Evaluation of a diode laser for use in induction of tendinopathy in the superficial digital flexor tendon of horses

Stuart A. Vallance Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Martin A. Vidal Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Mary Beth Whitcomb Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Brian G. Murphy Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Mathieu Spriet Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Larry D. Galuppo Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Abstract

Objective—To evaluate use of a diode laser to induce tendinopathy in the superficial digital flexor tendon (SDFT) of horses.

Animals—4 equine cadavers and 5 adult horses.

Procedures—Cadaveric SDFT samples were exposed to a diode laser at various energy settings to determine an appropriate energy for use in in vivo experiments; lesion size was assessed histologically. In vivo experiments involved laser energy induction of lesions in the SDFT (2 preliminary horses [0, 25, 75, and 87.5 J] and 3 study horses [0 and 125 J]) and assessment of lesions. Study duration was 21 days, and lesions were assessed clinically and via ultrasonography, MRI, and histologic evaluation.

Results—Lesion induction in cadaveric tissues resulted in a spherical cavitated core with surrounding tissue coagulation. Lesion size had a linear relationship (R2 = 0.9) with the energy administered. Size of in vivo lesions in preliminary horses indicated that larger lesions were required. In study horses, lesions induced with 125 J were ultrasonographically and histologically larger than were control lesions. At proximal and distal locations, pooled (preliminary and study horses) ultrasonographically assessed lesions were discrete and variable in size (mean ± SEM lesion percentage for control lesions, 8.5 ± 3%; for laser lesions, 12.2 ± 1.7%). Ultrasonography and MRI measurements were associated (R2 > 0.84) with cross-sectional area measurements.

Conclusions and Clinical Relevance—In vivo diode laser–induced lesions did not reflect cadaveric lesions in repeatable size. Further research is required before diode lasers can reliably be used for inducing tendinopathy.

Abstract

Objective—To evaluate use of a diode laser to induce tendinopathy in the superficial digital flexor tendon (SDFT) of horses.

Animals—4 equine cadavers and 5 adult horses.

Procedures—Cadaveric SDFT samples were exposed to a diode laser at various energy settings to determine an appropriate energy for use in in vivo experiments; lesion size was assessed histologically. In vivo experiments involved laser energy induction of lesions in the SDFT (2 preliminary horses [0, 25, 75, and 87.5 J] and 3 study horses [0 and 125 J]) and assessment of lesions. Study duration was 21 days, and lesions were assessed clinically and via ultrasonography, MRI, and histologic evaluation.

Results—Lesion induction in cadaveric tissues resulted in a spherical cavitated core with surrounding tissue coagulation. Lesion size had a linear relationship (R2 = 0.9) with the energy administered. Size of in vivo lesions in preliminary horses indicated that larger lesions were required. In study horses, lesions induced with 125 J were ultrasonographically and histologically larger than were control lesions. At proximal and distal locations, pooled (preliminary and study horses) ultrasonographically assessed lesions were discrete and variable in size (mean ± SEM lesion percentage for control lesions, 8.5 ± 3%; for laser lesions, 12.2 ± 1.7%). Ultrasonography and MRI measurements were associated (R2 > 0.84) with cross-sectional area measurements.

Conclusions and Clinical Relevance—In vivo diode laser–induced lesions did not reflect cadaveric lesions in repeatable size. Further research is required before diode lasers can reliably be used for inducing tendinopathy.

Equine tendon and ligament injuries remain a therapeutic challenge in performance horse practice.1 Many treatment modalities have been used to enhance healing with variable but mostly marginal success rates, with the rate of injury recurrence ranging from 23% to 43% in sport horses and 53% to 67% in racehorses.2–4 With the advent of new biological treatment options such as mesenchymal stem cells,5,6 growth factor technologies,7–11 and tissue scaffolding materials,12–14 there is a need for an appropriate method of inducing tendinopathy to test these therapeutic options.15

