JFAS 30(6): 529-533, 1991

Achilles Tendon Rupture in Athletes: Histochemistry of the Triceps Surae Muscle

Bilateral percutaneous muscle biopsies of the triceps surae were analyzed in 12 athletes
who had sustained a one-sided subcutaneous rupture of the Achilles tendon while
practicing their main sport. No necrosis, atrophy, or significant fiber grouping or
regeneration was evident. The soleus muscle in these athletes was composed of
approximately 70% of type I fibers in both the noninjured and injured sides, and no
significant differences were detected in their histochemical composition. Fiber areas were
within described values and not significantly different between the two sides. The
noninjured side revealed an insignificant trend to greater average capillary density and
average capillary/fiber ratio. Muscle abnormalities do not appear to be a significant factor
in determining Achilles tendon rupture in healthy athletes.

Nicola Maffulli, M.D.1

Vittorino Testa, M.D.2
Giovanni Capasso, M.D.3

Spontaneous rupture of the Achilles tendon is increasingly common (1). It causes serious
disability in athletes (2, 3), but its etiopathogenesis is still unclear. Intratendinous
injections of steroids are the most frequently incriminated causative agents (4-6).
Intensive training without appropriate recovery, muscle imbalance, and decrease in
flexibility and suppleness have also been advocated (3). In general, athletes do not suffer
from underlying metabolic conditions, which could predispose to tendon rupture.
Mucoid degeneration (7) and microtears (4) of the tendon can cause mechanical
failure. Chronic tendinous inflammation may result in nodular tendinitis and tendinosis
that may weaken the tendon, probably increasing the risk of spontaneous rupture (8).
Cases of acute or chronic tenosynovitis or tendinitis have been reported prior to
spontaneous ruptures among athletes (1, 9), but controversies still exist about tendon
degeneration being a possible causative factor in ruptures.
Kvist and Jarvinen (10, 11) found that, among 14 ruptured Achilles tendons, nine
were normal and five showed signs of degeneration. In the study of Jacobs et al. (2), 50%
of the tendons presented degenerative lesions. Fox et al. (12) showed that, of 22 patients
with chronic Achilles tendonitis, 10 suffered from a subsequent rupture.
While clear histopathologic alterations are generally visible in ruptured tendons in
athletes (13, 14), they are exceptional in autoptic studies of normal subjects (3). Probably,
tendon ruptures are not the consequence of pre-existing degeneration (15-17), even
though the association is found in several cases (3).
In absence of evident pathology, excessive tension may tear a normal Achilles
tendon at its weakest point, generally 2 to 6 cm. from its calcaneal insertion (18, 19). In
these cases, uncoordinated, possibly stiff calf muscles undergoing sudden eccentric
contraction while resisting forced dorsiflexion of the ankle may produce forces sufficient
to rupture the tendon. A recent paper suggested that structural changes in the triceps
muscle may indicate an underlying muscle metabolic dysfunction responsible for the
rupture (20). The authors report their experience in biopsies of the soleus muscle in a
group of previously healthy athletes who underwent surgery for ruptured Achilles tendon.

Patients and Methods

Patients

Twelve Caucasian patients (10 men and two women, eight right tendons), who
were operated on for acute subcutaneous midsubstance rupture of the Achilles tendon,
gave their informed consent to participate in the study. Average age was 34.1 + 5.5 years
(range: 27 to 43 years). All patients were injured while practicing a sporting activity
(three playing tennis, three soccer, two volleyball, one handball, one squash, and one
running).

____________________________________________________________
From the Newham General Hospital, Department of Orthopaedics,
Plaistow, London, England, and the First Institute of Orthopaedics,
University of Naples, First Medical School, Naples, Italy.
0449/2544/9 1/3006-0529$03.00/0
Copyright © 1991 by The American College of Foot Surgeons

529

All had been training at least three times per week and competing at regional and national
level for at least 2 years before the study (3.5 + 4.9 years, range 2 to 11 years). None had
any Achilles tendon problems for 1 year before the study, and only three patients had
ever seen a doctor for a transient Achillodynia. In this instance, they were treated with
physiotherapy and oral nonsteroidal anti-inflammatory drugs. None had ever received
any injections in the tendon area. None gave a positive medical history for or showed
evidence of metabolic, inflammatory, or rheumatological conditions.
All patients presented for observation within 24 hr. of the injury. Diagnosis was
formulated according to one or more of several clinical tests (21-24) and using high-
resolution real-time ultrasound scanning (25, 26). The patients were operated on under
general anesthesia and lying in the prone position. An end-to-end suture using chromic
catgut was carried out through a medial approach (26) within 12 hr. of admission, and 36
hr. from the trauma (median 26 hr.; average 27.3 + 8.1, range 13-36).

