarily due to a reduction in tissue
strains. Especially in the deep zones of the tissue a reduction of tissue compressive stress
in osteochondral versus chondral explants may produce more apoptosis at a given level of
applied loading. But if cell death is primarily due to excessive cell strains, the
distribution of apoptotic cells nearer the surface may be inﬂuenced by the fact that the
deeper zones of cartilage are stiffer (Schinagl, 1997). Studies by others using intact joints
also show that the location of cell apoptosis following blunt mechanical loading is

primarily in the upper zones of the tissue, similar to the current study using chondral

explants (D’Lima, 2001).

25

A study by another laboratory showed that a pan-caspase inhibitor, z-VAD.fmk
[benzyloxycarbonyl-val-ala-asp (Ome) ﬂouromethylketone] could help prevent
chondrocyte apoptosis in human articular cartilage explants compressed to 30% (D’Lima,
2001). The Caspase chain reaction is the enzymatic process the cell uses to facilitate
apoptosis. By interrupting this enzymatic chain reaction, it is believed that apoptosis can
be prevented in the cell. Caspase inhibition reduced chondrocyte apoptosis in human
chondral explants ﬁom 34% in non-treated specimens to 25% with z-VAD.fmk
intervention at 48 hours.

Further studies are needed to conclude that indeed necrosis, not apoptosis is the
primary cell death mode in cartilage trauma. An osteochondral model should be studied
to investigate the importance of subchondral bone in limiting the extent of necrosis and
apoptopsis. Further, an intervention in the necrotic pathway would be useful if 0A is to

be prevented. These ideas will be further developed in the subsequent chapters.

26

References

l.

Atkinson PJ, Haut RC. (1995) Subﬁ'acture insult to the human cadaver patellofemoral
joint produces occult injury. J Orthop Res. 13(6):936-44.

Atkinson TS, Haut RC, Altiero, NJ. (1998) Impact-induced ﬁssuring of articular
cartilage: An investigation of failure criteria. J Biomech Eng. 120:181-187.

Blanco FJ, Guitian R, Vazquez-Martul E, de Toro FJ, Galdo F. (1998) Osteoarthritis
chondrocytes die by apoptosis. A possible pathway for osteoarthritis Pathology.
Arthritis Rheum. 41(2):284-9.

Borrelli Jr. J, Torzilli PA, Grigiene R, Helfet DL. (1997) Effect of impact load on
articular cartilage: development of an intra-articular fracture model. J 0th Trauma.
11(5):319-326.

Chen C, Burton-Wurster N, Borden C, Hueﬂ‘er K, Bloom SE, Lust G. (2001)
Chondrocyte necrosis and apoptosis in impact damaged articular cartilage. J Orth
Res. 19:703-711.

Chen CY, Burton-Wurster N, Lust G, Borden C, Bloom S. (2000) Cell death in
articular cartilage aﬂer repeated impacts. 46“ Annual Meeting Orthopaedic Research
Society.

Costouros JG, Dang AC, Kim HT. (2003) Inhibition of chondrocyte apoptosis in vivo
following acute osteochondral injury. 0A Cart. 11(10) 756-9.

D’Lima DD, Hashimoto S, Chen PC, Colwell CW, Lotz MK. (2001) Impact of
mechanical trauma on matrix and cells. Clinical Orth Related Res. 391$:S90—S99.

Ewers BJ, Haut, RC. (2000) Polysulphated glycosaminoglycan Treatments can
mitigate decreases in stiﬁhess of articular cartilage in a traumatized animal joint. J
OrthRes. 18:756-761.

10. Ewers B, Krueger J, Dvoracek-Driksna D, Orth M, Haut R (2001) Chondrocyte

viability decreases over 24 hours post-impact in a mechanically traumatized cartilage
explant. 4 7" Annual Meeting Orthopaedics Research Society.

11. Flores RH, Hochberg MC. (1998) Deﬁnition and classiﬁcation of osteoarthritis. In:

Brandt KD, Doherty M, Lohmander LS (Eds). Osteoarthritis, Oxford University
Press, New York. Pp 1-12.

12. Grasl-Kraupp B, Ruttkay-Nedecky B, Koudelka H, Bukowska K, Bursch W, Schulte-

Hermann R (1995) In situ detection of ﬁagmented DNA (TUNEL assay) fails to
discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note.
Hepatology. 5:1465-8.

27

l3. Hashimoto S, Ochs RL, Komiya S, Lotz M. ( 1998) Linkage of chondrocyte
apoptosis and cartilage degradation in human osteoarthritis. Arthritis Rheum.
41(9): 1632-8.

14. Hashimoto S, Takahashi K, Amiel D, Coutts RD, Lotz M. ( 1998) Chondrocyte
apoptosis and nitric oxide production during experimentally induced osteoarthritis.
Arthritis Rheum. 41(7): 1266-74.

15. Heraud F, Heraud A, Harmand MF. (2000) Apoptosis in normal and osteoarthritic
human articular cartilage. Ann Rheum Dis. 59(12):959-65.

16. Kim HA, Lee YJ, Seong SC, Choc KW, Song YW. (2000) Apoptotic chondrocyte
death in human osteoarthritis. J Rheumatol. 27(2):455-62.

17. Kim HT, Lo MY, Pillarisetty R. (2002) Chondrocyte apoptosis following
intraarticular fracture in humans. Osteo Cart. 10(9):747-9.

18. Krueger JA, Thisse P, Ewers BJ, Dvoracek-Driksna D, Orth MW, Haut RC. (2003)
The extent and distribution of cell death and matrix damage in impacted chondral
explants varies with the presence of underlying bone. J Biomech Eng. 12521-6.

19. Levin A, Burton-Wurster N, Chen C, Lust G. (2001) Cellular signaling as a cause of
cell death in cyclically imapcted cartilage explants. 4 7‘” Annual Meeting
Orthopaedics Research Society.

20. Loening AM, James IE, Levenston ME, Badger AM, Frank EH, Kurtz B, Nuttall ME,
Hung H, Blake SM, Grodzinsky AJ, Lark MW. (2000) Injurious mechanical
compression of bovine articular cartilage induces chondrocyte apoptosis. Arch
Biochem and Biophys. 381(2):205-212.

21. Lucchinetti B, Adams CS, Horton Jr WE, Torzilli, PA (2002) Cartilage viability alter
repetitive loading: a preliminary report. Osteo and Cart. 10:71-81.

22. Schinagl R, Gurskis D, Chen A, Sah R. (1997) Depth-dependent conﬁned
compression modulus of full-thickness bovine articular cartilage. J Orth Res.
15(4):499-506

23. Tew SR, Kwan AP, Harm A, Thomson BM, Archer CW. (2000) The reactions of
articular cartilage to experimental wounding: role of apoptosis. Arthritis Rheum.
43(1):215-25.

24. US census bureau, 2000

28

Chapter Two

The Use of a Non-Ionic Surfactant (P188) to Save Chondrocytes from
Necrosis Following Impact Loading of Chondral Explants

Introduction

Injury of the lower extremities is a common occurrence in automobile accidents.
The consequence of these injuries is often the development of a chronic joint disease,
osteoarthritis (OA). 0A is a condition that develops as the cartilage of the joint wears
away, resulting in bone on bone contact accompanied by severe pain and loss of mobility.
It is believed that a loss of cellularity and damage of the cartilage matrix are the causes of
the degradation of the cartilage. Ewers et al. noted that both the amount of matrix
damage and cell death following trauma depends on the rate of loading applied to the
cartilage (Ewers et al., 2001). This study also observed that in a low rate of loading test,
cell death was more uniformly distributed throughout the matrix of the cartilage. In the
previous chapter the role of apoptotic cell death in bovine cartilage explants following a
low rate of blunt trauma was investigated. TUNEL positive (apoptotic) cells were
increased 48 hours post-impact, but only slightly above background levels. This ﬁnding
suggests that primary necrosis contributes to the degradation of cartilage following
trauma.

A deﬁning feature of cell necrosis is swelling, due to the inability of the injured
cell to maintain ionic gradients across the damaged plasma membrane. Ultimately, the

necrotic cell ruptures (Figure 1) (Duke et al., 1996).

29

.3-

 

Figure 1. Acellwithadamagedmembranewillswellandburst. Thismode
of cell death is called necrosis.
Within the ﬁrst 24 hours following blunt trauma, cell death increases steadily in explants
injured by blunt trauma (Ewers et al., 2000). These data suggest that a “window of
opportunity” may exist to intervene in the progressive disease process in the joint and
limit the degree of cell death following a single, blunt insult.

Because of their amphiphilic properties, some mild surfactants are able to interact
with the lipid bilayer of cell membranes to restore their integrity alter injury from
physical stress (Clarke et al., 1992, Papoutsakis et al., 1991), electroporation via electrical
trauma (Lee et al., 1999), and chemical and thermal stresses (Hellung-Larsen et al.,
2000). Recent studies of brain trauma also suggest that these surfactants, particularly
poloxamer 188 (Pl88) can help “save” severely traumatized neurons from developing
necrosis (Barbee et al., 1992, Marks et al., 2001). P188 is a triblock copolymer of the
form poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) with a molecular

weight of 8400 Daltons (Figure 2).

as
H —-<—OCH2 CH2 9:60 CH CH2 976009-12 CH2 92—014

Figure 2. Poloxamer 188 is a triblock copolymer. It consists of two
subunits of the hydrophilic poly(ethylene oxide) surrounding the hydrophobic subunit
poly(propylene oxide).

30

P188 is a highly surface active copolymer, which probably aids in its absorption and

facilitates its insertion into lipid monolayers (Figure 3) (Maskarinec et al., 2002, 2003).

 
 
  

restoration of
membrane inter: H II',’

”on
/
WW“?
P188moleculs
“fie,

Figur'e3.ltishypothesizedthatP188insertsiruodredamagedportionof
cennrembmnesmanestleakageandprevemceuularneaosis.1hecennnythen

repairitselfnatrnallyandtheP188issqueeeedout
The hypothesis of this study was that administration of the nonionic synthetic
surfactant P188 would be effective in limiting the development of cellular necrosis within

the ﬁrst 24 hours post trauma.

31

Methods:

Six bovine forelegs from 3 mature animals were obtained from a local abattoir
within six hours of slaughter. The legs were skinned and rinsed with water prior to
exposing the metacarpal joint in a laminar ﬂow hood. A biopsy punch (Miltex
Instrument Company, Bethpage, NY) was used to make 72 6mm diameter chondral
explants ﬁom the lower metacarpal surface of the limbs. The explants were separated
ﬁ'om the underlying bone using a scalpel. The specimens were washed three times in
Dulbecco’s Modiﬁed Eagle Medium: F12 (DMEM, Invitrogen Co., Grand Island, NY,
#12500-062). The explants were then equilibrated for 48 hours in DMEM:F12 (2
explants per well, approximately 1 mg of cartilage per 1 ml of media) supplemented with
10% fetal bovine serum (Invitrogen Co), 21.9 mg/ml glutamine, additional amino acids
and antibiotics in a humidity controlled incubator (37° C, 5% C02, 95% humidity).

Eight explants were randomly assigned to a non-impact, control group (Figure 4).