Investigators in many controlled trials5,6,8,13,14,16 have used the long-established collagenase-induced method for induction of tendinopathy.17 However, lesions caused by this method can be highly variable in size and extent from the site of injection and require approximately 3 weeks to stabilize before use in experiments. Thus, it has been suggested that they poorly represent clinical injuries.15,16 Histopathologically, bacterial collagenase results in dissolution of the tendon matrix and development of an inflammatory exudate (fibrin, erythrocytes, and polymorphonuclear neutrophilic leukocytes), followed by cellular infiltration (macrophages and leukocytes) and neovascularization that forms a connective tissue matrix. By 4 weeks after injection, no signs of necrosis, fibril lysis, or acute inflammation remain.17

More recently, multiple surgical techniques have been reported for use in the creation of tendinitis of the SDFT.11,15,16,18–23 Use of the surgical arthroscopic burr technique has the advantage of the creation of large, well-defined core lesions; however, it requires that horses be anesthetized and, depending on the surgical approach, the paratenon11,16,23 or proximal extent of the digital flexor tendon sheath may be disrupted.15 Histologically, the surgical techniques fail to induce an obvious inflammatory response that has been identified in naturally occurring tendinitis and collagenase-induced tendinitis.15,17,19,24 Coblation technology via radiofrequency energy has also been proposed to be a safe manner in which to create lesions in equine SDFTs.a However, there is considerable cost for this technique, and lesion size and location are unpredictable. A reproducible lesion size is vital to all tendinopathy methods because size of a clinical lesion influences prognosis.25 The ability to reliably create a lesion of a consistent size, with a cellular infiltrate that resembles naturally occurring disease, that is easy to induce would be ideal for studying regenerative medicine techniques for tendon repair and healing.

A 980-nm diode laser is a common surgical tool at many university-based large animal veterinary hospitals. A diode laser generates laser energy via a semiconductor chip (commonly composed of aluminium, gallium, and arsenide), with its wavelength dependent on the temperature, concentration of doping elements, electric current, and presence of a magnetic field. Diode lasers are available in wavelengths ranging from 405 to 1,470 nm. On the basis of its tissue absorption coefficients and Mie scattering properties, a 980-nm diode laser results in tissue coagulation and peripheral thermal injury that may extend 5 to 10 mm beyond the intended site of administration.26 Thus, it was hypothesized that a technique involving use of a diode laser in standing horses could be developed that would yield a repeatable size, location, and shape of lesion and maintain histologic similarity with naturally occurring tendinopathy.

The objective of the study reported here was to develop a technique for experimentally inducing tendinopathy in the SDFT of horses by use of a diode laser. We sought to establish a suitable energy level for tissue ablation with the diode laser in cadaveric SDFTs and to compare clinical, ultrasonographic, MRI, and histologic examination findings following in vivo induction of lesions at predetermined energy levels.

Materials and Methods

Cadaver experiment—The SDFTs of both hind limbs were harvested from each of 4 horses within 6 hours after death. The horses were three 3-year-old female Thoroughbreds that had been in race training and one 17-year-old Quarter Horse that had been retired from racing; all horses died of causes unrelated to hind limb musculoskeletal conditions. All SDFTs were palpably normal and appeared normal on gross inspection after dissection. Serial 2-cm-long SDFT tissue samples were sectioned from 15 cm distal to the point of the tarsus joint to the proximal extent of the digital flexor tendon sheath.

A 16-gauge, 1.5-inch hypodermic needle was placed in the lateral margin of the SDFT segment in a plane transverse to the bevel of the needle. The distal 5 mm of the outer coating of a 600-μm-diameter diode laser fiberb was removed, and the bare fiber tip was advanced through the needle until firmly inserted within the tendon. The laser fiber was stabilized, the needle retracted, and the tissue treated with predetermined amounts of energy. A 980-nm solid-state semiconductor diode laser,c with energy delivered in pulse mode (50% duty cycle, 15 Hz, 33 ms/pulse, and 12.5 or 25 W/pulse), was used to irradiate individual sites on the tendon segments through the optical fiber for specified exposure times (2, 4, 6, and 8 seconds). The total energy delivered for each exposure was 12.5, 25, 37.5, 50, 75, or 100 J. Two SDFT segments from each horse were used for each power and time setting. The laser fiber was retracted, a marker pend was used to indicate the point of the laser fiber entry, and the SDFT segments were placed in neutral-buffered 10% formalin. Needle (control) lesions (ie, 0 J) were not included as part of the cadaveric experiment.

