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HISGAN

.Laaamaa' IIIIIIIII
fe‘uchagan State 1 3 1293 00629 9303
University

 

 

 

 

 

                                                                                             

 

 

This is to certify that the

thesis entitled

THE EFFECTS OF GAMVIA IRRADIATION ON
PATELLAR TENJDN ALLOGRAFI‘S

presented by

Patrick De Deyne

has been accepted towards fulfillment
of the requirements for

Master . Science
degree m

 

 

    

Major professor

Date July 11, 1989

 

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

 

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MSU Is An Affirmative Action/Equal Opportunity Institution

 

 

 

THE EFFECTS OF GAMMA IRRADIATION ON PATELLAR TENDON
ALLOGRAFTS
BY

Patrick De Deyne

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Biomechanics

College of Osteopathic Medecine

1989.

@00184I

ABSTRACT

THE EFFECTS OF GAMMA IRRADIATION ON PATELLAR TENDON
ALLOGRAFTS
BY

Patrick De Deyne

The purpose of this study was to investigate the cause
of the altered mechanical properties of patellar tendon
allografts due to the sterilization with gamma rays used in
tissue banks. A Hydrothermal Isometric Tension device was
developed, solubility assays with acetic acid and pepsin
were used and electron microscopy was performed. A
significant lowering of the shrinkage temperature, unchanged
acetic acid fraction, an decreased pepsin released fraction
and a decrease in contrast in the banding pattern showed to
support the hypothesis that minimal additional cross-linking
from gamma irradiation occurs and that the main effect of

irradiation is chain scission, possibly at the telopeptide

ends of the collagen molecule.

ACKNOWLEDGEMENT S

The author wishes to express his deepest appreciation
to the following people for making the completion of his
Master of Science degree possible:

To Dr. Robert Wm. Soutas-Little, chairman, for his
encouragement.

To Dr. Roger Haut, his major professor, for his
invaluable assistance in the development of the study and
the preparation of this manuscript.

To Clifford Beckett, for his assistance with the
computer programming and the design process.

To Jane Walsh and Vance Kincaid for helping with the
electron microscopy.

To Valerie Smyth and Aimee Farqhar for the assistance
with the solubility assays.

To Marian De Deyne- De Backer for her excellent typing

skills.

iii

TABLE OF CONTENTS

Page

LIST OF TABLES.. ..... . ........................ ......V
LIST OF PICTURES ................................ ...VI
LIST OF FIGURES... ................................ VII

section

I. INTRODUCTION. .................................. 1
II. LITERATURE SURVEY.. ................ . .......... .3
III. MATERIALS AND METHODS ........... . ............. 18
IV. RESULTS AND DISCUSSION ............. ... ........ 31
BIBLIOGRAPHY ........................ .... ........... 44

iv

LIST OF TABLES

Table Page
1. H.I.T. results ................................ 32
2. Solubility results ........................... 35

LIST OF PICTURES

Picture Page
1. Mounting of test samples..... ....... . ..... ....19
2. H.I.T. test device. ........................... 22
3. Minimizing heat transfer ...................... 23
4. E.M. from control tissue ...................... 36
5. E.M. from irradiated tissue............ ....... 37

vi

LIST OF FIGURES

Figure Page
1. Enzymatic stages of collagen naturation ........ 7
2. End view representation of collagen ........... .8
3. Intra- and intermolecular cross-links .......... 9
4. Cross—link sites .............................. 10
5. General H.I.T. curve .......................... 13
6. Thermal shrinkage of collagen ................. 15
7. Force- temperature plot ......... . .......... ...17
8. a. Heating rate ............................... 20
b Calibration ................................ 21
9. H.I.T. parameters ............................. 25
10. H.I.T. curves ................................. 33

Vii

I In r i n

Replacement of the anterior cruciate ligament after
injury is a relatively common procedure. However, the
materials used to replace the anterior cruciate ligament are
still a topic of debate. Because no synthetic materials have
been able to meet the demands placed on them as ligament
substitutes (1). Only autogenous tissues (patellar tendon,
semitendinosus tendon, fascia) are widely accepted as
suitable substitutes for the anterior cruciate ligament. A
potential problem with autogenous tissues, however , is that
periarticular tissues must be removed from the knee to
function as graft. This loss of tissue may compromise other
structures of the knee. Thus the concept of using allografts
to replace the injured anterior cruciate ligament is of
major clinical significance (2). Current concepts and
research pertinent to allograft surgery such as
biocompatibility, biomechanical properties, biological fate,
procurements and preservation techniques are to be addressed
(3).

One of the techniques used by tissue banks that provide
tendon allografts is preservation by freeze-drying and/or
sterilization using gamma irradiation.

It is of primary importance, in order to guarantee high
quality allografts, that the currently used preservation and
sterilization procedures are investigated in order to detect
any detrimental effects on the strength or physical

integrity of the graft. The aim of this thesis is to study

2
the effect of gamma irradiation on patellar tendon

allografts by means of a hydrothermal isometric tension

procedure (H.I.T.)

I Li r r urv
A.Irradiation and collagen.