32

P188

treatment
znmn EEBzumM
n= 16
9 lHour
n= 16
No P1884

n=72

treatment

      

Flgure4. Diagramshowingexperimentalsetup. 64choudralexplaruswereinmacted
with25MPa 32explantsweretreatedwithP188and32wereunneated16explantsfmm
eachgroupwerestainedforcellviabilityatland24hours

The remaining explants were randomly assigned to two groups, poloxamer-treated and no
poloxamer treatment (n=32 each group). All explants, except the control group, were
loaded to 707 N (~25 MPa), following a 5 N preload, using a low rate of loading (ls to
peak). The loading was performed in unconﬁned compression between two highly
polished stainless steel plates using a haversine loading protocol with a servo-controlled
hydraulic testing machine (Instron, model 1331, Canton, MA). Eight mg/ml poloxamer
188 (Pl88, Pluronic F-68, Sigma Aldrich Co., St. Louis, Mo) was added to the media
within 2 minutes of impact for all 16 explants in the P188-treated group. This
concentration was chosen based on the in viva concentration used in human patients
(Orringer, 2001). The explants were manually submitted to 10 cycles of pressure at (~1
MPa) to encourage P188 uptake into the cartilage matrix. Sixteen explants from each

group were used to determine cell viability at either 1 or 24 hours post impact. The 24

33

hour explants were washed three times in DMEM:F 12 after loading and placed into
supplemented DMEM:F 12 media with P188 and incubated overnight. Following
incubation the explants were cut through the thickness into thin sections using a
specialized cutting tool with parallel blades spaced 0.5 mm The slices were stained with
Calcein AM and Ethidium Homodimer, according to the manufacturer’s speciﬁcations
(Live/Dead Cytotoxicity Kit, Molecular Probes, Eugene, OR). Brieﬂy, 10p] of Ethidium
Homodimer and 2.5 ul of Calcien AM were added to 10ml of PBS, vortexed and
approximately 500p] of the staining solution was added to each well containing explant
slices.

Approximately 3 slices from each explant were viewed across approximately 1
mm near the center in a ﬂuorescence microscope (Leitz Dialux 20 (frequency: 50-60 Hz),
Leitz MikroskOpie und Systeme GmgH, Wetlzar, Germany). Photographs were taken
using a digital camera (Polaroid DMC2, Polaroid Corporation, Waltham, MA, USA).
Each digital photo group ﬁom the explant slices was divided into three zones: superﬁcial,
middle and deep. Using a drawing program (Corel Draw, ) parallel lines were drawn on
either side of the explant through the thickness to outline the area of the explant to be
counted. These end lines were drawn perpendicular to the surface. Starting from the
superﬁcial surface of the explant, a line was started on one of the existing end lines at
20% distance from the superﬁcial surface, and extending to the other end line at the 20%
mark. The line was drawn to parallel the contours of the superﬁcial surface, as well as
the program would allow. Another line was drawn at 70% distance from the superﬁcial
surface, or 30% ﬁom the deep zone surface. These lines deﬁned the zones of the

cartilage: superﬁcial, middle and deep.

34

Cell viability was quantiﬁed in each zone of the explant using the software
described above. A live cell, with an intact membrane, will metabolize the calcien AM
resulting in a green ﬂuorescent byproduct. A necrotic cell, with a compromised cell
membrane, will not exclude the ethidium homodimer ﬁom its cytoplasm and its nuclear
material will be stained a red ﬂuorescent color.

The percentage of dead cells was quantiﬁed in each zone. The maximum applied
load, time to peak load and maximum explant compression were determined from the
instron data. A two-factor ANOVA was performed on these biomechanical parameters
and the percentage of total cell death in each group of explants. The two factors were
time (1 and 24 hours) and treatment (P188 vs. no P188). A Student-Newman-Keuls

(SNK) post-hoc test was used to determine differences between groups.

35

Results:

In the experiment, a random sampling of explants was administered 8 mg/rnl
P188 within 2 minutes of loading. An analysis of the biomechanical properties in the 4
impacted groups (1 and 24 hours, with and without P188 treatment, low rate of loading)
showed no signiﬁcant differences. The average maximum applied load, maximum
explant compression and time to peak load were 6440* 70.6 N, 0.298i 0.054 mm and
0.928t 0.017 sec, respectively (n = 64).

While the percentage of dead cells in control explants was 0.19t 0.17 % at 1 hour
(n = 4) and 1.24zt 0.76 % at 24 hours (n = 4), the levels of cell death were dramatically
increased with compressive loading (Figure 5). The percentage of dead cells signiﬁcantly

(p=0.005) increased between 1 hour and 24 hours post trauma (Figure 5).

 

 

 

 

50 _
,5, I Control -
w ‘°‘ No P188 Treatment
8: El P188 Treatment
E25
0”
Be 15 «
10
5
O

 

 

1 Hour 24 Hour

Figrres. Histogramshowingpercentageofcelldeathatland24hoursforeach
treatment group: control (black bar), no poloxamer 188 treatment (stripped bar) and P188
amm(whnebar).1hacwasasigniﬁcmnimeasemwudeammﬂowingimpaaﬁomlm
24hoursinbothtlretreatedandnon-treatedgroups(‘). Conversely,therewasadecreasein
oelldeathwiththeadditionofPlsstreatmentatbothland24hourspoaimpact(x).

Extensive cell death was speciﬁcally noted around impact-induced surface ﬁssures,

which were limited to the superﬁcial zone of the explant at 1 hour (Figure 6). A

36

r-I‘

signiﬁcantincreaseincelldeathbetween 1 and24hoursalsooccurredineachzoneof
the explant (p=0.007 superﬁcial, p<0.001 middle, p=0.005 deep) (Figure 6). While the
overall percentage increase in cell death between 1 and 24 hours was approximately 16
%, this increase was largely due to that occurring in the middle zone (approximately 80
%).

The percentage of dead cells in the P188 treated explants was signiﬁcantly less
thanthatinthenotreatmentgroupat1horn(p=0.001)and24horu's(p<0.001)(Figure
5). In the 1 hour group P188 treatment ofthe loaded chondral explants signiﬁcantly
reduced the percentage of cell death in the superﬁcial (p<0.001) and the middle zones
(p=0.022)versusthenotreatmentgroup,butdid notreducetheextent ofcelldeathinthe

deep zone of the explant (Figure 6).

37

Poloxamer

Paloxamer - -

1 Hour 24 Hours

Figureti. Cartilageexplarrtswereimpactedwithzsmaandexplarnsliceswere
stainedforcellviability. Selectedexplarnsweretreatedwiﬂrspg/mlPoloxamerlaa
iunnediatelyfollowingimpact Cellsstainedgreenweremeurbolicallyactiveandtherefore
ahvc,whilcoeﬂs9ﬁnedmdlmvewmpmmisedceﬂmmhmesandﬂraefomwemmcroﬁc

 
 

Twenty-four hours alter loading, however, there were signiﬁcant reductions in cell death

in all zones of the explant following P188 treatment (in the superﬁcial, middle and deep
zones p<0.001).

While early treatment of the loaded chondral explants with the non-ionic
surfactant (Pl88) signiﬁcantly decreased the percentage of dead cells, cellular death still
increased signiﬁcantly (p=0.047) intheseexplantsbetwecn 1 and24 hours(Figure 7).

90 1

our

70 <

 

 

 

 

é °°‘ laurel-rm
850 Imam.
.0 One-plans
C
O
a“
3
m.
m-r

 

 

 

o 6
II
a!“
:-

 

 

 

 

 

Figu'e7. Histogramshowingperocntageccﬂdeathineachzoneofcarﬁlageerquam
following blunt loading The explants were divided into three zones: superﬁcial (top 20%, black
bars), middle (50%, striped bars), deep (bottom 30%, white bars). At 1 hour post-impact, P188
neammdgrnﬁeanﬂy(+)Mccﬂdeammmcsupaﬁdaanndmemnesofﬂncmﬁhge
versustheuntreatcdgrwps. A124horuspost-inmact,P188ueatrnerlsigniﬁcantly(‘)rerhreedcell
deathinallzonesoftheexplarrtversustheuntreatcdgroupa

The apparent rate ofincrcase in the percentage ofcell death with time, however, tended
tobelessinthetreatrnent groupthaninthcnotreatmentgroup(Figurc 5).Bctween1
and 24 hours in the P188-treated group there was a signiﬁcant (p=0.008) increase in
percentage of cell death in the superﬁcial layer, but in contrast to explants without
treatment no signiﬁcant increases in the percentage ofcell death were noted between 1
and 24 hours in the middle (p=0.259) and the deep (p=0.725) zones of the treated

explants.

39

Discussion

The potential for “saving” traumatized chondrocytes was explored in this study
using the mild, nonionic surfactant P188. Bovine cartilage explants were loaded with 25
MPa of stress in unconﬁned compression, and assayed for cell viability at l or 24 hours.
The hypothesis was that chondrocytes damaged during blunt mechanical loading would
be "saved” from necrosis by early administration of P1 88.

Studies, by others, have documented the membrane restoring qualities of P1 88, as
this surfactant has been shown to be selectively absorbed into damaged regions of a cell
membrane. The “patch” then arrests the leakage of intracellular materials and helps
maintain ionic concentrations across this semi-permeable layer (Maskarinec et al, 2002).
In studies of chemical damage to cells with N-methyl-D—aspartatc, P188 treatment
normalizes membrane receptor and intracellular ﬁrnctions with fast (within 20s) action
(Marks ct al, 2001). In the current study the action ofcarly P188 treatment of
mechanically traumatized chondrocytes by 25 MPa of unconﬁned compressive pressure
was explored for the ﬁrst time. Studies by others have shown that P188 treatment has no
effect on the viability of apoptotic cells (Marks et al, 2001). While the applied pressure
in the viability study (25 MPa) was less than that used in the apoptosis study (30 MPa,
Chapter 1), cell necrosis was evident, even with the addition of P188 treatment, which
had a signiﬁcant effect on the percentage of cell death at 1 hour post trauma.

The current study also showed there was a "window of opportunity" to save cells
from death, as the percentage of cell death rose ﬁom approximately 17 % to 33 %
between 1 and 24 hrs without P188 treatment. The increase in the percentage of cell

death rose ﬁom approximately 12 % to 25 % in the middle zone of the tissue ﬁom 1 to 24

40

hours. While P188 treatment yielded a reduction in cell death of approximately 20% in
the superﬁcial zone at 1 and 24 hours, the reductions in cell death in the middle zone
were approximately 5% at 1 hour and 21% at 24 hours. The dramatic difference in the
ability of P188 to restore membrane integrity in the middle zone at 24 hours versus 1
hour may relate to the time required for the surfactant to penetrate the cartilage matrix.

In pilot studies we found the need to cyclically load the cartilage to allow P188 to have a
signiﬁcant cﬂ‘cct on the percentage of cell death. The cyclic loading, it was believed,
squeezed water out of the cartilage; when the cartilage relaxed, fresh water along with
P188 was sucked into the matrix and in contact with the cells. A limitation of the current
study was that this protocol was not ﬁrrthcr investigated.

Additionally, only one level of applied loading was investigated in the current
study. While approximately 25 MPa of pressure has been previously recorded on retro-
patcllar cartilage during blunt impact loading of the human knee (Haut et al, 1989), this
level of pressure applied to a chondral explant likely generated signiﬁcantly larger cell
strains than for an intact joint. With the degree of cellular strain reduced for an intact
joint, P188 treatment may be more effective.

A study by another laboratory showed that a pan-caspasc inhibitor, z-VAD.ﬁnk
[benzyloxycarbonyl-val-ala—asp (Ome) ﬂouromethylkctonc] could help prevent
chondrocyte apoptosis in human articular cartilage explants compressed to 30% (D’Lima
et al, 2001). While a reduction of apoptotic cell death within 48 hours may have a
signiﬁcant eﬁ‘cct on the long-term survival of articular cartilage, a higher percentage of
cell death by necrosis, as evidenced by the current study, increases the potential role of

P188 intervention clinically.

