Acta Orthop Scand 2004; 75 (1): 93–99 93
Platelet concentrate injection improves Achilles
Orthopedics and Sports Medicine, Department of Neuroscience and Locomotion, Faculty of Health Science, SE-581 85 Linköping, SwedenCorrespondence: PA [email protected] 02-12-30. Accepted 03-04-14
Background Blood platelets release a cocktail of growth molecules released by activated blood platelets. factors when activated, some of which are thought to initi-
In this study, we therefore hypothesized that
ate and stimulate repair.
the healing of Achilles tendon ruptures can be
Experiment and findings We studied whether a plate-
improved by the injection of a platelet concen-
let concentrate injection would improve Achilles tendon trate (PC). repair in an established rat model. The Achilles tendon was transected and a 3 mm segment removed. After 6h, a platelet concentrate was injected percutaneously into the hematoma. This increased tendon callus strength and stiff- ness by about 30% after 1 week, which persisted for as long Survey as 3 weeks after the injection. At this time, the mechanical 296 Sprague-Dawley rats were used. The plate- testing indicated an improvement in material character-
let concentrate was prepared from the blood of
istics—i.e., greater maturation of the tendon callus. This 30 rats that had been killed. We started with an was confirmed by blinded histological scoring.
experiment in which 20 rats were randomized
Interpretation Platelet concentrate may prove useful to receive the platelet concentrate or control for the treatment of Achilles tendon ruptures.
buffer injections. The findings were evaluated
mechanically at 8 days. This experiment showed
a significant improvement in tendon callus
strength and was followed by a repeat experi-
In previous experiments, we have noted an increase ment that did not reach statistical significance.
in bone formation in response to a blood platelet con- Therefore, a series of similar experiments was
centrate (Siebrecht et al. 2002). Since we regarded performed, each time with 10 rats receiving
this as a general stimulation of repair rather than a platelet concentrate and 10 rats receiving control
specific increase in bone formation, we now studied treatment (Table 1). After 5 such experiments,
whether an injection of platelet concentrate could we performed experiments with a healing time
also improve repair of the Achilles tendon. This of 11, 14, 21 or 28 days. In the final analysis,
possibility was further suggested by the observation data from the various experiments were pooled
that healing of the Achilles tendon can be acceler- into larger groups with identical treatment and
ated by a single injection of a growth factor from the follow-up times.
cartilage derived morphogenetic protein (CDMP)
family (Forslund and Aspenberg 2001, 2002), and Preparation of platelet concentrate
that another growth factor, platelet-derived growth Whole blood was collected from female
factor (PDGF), can stimulate ligament repair in rats Sprague-Dawley rats (200 g., M&B, Ry, Den-
(Batten et al. 1996, Hildebrand et al. 1998). PDGF is mark). Normally, 3 donor rats were sufficient for
included in the cocktail of growth factors and other an experiment on 10 recipient rats. The donor
Copyright Taylor & Francis 2004. ISSN 0001–6470. Printed in Sweden – all rights reserved.
94 Acta Orthop Scand 2004; 75 (1): 93–99
Acta Orthop Scand 2004; 75 (1): 93–99 95
consisted of 1 part CPD buffer as above, added to
One of the first experiments included another-
group that received the supernatant from the
second centrifugation—i.e., platelet-poor plasma,
The rats were anesthetized with 5% isoflurane in
an anesthetic induction chamber and then 3.5%
in a mask. The skin was shaved and the opera-
tion performed under aseptic conditions. A 3 mm
transverse incision was made in the skin lateral to
the right Achilles tendon. The surrounding fascia
was cut longitudinally and the Achilles tendon
exposed. The plantaris tendon was removed.
