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The effects of ultrasound treatment on flexor tendon healing in the chicken limb
THE EFFECTS OF ULTRASOUND TREATMENT ON FLEXOR TENDON HEALING IN THE CHICKEN LIMB B. S. GAN, S. HUYS, M. H. SHEREBRIN and C. G. SCILLEY
From the Departments of Plastic Surgery and Physiotherapy, VictoriaHospital, and the Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada We report the effects of early and late ultrasound treatment protocols on healing of surgically lacerated zone 2 flexor tendons in a chicken model. Ultrasound was administered directly using a coupling gel. Treatment was shown to increase range of movement, to advance scar maturation and to decrease the amount of inflammatory infiltrate around the repair site. No adverse effects on tensile strength were noted in either group. Early (starting 7 days post-operatively) administration was more effective than late (starting 42 days post-operatively) administration in achieving these effects. These results suggest that ultrasound therapy may be of benefit in the early healing process of flexor tendon lacerations. Journal of Hand Surgery (British and European Volume, 1995) 20B: 6:809-814
tendons, making it difficult to extrapolate the effects to the repaired human tendon (Turner et al, 1989). It is clear from these studies, performed in a wide variety of models that may have no resemblance to the human flexor tendon apparatus, that no clear conclusions can be drawn with respect to the possible benefit or harm of US treatment. In an effort to further investigate the effects of US on tendon healing the present study was undertaken. Because the chicken flexor tendon model is viewed as most closely approximating the human situation (Turner et al, 1989; Stevenson et al, 1986), and because the direct application of US most closely resembles its clinical administration, we have chosen to examine the effects of US, directly applied at different post-operative intervals, on flexor tendons surgically repaired in zone 2 in the chicken limb model.
Much of the investigative work on flexor tendon repair has focused on the improvement of suture materials, surgical techniques and post-operative immobilization or controlled mobilization (Steinberg, 1992; Stewart, 1991; Strickland, t989). Relatively little effort has been directed at elucidating the potential of adjunctive therapies, such as pharmacological or physiological modulation of tendon healing. Ultrasound (US) is used in some centres 6 to 8 weeks post-operatively in an effort to soften peritendinous adhesions which may limit range of movement of the tendon. It is not initiated earlier, because it has been suggested that it weakens the repair site (Stewart, 1991; Taylor Mullins, 1990; Roberts et al, 1982). There is, however, little experimental evidence to support either the benefits or the possible harmful effects of this treatment. Most studies on the effects of ultrasound (US) on tendon healing use it in the indirect or waterbath method. It is then difficult to determine the amount of US that is absorbed by the tissue and that actually reaches the repair site. Frieder et al (1988) found collagen maturation to be advanced in rat Achilles tendon treated with indirect US. Enwemeka et al (1990) reported an increase in tensile strength in rabbit Achilles tendons. Turner et al (1989) studied the effects of indirect US in a chicken model and noted no difference in strength of repair or range of motion. Stevenson et al (1986) in the same-model also found that US did not influence gap formation or breaking strength, but added that US did improve the functional recovery (flexion) of repaired tendons. Only one previously published report deals with the direct application of US. Roberts et al (1982), in their experiments on rabbit Achilles tendons, reported an adverse effect on the early healing process and a decrease in tensile strength. This study, however, has been criticized because of its lack of detail and the unusual timing, duration and frequency of the US treatments. It was also noted that the chemical composition of rabbit Achilles tendon differs greatly from that of human flexor
M E T H O D S A N D MATERIALS
76 White Leghorn hens (Reiman's Fur Ranch, St Agatha, Ontario, Canada), 24 weeks in age and weighing 1400 to 2000 grams were studied. The chickens were acclimatized for a minimum of 7 to 10 days and were allowed to roam freely in a room with a 12-hour light cycle which was kept at constant temperature. Food and water were freely provided. Study protocols were approved by the University of Western Ontario Subcommittee on Research and Teaching involving Animals (SURTIA) and comply with the Guidelines of the Canadian Council on Animal Care (CCAC). Each animal underwent FDP tendon repair to the right third toe just proximal to the middle IP joint. This site corresponds to zone 2 in the human hand and the anatomy and technique have been described previously in detail (Lindsay and Thomson, 1960; Farkas et al, 1974). The animals were anaesthetized with intramuscular injections of ketamine (50-100 mg/kg) and additional local anaesthetic (0.5% bupivacaine) was used as a digital block. Under sterile conditions and using magnification, the FDP tendon of the third toe was 809
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THE JOURNAL OF HAND SURGERY VOL. 20B No. 6 DECEMBER 1995
dissected free through a mid-lateral incision and subsequently transfixed using 26 gauge hypodermic needles. Care was taken to avoid damage to the neurovascular bundle. The tendon was then sharply divided proximal to the vinculum longum and repaired with a 4/0 KesslerTajima Mersilene suture. No running epitendinous suture was used and the flexor tendon sheath was not closed. The skin was approximated with a running 4/0 Vicryl suture, and topical antibiotic spray was administered. Post-operatively, the leg was immobilized in a thermoplast splint with the ankle (hock) joint in 60 ° of flexion and the IP joints in full extension. This position replicates immobilization of the wrist and hand after primary repair of human flexor tendons (Feehan and Beauchene, 1990; Fig 1). Post-operatively, the animals were numbered and randomly assigned to one of three groups:
Group 1 No US. Group 2 Early US treatment starting on day 7 Group 3 Late US treatment starting on day 42 US treatment was applied by the direct method using a coupling gel (0.5 cm 2 head AnstelEnraf Sonoplus 434 at 3 M H z and 0.8 W/cm 2 intensity, pulsed for 3 rain with a duty-cycle of 25%). The animals in the US groups received ten daily treatments. To avoid "mobilization artifacts" The splint was not removed for these treatments. Mock insonation was therefore not deemed necessary. At regular intervals, however, all chickens underwent removal of splints to inspect for pressure points and to allow modification of the splint, if required. Animals were sacrificed 4 and 8 weeks after surgery and both legs were amputated at the hock joint. In each