Tissues were fixed in neutral-buffered 10% formalin for approximately 24 hours. Serial 1-mm sagittal sections (n = 4 tendons) and transverse sections (4) were obtained through the lesions. Two or more representative sections were selected, placed in individual cassettes, and processed and embedded in paraffin in a routine manner (overnight processing). Immediately after removing the blocks from the processor, 5-μm-thick sections of freshly embedded tendon tissues were cut on a microtome, mounted on glass slides, and stained with H&E in accordance with standard protocols. Slides (1 to 4 slides/lesion) were examined microscopically at 20× to 100× magnifications and were digitally photographed under bright-field and polarized light by use of a microscopee and camera.f Image processing softwareg was used to quantify the lesion surface area.

In vivo experiment—The in vivo experiment was conducted with 2 preliminary horses (a 24-year-old Thoroughbred and a 23-year-old Quarter Horse) and 3 study horses (2 Thoroughbreds [age, 15 and 23 years] and a 13-year-old Quarter Horse). All were healthy female horses that were used for research at the University of California-Davis Center for Equine Health. An institutional animal care and use committee approved all procedures used in the study.

The 2 preliminary horses were used to verify in vitro findings and to determine the energy setting for the diode laser for in vivo experiments. In the first horse, 3 treatments (18-gauge needle [control] treatment, 25 J, and 75 J) were used at each of 3 sites (4, 10, and 18 cm DACB) in each forelimb SDFT on day 0. In the second horse, 2 treatments were used at each of 2 sites (12 and 18 cm DACB) in each forelimb SDFT on days 0 (18-gauge needle [control] treatment and 87.5 J), 7 (87.5 J), and 14 (87.5 J). Study duration was 21 days.

Results for the preliminary horses indicated that larger lesions were required for the method to be feasible for use in future experiments. Therefore, 3 study horses were used to test a control treatment (18-gauge needle only; day 0) and a laser energy setting of 125 J (25 W, 10 seconds, and 15 Hz) at 3 time points (day 0, 7, and 14). There were 4 lesions/horse (2 lesions/tendon). Because of variability in metacarpal length and location of carpal and digital flexor synovial sheaths among the horses, location for induction of the lesions was standardized to a proximal (10 to 12 cm DACB) or distal (18 to 20 cm DACB) region. All horses were euthanized by administration of a lethal dose (100 mg/kg) of bentobarbital sodiumh on day 21; thus, laser lesions were 7, 14, and 21 days old and control lesions were 21 days old.

Lesion induction technique

Horses were sedated with detomidine hydrochloridei (0.01 mg/kg) and butorphanol tartratej (0.01 mg/kg). Hair was clipped from both forelimbs. The skin was aseptically prepared, and high lateral and medial palmar nerve blocks were administered with 2% mepivacaine hydrochloride.k An 18-gauge, 1.5-inch needle was positioned via ultrasonographic guidance from the lateral aspect of the SDFT to the center of the tendon by use of a small curvilinear (4- to 8-MHz) transducer.l A 600-μm diode laser fiber (6 cm of the outer coating was removed from the distal end) was advanced through the needle until it was firmly inserted within the tendon, and the needle was retracted until the bevel exited the skin. The fiber was manually stabilized and the laserb was activated until the predetermined amount of energy had been delivered; the fiber then was retracted. The distal aspect of each hind limb was bandaged to protect the wounds. Bandages were maintained for 24 hours. Horses were confined to a stall or small yard, depending on the weather.

Clinical assessment

Circumference of each hind limb and signs of pain during palpation (assessed as positive if the limb was withdrawn during palpation at proximal, mid-metacarpal, and distal locations) were recorded. Lameness grade as determined in accordance with guidelines of the American Association of Equine Practitioners27 and physical examination findings were recorded on alternate days for 21 days.