The Achilles tendon was cut transversely 3 mm
proximal to its calcaneal insertion and a 3 mm long
segment was removed to enlarge the defect. The
wound was closed. Preoperatively, the rats were
a 10 for histology
given tetracycline 15 µg and buprenorphine 0.03
mg. After the operation, the rats were randomized
for treatment by taking colored marbles out of a
rats were anesthetized with a mixture of Rompun hat, randomizing groups of 10 rats at a time. The
and Ketalar (xylazine and ketamine 1 mL/kg rats were given a local injection 6 h postoperatively
and 1.5 mL/kg), but in the later experiments, we with 50 µL of the PC or control solution.
changed to isoflurane as described below. The
blood was collected by cardial puncture. The Evaluation
animals were then killed by an intracardiac injec- After 8, 11, 14, 21 or 28 days, the rats were
tion of an overdose of pentobarbital sodium. The anesthetized again with isoflurane and killed
blood was added immediately to an anticoagulant, by an intracardiac injection of pentobarbital
citrate phosphonate dextrose (CPD) buffer (0.15 sodium. The tendon with the attached calcaneal
mg CPD/mL) in a ratio of 1 mL of CPD buffer to bone was removed and dissected free from other
5 mL of blood. The blood was then centrifuged at tissues, wrapped in gauze soaked with saline
220 g for 20 minutes. The supernatant, contain- pending mechanical testing, that was performed
ing platelet-rich plasma, was used for a second within a few hours. In 29 rats, the tendon from
centrifugation at 480 g for 20 minutes. The the unoperated leg was taken for comparison.
pellet from the second centrifugation was saved Mechanical testing was done using a material test-
and diluted with supernatant until the platelet ing machine (100 R, DDL Inc. Eden Praire, MN,
PC concentration became 1.5 × 1012 platelets/L, USA). The tendon was fixed between two metal
measured in a Bürkner chamber. The PC was then clamps and pulled at a constant speed of 1 mm/sec
activated by adding thrombin 20 U per mL PC until failure. The angle between the calcaneus and
(USP Thrombostat Parke-Davis, Morris Plains, Achilles tendon during testing corresponded to
USA, 200 units/mL). To reduce the risk of a graft- 30º dorsiflexion of the foot. We recorded the peak
versus-host reaction, the PC was irradiated at 25 force, stiffness and energy uptake (until the force
Gy according to international blood banking stan- had fallen to 90% of maximum).
dards to inactivate the white blood cells. Up until
The tendons for histological examination
the operation, the platelet concentrate was stored were decalcified in EDTA, prepared with routine
at +4 ºC for a maximum of 24 h. Control solutions methods for paraffin sections and stained with
94 Acta Orthop Scand 2004; 75 (1): 93–99
Acta Orthop Scand 2004; 75 (1): 93–99 95
Ehrlichʼs hematoxylin and eosin. The specimens Table 2. Mechanical results of experiments with 8-day
were sectioned parallel to the longitudinal direc- follow-up
tion of the tendon. Only sections from the middle
of the tendon callus were made. 3–5 glass slides
per specimen comprising the entire length of the
tendon callus were prepared, blinded and labeled
with a specimen-related code number, so that
all slides from one specimen could be evaluated
together, without knowledge of the treatment and
duration of the follow-up. The specimens were
examined in random order, with a microscope,
mainly using a 12× objective. They were classified
according to an arbitrary scoring system from 1 to
5, where 1 represented an immature loose callus
and 5 a dense organized fibrous tissue with mostly
Biomechanical results were analyzed by two-way
ANOVA. Only buffer controls were included. In
the analysis of the 5 experiments with an 8-day
follow-up, the independent variables were treat-
ment (experiment vs control) and experiment
(1–5). In the analysis of the entire set of experi-
The force at failure was increased by PC treat-
ments with all follow-up times, the independent ment (p = 0.001) by an average of 27% (95%
variables were treatment (experiment vs control) confidence interval (CI) 10–44%). There was also
and duration of follow-up (8, 11, 14 or 28 days). a difference between the different experiments
Confidence intervals for comparisons between (p = 0.002). This difference was mainly caused
subgroups are based on t-statistics. Histological by differing responses to PC treatment, whereas
scores were compared using Kruskal-Wallis non- the controls did not differ significantly between
parametric ANOVA, followed by the Mann-Whit- experiments.