group, one half of the chickens not sacrificed at 4 weeks were allowed to mobilize freely and the other half were kept immobilized until week 8. After sacrifice, the tendons were examined for tensile strength, histological appearance and in situ range of movement of the repaired F D P tendon. At the end of the measurements, the animal code number was broken and the data were stratified according to treatment group. Measurement of in situ range of movement
Limitation in range of movement ( R O M ) of the repaired tendon was thought to reflect the severity of peritendinous adhesions. To measure this accurately, a special bench was constructed (Fig 2). Immediately after sacrifice, the amputated leg was secured in the appropriate position and the F D P tendon to the third toe was divided just distal to the trifurcation of the common F D P tendon. Using a system of pulleys, it was possible to measure the excursion of the isolated repaired tendon in an isotonic fashion. The force applied (65 g) was the minimum force required to obtain full reproducible excursion in unoperated tendons. The excursion of each tendon was measured and repeated four times and the average value was taken as the R O M of the particular tendon. The individual measurements never varied more than 0.5 mm and were due to a hysteresis effect. Measurements of tensile strength
Following ROM testing, the tendons were dissected, stored in 0.9% NaCt at 4°C and transported to the biophysics lab for tensile strength testing within 24 hours. The excised tendons were tested mechanically in
\ FDP muscle mass
Y
"+:
IE
flex
L", a
IP
°sf CHICKEN
Fig 1
Fig 2
b HUMAN
The position of post-operative immobilization after flexor tendon repair in the chicken (a) and m a n (b) (reproduced by the kind permission of Churchill Livingstone from F E E H A N , L. M., and B E A U C H E N E , J. G. (1990). Early tensile properties of healing chicken flexor tendons: Early controlled passive motion versus postoperative immobilization. Journal of H a n d Surgery. 15A: 63 68.)
Schematic diagram of the specially constructed bench to measure in situ R O M of the repaired flexor tendon. The weights (65 g), which were the m i n i m u m required load to obtain full flexion/extension in non-operated left 3rd toes, were alternatively unloaded to obtain the R O M measurement. Each tendon was taken four times through a range of motion and measured. The average values of the four individual measurements were taken as the ROM-value of the tendon. Proximal attachment to the tendon was just distal to the trifurcation of the c o m m o n F D P tendon and distal attachment was through the nail. In separate control experiments, the tolerance of our measurement system was shown to be better than 0.1 mm.
CHICKEN TENDON ULTRASOUND
an Instron model 1125 material testing machine (Instron Corp, Canton M A 02021-9949, USA) using a tensile load cell of 50 kg capacity. During testing the tendons were held in stainless steel clamps covered with grade 200 waterproof sand paper to decrease slipping. The measurements of load and elongation in tension were obtained while the tendon was immersed in physiological saline at a room temperature of 22°C. N o correction was made for the small change in buoyancy as the clampholder was withdrawn from the saline. Load in grams and elongation in millimetres was read from the strip chart recording. The thickness and width of the tendon scar was measured with a Starrett Dial Indicator Gauge model 25-881J (L. S. Starrett Company, Athol, Massachusetts, USA). The initial length was read with a millimetre ruler at zero load. The true stress and true strain were calculated using a Lotus 123 spreadsheet (Lotus 123, version 2.0, Lotus Development Corporation, Cambridge, Massachusetts, USA). The true stress was calculated from equation 1: = Force/Area = Load x Acceleration x Initial Length/Initial Volume = m x g x Lo/Vo and the true strain was calculated from equation 2: E = In (L/Lo) Where load is in kilograms, acceleration is 9.8 m/s 2, Lo is the initial length at zero load in metres, Vo the initial volume in m 3, L in meters is the given length at a given load. The stress is in SI units of Pascals when the load is in kilograms and the length in metres. The units of strain are non-dimensional (m/m). The strain rate used was approximately 0.001 strain units/s. We make the assumption that the tissue was noncompressible and therefore Poisson's ratio was 0.5. The stress is the force per unit cross-sectional area. Since this area changes with elongation, the true stress calculated with equation 1 corrects for the decrease in crosssectional area as the tendon is stretched assuming the volume is constant. The modulus of elasticity, also referred to as Young's modulus, was calculated from the slope of the curve of stress versus strain. The data points were read from the continuous curve of elongation versus load provided by the chart recorder on the Instron.
Histological analysis Two tendons from each group were submitted for histological analysis. Directly after R O M measurement these tendons were excised and immersed in 5% formaldehyde. The tendons were mounted in paraffin blocks and longitudinal sections with a thickness of 4 g were cut. The slides were processed with a haematoxilin-eosin stain. A senior pathologist then examined the slides, blinded to
811
the treatment group. The samples were graded on a scale of 1 to 4 with regards to the arrangement and orientation of the collagen fibres and on a scale of 0 to 3 with regards to the amount of inflammatory infiltrate. After histological assessment the identifying code was broken and the individual treatment groups compared. No statistical analysis was undertaken in view of the small numbers in each group.
Statistical analysis Students t-test was used for the statistical analysis of the R O M data (Minitab Release 7, State College, Pennsylvania, USA. The Instron data were analyzed using ANOVA in the Multivariate General Linear Hypothesis Module ( M G L H ; Systat version 3.0, Systat Incorporated, Evanston, Illinois, USA). RESULTS The observed complication rate was very low. Three animals developed pressure ulcerations proximal to the hock joint which were treated conservatively. This was not considered enough to warrant exclusion of these animals. There was one peri-operative death.