Ultrasonography

Ultrasonography was performed with a 13-MHz linear transducer. All lesions were ultrasonographically evaluated on days 0, 2, and 7 after lesion induction and on days 14 and 21, if applicable. The CSA measurements of the tendon and lesions were obtained in 2-cm increments from the proximal (4 to 6 cm DACB) to distal extent of the tendon (26 to 30 cm DACB). Ultrasonographic images were evaluated to determine tendon CSA, lesion CSA, lesion percentage (lesion CSA divided by tendon CSA), lesion length, lesion echogenicity (grade 1 to 3; 1 = mildly hypoechoic, 2 = moderately hypoechoic, and 3 = anechoic), presence of peritendinous edema (grade 0 to 3; 0 = none, 1 = minimal, 2 = moderate, and 3 = severe), and fiber pattern score (grade 1 to 3; 1 = mildly irregular, 2 = moderately irregular, and 3 = fiber disruption).

MRI

Immediately after horses were euthanized, MRI of each metacarpus was performed with a 1.5-T magnetic resonance systemm by use of a linearly polarized 56 × 22-cm general-purpose flexible-surface radiofrequency coil.n Images of the limbs were obtained parallel to the magnetic field by use of sagittal spin-echo T1-weighted sequences, sagittal 3-D spoiled-gradient echo sequences, and transverse spin-echo T1-weighted, T2-weighted, and STIR sequences. Images from MRI were evaluated by investigators who were not aware of the lesion induction method and age of lesions. Evaluations were performed with digital imaging and communications in medicine viewing softwareo on a flat-screen liquid crystal display monitor.p Measurements determined on transverse and sagittal spin-echo T1-weighted sequences20 included maximal width and length of each lesion, CSA of each lesion and tendon, and lesion percentage. Lesion volume was calculated by multiplying lesion area by lesion length. Shape of each lesion was characterized as a track (ie, linear shape) or track and core (ie, linear shape with round lesion at the extremity). The track and core lesions were further characterized as core larger than the track, core similar to the track, or track larger than the core. The signal intensity in the lesion was characterized for spin-echo T1-weighted, T2-weighted, and STIR images (grade 0 to 2; 0 = hypointense signal similar to grossly normal tendon, 1 = moderate hyperintensity similar to subcutaneous tissue, and 2 = markedly hyperintense and brighter than subcutaneous tissue).

Histologic evaluation

After horses were euthanized, the SDFT lesions were identified via ultrasonographic guidance. A 5-cm section of tendon was removed; the lateral aspect of the lesion was marked with a marker pen and fixed in neutral-buffered 10% formalin for 24 hours. Segments of the SDFT proximal and distal to the 5-cm section were also fixed in formalin. Fixed tendon tissues were transversely sectioned approximately 1.5 cm proximal and distal to the lesion site. Sagittal sections through the lesion were processed as described for the cadaver experiment. Sagittal lesion area included the peripheral coagulated tendon and central granulation tissue.

Statistical analysis—Cadaver tendon lesions were evaluated via an ANOVA with statistical softwareq to test the differences in lesion size and histologic sectioning technique in response to different amounts of laser energy. Lesions of the 5 horses for the in vivo experiments were evaluated by use of data obtained via ultrasonography, MRI, histologic evaluation, and circumferential limb measurements. Mixed models were used for the ANOVA to test the effect of laser energy, limb, lesion location, and lesion age on lesion size. Shapiro-Wilk analysis was used to test normality; as a result, lesion size measurements were logarithmically transformed when appropriate. Dunnett and least significant difference post hoc comparisons of the means were used when appropriate to compare laser and control treatments. Spearman correlations were made between ultrasonographic, MRI, and histologic measurements. Type I error was maintained at α = 0.05 for all comparisons.

Results

Cadaver experiment—Cadaver lesions were readily identifiable visually, during palpation, and during examination of histologic sections. Histologically, lesions were characterized by a central spherical core with variable cavitation surrounded by a variably thick hypereosinophilic rim of coagulated tissue (Figure 1). The demarcation between the coagulated region and unaffected tendon was most readily identifiable with polarized light. There was no significant difference in lesion measurements between transverse and sagittal sections, but transverse sections were prone to sectioning artifact (tissue fragmentation); therefore, only sagittal sections were used for the in vivo experiment.

Figure 1—
Figure 1—

Photomicrographs obtained by use of bright-field (A) and polarized light (B) microscopy of transverse sections of the SDFT from an equine cadaver after induction of lesions with a diode laser at 75 J. In panel A, the central spherical cavitated core is surrounded by a variably thick hypereosinophilic rim of coagulated tissue. In panel B, the demarcation between the region of coagulated tissue and unaffected tendon is most readily identifiable by use of polarized light. H&E stain; bar = 1 mm.