In the sixties some interest was centered on the
possibility of preserving raw meats by subjecting them to a
sterilizing dose of ionizing radiation, thereby inhibiting
spoilage due to the growth of micro organisms. The process
was accompanied by side effects resulting from the
interaction of the radiation with the various components of
the tissue. One of them was that meat became more tender and
investigations were focused on the connective tissue, more
specifically collagen.

Various authors have investigated the effects of gamma
irradiation on collagen. Person et al (4) used X-ray
diffraction patterns to study the phenomenon, and observed a
shrinkage of the wet irradiated, not the dry irradiated
collagen. J. Cassel (5) noted a decrease in shrinkage
temperature and increased water solubility of the collagen.
He also concluded the decrease in shrinkage temperature was
not influenced by moisture content of tendon during
irradiation (5 to 220 Mrad), and that the treatments caused
peptide chain scission. Kuntz et al (6) believed that
crosslinking was the main effect when the tissue was
irradiated wet, and an excess of breaks occurred in the
peptide chains when irradiated dry. Bailey et al (7) used
the hydrothermal shrinkage temperature to assess irradiation
effects, and found a decrease in shrinkage temperature

progressively with dose. He discussed the direct effect of

4
the gamma rays on the collagen molecule. Bowes et al (8)

elaborated on the differences between irradiation in the dry
state and the wet state of the samples. He contributed the
fall in shrinkage temperature and increased solubility to an
extensive loss of molecular structure. This was most obvious
with dry irradiated samples. The presence of water
determines the effects of irradiation on collagen, Bailey et
a1 (9) and (10) discussed the decrease in solubility with
increased tensile strength, and contributed that to the
formation of new crosslinks.

In the seventies more and more became known about the
nature of intramolecular crosslinks and the amino acid
sequence of collagen. For a good overview see (11,12) and
earlier work (13).

A better understanding of the biology of the collagen
molecule gave an impetus to the research to determine the
effects of gamma rays on the collagen structure. Grant et al
(14) found results consistent with the hypothesis that
electron irradiation of collagen in the dry state results in
scission of the polypeptide chains and that, in presence of
water, this is accompanied by the formation of
intermolecular bonds. Their methods consisted of
electronmicroscopy, differential enzyme digestion (elastase,
collagenase) and the use of glutaraldehyde. Van Caneghem et
a1 (15) deduced that those effects were irreversible and

oxygen dependent.

5
It was still unknown if radiation increased the inter-

or intramolecular crosslinks. Earlier work by Dancewicz (16)
was inconclusive. J.J.M. Labaut (17) proposed that gamma
irradiation in the wet state induced the formation of
aldehyde groups, which are of primary importance in the
production of intermolecular cross-links. Svojtkova et al
(18) studied the effects of gamma irradiation and induced
cross-linking in combination with high fat diets and
increasing age. M.M. Jelenska et a1 (19,20,21) studied the
effect of gamma irradiation at the level of the tropo
collagen molecule and concluded that there was an increase
in aldol cross-links and formation of new reducible
components of an unknown structure.

The experiments discussed above are in vitro
conditions. Data are available on in vivo effects of
irradiation on collagen (22,23) and report some changes in
its neutral salt solubility (22) are reported to be dose
dependent. Pickrell et al (24) notices radiation induced
changes in pulmonary collagen metabolism. Ohuchi (27) and De
Loecker (28) also reported changes in collagen metabolism.
Svojtkova et al (25) and Grant (26) have similar conclusions
regarding the effect of gamma irradiation on collagen:
depending on the experimental conditions (moisture content)
ionizing radiation may cause polymerization or
depolymerization of collagen. Irradiated in a wet state the
formation of new cross-links is the predominant process,

over a wide dose range (50 krad up to 5-10 Mrad),(5-10, 14-

6

18). The nature of the induced cross-links (intra- or inter
molecular) is unknown. There is also a shrinkage of the
tissue which is dose dependent (10,14). Irradiated in a dry
state depolymerization by peptide chain scission is more
predominant (10,14,15). It is also interesting to note that
Bailey (7) reported that lowering the temperature below the
freezing point during irradiation provides a protection to

chain scission.

B.Collagen and cross-links.

Cross-linking renders the collagen fibers stable and
provides them with an adequate degree of tensile strength
and visco-elasticity to perform there structural role. In
terms of its mechanical properties other parameters are
equally important, such as length and structure of the fiber
(29); metabolic interactions between its cells and the
extracellular matrix (30,31,32); maturation and development
(33) and mechanical testing conditions (34). Viewing the
interaction between gamma irradiation and cross-links (see
above) and thus mechanical strength (35), I will briefly
describe the current state of knowledge of cross-links in
collagen. For a general and historical overview of collagen
research see Mayne and Burgeson (36).

Structural collagen obtains its stability from its
unique molecular coil configuration, the quarter-staggered
packing of tropocollagen units (Fig.1), and its ability to

form covalent intra-molecular and inter-molecular cross-

links

7

(37,38). The former links occur between alpha chains

of the same tropocollagen molecule, the latter between

adjacent tropocollagen molecules

(Fig.2). Cross-links are

key to both tensile strength characteristics and resistance

to chemical or enzymatic breakdown.

Their absence causes the

collagen fiber to be extremely weak and friable.

Figure 1. Enzymatic stages in maturation of collagen.

