An introduction to FP4 laser with
Equine SDFT injuries in combination
with an ex vivo comparative study:
How to induce core lesions in Equine
Tendons in a standardized fashion.
Sanne Minnema | 3382796 | February 2013 – August 2014
Supervisors: Dr. S.M. Cokelaere and Prof. Dr. C.J.G. Delesalle
Department of Comparative Physiology, Ghent University, Belgium.
Faculty of Veterinary Medicine, Utrecht University.
Contents
Abstract ................................................................................................................................................... 2
Introduction ............................................................................................................................................ 3
Introduction to HLLT ............................................................................................................................ 4
Presentation of five cases, successfully treated with HLLT ............................................................... 5
Case 1.................................................................................................................................................... 6
Case 2 ................................................................................................................................................... 8
Case 3 ................................................................................................................................................. 10
Case 4 .................................................................................................................................................. 12
Case 5 .................................................................................................................................................. 13
Conclusion ......................................................................................................................................... 15
Inducing tendon lesions in horses ...................................................................................................... 16
Chemical models .............................................................................................................................. 16
Collagenase ................................................................................................................................... 16
Collagenase gel technique ........................................................................................................... 18
Mechanical model ............................................................................................................................ 19
Surgical method devised by M. Schramme 12............................................................................. 19
Advantages and disadvantages ............................................................................................................ 21
Collagenase .................................................................................................................................... 21
Mechanical models........................................................................................................................... 23
Surgical method devised by M. Schramme 12............................................................................. 23
Results practical assessment ............................................................................................................... 25
The Collagenase method ................................................................................................................. 25
The Collagenase gel method ........................................................................................................... 25
The Surgical method ........................................................................................................................ 26
Conclusion ............................................................................................................................................ 28
Discussion ............................................................................................................................................. 29
References ............................................................................................................................................. 30
1
Abstract
Equine tendon injuries tend to be a real challenge for clinicians, because of the slow healing
capacity of this inert and collagen rich tissue. There are many different medical treatment
approaches described, some of which have been objectively evaluated, while others are being
applied in a more empirical way. At this point there is great interest for regenerative therapies
to treat tendon injuries. In order to be able to evaluate these treatment options there is need for
a practical feasible and reproducible technique to induce standardized lesions in tendon tissue.
The aim of the current research project was to provide a literature overview of available peer
reviewed studies on induction of standardized lesions in equine tendons for research purposes
and secondly, to apply some of these techniques on horse legs obtained in the slaughterhouse.
This study fits into a larger project in which High Field Laser therapy for treatment of tendon
injuries is evaluated. Available techniques to induce standardized tendon lesions in horses are
the collagenase technique1,2, the collagenase gel technique by A. Watts et al, 2012 and the
surgical technique by Schramme et al, 2010. In our hands, and for the purpose of serving the
high field laser therapy study, the technique that most resembled natural occurring lesions and
that induced the most controllable, repeatable lesions and proved to be the safest and most
practical technique was the surgical technique by Schramme et al., 2010.
.
2
Introduction
Lameness is an important performance limiting factor in sports horses and one of the most
common causes of lameness in equine athletes is tendon injury. Most of these injuries occur at
the level of the flexor tendons. It is the primary cause for injury in racehorses with a prevalence
of 15% 4 and, although reliable figures for other equestrian disciplines are lacking 5, there are
indications that tendon injuries play an important role in those as well 6. Beside this, it has been
reported that 42,5 – 44,4% of horses with injured tendons and that are treated conservatively,
most likely will re-injure their tendons within 2 years after the original insult 5. There are a
whole range of different medical approaches to these problems and the ideal strategy for
tendon recovery has not been identified yet 7. Several treatment options have been proposed
and utilized, but most of these approaches have not been fully evaluated for safety or efficacy in
tendons or ligaments 8.
A novel treatment option that has been launched for tendon injury is High-Level laser
therapy(HLLT). Low-Level laser therapy(LLLT) has been used in human and veterinary
medicine for decades in the management of pain, wound healing and soft tissue injury but, next
to this, laser therapy is believed to have a bio stimulating effect as well 9,10. This effect supports
the application of laser therapy in promoting tissue repair, next to providing pain relief and
reducing inflammation. Laser therapy can promote healing in tissues, like tendons, that show
difficulties with healing. In addition to this it can be used as an anti-inflammatory and
analgesic treatment, without having the side effects that can occur with extended use of
NSAID’s 11. The main difference between LLLT and HLLT is the output-power. This higher
power output for HLLT reflects itself in a deeper penetration into treated tissue and the ability
to administer a larger amount of energy in a shorter amount of time with these devices.
HLLT has not been evaluated for safety and efficacy in horses, just like most of the treatment
options currently being applied for treatment of tendon injury. To be able to objectively
evaluate different treatment options, there is need for protocols to induce standardized and
reproducible tendon lesions. Ideally these lesions have the same configuration as the lesions
that are encountered under natural conditions. There are several different methods described
for inducing core lesions, most of them focus on the Superficial Digital Flexor Tendon(SDFT)
3,12,13
. These methods are divided into two major groups: models that induce tendon-injury
through a change in mechanical environment and models that induce tendon-injury by means
of deposition of a chemical agent 14. There is no general agreement about which method is most
successful and how these techniques should be executed.
The aim of this study was three fold:
First of all HLLT is described by presenting 5 different cases that were successfully treated with
this therapy. This study is part of a larger project in which HLLT is evaluated as a treatment
option for tendon injury.
Secondly, a literature overview is provided of all described techniques to experimentally induce
tendon injuries, paying attention to the different tendons/ligaments, in which these techniques
were applied and evaluated.
Thirdly, the aforementioned techniques were applied on cadaver legs and evaluated for their
suitability to be applied in the high field laser therapy study.
3
Introduction to HLLT
LLLT has been used for many different purposes both in human and veterinary medicine 10.
Therefore most scientific research on laser therapy has been focusing on LLLT and little is
known about the treatment effects of HLLT. However there does not seem to be a reason why
the effects of LLLT could not be extrapolated to HLLT. This because the only difference
between the two therapies lies in the output-power, which does not seem to have any effect on
the tissue itself, just on the penetration depth of the laser and the amount of energy that can be
delivered 15.
One of the major reasons for the use of laser in tendon injury is that laser therapy was found to
have a bio stimulating effect called “photostimulation”. This can be described as the ability of
light to stimulate living cells. This effect is based on the absorption of the visible red to near
infrared light by cytochrome-c-oxidase, which is one of the elements of the mitochondrial
respiratory chain 16. The complete mechanisms through which the absorption of light leads to
an increase in cellular metabolism and cel proliferation is not entirely clear 17.