Citation: American Journal of Veterinary Research 73, 9; 10.2460/ajvr.73.9.1435

Logarithmic transformation of the energy and histologic CSA data resulted in a normal data distribution. Lesion CSA had a linear relationship with the laser energy administered, both before and after logarithmic transformation (log10 CSA = [1.2774•log10 energy] −3.2668; R2 = 0.873). There were significant (P < 0.001) differences between each of the mean CSAs.

In vivo experiment—Ultrasonographic, MRI, and histologic measurements for preliminary and study horses revealed that the relationship between laser energy and lesion size in the cadaveric specimens could not be emulated (Table 1; Figure 2). Because of ethical considerations, the number of study horses was limited to 3 and results were combined with those of the preliminary horses for statistical comparisons.

Table 1—

Mean ± SEM values for ultrasonographic and MRI measurements in 2 preliminary horses for lesions at all SDFT locations on day 21 after induction of lesions.

VariableUltrasonographyMRI
ControlLaserControlLaser
No. of lesions3717
Tendon CSA (cm2)
   Day 00.61 ± 0.090.58 ± 0.05
   Day 210.78 ± 0.060.77 ± 0.060.940.81 ± 0.06
Lesion CSA on day 21 (cm2)0.13 ± 0.030.12 ± 0.020.110.13 ± 0.01
Lesion percentage on day 21 (%)20 ± 315 ± 311.217 ± 2
Lesion length on day 21 (cm)0.340.52 ± 0.14

Control lesions were induced by insertion of an 18-gauge needle (0 J), and laser lesions were induced with a diode laser at 25,75, or 87.5 J. Day of lesion induction was designated as day 0.

Lesion percentage was calculated as lesion CSA divided by tendon CSA.

— = Not determined.

Figure 2—
Figure 2—

Arithmetic mean ± SEM size of lesions in the SDFT of 4 equine cadavers (white circles) and 5 live horses (black triangles) after laser induction of lesions. Linear relationships for energy settings and histologic size of lesion area were significantly (P < 0.001) different between equine cadavers (y = [0.1508•x] − 1.366; R2 = 0.8) and live horses (y = [0.0264•x] + 3.301; R2 = 0.2). Cadaver histologic lesion areas differed significantly (P < 0.001) among energy levels. In the live horses, there were significant (P < 0.05) differences in lesion size between needle-induced (control) lesions (0 J) and lesions induced with 87.5 or 125 J.

Citation: American Journal of Veterinary Research 73, 9; 10.2460/ajvr.73.9.1435

Clinical variables

Two of 5 horses developed an increase in lameness score of 1 grade during the 21-day study period. Mild to moderate signs of pain could be elicited during palpation of the laser lesion sites for 7 to 21 days after lesion induction in 4 of 5 horses. Signs of pain were not elicited during palpation of control lesions. Laser-induced lesions resulted in a greater degree of palpable peritendinous edema, compared with that for control lesions (Figure 3). Limb circumference increased following laser induction of lesions but decreased significantly by days 14 (P = 0.001) and 21 (P = 0.027), compared with the circumference on day 7. Limb circumferences for lesions at proximal locations were significantly smaller for all laser energies, compared with circumferences for lesions at distal locations. Circumference ratios after correction for initial limb circumference also were affected by laser energy, and the mean ± SEM circumference ratios for 25 J (1.028 ± 0.006), 75 J (1.024 ± 0.006), 87.5 J (1.015 ± 0.006), and 125 J (1.009 ± 0.004) were all significantly larger than for needle-induced lesions (1.000 ± 0.004). Interestingly, the limb circumference ratios for areas where tendons were treated with 125 J were significantly (P = 0.01) smaller than ratios for areas treated with 25 or 75 J.

Figure 3—
Figure 3—

Photograph of the forelimb of a preliminary horse (A) and the corresponding sagittal image for T1-weighted fast spin-echo MRI (B) at day 21 after laser induction of lesions in the SDFT at proximal (25 J; arrow) and distal (75 J; arrowhead) locations. Peritendinous edema is visible grossly in the photograph and on the corresponding sagittal image at each lesion site. Notice the characteristic box-shaped lesion on the sagittal image.