From M.Nimni

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8
The enzyme of vital importance in the creation of cross-

links is lysyl oxidase (38,39). The crosslinks result from
enzyme-mediated reactions involving mainly lysine and
hydroxylysine. The lysine and hydroxylysine molecules have
secondary amine groups on terminal projections extending
lateral from alpha chain, which are available for cross-

linking reaction.

Figure.2. Representation of an end view of the peptide
backbone of the collagen helix.(From: Connective tissue
disease, molecular pathology of the extracellular matrix,

edited by J. Uitto, A.J. Perejda, Marcel dekker, Inc. 1988).

 

 

Collagen tnple helix

9
The initial reaction in cross-linking is the oxidatiye

deamination of the terminal to an aldehyde by the enzyme
lysyl oxidase. The resulting allysyl residues condense with
one another to form an aldol condensation product,
characteristic of ingramglegular cross-links.Intermolecular
cross-links characteristically form in the reaction of
allysine with lysine or hydroxylysine to form a Schiff;ba§e

(see Fig.3).

Figure.3. formation of intra- and intermolecular cross-

links.

NTRANOLECULAR
ALDOL — T YPE
CROSSLINK

 

NTERMCLECULAR I
scuvr"s BASE CH
cnossJNK

 

10

For basic mechanisms and pathways see Yameneuchi (40).

One must note that the hydroxylation necessary to stabilize
cross-links is an intracellular process, while the actual
cross-links are formed extracellulary (Fig.1).

Collagen intermolecular cross-linking reactions are
controlled by the stereospecificity of interacting
neighboring molecules (angular orientation and staggered
configuration). An important adjunct to the determination of
the specific molecular loci of the cross-links is the
primary sequence of collagen. In type I collagen, which has
1014 residues in the d.1 (I) chain, cross-linking sites are
characterized by low proline and hydroxyproline content and
the presence of the sequence Hyl-Gly—His-Arg, which appears
to be important as an attachment site for the enzyme lysyl
oxidase. Cross-links between residue 5N and the helical
residue 930 and between 16C and the helical residue 87 of

theci 1 (I) chain have been described (40,41) (Fig.4).

Figure 4. Schematic representation of cross-link sites.

 

 

 

5N 97 930 we
mlys A. Hyl IJ/A Hyl wiys—y
'~ ' /
mlys "VI 7/

5" e7

11
The enzyme mediated reaction mainly involves lysine and

hydroxylysine and the Schiff base reaction products are:

-H L N L (hydroxylysinonorleucine )

-D H L N L (dihydroxylysinonorleucine)

-and the more complex H H M D (histidino hydroxy

merodesmosine).
Most studies on cross-linking of collagen in the past decade
have used tritiated NaBH4 as a marker to the peptides which
contain reducible cross-links. For details see Bailey
(42,43).

The field of collagen cross-linking has been
significantly advanced during the last decade It is that
knowledge that will elucidate the geometry of packing and

organization of collagen molecules in various tissues.

C.Heat and collagen.

The denaturation of connective tissue, especially
collagen, by heat has been employed to assess the structural
qualities of its constituents. Native collagen is denatured
by heat. The protein loses its highly ordered crystalline
structure to a preferred random coil state (59) with higher
entropy. A variety of methodologies have been used on
different tissues. I will give a brief overview.

Intramuscular connective tissue was tested by Mohr et
al (44). Other tissues that were assessed by a form of

thermal denaturation were: Achilles tendon by Spadaro et a1

12
(45), Finch et al (46), Jong Jin Lim (47); wrist tendons by

Mitchell et al (48); extensor digitorum communis tendons by
Goldin et al (49); other tendons by Snowden et al (50) and
Mady Le Lous (51). Rat tail tendons, because of their almost
exclusive content of collagen, have been a frequent
substrate for testing used by: Viidik (52), Ward et al (53),
Magy et al (54), Burjanadze (55), Andreassen et al (56), Yue
et al (57,58), Andreassen et al (59), and Mitchell et a1
(48).Thermal stability of skin was assessed by Russell (60),
Allain et al (61), Danielsen (62), Gekko et a1 (63) and
Danielsen (64). A variety of other tissues have been under
investigation with hydrothermal procedures. Basal membrane
and type IV collagen was investigated by Gelman et al (65)
and Sank et a1 (66). Cartilage has been studied by Snowden
et al (50,67); scar tissue by Snowden et al (68), type X
collagen by Linsenmayer et al (69), and tropocollagen by
Berg et al (70).

Besides a diversity of tissues tested there are several
heating methodologies, especially with regard to the
determination of the initial shrinkage. Two general methods
have been employed most often:

-A temperature increase of an isometric tissue and

measuring the tissue response as force of contraction,

Mohr (44), Finch (46), Mitchell (48), Allain (61),

Goldin (49),Andreassen (56) and Le Lous (51).

-a constant temperature and observing with specific

instrumentation when the changes turbidity of the

l3

macromolecule solution, occur on a time basis. Spadaro

(45), Russell (60), Maggy (54).
For our purpose, the study of patellar tendon, the first
approach was chosen, this for practical and experimental
design reasons (control of heating rate, sensitivity of load
cell, the possibility of studying the rate of shrinkage).