Next to “photostimulation” laser therapy results in proliferation of fibroblasts 18,19, stimulation
of collagen production 20,21, an increase in tensile strength 22,23 increased development of
collateral circulation and angiogenesis 24,25, reduction of COX-2 11, decreased levels of proinflammatory mediators 26 and an improvement of collagen-fibre alignment 27,28. This last effect
could be very promising in the treatment of tendon injury because re-injury is a major problem
in horses 5. If a tendon could heal with a better structure and strength, this could prevent
occurrence of re-injury. Because of the effect on COX -2 and the pro-inflammatory mediators,
laser therapy has considerable pain reducing effects. Since long term NSAID’s use can cause
serious side effects, laser therapy could be considered as a good alternative for these drugs as a
painkiller.
These effects support the usage of laser therapy to stimulate tissue repair and reduce pain and
inflammation. However, based upon scientific research, one can expect differences in treatment
effects with application of different doses and wavelengths. The most effective laser dosage is
still not fully determined. The FP4 laser that is used in the cases below, combines four
wavelengths: 635nm, 660nm, 810nm and 980nm, both short and long wavelengths. Long
wavelengths are expected to penetrate deeper into the targeted tissues than short wavelengths
29
. Short wavelengths have a greater bio stimulative effect 30,31. This means that the FP4 laser
provides the option to combine the advantages effects of both wavelength types. Moreover,
Mendez et al., showed that a combination of wavelengths has a better effect on wound healing
when compared to one single wavelength 32.
To use HLLT in its full potential, more research is needed to determine the optimal therapeutic
dose and wavelengths to be applied at each laser session. The current study is part of a larger
project in which efficacy of the laser device will be determined and optimal stimulation
parameters will be determined.
4
Presentation of five cases, successfully treated with HLLT
The five cases that are displayed below, were taken from a large scale clinical study that is
researching the effects of HLLT on tendon injury in horses. In this clinical study all different
tendon injuries are researched but in this paper there are only SDFT injury cases displayed. This
has been done to coincide with the second part of the paper where different methods to
iatrogenically induce tendon lesions are examined, also only in the SDFT. These cases were
randomly chosen to show a scale of different patients with SDFT injuries.
NB. The ultrasound images that are shown in this paper are chosen to give an impression of the
cases. Not all cases show the same array of images.
Materials & methods
Table 1 AAEP Lameness score
When a horse was brought into the clinic, it was
0: Lameness not perceptible under any
clinically and ultrasonically assessed. When the
circumstances.
horse was suitable for being treated with HLLT and
1: Lameness is difficult to observe and is not
the owner consented, the horse was included in the
consistently apparent, regardless of
study. Next to the clinical assessment of the leg, the
circumstances (e.g. under saddle, circling,
lameness grade was ascertained, according to the
inclines, hard surface, etc.).
AAEP lameness scale(Table 1). The horses were
2: Lameness is difficult to observe at a walk or
trotted on a hard- and soft surface in a straight line
when trotting in a straight line but consistently
and on a circle to determine the score. This was done
apparent under certain circumstances (e.g.
by two objective observers, who gave there score
weight-carrying, circling, inclines, hard surface,
etc.).
separately. Next to the clinical evaluation, there was
3: Lameness is consistently observable at a trot
an ultrasonic evaluation. The ultrasonographic
under all circumstances.
examinations of the tendons were performed by the
4: Lameness is obvious at a walk.
same person each time. The ultrasonographic images
5: Lameness produces minimal weight bearing
33
were evaluated according to the scale used by
in motion and/or at rest or a complete inability
which uses a scale from 0 to 3 (0 = normal
to move.
echogenicity and homogenous fibrillar pattern; 1 =
regions of mild hypoechogenicity and/or subtle signs
of irregular fibrillar pattern; 2 = extensive regions of mild hypoechogenicity or regions of
moderate heterogenous echogenicity, and/or small focal disruptions of fibrillar pattern; 3 =
regions of marked disruption of fibrillar pattern, large anechoic ‘core’ defects).
Treatment
The horses were generally treated for 2 weeks, every day. The FP4 laser uses the four
wavelengths but there are also different settings available on the machine. This making it
possible to adjust the laser to the lesion or tendon that is treated. The settings and the way the
FP4 laser is used is beyond the scope of this paper.
Monitoring
The horses were clinically evaluated after the treatment had finished (mostly after 2 weeks, the
first re-check). The same clinical and ultrasound evaluation was performed as the one at the
beginning of the treatment and adjacent to that the percentage of improvement compared to
the original injury is reviewed. This is repeated after approximately one month (second recheck). After 6 months there was a long term follow up.
5
Case 1
A 6 year old Warmblood stallion came in on
the 21st of February 2014. Level of
performance before the injury occurred was
medium level dressage. On evaluation he
was not clinically lame but received an
ultrasound grade of 1. He had been lame in
the past, on his left front. The horse did not
receive any other treatment and did not have
any other lameness. The lesion in his left
front SDFT was located in region 1b - 2a on
the lateral side and had a sub0acute
appearance. The tendon structure had not
completely returned after the horse’s old
injury, which occurred on the 23rd of January,
2014. The gelding was admitted and treated
with the High Level laser therapy for two
weeks.
Fig. 1 Original injury, 23/01/2014
Fig. 2 Injury on 21/2/2014 when the horse was admitted
At the end of the treatment, after two weeks the horse had its first re-check. The gelding was
still sound on clinical evaluation and had the same ultrasound grade(1) but had improved by 70
% on ultrasound. There was very little swelling visible and the lesion was completely filled. The
structure of the lesion was still heterogeneous.
6
Fig. 3 First re-check
The second re-check was done after 5 weeks. The horse was still sound and had an ultrasound
grade of 0 – 1 with an improvement of 90%. The structure of the tendon was not completely
linearly organised in region 1b.
Figuur 4 Second re-check
Fig. 4 Second re-check
Long term follow up:
The horse was competing at its old level in dressage and had not had a re injury in the 6
months after this injury. Four months after the treatment the horse started training again.
7
Case 2
The second case involved a warmblood gelding of 14 years old that was competing at high level
show jumping. The horse came in on the 11th of March 2014. He was not clinically lame but had
a chronic swelling in his right front SDFT in region 1 – 3, which was not painful when touched.