ney test for comparison of controls versus PC at 11
The transverse area of the tendon callus differed
between the experiments (p = 0.006), but it was
also increased an average of 18% by PC (95% CI
The maximum stress was similar in the experi-
ments and was not significantly affected by PC
No rat was lost from mechanical or histological treatment (95% CI decrease by 20% to increase
follow-up, but sometimes 1 or 2 rats had to be by 20%).
taken from the original experimental groups of 10
The stiffness differed between the various
for the production of more PC. Thus, tendons from experiments (p < 0.001), but it was also increased
223 rats were used for mechanical analysis and 40 an average of 35% by PC (p < 0.001) (95% C
The energy uptake differed between the experi-
ments, but no effect of treatment could be shown
Mechanical results at 8 days (Table 2)
(95%CI decrease by 8% to increase by 43%).
Of 122 tested specimens, 112 ruptures occurred in
The effect of platelet-poor plasma (supernatant
the tendon callus, 7 at the proximal clamp and 3 at from the second centrifugation) was about the
same as that of the control solution and yielded
96 Acta Orthop Scand 2004; 75 (1): 93–99
Acta Orthop Scand 2004; 75 (1): 93–99 97
Table 3. Mechanical results in all groups
M – Midsubststance; C – Calcaneus; P – Proximal clamp
Mechanical results at all times (Tables 3 and 4)
With time, the number of tendons that failed at
the clamp or at the calcaneus increased so that at
28 days, 5 of 20 tendons had not ruptured in the
midsubstance. Of these, 3 were untreated tendons.
Area (mm2)
Exclusion of all tendons that did not rupture in
the midsubstance had little effect on the results
and did not change any of the conclusions based
on the statistical analysis. The PC-treated tendons
reached 84% of the force at failure of unoperated
tendons at 21 days. The controls had reached 63%
When the data at all times were analyzed
together, the force at failure was increased by PC
(p < 0.001). The average difference between PC-
treated specimens and controls was largest at 21
days after surgery (increase by 36%), but the 95%
less force at failure than PC (p = 0.01; one-way CI at 21 days was large (5–67% increase).
ANOVA of all data during 8 days, Fisherʼs post
The transverse area was increased by PC treat-
ment at 8 and 11 days after surgery, but after 14
��������������������
����������������
Force at failure and stiffness of PC (red) and control (blue) at 8, 11, 14, 21 and 28 days. Boxes indicate interquartile range,
whiskers 10–90 percentile range, and dots extreme values.
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96 Acta Orthop Scand 2004; 75 (1): 93–99
Acta Orthop Scand 2004; 75 (1): 93–99 97
Our results show that a single injection of platelet
concentrate can improve tendon repair in rats. This
effect is probably due to the growth factors that are
released from the platelets during activation. We
have previously found an improvement in tendon
repair in the same model, using recombinant car-
tilage-derived morphogenetic proteins (CDMPs)
days, the areas were similar in the PC-treated and (Aspenberg and Forslund 1999, Forslund and
control groups. Therefore, the maximum stress Aspenberg 2001, Forslund et al. 2003), but
was increased on days 14, 21 and 28. At 21 days, similar experiments using TGFß1 or FGF2, have
the maximum stress was increased by 31% (95% been unsuccessful (data not shown). The present
CI decrease by 14% to increase by 76%), and results seem to differ in several respects from those
even at 28 days by 23% (95% CI decrease by 4% obtained with CDMPs. With CDMP1, 2 or 3, we
to increase by 57%). In the analysis of all groups mainly studied the findings at 8 days. At that time,
together, the maximum stress was increased by PC CDMPs increase the transverse area by 250%, but
with platelet concentrate, we found only an 18%
The stiffness in all groups increased with time increase. The force at failure was increased 40%
(p < 0.001) and PC (p < 0.001). The effect of PC by CDMPs and 27% by the platelet concentrate.