Measurement of in situ range of movement Results of the R O M measurements of repaired tendons are summarized in Figure 3 and Table 1. Control values represent the R O M of 3rd F D P tendons in the nonoperated left leg. Repaired tendons in group 2 (early US) showed a statistically significant ( P < 0.05) greater ROM than in group 1 (no US) 8 weeks post-operatively. The average R O M of group 3 (Late US) falls in between. When R O M was measured 4 weeks postoperatively, the average R O M of group 2 (early US) was greater than group 1 (no US), but this did not attain statistical significance.
Measurement of tensile strength It appeared that the chickens which were sacrificed at week 8, but allowed to mobilize freely at week 4, proceeded to poor healing of the repair site. This expressed itself in breaking strengths which were on average 10 to 20 times lower than those of chickens which were kept immobilized until week 8. Most importTable 1--Numerical values of ROM testing Time
No US
Early US
Late US
4 wks
1.9+0.4 (n=9) 2.4_+0.4*
2.9_+0.3 ( n - 10) 4.0-t-0.5"
-3.3_+0.4
7.9_+0.3 (n=10) 7.7_+0.2
(n = 8)
(n = 10)
(n = 10)
(n = 10)
8 wks
R O M = r a n g e of movement. US - ultrasound. Values represent mean_+ SEM (mm). * Denotes P < 0.05.
Unoperated
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THE JOURNAL OF HAND SURGERY VOL. 20B No. 6 DECEMBER 1995
[ ] no U/S • lateU/S Q early U/S 0 conlrol ,k
5
p < 0.05
n,-
I
I
4
8
Time in weeks
Fig 3
Values of R O M measurements on the Y-axis versus time on the X-axis. Values represent mean _+SEM. There is a significant difference between R O M of early ultrasounded tendons ( O ) compared to non-ultrasounded tendons ([~).
1'1
10 g
g 7
6
WZ
S
4
3
2 1
0
Fig 4
0.02
0.04
0.06
0.09
Example of computer fitted curve (fifth order polynomial) with linear stress on Y-axis and strain on X-axis. Young's Modulus of elasticity was calculated from the slope of the curve. Note the close fit of the computer generated curve. This graph was obtained from a tendon at 8 weeks n o t treated with US.
CHICKEN TENDON ULTRASOUND
Fig 5
813
Representative photomicrographs (HE-stain) of early ultrasound-treated tendons (a and c) and non-ultrasound-treated tendons (b and d). At low power (original magnification 2.5 x), it is easy to note the smoother and more regular pattern of collagen fibres crossing the repair zone in an ultrasound-treated tendon (a), whereas the bundles in a non-treated tendon clearly end in scar tissue without a longitudinal orientation (b). At higher magnification (original magnification 10 x) in an area adjacent to the core-stitch, the pronounced decrease in inflammatory infiltrate in the treated tendon (c) compared to the. non-treated tendon (d) can easily be seen. At this magnification, it can also be appreciated that the fibre orientation and scar maturation are more advanced in the ultrasound-treated tendon.
antly, this occurred in all animals and was therefore independent of the assigned treatment group. For this reason, the 8-week results of the chickens which were allowed to move freely after 4 weeks were excluded from the final biomechanical analysis. The linear stress versus strain curve was sigmoidal in shape (Fig 4). Maximum stress, maximum strain and Young's modulus of elasticity were analyzed against treatment and time. No significant differences were found between the different US treatment protocols.
Histological analysis The blind histological analysis showed a marked decrease in inflammatory infiltrate and a more regular pattern of scar formation (Fig 5) in the US groups. These effects were more pronounced in the early US group than in the late US group.
DISCUSSION In spite of the advances that have taken place in flexor tendon repair, a significant percentage of patients still have a poor result. In an effort to improve the outcome in this group of patients, some receive ultrasound to soften scar adhesions in the later phases of rehabilitation. Reports of adverse effects of ultrasound on tendon healing, however, have tempered enthusiasm for its use in the early phase following repair. Using the chicken toe zone 2 flexor tendon model and direct ultrasound application with a coupling gel used in most clinical situations, we have been able to demonstrate a beneficial effect of ultrasound treatment (US) on R O M and histological appearance of repaired tendons without an adverse effect on tensile strength. Eight weeks post-operatively, there was a 67% increase in ROM in the early US treatment group compared to
814
the no US treatment group. The early US group achieved a final R O M exceeding 50% of that of unoperated control tendons. Tendons of chickens that were allowed to mobilize at 4 weeks were much weaker than those of chickens immobilized for the full 8 weeks. This contradicts previously published results indicating that early mobilization increases tensile strength (Hitchcock et al, 1987). A possible explanation is that unrestricted mobilization at 4 weeks is too early, and this in itself could have led to irreversible disruption at the repair site. The histological analysis confirmed previous reports that US has an effect on tendon wound healing. The alignment of collagen fibres in the scar was more regular and there was a marked decrease in inflammatory response following US treatment. Studies using systemic administration of indomethacin (Szabo and Younger, 1990) and ibuprofen (Kulick et al, 1986) have shown an increase in tendon ROM and it has been postulated that this effect was due to an alteration in peritendinous adhesions through suppression of inflammation. Our histological analysis showed a marked decrease in inflammatory infiltrate and is, therefore, consistent with such a hypothesis. The use of US in this study did not result in any decrease in tensile strength. This is in contrast to results published by Roberts et al (1982) who