Citation: American Journal of Veterinary Research 73, 9; 10.2460/ajvr.73.9.1435

Ultrasonography

Lesions were apparent at all induction sites, except for the site of 1 control lesion. Lesions were apparent within 48 hours after lesion induction, except for 1 control lesion and 1 lesion induced with 125 J, both of which were identified at day 7. Tendon and lesion size were not affected by increasing age of lesion; therefore, lesion size at completion of the study was recorded. At proximal and distal locations, lesions were small and variable (mean ± SEM lesion percentage was 8.5 ± 3% for the control lesion and 12.2 ± 1.7% for the laser-induced lesion; Tables 1 and 2). In contrast, control lesions at 4 cm DACB in 1 preliminary horse were large (lesion percentage was 22% and 24%). Because of these unexpectedly large control lesions and field-of-view constraints with the MRI protocol, the lesions at 4 cm DACB were not used after the first preliminary horse. Lesions induced at 125 J were significantly (P = 0.003) larger than needle-induced lesions at proximal and distal locations (Figure 4). Tendon CSA was not affected by laser energy and lesion site. Forty-eight hours after laser induction of lesions, fiber pattern, and echogenicity scores increased to a median of 2 (range, 0 to 3) and 3 (range, 0 to 3), respectively; these values did not differ significantly from values for the control lesions. Lesion location did not significantly alter ultrasonographic measurements. A moderate correlation (R2 = 0.57; P = 0.006) was detected between energy level and peritendinous edema scores.

Table 2—

Mean ± SEM values for ultrasonographic and MRI measurements in 3 study horses for lesions at all SDFT locations on day 21 after induction of lesions.

VariableUltrasonographyMRI
ControlLaserControlLaser
No. of lesions3939
Tendon CSA (cm2)
   Day 00.86 ± 0.190.80 ± 0.04
   Day 210.92 ± 0.10.90 ± 0.060.99 ± 0.160.95 ± 0.07
Lesion CSA on day 21 (cm2)0.05 ± 0.030.09 ± 0.020.05 ± 0.030.11 ± 0.02
Lesion percentage on day 21 (%)6 ± 310 ± 26 ± 312 ± 2
Lesion length on day 21 (cm)0.20 ± 0.110.48 ± 0.08

Laser lesions were induced at 125 J.

See Table 1 for remainder of key.

Figure 4—
Figure 4—

Transverse T1-weighted fast spin-echo MRI (left column) and corresponding transverse (middle column) and sagittal (right column) ultrasonographic images of lesions in an SDFT of each of 5 horses during the in vivo experiment. Treatments to induce lesions and age of lesions are as follows: A, 21-day-old needle-induced (control) lesion; B, 21-day-old lesion induced via a laser at 75 J; C, 21-day-old lesion induced via a laser at 75 J; D, 14-day-old lesion induced via a laser at 125 J; and E, 21-day-old lesion induced via a laser at 125 J. The lesions were categorized as a track lesion (A, D), track and core lesion (B, E), and double track lesion (C). In the middle column, A1 represents the cross-sectional area of the SDFT and A2 represents the cross-sectional area of the lesion, respectively.

Citation: American Journal of Veterinary Research 73, 9; 10.2460/ajvr.73.9.1435

MRI

Tendon lesions were observed at all proximal and distal locations, except for 1 control lesion for which only peritendinous changes were observed. The large control lesions at 4 cm DACB in 1 of the preliminary horses were not assessed with MRI. Of the 19 lesions identified, 10 (7 laser lesions and 3 control lesions) were classified as track lesions (Figure 4), 8 as track and core lesions (all laser lesions), and 1 as a double-track lesion (laser lesion). Among the 8 track and core lesions, 5 had a track wider than the core, 2 had a core wider than the track, and 1 had a track of similar width to that of the core. All lesions were moderately hyperintense on T1-weighted images and moderately to markedly hyperintense on T2-weighted and STIR images. Six of 8 track and core lesions had a markedly hyperintense core and a moderately hyperintense track on T2-weighted and STIR images; however, this was found for energy ranging from 25 to 125 J and lesion age ranging from 7 to 21 days.