On ultrasound he had a chronic swelling in
region 1-3, with an ultrasound grade 3. This
horse was treated for a longer amount of
time because of the chronic nature of the
swelling. In total he was treated for 3 weeks,
with a months between the first two and the
last week. This horse did also not receive any
other treatments and had no other lameness.
Fig. 5a Injury on admittance
Fig. 5b Injury on admittance (2)
After the first two weeks of laser therapy the swelling had not decreased.
Fig. 6a & b First re-check after first 2 weeks of laser
8
After a month of rest, the horse was evaluated again and another week of laser therapy was
started. Two weeks after this second period of laser therapy the horse had another re-check.
The gelding was still sound and had the same ultrasound grade(3) but did show a 40%
improvement on its ultrasound. Swelling had shrunk a small amount and there was a better
tendon structure in zone 1 and 3. In the second re-check the horse had improved another 10%
on ultrasound.
Fig. 7a First re-check after third week of laser
Fig. 7b First re-check after third week of laser (2)
Long term follow up
6 months after the injury the horse was back to its old level and did not reinjure. Five months
after the treatment the horse started training again.
9
Case 3
Another warmblood gelding(high
level show jumping) of 7 years old
came in on the 28th of February,
2014. He was not clinically lame but
had an acute lesion in his left front
SDFT in region 2-3 (ultrasound score
3). He had no other lameness but
was also treated with PRP for this
lesion. The gelding was admitted
and treated with laser therapy for 2
weeks.
Fig. 8a & b Injury on admittance
With the first re-check following the laser therapy the ultrasound image improved 70 % to an
ultrasound grade 2. There was a small amount of swelling visible and the lesion was completely
filled.
Fig. 9a First re-check
10
Fig. 9b First re-check (2)
With the second re-check, a months after the first, the gelding still had an ultrasound grade of
2 but the horse had improved 80%. The SDFT was mildly swollen, the lesion was smaller and
filled up. Two months after the second re-check this horse was checked again at the owners
request. There was still a small region with mild loss of tendon structure in zone 2 with
swelling.
Fig. 10a & b Extra check after two months
Long term follow up
After 6 months the gelding was back at his old
level in show jumping and had improved on
ultrasound as well. The horse had an ultrasound
grade 1 and had improved 90%. The gelding did
not suffer from re-injuries. Three months and
two weeks after the treatment the horse started
training again.
11
Case 4
The fourth case concerns another 7 year old warmblood gelding(medium level dressage). When
the horse came in on the 27th of May in 2014, it had a lameness grade of 3 and an ultrasound
grade of 1. His lower limb was swollen around the flexor tendons in zone 1 and this zone was
also painful when pressed. He had a sub-acute lesion in his left front SDFT in zone 1a with
swelling and loss of structure, the swelling extended to zone 1b. The horse received two weeks
of laser therapy.
Fig. 11 Injury on admittance
After the two weeks of laser therapy, the horse had its first re-check. The horse was not lame on
a straight line. Only on a circle to the left, there was a visible lameness of grade 1. On
ultrasound the horse had less swelling in zone 1b – 2 and the tendon structure was better but
the gelding still received an ultrasound grade of 1. No percentage of improvement was noted.
Fig. 12 First re-check
12
After one month the horse received its second re-check and the horse was completely sound.
On ultrasound there was no swelling and a better structure with an ultrasound grade of 1. There
is no long term follow up for this patient. Three months after the treatment the horse started
training again.
Case 5
The last case was an eight year old warmblood mare(high level show jumping) who came in on
the 5th of March in 2014. The mare had no clinical lameness but had on ultrasound a sub-acute
core lesion in her right front SDFT. It was located in zone 1a and the SDFT was
swollen(ultrasound grade 1). The horse was treated for two weeks.
Fig. 13a Injury on admittance
Fig. 13b Injury on admittance
13
On the first re-check the horse was still sound. On ultrasound the horse showed mild loss of
structure and mild swelling. The lesion was filled up and the horse had an ultrasound grade of 1
with 70% of improvement.
Fig. 14a First re-check
Fig. 14b First re-check (2)
After a month the horse was re-checked again (second re-check). On ultrasound there was a
small area with mild loss of structure and a small amount of swelling. The horse received an
ultrasound grade of 0.
14
Fig. 15 Second re-check
Long term follow up:
The mare was back at performance level after 6 months and had not reinjured in that period of
time.
Conclusion
These cases are meant as an introduction to HLLT. No conclusion can yet be determined if
laser therapy is a confirmed treatment for tendon injury in horses. It has properties that suggest
that it could be able to assist with quicker healing of tendon injury and to help the tendon to
gain a better structure and strength. This could prevent re-injury. Next to these properties, it is
also a non-invasive technique and is an easy technique to carry out. The large scale clinical
study will give more insight in the results of treating tendon injuries with HLLT.
15
Inducing tendon lesions in horses
To be able to evaluate this new laser technique, and other treatments that are used in tendon
injury in horses, there is a demand for a practical feasible technique to induce standardized
lesions in tendon tissue. Ideally these lesions mimic natural occurring tendon lesions, are
standardized, repeatable and safe and non-invasive for the horses. Next to these features, it is
also important that the technique is practical.
There are a lot of different methods to induce tendon lesions in animals. These methods can be
divided into two major groups: models that induce tendon injury through a change in
mechanical environment and models that induce tendon injury through a chemical agent. The
models that were used on horses in the past are explained in this paragraph.
Chemical models
Collagenase
Collagenase is the most frequent used research technique to induce core lesions by injecting it
into the tendon, and has been used in horses for over thirty years 34. Collagenase is an enzyme
that breaks down the native collagen that holds animal tissues together 35. It breaks the
collagen down by cleaving the peptide bonds in native, triple-helical collagen 36. There are six
different types of collagenases (α, β, γ, δ, ε and ζ) and these collagenase are used in science for
their potent hydrolytic action on connective tissue, a property that has made them the reagent
of choice for tissue dissociation experiments, one of which is the induction of lesions in equine
tendons 37. The most potent collagenase and often used in research is the “crude” collagenase,
secreted by the anaerobic bacteria Clostridium histolyticum. “Crude” collagenase alludes to the
fact that the material is in actual fact a mixture of several different enzymes besides collagenase
that act together to break down tissue 38.
Although there are, as pointed out, six different types of collagenase, the relative activities of
these enzymes toward collagen vs. synthetic peptides and all of the physiochemical properties
of these six collagenases enables them to be separated into two distinct classes.