reported an adverse effect of directly coupled US on tendon healing and tendon breaking strain. In that paper, crosssectional area of the repaired tendon was not considered, opening the possibility that breaking load was actually the variable measured. If this is true, then Roberts only measured the load at which the last remaining fibres fail. This value does not take into consideration the deformation (decrease in cross-sectional diameter during elongation of the tissue) during strength testing, nor does it tell anything about the first (weakest) fibres to fail, or the behaviour of the tissue in between these points. For example in Figure 4, at a strain value of 0.05 to 0.06 the curve becomes non-linear and the slope starts to decrease. This is the point at which the weakest fibres fail and irreversible damage starts to occur. Our method of data analysis allows for continuous assessment of true stress, true strain as well as Young's modulus of elasticity, and failed to show a difference in any of these parameters in tendons treated with US. Other investigators (Frieder et al, 1988; Enwemeka et al, 1990; Turner et al, 1989; Stevenson et al, 1986) also did not find an adverse effect of US on tendon strength, but their studies did not use direct coupling application of US, making direct comparison with this study difficult. The difference between the waterbath method and direct coupling has been investigated and direct administration was shown to be more effective (Forrest and Rosen, 1989). Moreover, in the clinical situation, US is administered using a coupling gel. The doses used in this study were calculated in a dosimetric analysis of the dimensions of the chicken leg, aiming for energy delivery to the tendon repair site equivalent to the dose adminis-
T H E J O U R N A L O F H A N D SURGERY VOL. 20B No. 6 DECEMBER 1995
tered in man, thus closely resembling the protocol applied in the clinical situation. Taken together, this study clearly suggests a potential benefit of direct coupling US when used as an adjunct in the early post-operative treatment of flexor tendon lacerations and provides encouragement for further investigation of the effects of US on healing of flexor tendons. We believe that US used in conjunction with existing methods of controlled mobilization may help in improving the outcome following injury to the flexor tendons. Acknowledgements We are indebted to Dr R. F. Armstrong for his invaluable help in the histological analysis of our samples. Parts of this paper have been presented at the 15th Annual Meeting of the American Society of H a n d Therapists, November 1992, Phoenix, Arizona, USA, and received the R.L. Petzoldt Award for Innovations in H a n d Therapy. It was also presented in part at the 47th Annual Meeting of the Canadian Society of Plastic Surgeons, June 1993, St John's, NewFoundland, Canada, and received the CSPS Educational Foundation Award for Best Research in the Basic Sciences.
References E N W E M E K A , C. S., R O D R I G U E Z , O. and M E N D O S A , S. (1990). The biomechanical effects of low-intensity ultrasound on healing tendons. Ultrasound in Medicine and Biology, 16: 801-807. FARKAS, L. G., THOMSON, H. G. and MARTIN, R. (1974). Some practical notes on the anatomy of the chicken toe for surgeon investigators. Plastic and Reconstructive Surgery, 54:452 458. FEEHAN, L. M. and BEAUCHENE, J. G. (1990). Early tensile properties of healing chicken flexor tendons: Early controlled passive motion versus postoperative immobilization. Journal of H a n d Surgery 15A: 63-68. FORREST, G. and ROSEN, K. (1989). Ultrasound: Effectiveness of treatments given under water. Archives of Physical Medicine and Rehabilitation 70: 28 29. F R I E D E R , S., WEISBERG, J., F L E M I N G , B. and STANEK, A. (1988). A pilot study: The therapeutic effect of ultrasound following partial rupture of Achilles tendons in male rats. Journal of Orthopaedic and Sports Physical Therapy, 10: 39-46. H I T C H C O C K , T. F., LIGHT, T. R., BUNCH, L. H. et al. (1987). The effects of immediate constrained digital motion on the strength of flexor tendon repairs in chickens. Journal of H a n d Surgery, 12A: 590-595. K U L I C K , M. I., SMITH, S. and H A D L E R , K. (1986). Oral ibuprofen: Evaluation of its effect on peritendinous adhesions and the breaking strength of a tenorrhaphy. Journal of Hand Surgery, l l A : 110 120. LINDSAY, W. K. and THOMSON, H. G. (1960). Digital flexor tendons: An experimental study. British Journal of PIastic Surgery, 12: 289-319. ROBERTS, M., R U T H E R F O R D , J. H. and HARRIS, D. (1982). The effect of ultrasound on flexor tendon repairs in the rabbit. The Hand, 14: 17-20. STEINBERG, D. R. (1992). Acute flexor tendon injuries. Orthopedic Clinics of North America, 23:125 140. STEVENSON, J. H., PANG, C. Y., LINDSAY, W. K. and ZUKER, R. M. (1986). Functional, mechanical and biochemical assessment of ultrasound therapy on tendon healing in the chicken toe. Plastic and Reconstructive Surgery, 77:965 972. STEWART, K. M. (1991). Review and comparison of current trends in the post-operative management of tendon repair. H a n d Clinics, 7:447 460. STRICKLAND, J. W. (1989). Flexor tendon surgery. Journal of Hand Surgery, 14B: 261 272. SZABO, R. M. and Y O U N G E R , E. (1990). Effects of indomethacin on adhesion formation after repair of zone II tendon lacerations in the rabbit. Journal of H a n d Surgery, 15A: 480-483. TAYLOR MULLINS, P. A. Use of Therapeutic Modalities in Upper Extremity Rehabilitation. In: Hunter, J. M., Schneider, L. H., Mackin, E. J., and Callahan, A. D. (Eds): Rehabilitation of the Hand." Surgery and Therapy., 1990. St Louis, Mosby, 195 220. T U R N E R , S. M., POWELL, E. S. and NG, C. S. S. (1989). The effect of ultrasound on the healing of repaired cockerel tendon: Is collagen cross-linkage a factor? Journal of Hand Surgery, 14B: 428-433.