41

References

l.

Barbee K, Ford C, Blackman B, Thibault L. Neural cell injury: Characterization and
treatment strategy. Yang, K. 2. (1992). 2nd Injury Prevention through Biomechanics
Symposium Proceedings: CDC.

Clarke MS, McNeil PL. (1992) Syringe loading introduces macromolecules into
living mammalian cell cytosol. J Cell Sci. 1022533-41.

D’Lima DD, Hashimoto S, Chen PC, Colwell CW, Lotz MK. (2001) Impact of
mechanical trauma on matrix and cells. Clinical Orth Related Res. 391$:S90—S99.

Duke RC, Ojcius DM, Young JD. (1996) Cell suicide in health and disease. Sci Am.
275(6):80-7.

Ewers B, Knrcgcr J, Dvoracck-Driksna D, Orth M, Haut R (2001) Chondrocyte
viability decreases over 24 hours post-impact in a mechanically traumatized cartilage
explant. 4 7‘" Annual Meeting Orthopaedics Research Society.

Ewers, BJ, Weaver, BT, Sevensma, ET, Haut, RC. (2002) Chronic changes in rabbit
rctro-patcllar cartilage and subchondral bone after blunt impact loading of the
patellofcmoral joint. J. Orth Res. 20(3):545-550.

Haut RC. (1989) Contact pressures in the patellofcmoral joint during impact loading
on the human ﬂexed knee. J Orthop Res. 7(2):272-80.

Hcllung-Larsen P, Assaad F, Pankratova S, Saictz BL, Skovgaard LT. (2000) Effects
of Pluronic F-68 on Tetrahymcna cells: protection against chemical and physical
stress and prolongation of survival under toxic conditions. J Biotechnol. 76(2-3):185-
95.

Lee RC, Rivcr LP, Pan F, Ji L, Wollmann RL. (1992) Surfactant-induced scaling of
electropermeabilized skeletal muscle membranes in viva. Proc Natl Acad Sci.
89:4524-4528.

10. Lee RC, Hannig J, Matthews KL, Mycrov A, Chen C. (1999) Pharmaceutical

therapies for sealing of permeabilized cell membranes in electrical injuries. Annals

New York Acad Sciences. 888:266-273.

11. Luchtrnan-Joncs L, Files B, Ballas SK. (1999) Phase I study of FLOCOR in patients

with acute chest syndrome of sickle cell disease. 1999 Meeting American Society

Hematology.

12. Marks JD, Pan C, Bushell T, Cromic W, Lee RC. (2001) Amphiphilic, tri-block

copolymers provide potent membrane-targeted neuroprotcction. FASEB J. 15:1107-
l 109.

42

13. Maskarinec SA, Hannig J, Lee RC, Lee KC. (2002) Direct Observation of Poloxamer
188 insertion into lipid monolayers. Biophysical J. 82:1453-1459.

l4. Maskarinec SA, Lee, KC. (2003) Comparative study of poloxamer insertion into lipid
monolayers. Langmuir. 19:1809-1815.

15. Papoutsakis ET. (1991) Media additives for protecting ﬁeely suspended animal cells
against agitation and aeration damage. UBYECH. 9:316-324.

16. Simon WH, Richardson S, Herman W, Parsons JR, Lane J. (1976) Long-term effects

of chondrocyte death on rabbit articular cartilage in vivo. J Bone Joint Surgery
58(4):517-526.

43

Chapter Three

The role of Poloxamer 188 in Saving Chondrocytes from Necrotic
Cell Death Following Various Mechanical Loads

Introduction

Osteoarthritis (0A), a disease of the articular cartilage, has been shown to be
induced by impact loading diarthroidal joints. It is thought that one factor that may cause
CA is a loss of cellularity due to chondrocyte death (D’Lima ct al., 2001). Chondrocytes
mediate the synthesis, secretion and degradation of matrix protcoglycans, glycoprotcins,
and collagen, and thus are integral to tissue homeostasis (Quinn ct al., 1998).

Tensile stresses developed in the membranes of cells during compressive loading
of cartilage can disrupt this semi-permeable membrane to cause an imbalance in the ionic
concentrations of the cell leading to cellular necrosis (Ewers ct al., 2000). Compressive
stresses as low as 14 MPa have been shown to kill cartilage cells, however the level of
applied stress necessary to damage cell membranes is at present unknown (Morel ct al.,
2002). In a previous study a 35 MPa impact stress signiﬁcantly decreased protcoglycan
biosynthcsis and increased water content in cartilage explants (Borrelli et al., 1997); the
study suggests that the decreased biosynthcsis could be due to either decreased
chondrocyte metabolism or chondrocyte death, or both. A similar study showed a stress
dependent inhibition in chondrocyte metabolic activity, an increase in water content, and
a decrease in viable cell population when cartilage is impacted with a single load
producing stresses of approximately 15 to 20 MPa (Torzilli ct al., 1999). This same study

observed that there was a correlation of the density of cell death with stress intensity.

Cell death appeared to be initiated in the explant at approximately 15 MPa with complete
cell death throughout the cxplant’s thickness at 2 20 MPa (Torzilli et al., 1999).

A non-ionic surfactant, Poloxamer 188 (P188), has been shown to repair cell
membranes (Phillips et al., 2004). At a stress of 25 MPa P188 can save nearly 50 % of
the chondrocytes from necrotic death. But at lower stress levels (less than 15 MPa), the
degree to which P188 is effective is unknown. The mechanism by which P188 functions
in saving cells is likely by patching holes in the cell membrane caused by tensile stresses.
P188 may be less effective at repairing cell membranes with greater degrees of damage.

At 25 MPa cell death occurs along the superﬁcial tangential zone and adjacent to
ﬁssures in the cartilage, which stretch into the middle zone. Shear stresses are generally
higher at the tips of propagating ﬁssures; therefore cell death is higher around these
cracks (Ewers et al., 2000). At lower stress levels, chondrocytes in the deeper zones may
not be affected by damaging shear stresses. Due to lower stresses developed in the
matrix, ﬁssures are generally smaller, and less common, than at higher stresses; therefore
the shear stresses that occur near these ﬁssures are smaller. Cell death is observed mostly
in the superﬁcial tangential zone of the cartilage at lower stresses and is only observed at
the higher levels of the middle zone of the cartilage.

We hypothesize that at lower stress levels P188 will be more effective in lowering
the amount of cell death than at higher stresses due to the lower tensile stresses
developing in the matrix. At lower stress levels, total cell death will be lower in the
cartilage, as well as the degree to which cell membranes are damaged will be lower,

therefore P188 will be more cﬂ‘cctivc.

45

Methods:

Two bovine forelegs ﬁom a mature animal were obtained ﬁom a local abattoir
within six hours of slaughter. The legs were skinned and rinsed with water prior to
exposing the metacarpal joint in a laminar ﬂow hood. A biopsy punch (Miltex
Instrument Company, Bethpage, NY) was used to make forty-four 6mm diameter
chondral explants from the lower metacarpal surface of the limbs. The explants were
separated from the underlying bone using a scalpel. The specimens were washed three
times in Dulbecco’s Modiﬁed Eagle Medium: F 12 (DMEM, Invitrogen Co., Grand
Island, NY, #12500-062). The explants were then split into two treatment groups,
poloxamer 188 (Pl88) pretreatment (n=18) and no P188 treatment (n=18), and the
remaining explants were assigned to a non-impact control group (n=8). The explants
were equilibrated for 48 hours in DMEM:F12 (2 explants per well, approximately 1 mg
of cartilage per 1 ml of media) supplemented with 10% fetal bovine serum (Invitrogen
Co), 21.9 mg/ml glutamine, additional amino acids and antibiotics in a humidity
controlled incubator (37° C, 5% C02, 95% humidity). Eight mg/ml P188 was added to
the media of the 18 explants in the P188-treated group. The explants were manually
submitted to 10 cycles of pressure (~1 MPa) to encourage P188 uptake into the cartilage
matrix.

The explants of the two treatment groups were then randomly assigned to one of
nine impact groups (n=2). Each impact group was loaded to one of nine different loads at

a low rate of loading (Is to peak). The nine load levels were 254.5N (9 MPa), 311.0N

(11 MPa), 367.6N (13 MPa), 424.1N (15 MPa), 480.7N (17 MPa), 537.2N (19 MPa),

593.8N (21 MPa), 650.3N (23 MPa), and 706.9N (25 MPa) (Figure 1).

 
 
  
  

9 MPa
Cell Viability
1 1 MPa determined at l
and 24 hours

  

13 MPa

98 Control

Figurel. Diagramdrowingthecxpcrimcntaldesign 36cxplantswcreimpactedat
oncofnincimpactlcvcls 'I‘wocxplantsfromeachimpactgroupwercincubatedinPl88,
andtwowcreuntreatcd. Cellviabilitywasdctcrmincdatland24hours

The explants were loaded in unconﬁned compression between two highly polished
stainless steel plates using a haversine loading protocol with a servo-controlled hydraulic
testing machine (Instron, model 1331, Canton, MA). The following biomechanical
parameters, peak load, time to peak, and maximum explant compression were

documented in each experiment.

47

Nine explants from each treatment group were used to determine cell viability at
either 1 or 24 hours post impact. The 24 hour explants were washed three times in
DMEM:F12 after loading and placed into DMEM:F12 media supplemented with 8ug/ml
P188 and incubated overnight. Following incubation the explants were cut through the
thickness into thin sections using a specialized cutting tool with parallel blades spaced 0.5
mm apart. The slices were stained with Calccin AM and Ethidium Homodimer,
according to the manufacturer’s speciﬁcations (Live/Dead Cytotoxicity Kit, Molecular
Probes, Eugene, OR). Approximately 2 slices ﬁom each explant were viewed across
approximately 1 mm near the center in a ﬂuorescence microscope (Leitz Dialux 20
(ﬁequency: 50-60 Hz), Leitz Mikroskopie und Systeme GmgH, Wetlzar, Germany) and
photographed using a digital camera (Polaroid DMC2, Polaroid Corporation, Waltham,
MA, USA). A blinded observer, using image analysis software (Sigma Scan, SPSS INC,
Chicago, IL), quantiﬁed cell viability of the explant.

A two-factor ANOVA was performed on the biomechanical parameters. A two-
factor ANOVA for total cell death in each explant was also performed. The two factors
were time (1 and 24 hours) and treatment (Pl88 and no P188). An SNK post-hoc test

was used to determine differences between groups.

48

Results

The experiment evaluated the cell death observed in bovine cartilage explants
following severe blunt impact at varying stress levels. Cartilage explants were subjected
to stresses ranging ﬁom 9 MPa to 25 MPa in increments of 2 MPa for a total of nine
stress levels and incubated for either 1 or 24 hours post impact. Halfof the explants were
treated with the surfactant poloxamer 188 (PlS8) immediately following impact.
Explants were then stained for cell viability using Calccin AM and Ethidium homodimer.

There was a trend towards increasing cell death found in the explants at 1 hour

with an increase in the stress applied. The slope of the linear regression line was 0.971

 

with an R2 value of 0.343 (P=O. 163).
50 '1
[We Poloxamer
4O 4
IPoloxamer

 

 

 

 

%COIIM
8
L

 

 

 

 

 

??.:.t1.1. . Ml

9MPa 11 MPa 13MPa 15MPa 17MPa 19MPa 21 MPa 23MPa 25MPa

Flare; Histogramshowingtheperccntageofoclldeathobservedinexplant
impactswithvariouslevclsofstressatlhourpostimpact. 'l‘hcwhitcbarsreprescntdata
ﬁomcxplaﬂsthﬂwcreunncatedwlnlcmeblackbamrcpresemmﬁommmm
weretmatedwith8mg/mlpoloxamcr188.