The class I collagenases (a, β and γ) have higher activities toward collagen and gelatine and
lower activities toward synthetic peptides. The class II collagenases (δ, ε, and ζ) have about onethird of the activity toward collagen and gelatine but have significantly higher activities toward
the synthetic peptides. And next to these properties, the effect of freeze-thawing on the activity
and the amino acid compositions serve as a distinction between the two 37,39. The different
relative activity toward native collagen and synthetic substrates is used in enzymatic essays to
distinguish which class of collagenase is most apparent in a collagenase preparation 36. In
“crude” collagenase preparations, both classes are present. This has been proven to be the most
efficient collagenase in digesting different types of collagen when compared to a purified
preparation of either class I or class II 38.
A lot of slightly different techniques and collagenase types are used to inject the collagenase
into the tendon, most often the SDFT. Horses can be sedated or put under general anaesthesia
in lateral or dorsal recumbancy. When the experiment uses a sedation to perform the
collagenase-injection, most articles pre-treat these horse with Non-Steroidal Anti-Inflammatory
drugs(NSAID), often Phenylbutazone(2,2 mg/kg – 4,4 mg/kg BW intravenously (IV)). The
horses are most often sedated with Detomidine hydrochloride (0,01 mg/kg BW) and to be able
to do further pain-management, regional nerve blocks are used, depending on the location of
the injection.
After the sedation of the horse or the general anaesthesia, the palmar aspect of the metacarpus
is clipped and aseptically prepared. Subsequently a needle, ranging from 22 to 27 gauge(G), is
inserted into the tendon and the collagenase is injected. This can be done under ultrasound
16
guidance. After the injection has been done, the limb is often bandaged to reduce swelling and
contamination. Horses usually receive Phenylbutazone orally, from 1 – 5 days following the
injection (4 – 4,4 mg/kg BW SID or 2,2 mg/kg BW BID). Various locations and tendons are
used, next to the different types of collagenase, the amount and the concentrations, as shown in
Table 2. Furthermore, in most articles details are often missing about the way the collagenase
has been prepared and what kind of solution is used.
Table 2
Article
(Williams et al., 1984)
(Clayton et al.,
2000)40
(Dahlgren et al.,
(Kersh et al.,
2002)41
2006)42
(Maia et al., 2009)
2
Type of collagenase Manufacturer
Injection volume Solution
Needle
Tendon
Unknown
Unknown
0,5 ml
Unknown
23G
SDFT
Type V11-s
Sigma Chemical Co
0,2 ml
3.000 IU/0,6 - 1,0 ml 0,9% NaCl 23G
SDFT
Type 1
Sigma
2640 IU
Unknown
Unknown SDFT
Type J-S, C1639
Sigma-Aldrich
1000 IU(0,1ml)
1000IU/0,1ml
26G
SDFT
Type 1: C-0130
Sigma Pharmaceutical 2,5 mg(1ml)
2,5 mg/1ml distilled water
Unknown SDFT
(Waguespack et al, 2011)1
Type 1
Sigma
2000 IU(0,5ml)
2000 IU/0,5ml sterile water
Unknown ALDDFT
(G Bosch et al., 2010)44
Type 1: C2674
Sigma-Aldrich
1500 IU(0,5ml)
1500IU/0,5 ml
Unknown SDFT
(Crovace et al., 2010)45
Type 1A
Sigma
4000 IU
Unknown
Unknown SDFT
(Donnelly et al., 2006)46
Type 1
Sigma-Aldrich
2097 IU(0,3ml)
2097 IU/0,30ml sterile water
Unknown SDFT
Unknown
Worthington
2000 IU
Unknown
25G
SDFT
Type I-s
Lateral Branch SL
(Karlin et al.,
2011)47
(Keg et al., 1996)
48
Sigma
1200 IU(0,4ml)
3000 IU/ml
23G
(Lacitignola et al., 2008)49 Unknown
Unknown
4000 IU
Unknown
Unknown SDFT
(Lure et al., 2004)50
Type I-S, C1639
Sigma-Aldrich
4000 IU(0,1ml)
4000 IU/0,1ml
22G
SL
Type 1: C-0130
Sigma
0,2ml
2,5 mg/1ml
23G
SDFT
Type 1
Sigma-Aldrich
0,15ml
2097 IU/0,30ml sterile water
Unknown SDFT
Type 1
Sigma
1000 IU
Unknown
Unknown SDFT
Unknown
Sigma-Aldrich
+/- 667
2000 IU/0,45m
27G
(Moraes et al., 2009)
(Nixon et al.,
51
2008)52
(Schnabel et al., 2009)
(Dowling et al.,
53
2002)54
* SDFT: Superficial flexor tendon
* DCAB : Distal to the accessory carpal bone
* ALDDFT: Accessory ligament of the Deep Digital Flexor Tendon
* SL: Suspensory Ligament
Injecting the collagenase into the centre of the tendon generates extensive degeneration of
collagen fibres and the interfibrillar matrix. The enzyme acts on collagen molecules at many
different sites along the helices, rapidly making them soluble 51. This results in acute swelling, a
rapid dissolution of fibres, matrix destruction, cell necrosis, vascular damage, extensive
haemorrhage and inflammation which mimics many aspects of the naturally occurring
traumatic injury (Dahlgren, van der Meulen, Bertram, Starrak, & Nixon, 2002; Lake, Ansorge, &
Soslowsky, 2008; Williams, I.F.; McCullagh, K.G.; Goodship, A.E.; Silver, 1984; Dirks & Warden,
2011)
The animals develop the characteristics of an acute tendonitis. After 12 hours following the
injection of the collagenase the horses show a diffuse warm swelling on the palmar metacarpus
and are painful on palpation for 7 to 12 days 2,50.
The horses can become lame within 6 hours after injection and the reaction continues to
develop for a considerable amount of time, up to 24 hours. Additionally, the horses remain
lame for a substantial amount of time, up to 3 weeks 2,34,50
17
SDFT
The lesion created by injecting collagenase into a tendon remains palpable up to 14 months.
The size of the enlargement gradually decreases but the tendon also remains bowed after 14
months 34. Histologically and by ultrasound the improvement of the tissue quality is visible and
demonstrated by a partial return of the tendons crimp pattern, which is a wavy pattern in the
collagen fibres. This pattern is responsible in part for the elasticity of the tendon 56. However,
the crimp pattern never fully returns to normal and the fibrils show a loss of parallel orientation
and a decrease in diameter. The cross-sectional area(CSA) of the tendon as a whole is generally
increased and does not return to normal as well 14.