Accepted: 2 May 1995 Christopher G. ScilleyMD, FRCS(C), Department of Plastic Surgery, Victoria Hospital, Box 5375, London, Ontario. Canada. N6A 405. © 1995 The British Society for Surgery of the Hand
From the Departments of Plastic Surgery and Physiotherapy, VictoriaHospital, and the Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada We report the effects of early and late ultrasound treatment protocols on healing of surgically lacerated zone 2 flexor tendons in a chicken model. Ultrasound was administered directly using a coupling gel. Treatment was shown to increase range of movement, to advance scar maturation and to decrease the amount of inflammatory infiltrate around the repair site. No adverse effects on tensile strength were noted in either group. Early (starting 7 days post-operatively) administration was more effective than late (starting 42 days post-operatively) administration in achieving these effects. These results suggest that ultrasound therapy may be of benefit in the early healing process of flexor tendon lacerations. Journal of Hand Surgery (British and European Volume, 1995) 20B: 6:809-814
tendons, making it difficult to extrapolate the effects to the repaired human tendon (Turner et al, 1989). It is clear from these studies, performed in a wide variety of models that may have no resemblance to the human flexor tendon apparatus, that no clear conclusions can be drawn with respect to the possible benefit or harm of US treatment. In an effort to further investigate the effects of US on tendon healing the present study was undertaken. Because the chicken flexor tendon model is viewed as most closely approximating the human situation (Turner et al, 1989; Stevenson et al, 1986), and because the direct application of US most closely resembles its clinical administration, we have chosen to examine the effects of US, directly applied at different post-operative intervals, on flexor tendons surgically repaired in zone 2 in the chicken limb model.
Much of the investigative work on flexor tendon repair has focused on the improvement of suture materials, surgical techniques and post-operative immobilization or controlled mobilization (Steinberg, 1992; Stewart, 1991; Strickland, t989). Relatively little effort has been directed at elucidating the potential of adjunctive therapies, such as pharmacological or physiological modulation of tendon healing. Ultrasound (US) is used in some centres 6 to 8 weeks post-operatively in an effort to soften peritendinous adhesions which may limit range of movement of the tendon. It is not initiated earlier, because it has been suggested that it weakens the repair site (Stewart, 1991; Taylor Mullins, 1990; Roberts et al, 1982). There is, however, little experimental evidence to support either the benefits or the possible harmful effects of this treatment. Most studies on the effects of ultrasound (US) on tendon healing use it in the indirect or waterbath method. It is then difficult to determine the amount of US that is absorbed by the tissue and that actually reaches the repair site. Frieder et al (1988) found collagen maturation to be advanced in rat Achilles tendon treated with indirect US. Enwemeka et al (1990) reported an increase in tensile strength in rabbit Achilles tendons. Turner et al (1989) studied the effects of indirect US in a chicken model and noted no difference in strength of repair or range of motion. Stevenson et al (1986) in the same-model also found that US did not influence gap formation or breaking strength, but added that US did improve the functional recovery (flexion) of repaired tendons. Only one previously published report deals with the direct application of US. Roberts et al (1982), in their experiments on rabbit Achilles tendons, reported an adverse effect on the early healing process and a decrease in tensile strength. This study, however, has been criticized because of its lack of detail and the unusual timing, duration and frequency of the US treatments. It was also noted that the chemical composition of rabbit Achilles tendon differs greatly from that of human flexor
M E T H O D S A N D MATERIALS
76 White Leghorn hens (Reiman's Fur Ranch, St Agatha, Ontario, Canada), 24 weeks in age and weighing 1400 to 2000 grams were studied. The chickens were acclimatized for a minimum of 7 to 10 days and were allowed to roam freely in a room with a 12-hour light cycle which was kept at constant temperature. Food and water were freely provided. Study protocols were approved by the University of Western Ontario Subcommittee on Research and Teaching involving Animals (SURTIA) and comply with the Guidelines of the Canadian Council on Animal Care (CCAC). Each animal underwent FDP tendon repair to the right third toe just proximal to the middle IP joint. This site corresponds to zone 2 in the human hand and the anatomy and technique have been described previously in detail (Lindsay and Thomson, 1960; Farkas et al, 1974). The animals were anaesthetized with intramuscular injections of ketamine (50-100 mg/kg) and additional local anaesthetic (0.5% bupivacaine) was used as a digital block. Under sterile conditions and using magnification, the FDP tendon of the third toe was 809
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THE JOURNAL OF HAND SURGERY VOL. 20B No. 6 DECEMBER 1995
dissected free through a mid-lateral incision and subsequently transfixed using 26 gauge hypodermic needles. Care was taken to avoid damage to the neurovascular bundle. The tendon was then sharply divided proximal to the vinculum longum and repaired with a 4/0 KesslerTajima Mersilene suture. No running epitendinous suture was used and the flexor tendon sheath was not closed. The skin was approximated with a running 4/0 Vicryl suture, and topical antibiotic spray was administered. Post-operatively, the leg was immobilized in a thermoplast splint with the ankle (hock) joint in 60 ° of flexion and the IP joints in full extension. This position replicates immobilization of the wrist and hand after primary repair of human flexor tendons (Feehan and Beauchene, 1990; Fig 1). Post-operatively, the animals were numbered and randomly assigned to one of three groups:
Group 1 No US. Group 2 Early US treatment starting on day 7 Group 3 Late US treatment starting on day 42 US treatment was applied by the direct method using a coupling gel (0.5 cm 2 head AnstelEnraf Sonoplus 434 at 3 M H z and 0.8 W/cm 2 intensity, pulsed for 3 rain with a duty-cycle of 25%). The animals in the US groups received ten daily treatments. To avoid "mobilization artifacts" The splint was not removed for these treatments. Mock insonation was therefore not deemed necessary. At regular intervals, however, all chickens underwent removal of splints to inspect for pressure points and to allow modification of the splint, if required. Animals were sacrificed 4 and 8 weeks after surgery and both legs were amputated at the hock joint. In each