The main effect of heat on collagen is'a shrinkage and
gradual development of tension in an isometrically held
tissue. This effect has been applied in surgical therapeutic
procedures (71). A generic curve for tension vs. temperature

was proposed by Mitchell (48) (Fig.5).

Figure 5. Diagram illustrating the main features of the
stress temperature curve, when a tendon is melted under

isometric conditions.

STRESS

 

 

 

TEM PE RATURE

14
The main characteristics are:

-Ts= shrinkage temperature: temperature at which the

tissue starts to shrink.

-The slope of the curve.

These two parameters are characteristic for each tissue, and
give an idea of the crystalline organization and stability
of the tissue’s constituents.

Due to its reproducibility and high sensitivity,
hydrothermal procedures have been used to assess the effect
of various chemicals on collagen (45,46,60,63);and the
effects of induced diabetes on collagen (56,57,58,59). The
above mentioned parameters have been correlated with the
amount of hydroxyproline incorporated in the collagen
molecule (47,53,55,62,70) and the influence of cleavage
enzymes (64) on shrinkage temperature has been assessed. The
most extensive study (more than 1000 samples) was performed

by Le Lous (51).

15
Figure.6. Thermal shrinkage of collagen and the role of the

intermolecular cross—links in providing for the contractile

force (as proposed by M.Nimni (39) p.43).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

°/o SHRINKAGE

 

60 I0 90
TEMPERATURE (°C)

 

 

 

 

 

 

 

 

 

 

 

16
Nimni (12) discusses the effect of heat on collagen and

concludes that denaturation by heat can produce a collapse
of the helical structure and a shrinkage in the direction of
the longitudinal axis of the fiber (fig.6). The isometric
tensions developed during thermal shrinkage are used to
evaluate the physical properties of the collagen networks.
This may quantify the degree of cross-linking of collagen
that occur with aging (48,54,57). The authors do not specify
the type of cross-links. One could distinguish between the
stabilization of the collagen by hydrogen bonds
(49,53,55,70), thermo labile vs thermo stabile bonds (51) or
reducible cross-links (HLNL, DHLNL, HHMD) (50,56,61).
Another perspective is brought by Danielsen (64) who focuses
more on structural continuation of the polymeric aspect of
collagen and its influence on shrinkage temperature.
Karlinski (72) mentions Treloar’s model for rubber
elasticity (Fig.7) to quantify the change in energy content

and increase in entropy with heat.

l7

Figure.7 Force-temperature plot for a pure rubber elastomer.

Slope et ,-
temperature ’’’’
f ' ’ ’

  
 

Entropy Contrlbutlen

 

IEnergy Contrtbutton

 

FORCE AT CONSTANT lENG‘lt-t

 

I
rmreuruns

 

-b-- o- - ----.---

We can conclude that a hydrothermal isometric tension
procedure based on the design by Goldin (49) can be a
sensitive, reliable, reproducible method to assess the
structural stability of collagen, and in our case any
potential damages or changes that occur with gamma
irradiation of graft tendons. The value of shrinkage
temperature, the slope of tension development with a linear
increase in temperature and how these relate to a
description of the effects of gamma irradiation on collagen

will be treated in the discussion.

III M ri l n m h
Te in r re

Three patellar tendons, from young males A, B, C,
respectively 26, 23, 28 years old, were donated by the
Michigan Tissue Bank. They were cut medio-sagitally. One
half was fresh frozen and served as a control, the other was
frozen at - 70°C and irradiated (2.0 MRad from a cobalt
source). The irradiation was performed by the Michigan
Tissue Bank using their standard irradiation procedure.The
tendons were shipped and stored (maximum 6 weeks) at - 20°C
until further experimentation.

Using a dissecting scope small patellar tendons strips
were cut out from the medial, central part of the half
tendon. Caution was given to dissect, at room temperature,
longitudinal intact fibers to have a continuity of fibrils.
After dissection the strips of tendon were frozen at -20°C
until testing. After being thawed in physiological solution,
the samples were mounted in the clamps on a frame to

standardize for length (12mm). See picture 1.

18

19
Picture 1. Test specimen mounted in clamps, length of the

sample is 12 mm.

 

The samples were placed in the testing device (picture
2) and a custom constructed pyrex cylinder (diameter: 7 cm,
length: 20 cm) was slid up so that the sample was completely
submerged in a 0.9% saline solution. A pre-tension, applied
by a thumb screw mechanism, of 0.16 N (monitored on a
digital multi meter) was given prior to the test. 750 ml of
0.9% saline solution was heated from 25°C to 95°C by a 209
W, 1.25 cm wide resistive heating tape (coated with silicone
rubber to increase the thermal contact area and to protect

from possible electrical shorts from solution spillage). A

20
variable A.C. transformer was used to drive the heating

element and the cylinder was surrounded with fiberglass
insulation to improve the linearity of the heating curve.
The heating rate was 1.5°C/min.Figure 8 shows the

relationship between time and temperature increase.

Figure 8a. Heating rate in the H.I.T. experiment.