Collagenase tendinopathy models demonstrate many important traits seen in clinical
tendinopathy including matrix degradation, increased cellularity and increased vascularity. The
severity of the lesion, the amount of swelling and lameness can be controlled, in theory, by the
concentration and volume of collagenase applied to the tendon 14.
Collagenase gel technique
Next to the injection of a collagenase solution there is a recent, not widely used, development
in using a polymerisation of injected collagenase in a fibrin gel. This to keep the collagenase
concentrated in the core of the tendon. The same type of collagenase is used, bacterial
collagenase type I. The injection technique can be performed with the horses under sedation
using 0.01 mg/kg BW Detomidine and 0.01 mg/kg BW Butorphanol. Next to the sedation, as in
the conventional collagenase technique, local anaesthesia was used for further painmanagement. The palmar aspect of the metacarpus is clipped and aseptically prepared before
the injection. For the injection a 45º curved 16 G 8,89 cm long epidural or catheter needle is
used, to create a columnar separation of collagen fibres by inserting it longitudinally under
ultrasonographic guidance. This technique has, so far, only been used in the SDFT. The needle
is passed from the palmar surface of the limb through the skin and subcutaneous tissues to the
centre of the SDFT, starting 12 cm DCAB, and then advanced distally following the long axis of
the SDFT centre until the tip reaches 18 cm DACB. The needle is then slowly withdrawn and a
dual injector syringe is used to deliver 1000 - 1300 units of collagenase in 500 units of bovine
thrombin, combined during injection with an equal volume of equine fibrinogen (30 mg/ml),
for a total injected volume of 1 ml over a distance of 2 cm (from 18–16 cm DACB) within the
mid-metacarpal SDFT. As the needle is withdrawn from 16 cm DACB back to the point of entry
at 12 cm DACB, the final 3.5 cm of the physical defect is injected with fibrinogen and thrombin
for a total volume of 0.2 ml. After the whole procedure the leg is bandaged and the horses
receive Phenylbutazone for a subsequent eight days (Carvalho et al., 2013; A. E. Watts, Nixon,
Yeager, & Mohammed, 2012; A. E. Watts, Yeager, Kopyov, & Nixon, 2011).
Over the first 24 hours after the collagenase gel injection, all horses developed lameness in
different severities. After the quick development of the lameness in those 24 hours, it
diminished rapidly and all horses are sound at a walk one week after the procedure. The limbs
developed mild diffuse swelling, heat, are painful on digital palpation and have oedema centred
around the palmar mid-metacarpal region after tendon lesion induction. These problems get
resolved over the first 5 days.
On ultrasound, all horses developed centrally located hypo-echoic core lesions with increased
CSA and thereafter the CSA increases linearly over the first 4 weeks. On histology, at 2 and 4
weeks after the procedure, there was complete disruption of collagen fibres and loss of collagen
crimp pattern, increased cell numbers, rounded cell morphology and haemorrhage. At 8 weeks,
there was no haemorrhage and there was a return of collagen crimp pattern. Tendon fibres
were not parallel to the surrounding normal tendon tissue in the majority of sections. By 16
weeks, all sections were well organised with obvious cellular and collagen fibre linearity that
complemented the surrounding normal tendon fibres. This return of tendon architecture was
coupled with collagen fibre co-linearity and a more normal crimp pattern ( A. E. Watts et al.,
2012).
18
Fig. 16 Collagenase gel technique, {Carvalho et al.}
Mechanical model
Surgical method devised by M. Schramme 12
This technique creates a core lesion in the SDFT of 6 -8 cm in length, using a synovial resector
or arthroscopic burr. Pre-operatively horses received an intravenous (IV) injection with a nonsteroidal anti-inflammatory drug (NSAID). Horses are placed under general anaesthesia and
positioned in lateral recumbency. The surgical site is clipped and prepared aseptically. A 5 mm
vertical skin incision is made with a number 15 scalpel blade, just proximal to the Digital
Tendon sheath on the palmar aspect of the fetlock. The incision is continued through the
palmar annular ligament and the palmar mesotenon into the palmar aspect of the SDFT. The
tendon is incised to a depth of 2 - 3mm, taking care not to penetrate the dorsal surface of the
SDFT. A 2.5 mm blunt obturator is then guided through the incision and lead through the
central part of the SDFT, parallel with the longitudinal fibre direction of the tendon. This is
done under ultrasonographic guidance to create a tunnel over a distance of approximately 10 12 cm. Penetration of the SDFT is avoided by ultrasonographic monitoring of the position of the
tip of the obturator. After the slow withdrawal of the obturator, a 3,5 – 4,5 mm synovial resector
or arthroscopic burr is inserted along the previously created tunnel. What size and what kind of
instrument is used, depends on the research. Most articles use a 3,5 mm arthroscopic burr but
there is only a small groups of researcher that have used this technique so far.
19
The shaver or burr is inserted, in inactivated state, into the tendon over the full length of the
instrument. When the shaver or burr is in position, the blade is activated. The shaver or burr is
then withdrawn from the SDFT, while manually rotating the cutting blade at the tip in order to
transect tendon fascicles on every side accept the palmar side. This to protect the thin margin
of tendon, that covers this side, from penetration. The cutting is ceased when the tip of the
shaver is located 2,5 cm proximal to the incision in the skin. This procedure is repeated up to 3
times to create a clear lesion of 10 % of the CSA on ultrasound. After the procedure the skin is
closed and the limbs are bandaged with a Robert Jones bandage. After the surgery horses
receive Phenylbutazone (2.2 mg/kg, PO, q12h) or oral meloxicam (0.6 mg/kg BW) for a total of
three days postoperatively 12,44,59,60.
Sole et al61 used a variation on this technique were they made two shorter lesions of 1-2 cm
each, one located 13 and one located 20 cm DCAB. Next to this they also used a combination of
a shaver and an arthroscopic burr with both a width of 4,5 mm which is the largest size used in
any research so far.
In the clinical assessment of these
horses, no lameness was seen 24
hours after the surgery. Two of four
horses became mildly lame in the
research of 12 but this was only seen
during or immediately following
the 6 day exercise period that was
done in the original version of this
technique. Lameness resolved
quickly within two to seven days
after the cessation of exercise. In
some horses there is a mild heat,
mild peritendinous swelling and
signs of mild pain on palpation of
the SDFT. Six weeks post-surgery
there is still a palpable irregular
thickening of the skin at the site of
injury and the tendons appear to be
tender and still swollen.