seemed to be largest at 11 days (increase by 41%; Thus, the CDMPs caused a dramatic proliferative
95% CI 12–70%), and had disappeared at 28 days. response, making the tendon callus larger, while
At this time, the stiffness was 57% of the unoper- the platelet concentrate mainly affected matura-
tion. We also noted an improvement in the maxi-
The energy uptake in all groups was affected mum stress with platelet concentrate as late as 21
by time (p < 0.001) and was increased by PC (p = and 28 days.
0.02). The effect appeared to be largest at 21 days
We were surprised that a single injection had
(increase by 18%; 95% CI decrease by 12% to an effect even 4 weeks later, and it remains to be
increase by 48%). The energy uptake exceeded seen whether the effects of CDMPs are similarly
that of the unoperated tendons from 14 days and long lasting. Recent results from a rabbit experi-
ment indicate that the initial increase in the size
of the tendon callus induced by CDMP treatment
lasts during remodeling and therefore increases the
Half of the unoperated tendons showed load defor- maximum strength at later times as well (Forslund
mation curves that indicated slipping of the clamps and Aspenberg 2002). Although CDMPs seem to
or other problems (this did not occur in operated be more potent, they are not available for clinical
tendons). The remainder (n = 14) had a force at use, but the patientʼs own platelets can be easily
failure of 56 N, sd 9.7, stiffness of 14 N/mm, sd prepared during routine care. We also believe that
5.7 and energy uptake of 78 Nmm, sd 30.
our platelet concentrates were not always optimally
prepared. The effects of PC varied significantly
between the groups with an 8-day follow-up. This
All specimens contained an almost homogeneous difference in response was probably due to differ-
mass of fibrous callus, with a few inclusions of ences in the platelet concentrate preparation. The
fat cells. The histological scores were higher platelets were obtained by percutaneous heart
at 21 days, and differed between the 4 groups puncture and it seems likely that trauma to the
(p < 0.001). PC had no detectable effect at 11 days endothelium caused premature platelet activation
(p = 0.4), but at 21 days, the tendon calluses were in some of our batches, so that the growth factors
were lost in the supernatant. We plan to improve
our methods of preparation in other experiments.
98 Acta Orthop Scand 2004; 75 (1): 93–99
Acta Orthop Scand 2004; 75 (1): 93–99 99
Moreover, the dose injected into the rat tendon repair (Lowery et al. 1999, Tischler 2002). How-
defects could be increased, for example, by simply ever, clinical applications of methods developed
doubling the injected volume. Thus, it might be in experimental animals have often been disap-
possible to increase the effect in this rat model.
pointing, and it remains unclear whether PC can
We had no controls which did not receive injec- improve tendon repair in humans. If the incidence
tions. In previous studies, we have repeatedly of rerupture were to be the effect variable, many
found that injection of an acidic buffer does not patients would have to be included in any study
affect healing in this model (Forslund et al. 2003, aimed at showing a beneficial effect. On the other
unpublished data). The only difference between hand, the risks of injecting an autologous platelet
PC and platelet-poor plasma preparations in this concentrate into,—e.g., the hematoma of an Achil-
study was the presence of platelets, and the effects les tendon rupture—would be minimal.
of PC differed from those of platelet-poor plasma
controls, but the effects of the latter were about the
Acknowledgements. Carina Forslund developed the animal
The tendon specimens were mounted so that model, Björn Skoglund assisted in surgery on rats and bio-
mechanical testing, Anna Fahlgren and Ali Sudeifi assisted
rupture usually occurred in the tendon substance in surgery on rats, Anders Kalén and Yawei Liu showed us
and not at its attachments. The unoperated tendons how to prepare the platelet concentrate.