 

9.94902

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0. 091MB
0. 00MB tine 0.2m“

ll SPllClllc: 108 t SPtCIllG: {I
0kl ac7t ( heating rate )

21
Figure 8b. H.I.T. calibration

 

0:96E'Ul

 

mince—-

 

 

 

 

 

-.32E+OB
RIDGE tenp 0.96302

ll SPACING: 4 t SPACING: 1
test3tl ( carbon calibration )

The H.I.T. device was calibrated. This was important
because of the not fully temperature compensated load cell.
The H.I.T. data beyond 80°C were not used in this study due
to the decrease in linearity of the heating rate (Fig.8a.)
and the sharp increase in negative readings from the load
cell (Fig.8b.). One could upgrade the H.I.T. device by using
a servo system for controling the heating rate and a
temperature compensated load cell (and mounting it away from

the heating source) to have valuable data beyond 80°C.

22
Picture 2. Hydrothermal Isometric Tension (H.I.T.)

Test device.

 

||||||l||||||I|l| -

“it

 

The temperature data were acquired using a type T
copper / constantan thermocouple placed in the center of the
saline bath. The reference electrode was placed in a Dewar
flask filled with ice mush. The signal from the thermocouple
was amplified with a Sensotec (model SA-4) strain gauge
amplifier. The voltage was then input into an A/D board
(METRABYTE model dash 16).

The load cell (Gould—Statham, model UC 3) gathered the
tension data. The load cell was not fully temperature
compensated. Heat transfer to the load cell was minimized

with a plexiglass shield for air current and with a 9 cm

23
long carbon composite fiber rod (connecting the load cell

with the top clamp holding the sample as can be seen on
picture 3). The output of the load cell was amplified using
a Sensotec (model SA-4) strain gauge amplifier. The signals
were then converted to digital data by a Metrabyte (model
dash 16) A/D board. The load cell was calibrated using 1.0 N

and 2.0 N loads weighed on a laboratory beam balance scale.

Picture 3. Plexiglass shield, carbon composite rod to

minimize heat transfer.

 

24
The loads generated in the experiments could reach

maximum values of 6.1 N but most were between 3 and 5 N. As
it was difficult to determine cross-sectional area for the
samples, the load data were normalized per dry weight.
Goldin et a1 (49) shows a good correlation between cross-
sectional area and dry weight. In my case where the length
of the sample remains constant being tested we can use the
same model.

Both signals were monitored during testing with digital
multi meters. The data were collected with a custom made
computer program at a sampling rate of 0.1 Hz for 2500 sec.
After testing the tendon specimen was carefully removed, cut
from in between the grips, placed in a test tube and
lyophilized. Dry weight of the samples was obtained by using
a Mettler (H8) laboratory balance.

Because the load cell was not fully temperature
compensated and the decreasing heating rate beyond 80°C
(Fig.8a,8b), the secondary slope (SL 2) was calculated from
the data available between 70 and 80 degrees C. The slopes
(SL 1 and SL 2) were manually calculated. Figure 9 shows the

parameters used for this study.

25

 

 

 

 

Figure 9.
I
!
N/
”'8
b.
Ts: The shrinkage temperature was obtained by the

SL

SL

projection of the intersection between the horizontal
through the deepest point of the curve and the vertical

through the point when initial rise occurred.

:The increase in tension in the segment between Ts and

Ts+3°C; the initial slope

:The secondary slope, the increase in tension between

the temperature interval from 70°C - 80°C.

26
The temperature , slope 1, slope 2 data of the

subgroups (A control n=9; B control n=10; C control n=10; A
irr.n=10; B irr.n=9; C irr.n=10) were tested with a two -
tailed ANOVA test to ascertain if they were from the same
population. As the data had a normal distribution (tested
for skewness and curtosis), the standard deviations were
homogeneous and the subgroup A,B,C were from the same
population a two tailed Student t test was used for
statistical analysis. There was a control group of 29 test

samples and an irradiated group of 29 samples.

Additional tests,

To have a better understanding of the mechanisms
involved in gamma irradiation of collagen, solubility
"studies and electron microscopy were performed.

Solubility data were based on hydroxyproline extraction
with 0.5 M acetic acid (referred to as acid soluble
fraction) and pepsin digestion (referred to as pepsin
released fraction); a method used by Schnider et al (73).
Hydroxyproline content was measured by the method of
Stegeman (74) and described also by Woessner (75).

1. Tendon samples were pulverized at liquid N2

temperature and freeze dried for 24 hours.

2. Three aliquots for solubility and two for totals

from approximately 30 mg were accurately weighed and
lipid removal was accomplished by extracting the

sample with 10 volumes of chloroform— methanol (2:1,

27

vzv). Samples were agitated overnight , spun 1 hour
at 2000 g, and supernatant was discarded. A total of
three extractions were performed. All samples were
lyophilized.

. Acid soluble fraction was obtained using 0.5 M
acetic acid, (for 24 hours at 4°C with agitation
then centrifuge 1.5 hour at 2000 g, pool
supernatant; a total of three extractions).

The pellet was then suspended in 0.5 M acetic acid
and digested with pepsin (P-7012, SIGMA, St.Louis,
M0) at a concentration of 1.0 mg/ml for 18 hours
with stirring at 4°C. NaCl in Tris HCl buffer

(pH 7.4) was added to the viscous digestion mixture,
after the incubation was completed, to a salt
concentration of 0.17 M. Only one digestion was
carried out. Collagen solubilized after pepsin was
collected by centrifuging the mixture at 5000 g

for 3 hours.