Fig. 17 A 3.5 mm synovial resector is advanced into the
On ultrasound, the images show a strong
core lesion of the SDFT (Schramme et al., 2010)
hypo-echogenicity and an altered
longitudinal fibre pattern in the centre of the
tendons. The organisational parameters based on histopathology revealed hypercellularity,
hypervascularity, loss of matrix organisation and rounding of cell nuclei in the induced lesions
12,59
. This relatively new surgical method has been used in a handful researches so far and
produces core lesions, representative of moderate tendonitis.
20
Advantages and disadvantages
All these different techniques suggest that there is not a one minded vision on the best way to
induce tendon lesion in horses. All the advantages and disadvantages mentioned in scientific
materials are outlined below.
Chemical models
Collagenase
Advantages
Collagenase is one of the most frequently used research tools to induce tendon lesions in horses
2,45,51
It is an easily performed injection technique and it can be performed with the horse under
sedation, there is no general anaesthesia needed. The model demonstrates many important
traits seen in clinical equine tendinopathy. It closely resembles naturally occurring tendon
injury in morphology and histology including matrix degradation, increased cellularity,
increased vascularity and loss of function. The severity of the lesion, the amount of swelling and
lameness can be controlled, in theory, by the concentration and volume of collagenase injected
into the tendon 14,54,55.
Disadvantages
Although it has been used over thirty years, there is no agreement about the dosage of
collagenase and the solution it is supposed to be used in. Many different kinds of collagenase,
quantities and different injection techniques are used (See Table 2). The different kinds of
collagenase are mostly different kinds of crude collagenase, a mixture of several different
enzymes besides collagenase that act together, as mentioned before and mostly the same
manufacturer is used. Because it is bacterial collagenase that is injected, there may be
dissimilarities from equine collagenase which may result in a modified response.
The collagenase model results in a strong inflammatory response through disrupting the
collagen fibres, with is not entirely consistent with the pathophysiology of a natural occurring
tendon lesion. Naturally occurring tendon lesions are mechanically induced causing micro
damage or alternatively caused by a single acute mechanical overload which is followed by an
acute inflammatory phase 56,62–64. The inflammation that is caused by the collagenase injection
often extends to the peritendinous tissues, either due to (excessive) leakage of collagenase from
within the paratenon through the injection site or because it causes a ‘melting’ tendonitis that
erodes from the center of the tendon outwards and destroys the superficial tendon layers and
paratenon. As a result the size, shape and location of these lesions are very difficult to control
and standardise between the different limbs. This causes an absence of ‘true’ core lesions
surrounded by a margin of undamaged tendon tissue. This may cause difficulties when
therapeutic medications administered by intralesional injection are tested, they possibly may
not remain within the injured tendon 3,12,54.
When using collagenase to induce tendon lesions, the enzyme takes some time to create the
defect and thereafter to stabilize the lesion. There is no conclusive time period for this
stabilisation. There are articles mentioning the lesions stabilizing between 2 – 6 weeks or even
up to 8 weeks after the injection 42,50,52. It is unknown if this expansion is solely due to the
collagenase. Natural occurring tendon lesion do also expand for up to 4 weeks and it has been
mentioned that bacterial collagenase loses its activity rapidly when in solution 3. When
incubated at room temperature, its activity declines in an almost parallel progressive line, in 24
hours almost half the starting activity is lost 37.
The clinical results of the collagenase method can be quit severe. Horses can be lame for up to 3
weeks and there are a few cases were the horses are not able to bear weight on the injected leg
after 24 hours34,48. The lesions remain clinically and ultrasonically visible for a long period of
21
time, up to 14 months after injection and the tendon will never regain a normal infrastructure.
The crimp pattern never fully returns to normal and the CSA of the tendon as a whole is
generally increased and does not return to normal as well 14. This does not correspond with
natural occurring lesions were normal tendon architecture was missing at 6 months 65,66
Collagenase gel technique
Advantages
Similar to the conventional collagenase technique, the collagenase gel technique can be
performed on horses that are just sedated. The alterations that are made in this technique are
used to improve the conventional method. By using a larger needle(16 G) and injecting the gel
while retracting this needle placed in a longitudinally direction to the tendon, creating a small
physical defect, the resistance is held down to a minimum. This is in direct contrast to the
conventional technique, were the collagenase is injected directly into the dense structure of the
tendon. This forces the collagenase in all different directions in the tendon or back through the
fine gauge needle tract. By creating this small defect and having a low injection pressure gives
the possibility for the bacterial collagenase to almost entirely remain within the defect.
Subsequent to this fact, the polymerisation of the collagenase in fibrin gel helps to keep it in
the centre of the tendon as well, and this combination gives the opportunity to use a smaller
amount of collagenase. Less collagenase is needed because the substance stays in position and
there is only minor extravasation of the enzyme to the epitendon. This results in a minimal
peritendinous reaction at the level of the core lesion and therefore, compared to the
conventional method, it develops into a mild swelling of the limbs, mild oedema and pain on
palpation 3.
The collagenase gel technique essentially creates a tendon lesion in the same manner as the
conventional collagenase technique and mimics an acute tendon lesion through induction of an
enzymatic core lesion with up regulation of matrix degrading enzymes. Although this
technique does not entirely resemble the etiopathogenese of natural occurring tendonitis with
the mechanical element, it was successful in creating consistent, centrally located, more
regularly shaped SDFT lesions confined to the mid-metacarpal SDFT. Subsequently it resulted
in minimal peritendinous reaction and obvious central matrix degradation, increased
cellularity, increased vascularity and increased CSA. The method did not rely upon post
induction exercise of the horse to develop the lesions 3,14,55,58. And the lesions that were created
continued to expand for the first 4 weeks after the procedure 3. This also resembles natural
occurring tendon lesions 65,67–69
Disadvantages
This recent addition to the possibilities to create tendon lesions has not yet been widely used.