group, one half of the chickens not sacrificed at 4 weeks were allowed to mobilize freely and the other half were kept immobilized until week 8. After sacrifice, the tendons were examined for tensile strength, histological appearance and in situ range of movement of the repaired F D P tendon. At the end of the measurements, the animal code number was broken and the data were stratified according to treatment group. Measurement of in situ range of movement
Limitation in range of movement ( R O M ) of the repaired tendon was thought to reflect the severity of peritendinous adhesions. To measure this accurately, a special bench was constructed (Fig 2). Immediately after sacrifice, the amputated leg was secured in the appropriate position and the F D P tendon to the third toe was divided just distal to the trifurcation of the common F D P tendon. Using a system of pulleys, it was possible to measure the excursion of the isolated repaired tendon in an isotonic fashion. The force applied (65 g) was the minimum force required to obtain full reproducible excursion in unoperated tendons. The excursion of each tendon was measured and repeated four times and the average value was taken as the R O M of the particular tendon. The individual measurements never varied more than 0.5 mm and were due to a hysteresis effect. Measurements of tensile strength
Following ROM testing, the tendons were dissected, stored in 0.9% NaCt at 4°C and transported to the biophysics lab for tensile strength testing within 24 hours. The excised tendons were tested mechanically in
\ FDP muscle mass
Y
"+:
IE
flex
L", a
IP
°sf CHICKEN
Fig 1
Fig 2
b HUMAN
The position of post-operative immobilization after flexor tendon repair in the chicken (a) and m a n (b) (reproduced by the kind permission of Churchill Livingstone from F E E H A N , L. M., and B E A U C H E N E , J. G. (1990). Early tensile properties of healing chicken flexor tendons: Early controlled passive motion versus postoperative immobilization. Journal of H a n d Surgery. 15A: 63 68.)
Schematic diagram of the specially constructed bench to measure in situ R O M of the repaired flexor tendon. The weights (65 g), which were the m i n i m u m required load to obtain full flexion/extension in non-operated left 3rd toes, were alternatively unloaded to obtain the R O M measurement. Each tendon was taken four times through a range of motion and measured. The average values of the four individual measurements were taken as the ROM-value of the tendon. Proximal attachment to the tendon was just distal to the trifurcation of the c o m m o n F D P tendon and distal attachment was through the nail. In separate control experiments, the tolerance of our measurement system was shown to be better than 0.1 mm.
CHICKEN TENDON ULTRASOUND
an Instron model 1125 material testing machine (Instron Corp, Canton M A 02021-9949, USA) using a tensile load cell of 50 kg capacity. During testing the tendons were held in stainless steel clamps covered with grade 200 waterproof sand paper to decrease slipping. The measurements of load and elongation in tension were obtained while the tendon was immersed in physiological saline at a room temperature of 22°C. N o correction was made for the small change in buoyancy as the clampholder was withdrawn from the saline. Load in grams and elongation in millimetres was read from the strip chart recording. The thickness and width of the tendon scar was measured with a Starrett Dial Indicator Gauge model 25-881J (L. S. Starrett Company, Athol, Massachusetts, USA). The initial length was read with a millimetre ruler at zero load. The true stress and true strain were calculated using a Lotus 123 spreadsheet (Lotus 123, version 2.0, Lotus Development Corporation, Cambridge, Massachusetts, USA). The true stress was calculated from equation 1: = Force/Area = Load x Acceleration x Initial Length/Initial Volume = m x g x Lo/Vo and the true strain was calculated from equation 2: E = In (L/Lo) Where load is in kilograms, acceleration is 9.8 m/s 2, Lo is the initial length at zero load in metres, Vo the initial volume in m 3, L in meters is the given length at a given load. The stress is in SI units of Pascals when the load is in kilograms and the length in metres. The units of strain are non-dimensional (m/m). The strain rate used was approximately 0.001 strain units/s. We make the assumption that the tissue was noncompressible and therefore Poisson's ratio was 0.5. The stress is the force per unit cross-sectional area. Since this area changes with elongation, the true stress calculated with equation 1 corrects for the decrease in crosssectional area as the tendon is stretched assuming the volume is constant. The modulus of elasticity, also referred to as Young's modulus, was calculated from the slope of the curve of stress versus strain. The data points were read from the continuous curve of elongation versus load provided by the chart recorder on the Instron.
Histological analysis Two tendons from each group were submitted for histological analysis. Directly after R O M measurement these tendons were excised and immersed in 5% formaldehyde. The tendons were mounted in paraffin blocks and longitudinal sections with a thickness of 4 g were cut. The slides were processed with a haematoxilin-eosin stain. A senior pathologist then examined the slides, blinded to
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the treatment group. The samples were graded on a scale of 1 to 4 with regards to the arrangement and orientation of the collagen fibres and on a scale of 0 to 3 with regards to the amount of inflammatory infiltrate. After histological assessment the identifying code was broken and the individual treatment groups compared. No statistical analysis was undertaken in view of the small numbers in each group.
Statistical analysis Students t-test was used for the statistical analysis of the R O M data (Minitab Release 7, State College, Pennsylvania, USA. The Instron data were analyzed using ANOVA in the Multivariate General Linear Hypothesis Module ( M G L H ; Systat version 3.0, Systat Incorporated, Evanston, Illinois, USA). RESULTS The observed complication rate was very low. Three animals developed pressure ulcerations proximal to the hock joint which were treated conservatively. This was not considered enough to warrant exclusion of these animals. There was one peri-operative death.