The collagen remaining after the acid and pepsin
treatment is considered insoluble. Pepsin released
fraction is dialyzed for 12 hours against 0.1 M

acetic acid.

. All the samples (pooled supernatants, totals,

insoluble fraction) were frozen in dry ice,
lyophilized;hydrolyzed with 2 ml of 6 M HCl at 105°C
for 16-18 hours; and neutralized with 2.5 M NaOH

(indicator was 0.02% methyl red).

28
In a timed manner 1 ml of neutralized sample was

added in marked test tube. Then add 1 ml of
chloramine-T solution (mix and let stand at room
temperature for 20 min.) Add 1 ml of HClO4
solution, mix and let stand at room temperature for
5 min. Finally, add 1 m1 of p-DAB solution, mix and
let stand at 60°C in a water bath for 15-20 min.
Cool tubes to 20°C and read in spectrophotometer at
550 nm. Hydroxyproline content is determined by

using a standard curve (from stock hydroxyproline)

Reagents used in assay are:

0.03 M chloramine -T (C 9887, SIGMA) solution.
Dissolve 0.845 chloramine -T in mixture A. Mixture
A: 20 ml H20, 30 ml propanol, 50 ml citrate-acetate
buffer at pH 6.

3 M perchloric acid (25.5 ml 70% HC1O4 H20 to

100 ml)

5 % solution of p- dimethylaminobenzaldehyde(p-DAB),
(KODAK Co.) in propanol, dissolve under heating,
store covered.

standards: 0-5 ug Hyp/ml.

Besides acid-soluble, pepsin released fraction, and

insoluble fraction, total hydroxyproline content was also

determined.

Electron microscopy protocol was based on the procedure

by Lendis (76) and the following steps were performed.

1.

When excised the samples were placed in cold (4°C)

29
4% glutaraldehyde in 0.1 M phosphate buffer with pH

7.4. No additives were used.
Longitudinal samples of 1 mm were put for the second
time in fresh fix for 1.5 hours at 4°C.
The samples were washed 3x in cold 0.1 M phosphate
buffer for 20 minutes each wash.
Post fixation in 0.1% OsO4 in the phosphate buffer
for 1.5 hours at room temperature.
The samples were washed 2x in buffer for 15 minutes,
each wash at room temperature.
The samples were dehydrated at room temperature in
graded ethanol

25% - 30 minutes

50% - 30 minutes

75% - 30 minutes

100% - 30 minutes gently agitate.

100% - 30 minutes " "

100% - 30 minutes " "
Samples were embedded into Spur epoxy (Polysciences,
Inc. Warrington, Pa.)with graded steps at room

temperature.

1:3 resinzethanol 8 hours gently agitate
1:1 resinzethanol 8 hours " "
100% resin 8 hours " "

Embedded samples were oriented for longitudinal

sectioning. Molds were placed in desiccator 2 hours

10.

30
before curing at 70°C overnight (000 gelatin

capsules).
Silver section on 200 mesh Cu grids.

Stain with 3% uranyl acetate in ethanol solution

for 45 minutes and 3 minutes in Reynolds Pb.

31
V n i i n

Collagen shrinks when exposed to heat as a result of
the breakdown of the ordered crystalline structure of the
collagen to a preferred random coil state (59). In
determining the parameters used in this study care must be
given to the heating rate. Mitchell and Rigby (48)
demonstrated that the shape of the tension curve and the
shrinkage temperature are dependent upon the rate of heating
during the isometric melting experiment. They conclude that
in general, 1) an increase in the heating rate increases the
stresses and the shrinkage temperature (Ts), 2) the changes
in the heating rate become less marked with in vitro aging.

The data collected from the HIT experiment are shown in
Table 1. For the control tissue (n=29) the average shrinkage
temperature was 62.4°C (S.D. 0.885), the initial slope (SL
1) had a value of 2.806 E-2 N/mg.°C (S.D. 8.67 E-3) and the
secondary slope (SL 2) had a value of 1.105 E-2 N/mg.°C
(S.D. 3.24 E-3). The irradiated tendon samples (n=29) had a
shrinkage temperature of 58'4°C (S.D. 0.96) and an initial
slope of 3.038 N/mg.°C (S.D. 6.68 E-3). The secondary slope
had a value of 1.644 E-2 N/mg.°C ( 5.0. 2.81 E-3). The
average dry weight was 7.06 mg (S.D. 2.66) for the control

tissues and 6.48 mg (S.D. 2.25) for the irradiated samples.

Table 1.

experiment.

Ts (0C)

SL

SL

XI

XI

xl

(N/mg.°C)

(N/mg.°C)

.D.

32

Shrinkage temperature,

CONTROL

62.4

0.885

2.806 E-2
8.67 E-3

1.105 E-2

3.24 E-3

slope 1, slope 2, from HIT

IRRADIATED t-test
58.4 p< 0.005
0.96

3.308 E-2 n.s.
6.68 E—3

1.644 E-2 p< 0.20
2.81 E-3

Figure 10 shows a general view of the H.I.T. curves. One can

observe that the irradiated curve is shifted to the left

while the secondary slope is steeper, although there was no

statistically significant difference from controls.