As mentioned with the conventional method, the collagenase does not create a tendon lesion
that is entirely consistent with the pathophysiology of a natural occurring tendon lesion. It
lacks the mechanical element, which causes microdamage, creating a tendon lesion. This is one
of the reasons that the relevance of collagenase induced tendon lesions compared to
mechanical overload injury is unsure and has progressively been questioned 12. The stabilisation
time of the lesions, the use of bacterial collagenase and how long the collagenase keeps working
is also an issue with this technique. There is no conclusive stabilisation time known as
mentioned before and, even when there are articles mentioning a stabilisation time of up to 8
weeks, the two articles that used this technique started their treatment 1 and 2 weeks after the
lesion induction57,58. Although this technique is a huge improvement from the conventional
collagenase method and creates consistent, centrally located SDFT lesions confined to the midmetacarpal SDFT, there is still a possibility of a minimal amount of extravasation of the enzyme
and therefore a peritendinous reaction. In addition the CSA of the lesions that are created
confirmed that there is a minimal proximal migration of the collagenase which gives an
22
uncertainty regarding the consistency and the size of the lesions that are created using this
method 3.
Mechanical models
Surgical method devised by M. Schramme 12
Advantages
By using this minimal invasive approach and creating traumatic fibre damage confined to the
core of the SDFT, considerable core lesions were created using this technique. This was
achieved without splitting the paratenon and the outer layer of the tendon except at the site
were the instrument were inserted into the limb. Because there are no enzymes involved and
the creation of the lesions can be controlled when the technique is performed, the lesions that
are created by this technique are very controllable and reproducible. By measuring the
proportion of the CSA that the lesion was covering during the technique, every lesion created
can be managed by the percentage of CSA they were covering, for example 10% 12.
The lesions that were created continued to increase in size in the first 4 weeks 12. This resembles
natural occurring tendon lesions, as mentioned before 65,67–69. The progression of healing in
these surgically created defects was slow, there was only minimal reconstruction of normal
tendon architecture at 12 weeks 12. Different researches of this same technique showed that the
biomechanical properties were still altered at 24 weeks 44 and that the healing process remained
visible 70. An older, less used and explored surgical technique showed that normal tendon
architecture was still missing at 5 – 6 months 71. This corresponds with natural occurring
lesions, were normal tendon architecture was also still missing at 6 months 65,66. Collectively
these studies demonstrate that the method captures the defective healing response to the
microdamage in the tendon, characteristic for tendon injury. The reasons why the tendon in
unable to effectively repair itself is largely unknown 72
This surgical technique is able to induce standardised core lesions and creating a moderate
tendonitis that shows similarities in ultrasonographic progression and histological
characteristics to core lesions in naturally occurring tendon injury 12.
Disadvantages
These surgical boring lesions do not entirely resemble the natural pathogenesis of tendon
injury were a build-up of degenerative changes in the tendon are followed by a partial tendon
rupture and an elevation in degradative enzymes. This technique result in a per acute
disruption of the tendon fibres, and there is only a very small amount of inflammation present
3,12,59,73
. This is not consistent with naturally occurring tendonitis were signs of inflammation are
visible after 3 weeks 65. Subsequently, this technique does not allow the lesion to be localised in
the region of the tendon were most injuries occur, the tensile region of the SDFT: the mid
metacarpal region 3,56.
The severity of the clinical signs of this method is not only dependant on the physical
technique, the horses need to be exercised before clinical tendonitis may be visible. The
exercise only resulted in mild lameness and mild peritendinous swelling. A consistent model of
a more severe tendonitis with a larger core lesion will be required to use in treatment effect
testing to show a significant effect 12. As shown in Tabel 3, there have been some researchers
who used a larger instrument, up to 4,5mm and some shifted to an arthroscopic burr. This
resulted in a more obvious clinical tendonitis with a moderate peritendinous swelling and pain
on palpation but no lameness was recorded 59,61.
23
Table 3
Article
(M. Schramme et al.,
(G. Bosch,.
2010)12
2010)43
Instrument
Size instrument
Length of lesion
Synovial resector
3,5 mm
8 cm
Arthroscopic burr
3,5 mm
7-8 cm
(Gerco Bosch et al., 2011)60
Arthroscopic burr
3,5 mm
7 cm
2010)44
Arthroscopic burr
3,5 mm
7-8 cm
Arthoscopic burr
4,0 mm
6 cm
Synovial resector
Unknown
Unknown
Arthroscopic burr
4,0 mm
6 cm
Synovial resector
3,5 mm
8 cm
Synovial resector & rotating burr
4,5 mm
1 -2 cm
(Gerco Bosch et al.,
(Cadby et al.,
2013)59
(Caniglia et al., 2012)73
(David et al.,
2012)70
(Schramme, Kerekes et al.,
(Sole et al, 2013)61
2010)74
As a last note, the technique can only be performed on horses under general anaesthesia. This
brings in extra costs but most of all extra risks for the horses.
24
Results practical assessment
To evaluate how these techniques perform when carried out and if the lesions are practicable
and repeatable, these three techniques were executed on cadaver limbs to monitor them. Next
to the practicability of the technique, ultrasound images of the created lesion were evaluated
and the limb was then macroscopically assessed.
The Collagenase method
To carry out the collagenase method the collagenase solution was switched with a methylene
blue solution. This change was made to be able to locate where the solution ended up after it
had been injected into the tendon. The rest of the procedure was carried out with a 23 G needle
and 0,35 ml methylene blue solution, using the needle size that was used in most of the
researches and the mean of the amount of solution that was used. The remainder of the
procedure was carried out as mentioned above.
Procedure: With ultrasound guidance the needle was easily inserted into the tendon but
injecting the solution gave a lot of resistance.
Ultrasound: No clear lesion was visible on ultrasound. This is due to the fact that no collagenase
was used in this experiment.
Macroscopically: The injected methylene blue was not centrally located in the tendon but had
mostly leaked into the surrounding areas. It had not created a clear visible lesion on cross
section. As mentioned before, this is due to the fact that no real collagenase was used.
The Collagenase gel method
The gel that was used to carry out this technique was an ultrasound gel, tinted with betadine to
give it a distinct colour. This, just as with the collagenase method, to be able to locate the gel
after the injection. The needle that was used was a 12 cm long 14 G needle, that was bend to an
45º angle. The rest of the procedure was carried out as mentioned in the description before.
Procedure: The needle was easily inserted into the tendon under ultrasound guidance and
continued on following the long axis of the SDFT. The injecting of the gel while retracting the
needle could be done without a lot of resistance.
Ultrasound: Clearly visible equally shaped lesion apparent on ultrasound after injection.
Macroscopically: No leakage of gel in the surrounding areas. Clearly visible lesion on cross
section of the SDFT.
Fig. 18: Collagenase gel method -> lesion
on cross section (Thanks to Mathilde
Pluim).