Measurement of in situ range of movement Results of the R O M measurements of repaired tendons are summarized in Figure 3 and Table 1. Control values represent the R O M of 3rd F D P tendons in the nonoperated left leg. Repaired tendons in group 2 (early US) showed a statistically significant ( P < 0.05) greater ROM than in group 1 (no US) 8 weeks post-operatively. The average R O M of group 3 (Late US) falls in between. When R O M was measured 4 weeks postoperatively, the average R O M of group 2 (early US) was greater than group 1 (no US), but this did not attain statistical significance.
Measurement of tensile strength It appeared that the chickens which were sacrificed at week 8, but allowed to mobilize freely at week 4, proceeded to poor healing of the repair site. This expressed itself in breaking strengths which were on average 10 to 20 times lower than those of chickens which were kept immobilized until week 8. Most importTable 1--Numerical values of ROM testing Time
No US
Early US
Late US
4 wks
1.9+0.4 (n=9) 2.4_+0.4*
2.9_+0.3 ( n - 10) 4.0-t-0.5"
-3.3_+0.4
7.9_+0.3 (n=10) 7.7_+0.2
(n = 8)
(n = 10)
(n = 10)
(n = 10)
8 wks
R O M = r a n g e of movement. US - ultrasound. Values represent mean_+ SEM (mm). * Denotes P < 0.05.
Unoperated
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THE JOURNAL OF HAND SURGERY VOL. 20B No. 6 DECEMBER 1995
[ ] no U/S • lateU/S Q early U/S 0 conlrol ,k
5
p < 0.05
n,-
I
I
4
8
Time in weeks
Fig 3
Values of R O M measurements on the Y-axis versus time on the X-axis. Values represent mean _+SEM. There is a significant difference between R O M of early ultrasounded tendons ( O ) compared to non-ultrasounded tendons ([~).
1'1
10 g
g 7
6
WZ
S
4
3
2 1
0
Fig 4
0.02
0.04
0.06
0.09
Example of computer fitted curve (fifth order polynomial) with linear stress on Y-axis and strain on X-axis. Young's Modulus of elasticity was calculated from the slope of the curve. Note the close fit of the computer generated curve. This graph was obtained from a tendon at 8 weeks n o t treated with US.
CHICKEN TENDON ULTRASOUND
Fig 5
813
Representative photomicrographs (HE-stain) of early ultrasound-treated tendons (a and c) and non-ultrasound-treated tendons (b and d). At low power (original magnification 2.5 x), it is easy to note the smoother and more regular pattern of collagen fibres crossing the repair zone in an ultrasound-treated tendon (a), whereas the bundles in a non-treated tendon clearly end in scar tissue without a longitudinal orientation (b). At higher magnification (original magnification 10 x) in an area adjacent to the core-stitch, the pronounced decrease in inflammatory infiltrate in the treated tendon (c) compared to the. non-treated tendon (d) can easily be seen. At this magnification, it can also be appreciated that the fibre orientation and scar maturation are more advanced in the ultrasound-treated tendon.
antly, this occurred in all animals and was therefore independent of the assigned treatment group. For this reason, the 8-week results of the chickens which were allowed to move freely after 4 weeks were excluded from the final biomechanical analysis. The linear stress versus strain curve was sigmoidal in shape (Fig 4). Maximum stress, maximum strain and Young's modulus of elasticity were analyzed against treatment and time. No significant differences were found between the different US treatment protocols.
Histological analysis The blind histological analysis showed a marked decrease in inflammatory infiltrate and a more regular pattern of scar formation (Fig 5) in the US groups. These effects were more pronounced in the early US group than in the late US group.
DISCUSSION In spite of the advances that have taken place in flexor tendon repair, a significant percentage of patients still have a poor result. In an effort to improve the outcome in this group of patients, some receive ultrasound to soften scar adhesions in the later phases of rehabilitation. Reports of adverse effects of ultrasound on tendon healing, however, have tempered enthusiasm for its use in the early phase following repair. Using the chicken toe zone 2 flexor tendon model and direct ultrasound application with a coupling gel used in most clinical situations, we have been able to demonstrate a beneficial effect of ultrasound treatment (US) on R O M and histological appearance of repaired tendons without an adverse effect on tensile strength. Eight weeks post-operatively, there was a 67% increase in ROM in the early US treatment group compared to
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the no US treatment group. The early US group achieved a final R O M exceeding 50% of that of unoperated control tendons. Tendons of chickens that were allowed to mobilize at 4 weeks were much weaker than those of chickens immobilized for the full 8 weeks. This contradicts previously published results indicating that early mobilization increases tensile strength (Hitchcock et al, 1987). A possible explanation is that unrestricted mobilization at 4 weeks is too early, and this in itself could have led to irreversible disruption at the repair site. The histological analysis confirmed previous reports that US has an effect on tendon wound healing. The alignment of collagen fibres in the scar was more regular and there was a marked decrease in inflammatory response following US treatment. Studies using systemic administration of indomethacin (Szabo and Younger, 1990) and ibuprofen (Kulick et al, 1986) have shown an increase in tendon ROM and it has been postulated that this effect was due to an alteration in peritendinous adhesions through suppression of inflammation. Our histological analysis showed a marked decrease in inflammatory infiltrate and is, therefore, consistent with such a hypothesis. The use of US in this study did not result in any decrease in tensile strength. This is in contrast to results published by Roberts et al (1982) who reported an adverse effect of directly coupled US on tendon healing and tendon