33

Figure 10. H.I.T. curves for control (C) tissue and

irradiated (I) tissue.

”/Ma, }
0.3 . I

0.2. .

0.! .

 

 

All the samples showed an initial relaxation up to the
shrinkage temperature. I did not try to quantify that
observation. Rigby et al (77) also reported a thermal
expansion prior to the sudden development in tension at the
shrinkage temperature. At the shrinkage temperature a rapid
increase in tension occurs (SL 1); in the range from Ts to
Ts + 3°C. After Ts +3°C the tension levelled off (SL 2) and
developed steadily to the end of the test (80°C). In some
samples (3) mechanical failure occurred between 90 and 95
degrees C.

The development of the slopes and the shape of the curves
are consistent with the data found by Mitchell et al (48),
Le Lous et a1 (51), Goldin et al (49) and Allain et al (61).
Andreassen et a1 (56) uses a different experimental protocol

but also reports the particularity of the slopes.

34
Solubility changes have been observed with increased

aging (80) and the increased insolubility has been
correlated with increased tensile strength (80,81) and
increased cross-linking (9,10). The current solubility
studies were performed in addition to the H.I.T. experiment
to have a better understanding of the effects of gamma
irradiation. The results are shown in Table 2. They show a
major increase in insoluble fraction with a concomittant
decrease in pepsin released fraction. The total
hydroxyproline content for control and irradiated tissue was
almost identical (Table 2). As mentioned earlier
hydroxyproline content alters the shrinkage temperature
(47,53,55,62,70). There is also no change in acid
solubility. The main difference is found in the pepsin
solubility. It is to be noted that in the experiments some
of the pepsin released fraction was lost, most likely in a
dialysis step in the assay, and that the actual numbers
should be higher. This does not affect the amount of

insoluble fraction.

35
Table 2. Solubility of irradiated and control tendons

(in ug Hyp/mg).

Control Irradiated t-test
Totals
i 84.0 96.0
S.D. 13.3 19.8 n.s.
n 6 6

Insoluble fraction

2 11.0 76.3 p < 0.001
S.D. 4.82 11.3
n 9 7

Pepsin released fraction

2 37.4 7.86 p < 0.001
S.D. 7.0 1.86
n 7 7

Acid soluble fraction

Ԥ 0.343 0.26 n.s.
S.D. 0.37 0.24
n 7 5

The electronmicroscopy (pictures 4,5) did not show any
structural changes in the irradiated collagen, nor was there
a change in banding pattern; but a decrease in contrast of
the cross-bands was noticable (picture 5). Bailey (9)
reports change in structural features with an irradiation

dose higher than 10 Mrads.

36

(70,000 X).

Collagen from normal patellar tendon

Picture 4.

It... luv)» .17..

 

..vOarIIJJatJa

 

 

37

Picture 5. Irradiated (2 M rad.) collagen from patellar
tendon (70,000 X). No change in banding pattern, but some

decrease in contrast of the cross-bands.

 

 

 

38

Discussion

Although the population was small one can observe from
Table 2 the obvious changes in solubility. As mentioned
earlier a decrease in acid soluble fraction has been
correlated with increased cross-linking (9,10), although
until now the nature of the cross-links susceptible to acid
solubilization has not been determined. Also the shape of
the H.I.T. curve (SL 2) has been correlated with cross-
linking (48,56,61).
Thermal agitation disrupts these chemical bonds producing
fiber shortening due to folding of the collagen chains and a
rise in isometric thermic denaturation tension. Isometric
denaturation behavior beyond the shrinkage temperature is a
reaction to disruption of high energy covalent bonds (49),
which progressively form during fibrogenesis. Mitchell et al
(48) found an increase in thermal stability (steeper slopes)
with aging; they infer two possibilities: 1) an increase in
cross-linking density with age or 2) an increase in cross-
links that are thermally stable. Allain et a1 (61)
determined the type of cross-link that is responsible for
the tension development. The experiments show that with
aging there is an increase in the number of HLNL and HHMD
cross-links while the number of DHLNL cross-links decrease.
They explain the type of slope of the H.I.T. curves in terms
of the type of intermolecular cross-link; and contribute the
tension development beyond shrinkage temperature (SL 2) to

thermally stable DHLNL cross—links and a yet unknown

39
aldimine cross-link. Although the HHMD cross-link is the

major cross-link in the native collagen fiber, Allain et al
states that it is thermally labile. Goldin et al (49) draws
similar conclusions.
In view of assessing the effects of irradiation on patellar
tendon allografts the H.I.T. experiment will assess the
amount of induced cross-link formation. As discussed,
earlier authors (7,10,26) have reported an increase in
cross-linking and an increase in aldehyde content (20) with
irradiation, due to the high probability that slope 2 from
controls and irradiated samples are from the same population
we may infer that there is minimal, induced cross-linking.
Although one could report that all the values of slope 2
were consistently higher than the control values for slope
2, but not significantly. Also the major increase in pepsin
insolubilty (Table 2) can be explained in function of
induced cross-linking from gamma irradiation. But one can
ask if a sevenfold increase in pepsin insolubilty should not
be more obvious in the H.I.T. curve (SL 2) if it was only
caused by new cross-link formation from gamma irradiation.
The unchanged acid soluble fraction also points to the
same conclusion that minimal induced cross-linking occurs.
The development of tension in an H.I.T. experiment has only
been discussed as a function of the quantity and quality of
the cross-links (48,56,61), but this does not take into

account a gradual straightening of the collagen fibrils and

40
removal of the waveform (29), nor the slip or glide between

the components (fibrils) (12) as a reaction to the tension.