25
The Surgical method
The procedure was carried out as in the description before. Because there are two kinds of
instruments being used in literature and a few sizes, ranging from 2,5 – 4,5 mm, the technique
was completed with a few different kinds of shavers:
* 2,5 mm Synovial resector
* 3,5 mm Synovial resector(Slotted Whisker)
* 4,0 mm Arthroscopic Burr
Next to this, there was speculation about which direction would be best to insert the shaver. As
in the literature, from distal to proximal or from proximal to distal because the procedure can
be performed more easily and it makes it easier to create a linear lesion. Both directions were
tested with the 3,5 mm synovial resector. The other two instruments were used form distal to
proximal.
Procedure: All procedures were carried out under ultrasound guidance and with a blunt
obturator.
* 2,5 mm Synovial resector: with this shaver the technique is moderately difficult to perform. It
is mainly difficult to create a linear lesion.
* 3,5 mm Synovial resector(Slotted Whisker) – Distal to Proximal: moderately difficult to
perform, difficult to create a linear lesion.
* 3,5 mm Synovial resector(Slotted Whisker) – Proximal to Distal: easy to perform and to create
a linear lesion.
* 4,0 mm Arthroscopic Burr: the technique is hard to perform, and there is a greater risk of
penetrating the SDFT.
Ultrasound:
* 2,5 mm Synovial resector: lesion barely visible and not clearly linear on ultrasound.
* 3,5 mm Synovial resector(Slotted Whisker) – Distal to Proximal: lesion clearly visible and
equal in aspect over the entire length of the lesion.
* 3,5 mm Synovial resector(Slotted Whisker) – Proximal to Distal: lesion clearly visible and
equal in aspect over the entire length of the lesion.
* 4,0 mm Arthroscopic Burr: lesion clearly visible but irregular in shape and aspect.
Fig. 19 3,5 mm Slotted Whisker being inserted into the SDFT (Thanks to Mathilde Pluim).
26
Macroscopically:
* 2,5 mm Synovial resector: Lesion barely visible and hard to identify on cross section.
* 3,5 mm Synovial resector(Slotted Whisker) – Distal to Proximal: lesion clearly visible on cross
section
* 3,5 mm Synovial resector(Slotted Whisker) – Proximal to Distal: lesion clearly visible on cross
section
* 4,0 mm Arthroscopic Burr: lesion clearly visible on cross section but irregularly shaped.
Fig. 20 Visible lesion on cross section, after inserting slotted whisker (Thanks to Mathilde Pluim).
27
Conclusion
To compare the three models to induce tendon lesions that are displayed in this paper, we
combine the knowledge found in scientific materials and the practical assessment. Aspects that
were important in comparing the three models were mostly focused on the lesion. The lesions
that were created had to be as close to natural occurring tendon lesions as possible. Next to this
it was important that the lesions were standardized, repeatable and safe for the horses.
Alongside these features, the technique that was used needed to be practical.
The collagenase technique is a technique that is easily carried out and the horse only needs to
be under sedation but this technique does not create lesions that are repeatable and
standardized which is an important factor. In addition to this there is no general agreement,
even though this technique has been used for over thirty years, about the dosage and type of
collagenase that is utilized and the clinical results can be quit severe. As a conclusion, this
technique is not suitable to use as a tool in evaluating new treatment options for tendon injury
in horses. The collagenase gel technique is a technique that, as the collagenase technique, is a
technique that is easily performed, only the preparation of the injection is quite sophisticated,
and the horse only needs to be sedated. By using the gel, the collagenase gel technique does
create repeatable and standardized lesions and the clinical effects are much less severe than the
standard collagenase method. The defects that are created do not entirely mimic natural
occurring lesions. They lack the mechanical element but do resemble an acute tendon lesion. In
addition to this there is some uncertainty surrounding the use of collagenase. Because there is
no real data on how long the collagenase is active and how long it takes for the lesions to
stabilize, this makes the lesions less dependable. There is also some uncertainty about the use
of the “crude” bacterial collagenase. Because it is bacterial, there could be dissimilarities from
equine collagenase which can result in a modified response. Because of these uncertainties
regarding the activity of collagenase, the collagenase gel technique is less reliable than would
be suitable for use in evaluating new treatment options. If this data concerning the activity of
collagenase would be available, this technique could be a good option to use in researches. The
surgical method by 12 is a technique that is a moderately difficult procedure to perform. The
horses need to be under general anaesthesia and more sophisticated instruments are needed.
The core lesions that are created are very controllable and reproducible because no enzymes
are involved. This does mean that the lesions do not entirely resemble natural occurring
lesions. They do resemble the ultrasonographic progression and histological characteristics but
there is only a small amount of inflammation present. Furthermore, this technique does not
allow the core lesion to be in the tensile region of the tendon. Because this technique makes it
possible to create lesions that are very controllable, this method would be a good option to use
in evaluating new treatment options.
For evaluating the HLLT technique and the FP4 laser, the choice was made to use the surgical
method. This due to the fact that it was felt that it is a more controllable method and although
the horses need to be anesthetised for the procedure which brings in extra risks, it was believed
that this would be the best option. And although the model cannot be used to study the
inflammation that is present in an acute tendinitis, this equine tendinopathy model provides a
satisfactory standardised lesion that may serve well to study the healing mechanisms of tendon
tissue. In addition to this, the researchers of the FP4 laser wanted to focus their study on the
bio-stimulative effect and not focus on the anti-inflammatory effect that the laser also
possesses.
Both the surgical and the collagenase gel method are good options to use in evaluating new
treatment option for tendonitis in horses, when more data will be available on the activity of
collagenase.
28
Discussion
In the practical assessment that was carried out, there was a limited amount of monitoring
possible, only ultrasound and a macroscopic evaluation. To be able to validate these models for
evaluating treatment options, a clear assessment must be established. Most of this was done
through research in scientific literature but the amount of monitoring in this practical
assessment was limited. This could give a less detailed estimation of how well the methods
would work and which method would suit the HLLT research best.
Because all the techniques were carried out on cadaver legs, the reaction of the tissue to the
methods could not be evaluated. And the amount of legs were the methods were tested on was
low. This meant that all the techniques could only be evaluated two or three times. In addition
to this, both the collagenase techniques were carried out with other substances. These were
tinted so they could be located easily in the macroscopic evaluation. Because of the absence of
the active enzyme, the reaction of the tissue to the collagenase was not visible.
29
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