 

 

 

A stress of 9 MPa resulted in approximately 8% cell death at 1 hour post-impact, whereas
a stress of 25 MPa resulted in approximately 28% cell death at 1 hour post-impact

(Figure 2). At 24 hours post-impact this trend increased with the slope of the regression

49

line of 1.148 and an R2 value of 0.827 (P<0.05). A stress of 9 MPa resulted in
approximately 7.3% cell death at 24 hours post-impact, and a stress of 25 MPa resulted in

approximately 25% cell death (Figure 3).

 

35, DNoPoloxamar
IPoloxamer

 

 

 

304
1:25,
3...
32154

104
51 I I
o.

9MPa 11MPa 13MPa 15MPa 17MPa 19MPa 21MPa 23MPa 25MPa

Flgure3. Histogramshowingtheperoerrtageofcclldeathobscrvedinexplants
impaetwithvariouslevelsofsuessatuhourpostimpaet Thcwhitebarsrepresentdata
ﬁomermmmatwucrmneatcdwhiletheblackbmsreprlscmdataﬁomcxplmdm
wereueatcdwith8mg/mlpoloxamer188.

 

 

 

 

 

 

 

With the addition of P1 88 the slope of the regression line was reduced to 0.826
(R’=.750, P<0.05) at 1 hour post-impact, though it was not signiﬁcantly different then
from the no P188 group. At a stress of9 MPa, cell death was reduced to 3.6%, a
reduction of greater than 50% versus the no P188 group, while at 25 MPa cell death was
reduced to 15.6%, nearly a 50% reduction (Figure 2). At 24 hours, the slope of the
regression line was greatly reduced to 0.638, a signiﬁcant difference ﬁ'om the no P188
group (R’=.609, P<0.05). At a stress of9 MPa, cell death was reduced to 5.6% versus

the no P188 group, and at 25 MPa cell death was reduced to 15.8% versus the no P188

group (Figure 3).

50

Discussion

The objective of the study was to determine the effectiveness of P1 88 following
various levels of mechanical stresses. Cartilage explants were loaded to one of 9 stress
levels ranging ﬁ'om 9 MPa to 25 MPa and cell viability was assessed at either 1 or 24
hours. Half of the explants from each stress level were administered P188 immediately
following impact.

The hypothesis of the study was that an increase in the stress applied to chondral
explants would correspond to an increase in cell death. The amount of cell death
correlates with the degree of mechanical disruption of the cartilage surface (Lewis et al, ),
and a higher applied load would yield more surface disruption. Also, P188
administration is hypothesized to have an effect in decreasing the percentage of
chondrocytes killed following various levels of applied stress. Previously, we
demonstrated that the surfactant had the ability to save chondrocytes ﬁ'om necrotic cell
death following a mechanical load of 25 MPa (Phillips et al, 2004). It is also theorized
that at the lower stresses, P188 would have a greater result in preserving cell viability due
to the lower shear forces that occur under these mechanical stimuli.

The percentage of cell death increased as the applied stress increased at both 1
and 24 hours in the untreated groups (Figure 2). This ﬁnding agrees with the original
hypothesis. With the addition of P188, the trend of increasing cell death with increasing
stress continued, but the relationship between cell death and stress lessened. At 1 hour,
P188 treatment was reduced at every stress level versus the untreated groups with the
larger effect observed at the lower stresses (9 MPa - 17 MPa). The effectiveness of P188

at 1 hour may possibly be due to chondrocytes that are less severely injured being

51

rescued by the surfactant. At higher stress levels, these cells may be fewer in number
compared to more severely injured cells.

At 24 hour, P188 treatment reduced cell death at every stress level as well, but it
appeared to have a greater effect at the higher stress levels (17 MPa — 25 MPa) as
opposed to the groups at 1 hour. This may be due to the time it takes for P188 to repair
the more severely damaged cells, of which there is a higher number than at the lower
stress levels.

There appeared to be a threshold stress level above 17 MPa in which cell death
was greatly increased above the lower stress levels at both 1 and 24 hours. This threshold
level of stress also appeared in the poloxamer treated groups as well. This threshold
could represent an increased level of shear stresses that develop within the matrix, above
the levels of shear stresses present at lower stresses. This increase in shear stress may be
responsible for the death of a certain population of cells not injured at lower stresses, but
susceptible to a higher level of shear stresses. It is this population of cells in which P188

is most eﬂ‘ective as demonstrated by the larger reduction in cell death with treatment

observed above 17 MPa.

52

.1;

References

l.

Borrelli I, Torzilli PA, Grigiene R, Helfet DL. (1997) Eﬁ‘ect of impact load on
articular cartilage: development of an intra-articular fracture model. J 0th Trauma.
11(5):319—326.

Clements KM, Bee ZC, Crossingham GV, Adams MA, Sharif M. (2001) How severe
must repetitive loading be to kill chondrocytes in articular cartilage? Osteo and Cart.
9:499-507.

. D’Lima DD, Hashimoto S, Chen PC, Colwell CW, Lotz MK (2001) Impact of

mechanical trauma on matrix and cells. Clinical Orth Related Res. 391$:S90-S99.

Ewers B, Dvoracek-Driksna D, Orth M, Altiero N, Haut R (2000) Matrix damage
and chondrocyte death in articular cartilage depends upon loading rate. 46‘” Annual
Meeting Orthopaedic Research Society.

Lee RC, River LP, Pan F, Ji L, Wollmann RL. (1992) Surfactant-induced sealing of
electropermeabilized skeletal muscle membranes in vivo. Proc Natl Acad Sci.
89:4524-4528.

Lee RC, Hannig J, Matthews KL, Mycrov A, Chen C. (1999) Pharmaceutical
therapies for sealing of permeabilized cell membranes in electrical injuries. Annals
New York Acad Sciences. 888:266—273.

Maskarinec SA, Hannig J, Lee RC, Lee KC. (2002) Direct Observation of Poloxamer
188 insertion into lipid monolayers. Biophysical J. 82:1453-1459.

Maskarinec SA, Lee, KC. (2003) Comparative study of poloxamer insertion into lipid
monolayers. Langmuir. 19:1809-1815.

Morel V, Quinn TM. (2002) Micromechanical factors in cartilage injury under ramp
compression of explants for deformations applied near the “gel difﬁision” rate. 48‘”
Annual Meeting Orthopaedic Research Society.

10. Phillips DM, Haut RC. (2004) The use of a nonionic surfactant (P188) to save

chondrocytes from necrosis following impact loading of chondral explants J Orth
Res. (In Print).

11. Quinn TM, Grodzinsky A], Hunzinker EB, Sandy JD. ( 1998) Effects of injurious

compression on matrix turnover anround individual cells in calf articular cartilage
explants. J 0th Res. 16:490-499.

12. Togo T, Alderton JM, Steinhardt RA. (2000) The mechanism of cell membrane

repair. Zygote. 8(Supplement):S31-S32.

53

l3. Torzilli PA, Grigiene R, Borrelli J, Helfet DL. (1999) Effect of impact load on

articular cartilage: cell metabolism and viability, and matrix water content. JBiom
Eng.121:433-441.

54

Chapter Four

The Effect of Pretreatment Versus Post-treatment of
Poloxamer 188 in Treating Chondrocytes Damaged
by Mechanical Loads

Introduction

Traumatic injury is one of the most common causes of secondary osteoarthritis.
Osteoarthritis (0A) is classiﬁed by the degeneration of articular cartilage in diarthroidal
, joints, resulting in bone on bone contact that causes pain and loss of mobility. Blunt
trauma has been shown to cause chondrocyte cell death, which may lead to 0A Tensile
stresses caused by impact loading damage cell membranes, which disrupt ion gradients
across the cell membrane and cause the cell to absorb water (Torzilli et al., 1999).
Necrosis, a mode of cell death, occurs as the cell swells with water and then bursts. The
prevention of a post-traumatic 0A may start with the inhibition of cell death by repairing
damaged cell membranes.

Administration of poloxamer 188 (Pl88) can save chondrocytes damaged by
severe blunt trauma from death by necrosis (Phillips et al., 2004). P188 is a triblock
copolymer of the form poly(ethylene oxide)-poly(propylene oxide)—poly(ethylene oxide)
with a molecular weight of 8400 Daltons; it is a highly surface active copolymer, which
probably aids in its absorption and facilitates its insertion into lipid monolayers
(Maskarinec et al., 2002, 2003). The surfactant is similar in structure to the lipid bi-layer
of the cell membrane, and is incorporated into the damaged portion when trauma occurs,
thereby sealing the tear in the membrane. As recent injection experiments have shown,
when surface pressure of an artiﬁcial lipid monolayer was decreased below a threshold

level, therefore representing damage, P188 was easily inserted into the monolayer. On

55

the other hand, at high surface pressures, P188 did not insert itself into the artiﬁcial
monolayer, therefore it was concluded that P188 could only insert itself into the damaged
portions of membranes (Maskarinec et al., 2002, 2003). At membrane surface pressures
of 25 lem or higher, the P188 is squeezed out of the membrane (Maskarinec et al.,
2002). Some studies suggest that P188 also has the ability to strengthen the matrix of the
cartilage whereby lowering the shear stresses resulting from blunt trauma.

Chapter 2 investigated the ability of P188 to save cells when added post-impact.
The percentage of cell death was greatly reduced in the explants treated with P188. The
current study investigates the role of P188 applied to the cartilage pre-impact versus post-
impact. The application of P188 as a supplement used prior to trauma could be an
important step in preventing the initial stages of 0A It is unknown how long P188 may
last in the cartilage matrix when used as a supplement and whether it is degraded over
time. However, recent investigations on the use of poloxamer in protecting model drug
carriers from phogocytosis indicate that surfactant micelles themselves possibly have a
longer in viva half-life time and thereby can act as a slow release vehicle (Lee et al.,
1999). The effects of pre-treating cartilage with P188 are important in order to use P188
as a supplement instead of an immediate treatment following blunt trauma. The
hypothesis of the current study was that pretreatment of chondral explants with P188
surfactant would reduce the degree of necrosis that develops in the initial 24 hours

following a severe impact trauma to joint cartilage.

56

Methods:

Two bovine forelegs ﬁ'om a mature animal were obtained ﬁom a local abattoir
within six hours of slaughter. The legs were skinned and rinsed with water prior to
exposing the metacarpal joint in a laminar ﬂow hood. A biopsy punch (Miltex
Instrument Company, Bethpage, NY) was used to make seventy-six 6mm diameter
chondral explants ﬁ'om the lower metacarpal surface of the limbs. The explants were
separated from the underlying bone using a scalpel. The specimens were washed three
times in Dulbecco’s Modiﬁed Eagle Medium: F12 (DMEM, Invitrogen Co., Grand
Island, NY, #12500-062). The explants were then split into two treatment groups (3
groups total). All treatment groups and controls were equilibrated for 48 hours in
DMEM:F12 (2 explants per well, approximately 1 mg of cartilage per 1 ml of media)
supplemented with 10% fetal bovine serum (Invitrogen Co), 21.9 mg/ml glutamine,
additional amino acids and antibiotics in a humidity controlled incubator (37° C, 5% C02,
95% humidity). The equilibration media of the ﬁrst group was supplemented with 8
mg/ml poloxamer 188 (Pl88). The explants were submitted to 10 manual cycles of
loading (~ lMPa) prior to impacting to encourage P188 uptake into the matrix (n=20).
The media of the second group was also supplemented with P188 during equilibration
and following impact. The explants of the second treatment group were submitted to 10
manual cycles of loading prior to impacting and 10 cycles of manual loading following
impacting (n=20). The ﬁnal treatment group received no P188 treatment (n=20). The
remaining sixteen explants were assigned to a non-impact control group that also

underwent 10 cycles of manual loading.