Muscle Sampling

Muscle biopsies were taken from the medial aspect of the soleus muscles
bilaterally using the needle (27) or conchotome (28) technique,4 before exsanguinating
the limb, applying the tourniquet, and starting the operation. The muscles were sampled
at a depth of 2 to 3 cm. from the skin, immediately above the border between the upper
one third and lower two thirds of the distance between the popliteal fossa and insertion of
the intact Achilles tendon, measured with the foot in neutral position. After having been
oriented, the samples were mounted in an embedding medium (Ames, OCT
Compound),5 frozen in isopentane cooled to the freezing point with liquid nitrogen, and
stored at -80°C until subsequent analyses were performed (29).

Histochemical Analyses

Serial transverse sections, 10 to 16 µm thick according to the staining to be

performed, were cut with a microtome refrigerated at -18'C, and stained for myofibrillar
ATPase (30), NADH-tetrazolium reductase (31), and by the amylase-PAS method to
visualize capillaries (32). All stained specimens were photographed, and
microphotographs were analyzed in a random sequence. Subject identity, sport, and side
were not disclosed until all specimens had been analyzed.
Fibers were classified into type I and II, and in subtypes IIA, IIB, and IIC (30, 33-
36). The percentage of fiber distribution was determined from microphotographs of all
fibers from biopsy specimens (at least 600 fibers/specimen, average 721 + 102 fibers per
biopsy). A minimum of 400 fibers for each section were counted (37).
Capillary density (cap. per mm2) was determined on microphotographs of the
amylase-PAS method stained sections (38). Fiber areas were determined on
microphotographs of the myosin ATPase method stained sections (39) with a computed-
assisted planimetre.6 The area of a minimum of 200 type I fibers and 200 type II fibers
for each specimen was determined (40).

Statistics

Results were fed into an IBM compatible PC, and analyzed using the Systat
statistical package (41). Descriptive statistics + 1 standard deviation (SD) are given. Data
were analyzed using one-way or two-way analysis of variance (ANOVA) of no
difference in mean to the data. If ANOVA revealed an overall significance, comparisons
among means were applied (42). When appropriate, two-tailed Student's t-test for paired
data was applied. Significance was set at the 0.05 level.

Results

Myofibrillar ATPase staining did not show necrotic, atrophic, or regenerating

fibers (Fig. 1). Fiber-grouping was not evident in any biopsy. The average fiber type
distribution was 68% type I, 19% type IIA, 12% type IIB, and 1% type IIC on the injured
side, and 66% type I, 18% type IIA, 15% type IIB, and 1% type IIC on the noninjured
side (NS).
Type IIA fibers had an average area of 4500 µm2, which was significantly greater
than the area of type IIB (3900 µm2) and type 1 (3550 µm2) fibers. The two sides were
not significantly different.
Morphological analysis of NADH tetrazolium reductase stained sections revealed
a typical pattern, with type I fibers staining more darkly than type II (Fig. 2).
 The average capillary density was 299 cap. per mm2 (injured side) and 312 cap.
per mm2 (noninjured side) (NS), and the average capillary/fiber ratio was 1.52 (injured
side) and 1.63 (noninjured side) (NS) (Table 1) (Fig. 3).

530

Discussion

The cause of spontaneous rupture of the Achilles tendon in sportsmen remains

unknown (1). In this study, histochemical analysis of the soleus muscle in previously
healthy athletes showed normal findings both in the soleus muscle of the affected leg and
in the contralateral one. This contrasts with recent work, which showed accumulation of
intracellular lipid droplets in the leg muscles examined (20). These are generally found in
hypothyroidism (43), and in rare metabolic disorders, such as carnitine or carnitine-
palmytyltransferase deficiency (44). The authors did not actively look for such
conditions, which are generally not compatible with strenuous physical activity, and there
was no clinical sign of them in the patients examined.
All patients were athletes who had been actively practicing their sport for at least
2 years. They were well trained and routinely performed warming up and stretching
exercises. They had never had previous Achilles tendon problems requiring other
medications than oral nonsteroid anti-inflammatory drugs, and this only happened in
three of them. Also, the patients entering this study were relatively young and first seen
within 24 hr. of the trauma; a biopsy was taken within 36 hr. of it. Given the timing of
biopsy sampling, the changes shown in previous studies (20) could have been secondary
to the rupture, representing the local metabolic response to severe injury, and not the
cause of it.
If an ill-conditioned muscle were exposed to intense exercise, the oxidative
metabolism would have to be supplemented by glycolysis. These metabolic demands may
be associated with relative ischemia and overloading, which may have caused the
mitochondrial lesions found in other reports (20). In this case, when these muscles are
required to exercise intensively, they may possibly react in an uncoordinated way,
leading to abnormal stiffness in the triceps muscle, resulting in excessive strain of the
Achilles tendon, eventually resulting in a rupture (45).