also usually ruptured in the midsubstance. The This study was supported by the Swedish Research Council,
uninjured tendon may be more sensitive to uneven project 2031.
loading during testing, so that a few of the well-
organized fiber bundles may have taken most of
the initial load in an unphysiological way, which No competing interests declared.
would cause premature initiation of rupture. With
the more diffuse arrangement of fibers in the tendon
calluses, this effect should be less in the operated Aspenberg P, Forslund C. Enhanced tendon healing with
tendons. Therefore, we believe that the strength of
GDF 5 and 6. Acta Orthop Scand 1999; 70 (1): 51-4.
the intact tendon in the physiological situation is Batten M L, Hansen J C, Dahners L E. Influence of dosage
and timing of application of platelet-derived growth
greater than our measurements had indicated. The
factor on early healing of the rat medial collateral liga-
comparisons with unoperated tendons in the results
ment. J Orthop Res 1996; 14 (5): 736-41.
section are intended to give a general impression Forslund C, Aspenberg P. Tendon healing stimulated by
injected CDMP-2. Med Sci Sports Exerc 2001; 33 (5):
Platelet concentrate has been used for many Forslund C, Aspenberg P. Improved healing of transsected
purposes in orthopedics, but to our knowledge
rabbit Achilles tendon by a single injection of CDMP-2.
only one controlled clinical study has evaluated its
Am J Sports Med. Accepted for publication.
effects. Marx et al. (1998) implanted maxillofacial Forslund C, Rueger D, Aspenberg P. A comparative dose-
bone grafts enhanced with PC and found a higher
response study of CDMP-1, 2 and 3 for tendon healing in
rats. J Orthop Res. Accepted for publication.
rate of bone formation and more bone in the PC- Hildebrand K A, Woo S L, Smith D W, Allen C R, Deie M,
treated grafts than in controls. Human PC improved
Taylor B J, et al. The effects of platelet-derived growth
bone ingrowth into porous hydroxyapatite in nude
factor-BB on healing of the rabbit medial collateral liga-
rats (Siebrecht et al. 2002). Muscle regeneration in
ment. An in vivo study. Am J Sports Med 1998; 26 (4):
rabbits was improved by rabbit PC (Jodczyk et al. Jodczyk K J, Bankowski E, Borys A. Stimulatory effect
1986), and in skin wounds, PC increased granula-
of platelet-breakdown products on muscle regeneration.
tion tissue and fibrous tissue formation (Ksander et
Zentralbl Allg Pathol 1986; 131 (4): 357-61.
al. 1990). One of the growth factors in platelets is Ksander G A, Sawamura S J, Ogawa Y, Sundsmo J, McPher-
son J M. The effect of platelet releasate on wound healing
platelet-derived growth factor (PDGF). This factor
in animal models. J Am Acad Dermatol 1990; 22 (5 Pt
has improved regeneration in rabbit experiments
on ligament repair (Batten et al. 1996).
Lowery G L, Kulkarni S, Pennisi A E. Use of autologous
Many uncontrolled clinical studies have reported
growth factors in lumbar spinal fusion. Bone (2 Suppl)
that PC has a good effect on bone and soft tissue
98 Acta Orthop Scand 2004; 75 (1): 93–99
Acta Orthop Scand 2004; 75 (1): 93–99 99
Marx R E, Carlson E R, Eichstaedt R M, Schimmele S R, Tischler M. Platelet-rich plasma. The use of autologous
Strauss J E, Georgeff K R. Platelet-rich plasma: Growth
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The Dread Pirate’s Summer Beach Bash Topics: Pirates, beach, party & fuzzy dice. This ebook may not be reproduced in whole or in part, CREATED & PUBLISHED IN THE UNITED STATES OF AMERICA. It had been a long time in coming. I had not had a vacation in years and then my therapist told me to take a break, get away, have some fun or be committed to a life of stress, Prozac and