57

l
r
.
. .. .
h-‘
r
.. r r
all v
,.
1.
bl
'4
..
3x
‘
..
'a
f

I

All explants from the treatment groups were loaded to 706 N (~25 MPa), at a low

rate ofloading (Is to peak) (Figure 1).

Cell viability
determined at:
P1 88
post. ’ l and 24
treatment 11 = 20 hours

P188 _
pretreatment

25 MPa

9 land24

n=20 hours

-%‘ 9 1 and 24

n = 20 hours

 

9 Control

n=l6

Figure 1. Diagram showing the experimental design. 40 chondral explants were
pre—treatedwithP188,andwerethenloadedt025MPa 20explamswcreuntreatedand
loaded to 25 MPa 20 explants from the pretreated group were post-treated with P188. Cell
viability was determined at I and 24 hours.

The explants were loaded in unconﬁned compression between two highly polished
stainless steel plates using a haversine loading protocol with a servo-controlled hydraulic
testing machine (Instron, model 1331, Canton, MA). Peak load, time to peak and
maximum explant compression were documented in each experiment. The explants were
then incubated for l or 24 hours following impact, in fresh DMEM:F12 supplemented

medium according to treatment group.

58

Six explants from each treatment group were used to determine cell viability at
either 1 or 24 hours post impact. The 24 hour explants were washed three times in
DMEM:F 12 after loading and placed into DMEM:F12 supplemented media and
incubated for 24 hours. The media of the second treatment group was supplemented with
8 mg/ml P188. Following incubation the explants were cut through the thickness into
thin sections using a specialized cutting tool with parallel blades spaced 0.5 mm. The
slices were stained with Calccin AM and Ethidium Homodimer, according to the
manufacturer’s speciﬁcations (Live/Dead Cytotoxicity Kit, Molecular Probes, Eugene,
OR). Approximately 2 slices ﬁ'om each explant were viewed across approximately 1 mm
near the center in a ﬂuorescence microscope (Leitz Dialux 20 (frequency: 50-60 Hz),
Leitz Mikroskopie und Systeme GmgH, Wetlzar, Germany) and photographed using a
digital camera (Spot Digital Camera, Diagnostic Instruments Inc). A blinded observer,
using image analysis software (Polaroid DMC2, Polaroid Corporation, Waltham, MA,
USA), quantiﬁed cell viability of the explant. The data from the two explant slices from
a single explant were averaged prior to statistical analysis.

The data in this chapter represents three experiments, the experiment described
was repeated to increase the power of the study, and a pilot study was performed prior to
investigate P188 pre-treatrnent. The second experiment was performed similar to the ﬁrst
with a total of 28 explants and group sizes of n=4. The third experiment was a pilot
experiment and only consisted of two treatment groups, P188 pro-treatment (n=14) and
no P188 treatment (n=14). Both groups were treated according to the treatment groups

described above.

59

The maximum applied load, time to peak load and maximum explant compression
were determined from each experiment. A two-factor ANOVA was performed on these
biomechanical parameters. The percentage of dead cells in each explant was quantiﬁed
using the image software. A two-factor ANOVA for total cell death in each explant was
performed. The two factors were time (1 and 24 hours) and treatment (Pl88 pretreatment
and P188 pre and post treatment and no P188). An SNK post-hoe test was used to

determine differences between groups.

Results

The study was designed to compare untreated explants with explants pre-treated
with 8 mg/ml P188 prior to loading and explants pre-treated with P188 as well as
incubated in P188 post impact. An analysis of the biomechanical properties in the 6
impacted groups (1 and 24 hours, no poloxamer, P188 pre-treatment, and P188 pre- and
post-treatment) showed no signiﬁcant differences. The average maximum applied load
and maximum explant compression were 713.84i 7.03 N and 0.3041 0.04 mm,
respectively (n = 60).

The percentage of dead cells in the control explants was 5.87: 2.19% at 1 hour

(n=8), and 9.71: 4.29% at 24 hours (n=8) (Figure 2).

 

    
    

 

 

  

 

 

 

 

 

45 «
Econtrol
4o _
Elno poloxamer
35 ‘ Dpoloxamer pre-treatment
30 _ I poloxamer pre- and post-
5 treatment
8
u 25 -
a 2°~
at
15 ,
10 «
5 r
0 J

 

1hour 24hours
Fignre2. Histogramshowingthepercentageofcelldeathdocumentcdatland24

hours post-trauma in each ofthe treatment groups: control (striped bar), no poloxamer (white
bar), poloxamer pre-treatment (gray bar), and poloxama pre-and post-treatment (black bar).

61

As previously noted, cell death was increased around ﬁssures which occurred on the
superﬁcial surface of the explants (Phillips, 2004). On several explants a characteristic
‘butterﬂy’ pattern of cell death occurred around the ﬁssures. There was a trend of
increasing cell death in the explants from 1 to 24 hours following impact, though it was
not statistically signiﬁcant (p=0.216). Following a 25 MPa impact, cell death in
untreated explants was 27.5: 10.3% at 1 hour post impact (n=10), and 30.5: 10.1% at 24
hours (n=10).

With the addition of 8 mg/ml P188 to the media of the explants during the 48
equilibrium period, cell death was signiﬁcantly reduced ﬁ'om the untreated groups at both
1 (p=0.020) and 24 (p=0.035) hours post impact. At 1 hour cell death in the pre-treated
group of explants was 21.75: 7.84% (n=10), and at 24 hours cell death was 25.5: 7.06%
(n=10).

When explants were pre-treated with P188, and incubated in media supplemented
with P188 following impact, cell death was reduced from the untreated group of explants
at both 1 (p<0.001) and 24 hours (p<0.001), as well as the pre-treated group of explants
at both 1 (p=0.002) and 24 hours (p<0.001). At 1 hour post impact cell death in the pre-
and post-treatment group was 14.1: 4.4% (n=10) and at 24 hours the cell death was

14.7: 6.4% (n=10).

62

J»

Discussion

The study documented the use of a surfactant, poloxamer 188, in the intervention
of cell death following compressive blunt trauma. Bovine cartilage explants were
impacted with 25 MPa of impact stress, and cell viability was assessed at either 1 or 24
hours. In one group of explants, the surfactant was added prior to impact to investigate
the role of pretreatment. In a second group the surfactant was added both prior to impact
as well as following the impact.

Cell death increased in untreated explants from 1 to 24 hours, though not
statistically (p=0.22), which supports the data reported in Chapter 2. In Chapter 1, it was
reported that following blunt trauma, apoptosis was not observed until 48 hours; this
point along with the current data suggests that the increase in cell death in this study is
through the mechanism of necrosis. The observed increase in cell death in the ﬁrst 24
hours following trauma is a vital event if intervention is desired. It is within the ﬁrst 24
hours that this study focused its attempt at saving cells.

The spread of necrotic cell death can be by several methods. First, cells damaged
during impact, may remain viable at 1 hour, but may lose the ability to maintain
membrane integrity over time, resulting in the onset of necrosis. Second, cells in the area
of necrotic cells may be damaged by the contents of the cytoplasm spilled into the
cellular milieu by damaged cells. Lastly, cells damaged initially by the trauma may
release a cell signaling molecule alerting other cells of its damaged, and initializing a
necrotic pathway in neighboring cells.

With the addition of P188 treatment prior to impact, cell death was reduced

signiﬁcamly at both 1 and 24 hours post impact. The mechanism by which P188 works

63

 

 

is not yet ﬁrlly understood, but according to studies by others, P188 repairs defects in the
membranes of cells damaged by mechanical stresses (Lee et al, 1992, Maskarinec et al,
2002). In the plasma membrane of living cells, the rigid cytoskeleton greatly increases
the surface tension of the membrane (Togo et al, 2000). When membrane integrity is
lost, a drop in surface tension occurs. As shown by recent injection experiments, when
surface pressure of an artiﬁcial lipid monolayer was lowered below a threshold level, as
occurs during membrane damage, P188 was easily inserted into the monolayer.
Conversely, at high surface pressures, P188 did not insert itself into the artiﬁcial
monolayer, therefore it was concluded that P188 could only insert itself into the damaged
portions of membranes (Maskarinec et al, 2002, 2003). Apoptosis, a cellular event in
which the cell ‘self-destructs’ emu within, does not exhibit compromised cell
membranes, and therefore would not be aﬁ‘ected by P188 treatment. On the other band,
the repair of cell membranes may prevent necrotic cell death if the process occurs early
enough in the necrotic event. Recent investigations on the use of poloxamer in protecting
model drug carriers ﬁ'om phagocytosis indicate that surfactant micelles themselves
possibly have a longer in vivo half-life time and thereby can act as a slow release vehicle
(Lee et al, 1999).

The objective of the current study was to document the timeframe for p188
treatment most effective in preventing cell death following mechanical trauma. P188
pre-treated explants and P188 pre- and post-treated explants were compared with
untreated explants to determine the scenario in which P188 was most effective in
preserving cell viability. The present experiment shows that while pre-treatment of P188

is effective in preserving cell viability, pre- and post-treatment of cartilage was more

effective. Pre- and post-treatment with P188 reduced cell death signiﬁcantly versus the
untreated group, and the pre-treatment only group at both 1 and 24 hours.

Comparing these data with data presented in chapter two shows a similar
reduction in cell death for explants that were only treated post-impact. In chapter two,
data was presented showing the ability of P188 to preserve cell viability when added
post-impact only; a reduction in cell death of approximately 50% at both 1 and 24 hours
versus an un-treated group showed that following injury, addition of P1 88 would help to
preserve cell viability threatened by mechanical trauma.

A possible conclusion is that at the time of impact, a portion of P188 may be
squeezed out of the explants, and would not be available to the cells to repair damage.

By post-treating the explants, P188 is reintroduced to the matrix, and therefore is present
to preserve cell viability. A second possible conclusion that can be made ﬁ'om these data
is that it is more important to have a dynamic inﬂux of p188 available to the cells, than to
have the P188 present at the time of injury. There may be a physical mechanism
associated with the insertion of P1 88 into membranes, and this could lave been
facilitated by the ten cycles of 1 MPa loading that the explants were subjected to

following P188 addition.

65

References

1.

Lee RC, Hannig J, Matthews KL, Myerov A, Chen C. (1999) Pharmaceutical
therapies for sealing of permeabilized cell membranes in electrical injuries. Annals
New York Acad Sciences. 888:266—273.

Maskarinec SA, Hannig J, Lee RC, Lee KC. (2002) Direct Observation of Poloxamer
188 insertion into lipid monolayers. Biophysical J. 82:1453-1459.

Maskarinec SA, Lee, KC. (2003) Comparative study of poloxamer insertion into lipid
monolayers. Langmuir. 19:1809-1815.

Oyen-Tiesma M, Deloria LB, Meglitsch T, Oegema TR, Lewis JL. (1999) Cell death
after cartilage impact depends on matrix damage. 45” Annual Meeting Orthopaedic
Research Society.