TABLE 1. Capillary supply of the different fiber types (N = 12)

———————————————————————
Injured Side Uninjured Side
I IIA IIB I IIA IIB
————————————————————————————
Capillaries in 4 4.1 2.8 3.9 4.2 3.0
contact with
each fiber (average)
———————————————————————
 Figure 1. Myosin ATPase staining, x50. A normal pattern of distribution of type 1
and type 11 fiber is shown. There is a predominance of type 1 fibers.

Figure 2. NADH tetrazolium reductase. A pattern revealed by the myosin ATPase

staining is evident, with type 1 being darker than type 11 fibers.

Figure 3. Amylase-PAS for capillaries staining, x250. The capillaries appear as

small darker dots between the borders of the fibers.

531

The group of patients presented, instead, was well trained, and so the above hypothesis
cannot be applied in full. It cannot be excluded that a sudden uncoordinated contraction
could have taken place, exceeding the loading capacity of the tendon. This is possible,
given the nature of the sports practiced, which, except in one case, were all ball sports,
characterized by their acyclical nature and the extreme differences in playing situations.
Another difference between this and previous studies is the timing and technique
of the biopsy sampling. Hoffmeyer et al. (20) studied patients operated on within 5 days
of the rupture, when diffuse inflammatory response was present around the tendon (46,
47) and significant amounts of plasma proteins surrounded the ruptured area (13). Also,
this study used the percutaneous, not the open biopsy technique. Although it has been
shown that a percutaneous biopsy is well representative of the conditions of the whole
muscle examined (37), this may account for some of the differences found. The above
findings do not rule out that even gentle training and warm up may help sports
enthusiasts to lessen the risk of an Achilles tendon rupture by enhancing the aerobic
metabolism and the mechanical characteristics of their triceps surae muscles (48).