The initial slope, slope 1, which is observed
immediately in the interval Ts, Ts+3°C, is not considered by
Mitchell et al (48) as being involved in co-valent cross-
links. They attribute it to Van Der Waals, hydrogen and
electrostatic bonding and hydrophobic bonding (66). One has
to note, as recently reported (78) that the d.helices that
form the tropo collagen molecule are stabilized intra-
cellularly by disulfide and hydrogen bonds. The cross-
linking process itself is an extra cellular event induced by
the enzyme lysyloxidase and the presence of 02. The
hydroxylisation of lysine and proline is important for the
stabilization of the triple helix through hydrogen bonding.
It is reported (53,55,65,66) that an increase in
hydroxyproline content increases the melting temperature
(Fig.5).

The significant decrease in shrinkage temperature of
the irradiated tissue during the test, relates to some
change in structural integrity of the collagen molecule.
Danielsen (64) determined the shrinkage temperatures of
collagenase treated rat skin and states that the shrinkage
temperature of the uncleaved molecule and the N-terminal
fragment are independent of the extent of N-terminal cross-
linking. He inferred that intra molecular cross-linking had
no effect on the shrinkage temperature (62). His data

suggest that the shrinkage temperature is a measure of

41
structural integrity; the shrinkage temperature of the

fragments (TCA, TCB) were found to be 4-5°C lower than the
uncleaved molecule. If we follow the same analogy this model
could explain the decrease in shrinkage temperature as a
result of chain scission.

The loss in mechanical strength of irradiated patellar
tendon allografts that is reported by Haut et al (35) and
Gibbons et al (79) must be due to changes of the factors
that determine the tensile strength. The tensile strength of
the collagen is due to its unique molecular conformation
(33) which is bestowed upon it by the regular repeating
units in amino acid sequence, the highly specific alignment
and packing of the molecules in the fibrils, and the axial
and lateral cohension afforded by the formation of
intermolecular covalent cross-links. The changes in
mechanical properties could be a result of chain scission.
Although formation of new cross-links occurs simultaneously
with chain scission (9,10,14) from irradiation, One can
presume that chain scission could be predominant with the
current sterilization procedures. The unaltered acid
solubility and the virtually identical slopes (SL 2) of the
H.I.T. data would infer no induced cross-linking, as an
effect of gamma irradiation at 2 Mrads; but the rise in
pepsin insolubility and the "trend" (p <0.20) that SL 2 is
higher for the irradiated tendons leaves to believe that
induced cross-linking may not be ruled out. This would infer

that the H.I.T. slopes give a global picture of structural

42
stability rather than only information about the cross-

links.

As reported by Danielsen (62,64) the shrinkage
temperature can be interpreted as a parameter for structural
integrity, independently from covalent cross-linking. The
significant decrease (4°C) in shrinkage temperature in
irradiated samples could be explained as result of
predominant chain scission. The loss of structural integrity
or continuity can explain the reported (35,79) mechanical
changes. One can refer hereby to the decreased contrast in
the electron microscopy preparations (pict.5); while the
banding pattern remained unchanged.

Pepsin can only attack collagen when the latter is in a
denatured state (random coil); pepsin digestion of collagen
will act on the telopeptide ends (first on the NHZ terminal,
the COOH terminal is more slowly digested) of the molecule
(82). Pepsin cleaves specifically the peptide bonds
indicated (*) in the following aminoacid sequences:
Gly-Tyr*—Asp-Glu, Ala-Gly*—Val-Ala. Collagen becomes
resistant to pepsin digestion with age and increased cross-
linking hinders pepsin digestion reported by Weiss (82).The
decreased pepsin released fraction (table 2) can be
explained as a result of a decrease in available sites where
pepsine can act as proteolytic enzyme. That is consistent
with the reported (9,10,14) chain scission from gamma

irradiation. This would mean that the chain scission occurs

43
at the telopeptide end of the collagen molecule, and that it

would hinder the proteolitic activity of pepsin.

I cannot conclude that cross-link sites are not
affected; as shown on fig.4 the cross-links are between the
telopeptide end and the helical region of the adjacent
molecule. The loss in mechanical properties can be explained
as discontinuity in the polypeptide chain. The cross-links
would not be connected in series with the adjacent collagen
molecule.

There is reason to believe that the currently used
method to preserve and sterilize tendon allografts by gamma
irradiation has chain scission for effect. The decreased
mechanical qualities should be seen in the light of
disruption of the collagen "polymer" at its peptide bonds
and more specifically at the telopeptide end of the
molecule. With the current procedure there is minimal
concomitant cross-linking (which is dependent on the
moisture content). Interesting to know is that Bailey et al
(7) reports some protective effects against collagen damage
by irradiating at lower temperatures (—200°C).This could be
directly applicable for tissue banks which are concerned

with maintaining the quality of prospective grafts.

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