Phillips DM, Haut RC. (2004) The use of a nonionic surfactant (PI 88) to save
chondrocytes ﬁ'om necrosis following impact loading of chondral explants J Orth
Res. (In Print).

Torzilli PA, Grigiene R, Borrelli J, Helfet DL. (1999) Effect of impact load on
articular cartilage: cell metabolism and viability, and matrix water content. J Biom
Eng. 121 :433-441.

Chapter Five

The Use of a Rabbit Animal Model to Determine the
in viva Effectiveness of Poloxamer 188 in Saving
Chondrocytes Damaged by Mechanical Trauma

Introduction

Osteoarthritis (OA), a disease of diarthrodial joints, has been shown to be induced
by blunt trauma (Gelber et al., 2000, Nagel et al., 1977). 0A is characterized by cartilage
loss on the ends of weight bearing bones, resulting in pain and loss of function (Flores et
al., 1998). It is believed that a loss of cellularity within cartilage is a major factor in the
onset of OA (Simon, et al., 1976). Chondrocytes, the resident population of cells in
cartilage, are damaged by tensile stresses in membranes that arise from blunt trauma
(Ewers et al., 2001). The cell membrane of chondrocytes is compromised and delicate
ion balances are interrupted within the cell leading to a necrotic cell death.

Poloxamer 188 (P188), a nonionic synthetic surfactant, has been shown to repair
the membranes of cells damaged by physical stresses, electrical trauma and chemical and
thermal stresses (Hannig et al., 2000, Lee et al., 1992, Maskarinec et al., 2002, 2003,
Chapters 2, 3, 4). The surfactant inserts itself into damaged portions of cell membranes
(Chapter 4) and repairs the defect, thereby saving the cell from necrotic cell death
(Maskarinec et al., 2002). The effectiveness of P188 in preventing cell death in cartilage
explants has been documented (Phillips et al., 2004), but the role that P188 may play in
the in viva environment remains unknown. This chapter will try to elucidate the role of
P188 in vivo.

In the previous chapters, the role of P1 88 in preventing cell death in bovine

cartilage explants was documented. P188 reduced the amount of cell death in the

67

explants at 1 and 24 hours post impact, though a long-term study would be needed to
elucidate the chronic effects of this treatment. In the current study, a live animal model

was used to investigate the eﬂ‘ectiveness of P188 in viva (Figure 1).

 

 

 

 

 

 

 

 

   

Figure l. Flemishgiantrabbitsreceivedabluntimpacttotheleﬁpatellofemoral
joint. SeveralrabbitsreceivedanmjecﬁonofPl88hnmediatelyfollowingimpact

Flemish Giant rabbits were used as part of an animal model that was developed in our lab
to study the long-term eﬁ‘ects of blunt trauma to the patellofcmoral joint (Haut et al.,

1995). The effectiveness of P188 in preventing cell death at 96 hours post impact will be

investigated.

68

Methods

Fourteen skeletally mature Flemish Giant rabbits (age range 8-11 months,
average weight 5.7: 0.34 kg) were used for this study. Seven rabbits were impacted
with 6 Joules (J) of energy to the patello-femoral joint and seven received an impact
of 10] of energy. Three rabbits from each impact group received a 1.5m] injection of
poloxamer 188 (8mg/ml) to the knee joint immediately following impact. Four
animals were sacriﬁced at time zero and the remaining animals at 96 hours post
impact. Michigan State Universities All-University Committee on Animal Use and

Care approved the study.

 

sigma Theapparatususedtonuintaintbeanimarsmdergenerarmsthesia
‘ The rabbits were maintained under general anesthesia using a 2% isoﬂurane
and oxygen mixture (Figure 2). The right hind limb was arbitrarily chosen to be
impacted over the left limb for every rabbit. The animal’s right hind limb was shaved
and the animal was placed in a specially designed chair that held the right hind limb

rigid and ﬂexed at a 120° angle with the animal laying on its back. Special care was

69

mkenmahgnthefemmvaticaLwhhthepatdhresﬁngmthefemoralgrooveandthe

impactheadawayﬁ'omthetibia Acanvasstrapwasfastenedacrosstheanimalsleﬁ
hindlimbtokeepthepelvisfromrotatingduringimpact.

Sacriﬁced at time
{a ...
(ta Sacriﬁced at 96

hours post-impact

.231...

Figure3. Diagramshowingtheexpaimmtaldesign. Fourteenanimalswerensed
fmﬂnstudyzdglﬂadmlseachwuemmaaedwnheitha6m10hﬂesofimpaamergy.
'I‘hreeaninnlsfi'omeachgmupweregivena1.50cinjectionof8mglmlPoloxamer188.

The impacts were delivered to the patello—femoral impact of the rabbit via a

 

gravity-driven drop ﬁxture (Figure 5). The experiment was performed by dropping a
weight ofeither 1kg or 1.33kg ﬁom a height of 1m or 0.46m to generate a 10] or 61
of impact energy load. The dropped sled was prevented ﬁ'om making multiple

impacts electronically, by arresting the sled following the ﬁrst impact. A load

70

transducer (model 31/1432: Sensotec, Columbus, OH) with a 2,224 N capacity was in
serieswiththeimpactheadattachedtothedrop sledtoacquiretheimpactloads

generated. Using a PC

 

ﬂame 'I‘heimpactchair(holdingananiml)thatorientedthefenmr
verﬁcalandaﬂowedasolidhnpacttoﬂrepateﬂofemoraljoirtofﬂrearﬁnnl

equipped with an analog-to-digital board, the loads generated were collected at 10
kHz for 1 sec prior to impact (100 samples), and following a 30 N analog trigger, 400

samples

 

ﬂnﬁrmnewithﬂreamm’sfenunvuﬁcalandthepatelladimlyrmdameimpmﬂed
Nowtheimpaasledstoppedameoftbedecuomchahngﬁxuuesﬂntprwanedmﬂdple

71

ofdataweresaved. Thepeakloadandtimetopeakwererecordedﬁ'omtheload
versus time curve.

Following impact, three animals from each impact group were administered a
1.5m] injection of P188 (in phosphate buﬁ'ered saline, PBS at a concentration of
Sing/ml) to the patello-femoral joint-capsule. The P188 was ﬁlter sterilized using a
0.2m vacuum ﬁlter (Nalgene, Nalge Nunc Int, Rochester, New York, USA). The
animal’s limb was exercised mamrally to incorporate the P188 into the patellar
cartilage. All animals were exercised on a treadmill for 10 minutes a day at 0.3 mph
starting the day ofimpact. Animals were housed in individual cages (122 cm x 61cm
x 49 cm) when not exercising. At 96 hours post-impact (four days), the animals were
euthanized with a lethal injection of Pentobarbital.

Following sacriﬁce, the patellas were excised ﬂ'om the animal using a scalpel.
The skin was cut away ﬁom the knee with the knee ﬂexed. The paellar tendon was

then cut using a scalpel and the proximal end of the tendon was pulled distally using

 

Figure6. Pictnneshowingarninnlsriglnhindlimbsituatedbeneathirnpactorhead
priortoimpact 'I'heanimal’sfemrn’wupositionedvertically, andtheirnpactorheadwas
alignedtoimpactthepatella

72

 

forceps. The soft tissue on both sides of the patella was removed and the patella was
removed by cutting the quadriceps tendon. The patellas were wrapped in PBS soaked
gauzetomaintainhydration. Thepatellaswerethenstainedwithlndiainkto
highlight surface ﬁssures and were photographed using a digital camera (Polaroid
DMC2, Polaroid Corporation, Waltham, MA, USA) under a dissecting microscope
(Wild TYP 374590,

Heerbrugg, Switzerland). The soft tissue was removed ﬁ'orn the back of the patella,
and the patella was glued to a custom cylindrical base using cyanoacrylate glue.
Approximately half of the bone was removed from the back of the patellas using an
Isomet low speed saw (model# 11-1180—170, Buehler, Inc, Lake Bhrﬂ‘, IL) by
attaching the cylindrical base to the lever arm of the saw. The patella was placed on
the edge of the blade, while the saw was turned oﬁ‘. After aligning the patella on the
bladetoensuretbatthecartilagefaceofthepatellawastotherightofthebladethe
sawwasstartedataverylowspeed, andtlne speedwas increased slowly, astheblade
cut through the bone (Figure 7).

 

Figure7. Theexcisedpatellaewerecutﬂnougntheirthidmessnsinganlsometmwas
showninthephoto. Patellaewereghredtoannetalbase,andkeptmoistwithPBSdming
cutting.

73

Whenthebladewascompletelythroughthebone, ascalpelwasusedtocutthesoﬁ
tissue holding the patella together. The remaining portion of the patella was sliced
into sections using a custom cutting tool with steel blades spaced 0.5m with ﬁxed
vertical movement using linear ball bearings (Figure 8). The slices were placed into a

24-well plate containing approximately 1 ml of PBS.

 

rig-res. Pateﬂaewucslicedhno0.5mmsectionspriortoaanﬁngusingthedcviccshown

The patellar slices were stained with Calccin AM and Ethidium Homodimer,
according to the mamifacturer’s speciﬁcations (Live/Dead Cytotoxicity Kit,
Molecular Probes, Eugene, OR, USA). Brieﬂy, will of Ethidium Homodimer and
2.5.11 of Calccin AM were added to 5rd of PBS and vortexed brieﬂy. The staining
mixture was then added to the slices in the 24-well plate after removing the PBS from
the wells. The slices were then incubated at room temperature for 15 minutes,
followed by several washes with PBS. Approximately 5 slices from each patella were
viewed acrossthecenter l mmofthesliceinaﬂuorescencemicroscopemeitzDialux

20 (frequency: 50-60 Hz), Leitz Mikroskopie und Systeme GmgH, Wetlnr,

74

Germany) and photographed using a digital camera (Polaroid DMC2, Polaroid
Corporation, Waltham, MA, USA). A blinded observer, using image analysis
software (Sigma Scan, SPSS INC., Chicago, IL, USA), quantiﬁed cell viability of the
retropatellar cartilage.

75

Results

This study was performed to investigate the efﬁcacy of poloxamer 188 (1’188) in
mitigating cell death in Flemish giant rabbits in viva. Fourteen animals received a blunt
impact to the right patellofcmoral joint using a gravity driven drop ﬁxture at an impact
energy ofeither 6 J or 10 J. Six (3 @ 6], 3 @ 10]) rabbits received 1.5 ml of8 mg/ml
P188 into the patellofcmoral joint immediately post impact. Cell viability was assessed
at 96 hours post impact for 10 animals, and at approximately 1 hour post impact for four
animals. All rabbits appeared healthy at time of sacriﬁce, and did not favor one leg over
the other.

The average peak load of the 6 J impact animals was 686.6: 120.0 N, and the
average time to peak was 0.003: 0.0008 seconds. The average peak load for the 10 J
impacts was 996.3i 123.4 N, and the average time to peak was 0.00241L 0.0005 seconds.
The documentation of the extent of surface ﬁssuring was not completed for the writing of
this thesis. These data will be utilized in subsequent peer-reviewed publications. Brieﬂy,
no difference has been shown, to date, between subsequent groups. The average
percentage of cell death in the retropatellar cartilage for the 10 J time zero rabbits was
8.3: 5.8% for control patellae and 9.3: 5.2% for the test patellae (Figure 9).

25.0.
20°- Itest
no.
no.