breaking strain. In that paper, crosssectional area of the repaired tendon was not considered, opening the possibility that breaking load was actually the variable measured. If this is true, then Roberts only measured the load at which the last remaining fibres fail. This value does not take into consideration the deformation (decrease in cross-sectional diameter during elongation of the tissue) during strength testing, nor does it tell anything about the first (weakest) fibres to fail, or the behaviour of the tissue in between these points. For example in Figure 4, at a strain value of 0.05 to 0.06 the curve becomes non-linear and the slope starts to decrease. This is the point at which the weakest fibres fail and irreversible damage starts to occur. Our method of data analysis allows for continuous assessment of true stress, true strain as well as Young's modulus of elasticity, and failed to show a difference in any of these parameters in tendons treated with US. Other investigators (Frieder et al, 1988; Enwemeka et al, 1990; Turner et al, 1989; Stevenson et al, 1986) also did not find an adverse effect of US on tendon strength, but their studies did not use direct coupling application of US, making direct comparison with this study difficult. The difference between the waterbath method and direct coupling has been investigated and direct administration was shown to be more effective (Forrest and Rosen, 1989). Moreover, in the clinical situation, US is administered using a coupling gel. The doses used in this study were calculated in a dosimetric analysis of the dimensions of the chicken leg, aiming for energy delivery to the tendon repair site equivalent to the dose adminis-
T H E J O U R N A L O F H A N D SURGERY VOL. 20B No. 6 DECEMBER 1995
tered in man, thus closely resembling the protocol applied in the clinical situation. Taken together, this study clearly suggests a potential benefit of direct coupling US when used as an adjunct in the early post-operative treatment of flexor tendon lacerations and provides encouragement for further investigation of the effects of US on healing of flexor tendons. We believe that US used in conjunction with existing methods of controlled mobilization may help in improving the outcome following injury to the flexor tendons. Acknowledgements We are indebted to Dr R. F. Armstrong for his invaluable help in the histological analysis of our samples. Parts of this paper have been presented at the 15th Annual Meeting of the American Society of H a n d Therapists, November 1992, Phoenix, Arizona, USA, and received the R.L. Petzoldt Award for Innovations in H a n d Therapy. It was also presented in part at the 47th Annual Meeting of the Canadian Society of Plastic Surgeons, June 1993, St John's, NewFoundland, Canada, and received the CSPS Educational Foundation Award for Best Research in the Basic Sciences.
References E N W E M E K A , C. S., R O D R I G U E Z , O. and M E N D O S A , S. (1990). The biomechanical effects of low-intensity ultrasound on healing tendons. Ultrasound in Medicine and Biology, 16: 801-807. FARKAS, L. G., THOMSON, H. G. and MARTIN, R. (1974). Some practical notes on the anatomy of the chicken toe for surgeon investigators. Plastic and Reconstructive Surgery, 54:452 458. FEEHAN, L. M. and BEAUCHENE, J. G. (1990). Early tensile properties of healing chicken flexor tendons: Early controlled passive motion versus postoperative immobilization. Journal of H a n d Surgery 15A: 63-68. FORREST, G. and ROSEN, K. (1989). Ultrasound: Effectiveness of treatments given under water. Archives of Physical Medicine and Rehabilitation 70: 28 29. F R I E D E R , S., WEISBERG, J., F L E M I N G , B. and STANEK, A. (1988). A pilot study: The therapeutic effect of ultrasound following partial rupture of Achilles tendons in male rats. Journal of Orthopaedic and Sports Physical Therapy, 10: 39-46. H I T C H C O C K , T. F., LIGHT, T. R., BUNCH, L. H. et al. (1987). The effects of immediate constrained digital motion on the strength of flexor tendon repairs in chickens. Journal of H a n d Surgery, 12A: 590-595. K U L I C K , M. I., SMITH, S. and H A D L E R , K. (1986). Oral ibuprofen: Evaluation of its effect on peritendinous adhesions and the breaking strength of a tenorrhaphy. Journal of Hand Surgery, l l A : 110 120. LINDSAY, W. K. and THOMSON, H. G. (1960). Digital flexor tendons: An experimental study. British Journal of PIastic Surgery, 12: 289-319. ROBERTS, M., R U T H E R F O R D , J. H. and HARRIS, D. (1982). The effect of ultrasound on flexor tendon repairs in the rabbit. The Hand, 14: 17-20. STEINBERG, D. R. (1992). Acute flexor tendon injuries. Orthopedic Clinics of North America, 23:125 140. STEVENSON, J. H., PANG, C. Y., LINDSAY, W. K. and ZUKER, R. M. (1986). Functional, mechanical and biochemical assessment of ultrasound therapy on tendon healing in the chicken toe. Plastic and Reconstructive Surgery, 77:965 972. STEWART, K. M. (1991). Review and comparison of current trends in the post-operative management of tendon repair. H a n d Clinics, 7:447 460. STRICKLAND, J. W. (1989). Flexor tendon surgery. Journal of Hand Surgery, 14B: 261 272. SZABO, R. M. and Y O U N G E R , E. (1990). Effects of indomethacin on adhesion formation after repair of zone II tendon lacerations in the rabbit. Journal of H a n d Surgery, 15A: 480-483. TAYLOR MULLINS, P. A. Use of Therapeutic Modalities in Upper Extremity Rehabilitation. In: Hunter, J. M., Schneider, L. H., Mackin, E. J., and Callahan, A. D. (Eds): Rehabilitation of the Hand." Surgery and Therapy., 1990. St Louis, Mosby, 195 220. T U R N E R , S. M., POWELL, E. S. and NG, C. S. S. (1989). The effect of ultrasound on the healing of repaired cockerel tendon: Is collagen cross-linkage a factor? Journal of Hand Surgery, 14B: 428-433.
Accepted: 2 May 1995 Christopher G. ScilleyMD, FRCS(C), Department of Plastic Surgery, Victoria Hospital, Box 5375, London, Ontario. Canada. N6A 405. © 1995 The British Society for Surgery of the Hand