References

1. Smart, G. W., Taunton, J. E., Clements, D. B. Achilles tendon disorders in

runners: a review. Med. Sci. Sports Exerc. 12:231-243, 1980.
2. Jacobs, D., Martens, M., Van Audekercke, R., Mulier, J. C., Mulier, F.
Comparison of conservative and operative treatment of Achilles tendon rupture. Am. J.
Sports Med. 6:107-111, 1978.
3. Williams, J. G. P. Achilles tendon lesions in sport. Sports Med. 3:114-135,
1986.
4. Kleinman, M., Gross, A. E. Achilles tendon rupture following steroid injection.
A report of three cases. J. Bone Joint Surg. 65A:345-347, 1983.
5. Lee, Ii. B. Avulsion and rupture of the tendon calcaneus after injection of
hydrocortisone. Br. Med. J. 2:395, 1957.
 6. Matthews, L. S., Sonstegard, D. A., Phelps, D. B. A biomechanical study of
rabbit patellar tendon: effects of steroid injection. J. Sports Med. Phys. Fitness 2:339-341,
1975. 7. Mornet, J., Doliveux, P. La maladie des tendons d'Achilles et sa complication:
la rupture. Rev. Rhum. Mol. Osteoartic. 40:607611, 1973.
8. Maffulli, N., Regine, R., Angelillo, M., Capasso, G., Filice, S. Ultrasound
diagnosis of Achilles tendinopathy in runners. Br. J. Sports Med. 21:158-162, 1987.
9. Stanish, W. W. D., Curwin, S., Rubinovich, M. Tendinitis: the analysis and
treatment for running. Clin. Sports Med. 4:593-609, 1985.
10. Kvist, M., Jarvinen, M. Biological and biochemical features in repair of
muscle and tendon injuries. Tnt. J. Sports Med. 2:187188, 1981.
11. Kvist, M., Jarvinen, M. Clinical, histochemical and biomechanical features in
repair of muscle and tendon injuries. Int. J. Sports Med. 3:12-14, 1982.
12. Fox, J. M., Blazina, M. E., Jobe, F. W., Kerlan, R. K., Carter, V. S., Shields,
C. L., Jr., Carlson, G. J. Degeneration and rupture of the Achilles tendon. Clin. Orthop.
107:221-224, 1975.
13. Jozsa, L., Lehto, M., Kannus, P., Kvist, M., Reffy, A., Vieno, T., Jarvinen, M.,
Demel, S., Elek, E. Fibronectin and laminin in Achilles tendon. Acta. Orthop. Scand.
60:469-471, 1989.
14. Jozsa, L., Reffy, A., Kannus, P., Elek, E., Demel, S. Pathological alterations in
human tendons. Arch. Orthop. Trauma Surg. 110:15-21, 1990.
15. Arner, O., Lindholm, A. Subcutaneous rupture of the Achilles tendon: a study
of 92 cases. Acta. Chir. Scand. 239(Suppl): 1-51, 1959.
16. Lawrence, G. H., Cave, E. F., O'Connor, H. Injury to the Achilles tendon. Am.
J. Surg. 89:795-802, 1955.
17. Ljungqvist, R. R. Subcutaneous partial rupture of the Achilles tendon. Acta.
Orthop. Scand 113(Suppl): 1-68, 1968.
18. Barfred, T. Experimental rupture of the Achilles tendon. Comparison of
various types of experimental rupture in rats. Acta. Orthop. Scand. 42:528-543, 1971.
19. Barfred, T. Achilles tendon rupture. Acta. Orthop. Scand. 152(Suppl):1-34,
1973.
20. Hoffmeyer, P., Freuler, C., Cox, J. N. Pathological changes in the triceps
surae muscle after rupture of Achilles tendon. Int. Orthop. 14:183-188, 1990.
21. Matles, A. L. Rupture of the tendo achillis. Another diagnostic sign. Bull.
Hosp. Jt. Dis. Orthop. Inst. 36:48-51, 1975.
22. O'Brien, T. The needle test for complete rupture of the Achilles tendon. J.
Bone Joint Surg. 66A:1099-1101, 1984.
23. Simmonds, F. A. The diagnosis of the ruptured Achilles tendon. Practitioner
179:56-58, 1957.
24. Thompson, T. C., Doherty, J. C Spontaneous rupture of tendon of Achilles: a
new clinical diagnostic test. J. Trauma 2:126-129, 1962.
25. Maffulli, N., Dymond, N. P., Capasso, G. Ultrasonographic findings in
subcutaneous rupture of Achilles tendon. J. Sports Med. Phys. Fitness 29:365-368, 1989.
26. Maffulli, N., Dymond, N. P., Regine, R. Surgical repair of ruptured Achilles
tendon in sportsmen and sedentary patients: a longitudinal ultrasound assessment. Int. J.
Sports Med. 11:7884, 1990.
 27. Bergstrom, J. Muscle electrolytes in man determined by neutron activation
analysis on needle biopsy specimen: a study in normal subjects. Scand. J. Clin. Invest.
14(Suppl): 1-110, 1962.
28. Henriksson, J. "Semi-open" muscle biopsy technique. A simple outpatient
procedure. Acta. Neurol. Scand. 59:317-323, 1979.
29. Maffulli, N., Capasso, G., Auzino, G. The needle muscle biopsy. Technique
and methods. Ital. J. Sports Traumatol. 9:1-7, 1987.
30. Padykula, H. A., Herman, E. The specificity of the histochemical method for
adenosine triphosphatase. J. Histochem. Cytochem. 3:170-195, 1955.
31. Novikoff, A. B., Shin, W., Druckner, J. Mitochondrial localization of
oxidation enzymes. Staining results with two tetrazolium salts. J. Biophys. Biochem.
Cytol. 9:47-61, 1961.
32. Andersen, P. Capillary density in skeletal muscle of man. Acta. Physiol.
Scand. 95:203-205, 1975.
33. Dubowitz, V., Brooke, M. Muscle Biopsy: A Modern Approach, pp. 44-51,
W. B. Saunders, London, 1973.
34. Engel, W. The essentiality of histo- and cyto-chemical studies of skeletal
muscles in the investigation of neuromuscular disease. Neurology 12:778-794, 1962.
35. Henriksson, J. Human Skeletal Muscle Adaptation to Physical Activity.
Thesis. Stockholm, 1976, pp. 1-53.
36. Schantz, P. G. Plasticity of human skeletal muscle. Acta. Physiol. Scand.
558(Suppl):1-62, 1986.
37. Blomstrand, E., Ekblom, B. The needle biopsy technique for fibre type
determination in human skeletal muscle. A methodological study. Acta. Physiol. Scand.
116:437-442, 1982.
38. Andersen, P., Henriksson, J. Capillary supply of the quadriceps femoris
muscle of man. Adaptive response to exercise. J. Physiol. 270:677-690, 1977.

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