5.0 <

 

 

 

 

0.0
1)
Figure 9. Histogramushowing the percentage of cell death in rabbit patellas
administeredeitherélorlOJofimpactenergy. Celldeathwasdetermined lhourpost
impactforbothcontmlandtestpatellas. 76

 

The percentage of cell death for the 6 J time zero rabbits was 6.2: 3.6% for the
control patellae and 13.4: 7 .3% for the test patellae. At four days post impact, there was
a signiﬁcant difference between control and test patellae in the 10 J rabbits. The
percentage of cell death for the 10 I control patellae was 8.5: 4.5% and 22.9: 17.3% for
the test patellae. There was not a signiﬁcant diﬁ‘erence in cell death between control and
test patella in the 6 J rabbits at 96 hours post impact, however there was a strong trend
(p=0.051). The cell death in the 6 J patellae was 14.8: 8.1% for the control side and

24.8: 17.3% for the test side (Figure 10).

 

 

 

 

 

 

 

 

6.1 ‘DJ
Fig-rem. I-Iistogramshowingperoemagccelldeathinuntreatedrabbitpatellas
administeredeither6lor1010fimpactenergy. Celldeathwasasseesedawéhmnspost
impactforbothcontrolandtestpatellas 'lhereisasigniﬁcantdiﬂ'erenceinpercentageof
celldeathinthecrmmilversnstestnatellaoftheIOJanimals.

There was a small amount of background cell death observed in the patellae of the
animals used in the study. This could be due to the genetics of the animals, or the
conditions in which the animals were raised. After P188 treatment, the percentage of cell
death in the 10 .1 animals was 10.9: 6.1% in the control patellae and 12.7: 8.1% in the

test patellae. In the 6 .1 animals cell death was 20.4: 15.9% in the control patellae and

77

17.5: 8.1% in the test patellae. There was not a signiﬁcant difference between the
percentage of cell death in the control limb versus the test limb in either the 6 J or 10 J
animals. Likewise, there was no signiﬁcant difference between the test limbs of the 6 J

and 10 J animals (Figure 11).

4000<

$.m . Boonlml
IN
3000~

2500-
:; 2Q00~
IF 15001

1000—

 

 

 

 

OJ 10J

Figure 11. Histograrnshowingthepercentageofcelldeathinrabbitpatellas
treatedwithpoloxamer188invivoandadministeredeither6Jor10Jofimpact
energy. Celldeathwasdetermined96hourspostirnpactforbothcontrolandtest
patellas.

78

Discussion

The objective of the experiment was to investigate the efﬁcacy of P188 in
preserving chondrocyte cell viability in an in viva rabbit model. Fourteen Flemish giant
rabbits were administered a blunt trauma to their right patellofcmoral joint of either 6
Joule or 10 Joule (n=7 each). Six of the impacted rabbits (3 @ 6], 3 @ 10]) received 1.5
ml injection of sterile P188 at a concentration of 8 mg/ml immediately post-impact. At
96 hours post-impact, the patellae of the animals were stained with calcein AM and
ethidium homodimer to assess cell viability.

Our laboratory has previously documented surface lesions on the retropatellar
cartilage of the patella following blunt impact in a rabbit model, although not reported in
this study, surface lesions were observed on the impacted patellae of the animals
(Newberry et al, 1997). The study by Newberry also documented thickening of the
subchondral bone and degeneration of the overlying cartilage at 12 months post-trauma,
both early signs of 0A Our laboratory and others believe that cell death may play a
signiﬁcant role in the onset of 0A. In Chapter 2 of this thesis, cell death was reduced in
cartilage explants treated with P188 versus untreated explants following a blunt impact.
It was therefore the hypothesis of this study that cell death would be reduced in the
patellae of animals treated with P188 versus those that were untreated. The degradation
of cartilage that occurs during 0A may arise ﬁ‘om the death of chondrocytes within the
cartilage and ﬁssuring ofthe surface ofthe cartilage matrix caused by a blunt impact to
the joint (Simon et al, 1976, D’Lima et al, 2001, Borrelli et al, 1997). The prevention of

chondrocyte death may prevent the 0A like changes that occur following a blunt impact.

79

In the 4 animals sacriﬁced at time zero, cell death was increased in the 6 Joule
impacted rabbits versus their control limbs, but no increase was seen in the 10 Joule
rabbits over their control limbs. The amount of cell death in the test limb of the 6 Joule
animals was also higher than in the test limb of the 10 J animals. In a previous study by
our laboratory, a 6 Joule impact delivered to the patello-femoral joint of Flemish Giant
rabbits produced a peak average contact pressure of 25 MPa, while a 4.2 Joule impact
produced 16 MPa (Haut et al, 1995). It can reasonably be stated that a 10 Joule impact
would therefore produce contact pressures of greater than 25 MPa However, the higher
impact did not produce greater cell death in the retropatellar cartilage. The increase in
cell death for the 6 Joule impact may be due to an uneven distribution of the forces on the
retropatellar cartilage, while the 10 Joule impact was more evenly spread across the
patella, thereby more evenly distributing the impact energies (unpublished data). The
development of high hydrostatic pressures in the 10 Joule versus the 6 Joule animals may
help protect cartilage cells.

After 96 hours post-impact, there was an increase in cell death in the test limbs of
both the 6 Joule and 10 Joule animals versus their controls. This increase ﬁrrther expands
a conclusion of Chapter 2 that cell death increases in the ﬁrst 24 hours following a blunt
impact (Phillips et al, 2004, Ewers et al, 2001). Damaged chondrocytes may stain viable
within the ﬁrst hour following impact, but succumb to the injuries and lose membrane
integrity aﬁer the ﬁrst hour. It may also be that degradative enzymes are released from
cells heavily damaged by the impact, and spread causing further damage to surrounding

undamaged cells.

80

 

With the addition of the P188 injection, cell death was reduced in both the 6 J and
10 J animals versus those animals not treated with P188. P188 therefore had a positive
eﬁ‘ect in preserving cell viability. This result supports a previous study by our laboratory
that showed a 41 % reduction in cell death in bovine cartilage explants treated with
Poloxamer following blunt trauma (34 % in untreated explants and 14 % in P188 treated
explants; Phillips et al, 2004). An important difference between this previous study and
the current study was the active delivery of the surfactant to the deeper layers of the
cartilage matrix. Although zonal data was not collected in the current study, in the
previous study ‘pumping’ of the cartilage allowed the surfactant to reach the deeper
zones of the cartilage. The cartilage explants received 10 cycles of 1 MPa of pressure
immediately following impact as well as approximately 22 hours later. Following
injection of P188 in the current study, the animal joint was ﬂexed several times, and
regular exercise was initiated on the date of impact. It was believed that this exercise
would accomplish the same goal as pumping the explants. It is possible that ﬁrrther
mechanical pumping of the patellofcmoral joint in vivo may show P188 to be even more
effective at saving chondrocytes ﬁom mechanical trauma

A limitation of the current study was that the impacted, control animals were not
given a placebo injection of phosphate buffered saline without the surfactant. This
control procedure would show the effect ofthe injection on the joint capsule and whether
an inﬂammatory reaction occurs. The increase in cell death between the test and control
limbs of the untreated animals in this study may have been affected.

The chondrocyte cells that stain viable at 96 hours are not necessarily viable after

this time point. The effect of P188 on chondrocytes in the long term is not known and

81

needs ﬁrrther study. It may be found that chondrocytes saved by P188 in the ﬁrst 96
hours following impact die by apoptosis at a later time due to further damage within the
cell not repaired by the surfactant. A possible treatment would therefore be an apoptosis
inhibitor such as the z-VAD.ﬁnk pan-caspase inhibitor that interrupts the apoptosis
cascade and prevents cell death (D’Lima et al., 2001). Further study is needed to study
this theory.

The next step in studying the effects of the surfactant would be an animal study to
determine the long-term success of the surfactant in preserving cell viability. In previous
work published by our laboratory, positive eﬁ‘ects were observed for the mechanical
stiffness of retropatellar cartilage following early polysulfated glycosaminoglycan
treatments in our rabbit model (Ewers et al, 2000). However, the gross appearance of
surface lesions on the retropatellar surface was unaffected at the time of sacriﬁce. It was
thought that the loss in the number of chondrocytes at the time of impact caused the
degradation of the cartilage. It is possible that a combination of P1 88 and polysulfated
glysosaminoglycan treatment could ﬁrrther limit OA changes to cartilage in vivo, by
limiting cell death and stiffening the cartilage to lessen surface disruptions such as
ﬁssures. Our laboratory has developed a 7 month and 1 year chronic animal model to
study the 0A changes found following severe blunt impacts. This length of time would
give a deﬁnitive answer as to the long-term progress that chondrocytes make following
repair with P188.

The conclusions to this study are that an increase in cell death shown following
both a 6 and 10 Joule impact to the patella—femoral joint of rabbits. Following P188

treatment, cell death was reduced in both the 6 and 10 Joule impacts. The long-term

82

I t

success of this treatment is unknown and must be investigated. This treatment along with
others may severely limit the death of chondrocytes in retropatellar cartilage following

blunt trauma and may postpone or eliminate the onset of 0A

83

References

l.

10.

11.

12.

Borrelli Jr. J, Torzilli PA, Grigiene R, Helfet DL. (1997) Effect of impact load on
articular cartilage: development of an intra-articular fracture model. J Orth Trauma.
11(5):319-326.

D’Lima DD, Hashimoto S, Chen PC, Colwell CW, Lotz MK. (2001) Impact of
mechanical trauma on matrix and cells. Clinical Orth Related Res. 39IS:S90-S99.

Ewers B, Dvoracek-Driksna D, Orth M, Haut R (2001) The extent of matrix damage
and chondrocyte death in mechanically traumatized articular cartilage depends on rate
of loading. .1. Oriha. Res. 19(5)779.

Ewers B, Haut RC. (2000) Polysulfated glycosaminoglycan treatment can mitigate
decreases in stiffness of articular cartilage in a traumatized animal joint. J. Ortlra.
Res. 18(5)756-76l

Flores RH, Hochberg MC. (1998) Deﬁnition and classiﬁcation of osteoarthritis. In:
Brandt KD, Doherty M, Lohmander LS (Eds). Osteoarthritis, Oxford University
Press, New York. Pp 1-12.

Gelber AC, Hochberg MC, Mead LA, Wang NY, Wigley FM, Klag MJ. (2000) Joint
injury in young adults and risk for subsequent knee and hip osteoarthritis. Ann Intern
Med. 133(5):321-8.

Hannig J, Zhang D, Canaday DJ, Beckett MA, Astumian RD, Weichselbaum RR, Lee
RC. (2000) Surfactant sealing of membranes permeabilized by ionizing radiation.
RadRes. 154(2): 171-177.

Haut RC, Ide TM, DeCamp CE. (1995) Meclmnical responses of the rabbit patello-
fcmoral joint to blunt impact. J Biomech Eng. 117:402-8.

Lee RC, River LP, Pan F, Ji L, Wollmann RL. (1992) Surfactant-induced sealing of
electropermeabilized skeletal muscle membranes in viva. Proc Natl Acad Sci.
89:4524-4528.

Maskarinec SA, Hannig J, Lee RC, Lee KC. (2002) Direct Observation of Poloxamer
188 insertion into lipid monolayers. Biophysical J. 82:1453-1459.

Maskarinec SA, Lee, KC. (2003) Comparative study of poloxamer insertion into lipid
monolayers. Iangmuir. 19: 1809-1815.

Nagel DA, Burton DS, Manning J. (1977) The dashboard knee injury. Clin Orthop.
126:203-8.

13. Newberry WN, Zukosky DK, Haut RC. (1997) Subfracture insult to a knee joint
causes alterations in the bones and in the functional stiffness of overlying cartilage. J
Orth Res. 15:450-455.

14. Phillips DM, Haut RC. (2004) The use of a nonionic surfactant (Pl88) to save
chondrocytes ﬁom necrosis following impact loading of chondral explants. J Orth
Res. 22: 1 135-1 142.

15. Simon WH, Richardson S, Herman W, Parsons JR, Lane J. (1976) Long-term eﬁ‘ects
of chondrocyte death on rabbit articular cartilage in vivo. J Bone Joint Surgery
58(4):517-526.

85

 

APPENDIX A

STANDARD OPERATING PROCEDURE

Siain' a c e (ants or tosisusin thePro akit

Kit: Promega Apoptosis Detection System, Fluorescein

Materials needed: PBS, Propidium iodide, APES coated microscope slides, 4%
formaldehyde in PBS, dH20, incubator, ﬂuorescence microscope, covered tray

Prior to day of experimem

1. Remove each explant from the 24 well plate and embed the explant in OCT ﬁeeze

media on a cork disk. This should be done on dry ice. Wrap each OCT

 

embedded explant in Aluminum foil along with a label, and place at -80C.

2. Using the cryotome make slices of the explant and attach the slice to an APES
coated microscope slide. Warm back of slide with ﬁnger to attach slice to slide.
Place slide into microscope box and place box at -80C. Explants can remain at -
80C for several weeks.

Day of Experiment

1. Prepare all solutions including the 4% formaldehyde and PBS. Remove kit from
freezer and thaw nucleotide mix, equilibration buffer, and TdT enzyme on ice.
Place all microscope slides on a tray covered with paper towels.

2. Fix the explants by applying an amount of 4% formaldehyde sufﬁcient to cover
the slices. Incubate for 15 minutes at room temperature. Gently remove liquid.

3. Wash the slices 2x with PBS for 5 minutes each at room temp. Gently remove
liquid.

4. Prepare a 20ug/ml Proteinase K solution ﬁom the reconstituted Proteinase K.

87

 

C V = C v
10x10'3g/ml * x = 20x10'6g/ml * lOOul
X = 0.2ul of stock Proteinase K per 100ul needed

Add approximately IOOul of Proteinase K per slide or enough to cover tissue

sections. Incubate slides at room temperature for 8 minutes. Longer incubations may

release sections ﬁom slide.

5.

6.

7.

10.

11.

Wash the samples with PBS for 5 minutes at room temperature.

Fix the sections with 4% formaldehyde for 5 minutes at room temperature.
Wash samples with PBS for 5 minutes at room temperature.

Be sure nucleotide mix is thawed on ice.

Remove as much liquid from the slides as possible. Cover the tissues with

approximately lOOul of Equilibration buffer. Equilibrate at room temperature for

 

 

10 minutes.

Prepare TdT incubation buffer according to following chart while tissues are

equilibrating.

Buﬂ‘er component Component volume # of reactions Component

volume

Equilibration buffer 45 ul = ul

Nucleotide Mix Sul = ul

TdT enzyme lul = pl
Total TdT Incubation buffer volume = ul

Remove all liquid and blot with tissue paper around tissues. Add 50p] of TdT

incubation buffer to the tissue sections and be sure sections are completely

88

12.

13.

14.

15.

16.

17.

covered. Cover the slides with the plastic coverslips provided to ensure even
distribution of reagent. BE SURE TO WORK IN THE DARK FROM THIS
STEP ON!!! 1!

Wet the paper towels that line the tray and cover the tray. Cover the container
with aluminum foil to protect from direct light. Place into 37°C incubator and
incubate for 60 minutes to allow reaction to occur. Be sure that a tray of water is
in the bottom of the incubator to provide moisture.

Dilute the 20X SSC 1/ 10 with ngO and add enough of the resulting 2X SSC to
cover the tissue sections and terminate the reaction. Incubate the slides for 15
minutes at room temperature in the dark.

Wash tissues with PBS for 5 minutes at room temperature. Repeat 2 times for a
total of three washes.

Prepare a solution of lug/ml propidium iodide. Add 1mg of P1 to a ml of PBS.
Vortex to mix. Add In] of PI solution to lml of PBS. Vortex to mix. Add
enough P1 to completely cover tissue sections. Incubate for 15 minutes at room
temperature in the dark.

Wash the samples with deo for 5 minutes at room temperature. Repeat 2 times
for a total of 3 washes.

Add approximately 501.11 of deO to the tissue sections to keep tissues from
drying out. Cover the slide tray with foil to protect from light. Analyze slides on
ﬂuorescence microscope using a ﬂuorescein ﬁlter set to view green ﬂuorescence
at 520 : 20nm. View red ﬂuorescence of PI at >620nm. On Orth’s scope, set

dial to ﬁlter 3.

89

Procedure for obtaining Cartilage Explantsﬁom Bovine Metacarpal
Joints

1. Call Bellinger’s meat packaging/slaughter house Monday’s and ask for ('2) pairs
of Bovine Knees (1-800-778-4577). Pick up the knees Tuesday.

2. Prepare all media and make sure of supplies.

3. Aﬁer obtaining the knees, wash the outside of the leg in a sink to remove mud.
Use a scalpel to remove skin ﬁ'om leg down to hoof. Place skin in biohazard bag.
Wrap plastic bag around hoof and leg up to joint.

4. Set up the Laminar ﬂow hood. Lay down laboratory paper in hood, place scalpel,
biopsy punches, tweezers, a beaker with ethanol soaked kim wipe, and a petri dish
with media (no FBS) in hood.

5. Take leg into hood. Dissect open the metacarpal joint (lower of two joints at top
of removed leg) using a scalpel.

6. Using a biopsy punch, punch several explants in the lower (articular) surface of
cartilage of the joint. Use the scalpel to remove the explants by slowly cutting
under the explants along the bone surface. Use the tweezers to place the explant
in the petri dish of media. Repeat until you have more than enough explants.

7. Take petri dish of explants into Laminar ﬂow hood. Perform three washes by
using tweezers to place the explants in a new petri dish with media. Allow 5 min
incubation for each wash. Place two explants into each well of a 24 well plate
with 1 ml of media (w/ 10% FBS) in each well. Explants may (or not) be cultured
for two days before impacting. Ifexplants are cultured for more than 2 days,
change media every two days.

8. Take cultures to Instron room. Impact each explant.

9. Place 24 well plate of explants into the hood. Label three petri plates with the
impact categories of the explants and add media to the plates. Transfer the
explants from the 24 well plate to the ﬁrst petri plate using tweezers and incubate
for 5 minutes. Repeat for total of three washes.

10. Explants can now be examined for ﬁssures, Live/Dead cell assay, or ﬁozen for

Apoptosis study.

91

 

 

RABBIT PATELLOFEMORAL IMPACT SOP

 

What to bring with you:

1.

LPS lubricant, ethanol, meter stick, rabbit data sheets, program disk, white silicon

grease, duct tape, and crescent wrench.

Pre-test set up:
Is Plug in Valadyne strain gage amp, attach load cell plug to plug on back of resistor

box, and plug resistor box into back of computer.

1b. Turn on and start up computer, then insert program disk

1.

Turn on the Valadyne resistor box and insure that the trigger release switch is
turned off to protect from accidental triggering.

Connect the channel #2 output A of the Valadyne strain gage amp, to the A/D box
channel #1 input (Load cell output). This should be done already.

Connect the channel #3 output B of the Valadyne strain gage amp, to the A/D
trigger input signal.

Wait for 15 minutes before calibrating the load cell to allow the electronics to
stabilize.

Spray some LPS greaseless lubrication on a rag and wipe down the rail of the
impact cart.

6. Clean, with alcohol, the side of the impact cart that is gripped by the brakes.
7. Hook the volt meter up to the strain gage amp. Put the volt meter probes into the

holes to the right and above and below the black dial. Switch the volt meter on
and set to voltage.

Using the small screwdriver, zero the load cell on the Valadyne strain gage amp.
Use the opening to the top left of the black dial. Use the readout of the volt meter.
Calibrate the load cell by depressing the shunt cal on the Valadyne strain gage
amp. If needed readjust the set point to :1,_5_3_ by using the Gain knob (black
knob).

10. Double check to make sure the load cell is still zeroed, adjust if necessary.
Rabbit preparation:

92

1. On the Data Sheet, record the Rabbit name, weight (kg), and sex.

2. Once the rabbit is ﬁrlly sedated, pull the left hind foot through the very bottom
hole of the leather strap of the holding chair and tuck the rest of the strap under
the rabbit so it can be ﬁxated to the underside of the chair.

3. Position the right leg so that the femur is pointing vertical and the impactor is
directed to hit the middle of the patella.

4. Place the black strap around the right hind foot and pull tight to insure the foot is
fully constrained. Fix the end of the strap, as well as the leather strap, to the
Velcro pad on the underside of the chair.

5. Move the clamping bar into position and attach the ﬂee end to the distal clamp.

6. Slowly apply even pressure to both clamps until the clamps lock in place.

7. Slide the chair into position so the patella is directly under the head of the
impacting cart.

8. Lower the cart so the head of the impacting cart is just above the patella, checking
to insure the patella is centered under the head.

9. Raise the cart to the desired height.

10. The position of the beam holding the upper brake may need repositioning to
accommodate the desired height. Do this by removing the screws of the beam
and moving the beam to the desired height above the patella.

11. The height and weight of the sled should be:

Energy Height Mass
6 J 0.46meters 1.33kg
10 J 1 meter 1 kg
Computer set-up:
1. Start the “CDC RABBIT DROP TEST” program by double clicking on the icon
in the Windows atmosphere.

2. Answer the AID set-up questions ﬁ'om the computer

Start channel = 1
End channel = 1

Time as ref. = “Y’

9

93

3.

4.

.‘°.°°."P‘.V‘

Inverting load cell = “N”
Rotary encoder = “N”

Change the ﬁlename.
Type “F” [enter]
File to change = 1
F ilename = rabbit’s name (ID).csv

Enter

Check the plot control ﬁle data

# of channels to plot = 1
X label = time (seconds)
Y label = Newtons
Data ﬁle name = rabbit’s name being tested
Channel number 0 cal factor = 1

(time calibration must be set to 1)
Channel mrmber 1 cal factor = 444.8 NN

Press E for exit when all values are set correctly
Sample rate 10,000 samples / sec (Hz), Type y for yes
Number “N” = 10,000, Type y for yes

Trigger level for data acquisition = 30(N)

Number of samples to save after the trigger = 400

10. Press space bar
Impacting the rabbit:

l.
2. Press the red release button on the Valadyne resistor box to drop the cart.
3.
4
5

Turn on the trigger enable switch on the Valadyne strain gage amp.

Impact cart will drop and the A/D board will trigger and save the data

. Check status of D.M.A operation = “N”
. If you select “Y” then it should read:

94

0ptype=1
Status=0
Word count = 10000

. Plot using line mode = “L”

. After the data scrolls to screen, it will be plotted. On the data sheet note the peak
load and the time to peak.

. Sketch the graph on the data sheet and write any comments about the test that
were unusual, i.e. if the impact cart hit the rubber stoppers during the test, etc.

. F LLO MACTYON ALLANMLSREM VED FR M

DRIVE a:l AND COPY ALL FEES T THE :luserlbi
DIRECT RY F RPERAMNENT A !

95