Laser energy, lesion location, and lesion age did not affect lesion CSA, maximum lesion length and width, tendon CSA, or lesion percentage (mean ± SEM; control lesion, 7.3 ± 2.6%; laser-induced lesion, 13.8 ± 1.4%; Tables 1 and 2). Lesion volume was not affected by energy level; therefore, the cumulative mean ± SEM lesion volume for all diode laser lesions was 0.083 ± 0.012 cm3.

Correlations between imaging techniques and histologic evaluation

Tendon CSA, lesion CSA, and lesion percentage correlated well between ultrasonography and MRI (R2 > 0.84; P < 0.001; Table 1; Figure 4). However, histologic measurements of sagittal CSA measurements were only moderately correlated with transverse CSA measurements obtained via ultrasonography (R2 = 0.49; P = 0.02) and MRI (R2 = 0.54; P = 0.01). Similar results were obtained for ultrasonographic and MRI measurements of lesion length.

Gross pathological changes

After blunt dissection of the skin was completed, laser lesions were readily identified as reddened, edematous, and thickened subcutaneous and paratenon tissue (chronic-active inflammation). Control lesions had less pathological change in the peritendinous tissue. The point of needle entry was readily distinguished as a red defect in the epitenon in control and laser lesions and as a red-brown focal lesion on cut section following fixation.

Histologic evaluation

The histologic appearance of the laser lesions in the tendons varied depending on chronicity and energy level. On sagittal section, the laser lesions often had a box or tailed-box conformation with a central region of cavitating coagulation necrosis (Figure 5). The necrotic foci often contained small numbers of interstitial RBCs (microhemorrhage), infiltrating neutrophils, macrophages, reactive fibroblasts, and variable amounts of fibrin. Depending on chronicity, the central cavitations often contained variable amounts of granulation tissue to loose connective tissue along with evidence of neovascularization. By 21 days, control lesions consisted almost exclusively of granulation tissue, whereas laser-induced lesions had a centrally cavitated region that consisted of eosinophilic acellular debris, hemorrhage, and vestiges of poorly organized fibroplasia. The extent of fibroplasia in lesions became progressively more pronounced from 7 to 14 to 21 days. Laser-induced foci were flanked proximally and distally by regions of coagulation necrosis, whereas control lesions did not have flanking coagulation necrosis. As was identified during the cadaver portion of the study, the demarcation between the laser-induced lesion and the surrounding intact tissue was most readily identifiable with polarized light.

Figure 5—
Figure 5—

Photomicrographs of sagittal images obtained by use of bright-field (A and C) and polarized (B and D) microscopy of 21-day-old lesions in the SDFT of horses during the in vivo experiment. Lesions were induced with a needle (A and B) and laser at 87.5 J (C and D). The laser-induced lesion (C and D) has prominent eosinophilic coagulation of necrotic material (white arrows) flanking a central rectangular region of granulation tissue (asterisk). Flanking coagulation was not identified in the lesion induced by insertion of the needle alone (A and B). H&E stain; bar = 1 mm.

Citation: American Journal of Veterinary Research 73, 9; 10.2460/ajvr.73.9.1435

Lesion location and lesion age did not affect CSA measurements of histologic specimens (Figure 2). In the in vivo experiment, significant differences in mean ± SEM lesion size were detected between needle-induced lesions (3.2 ± 1 mm2) and those induced with 87.5 J (6.1 ± 1.3 mm2; P < 0.05) or 125 J (6.6 ± 1.4 mm2; P = 0.01). However, no significant differences in lesion size were detected among the laser energy levels.

Discussion

Analysis of results of the present study suggested that application of the diode laser technique induced tendinitis in the SDFT with small, focal, and variably shaped lesions and therefore was not suitable for testing intralesional treatments. The linear relationship found between lesion area determined histologically and laser energy in the cadaver experiment could not be emulated in the in vivo experiment, and only the lesions induced with the diode laser at 125 J were larger than the needle-control lesions, as measured via ultrasonography and histologic evaluation. Limitations to the present study were predominantly related to the small number of experimental units; however, the authors considered it unethical to continue this terminal experiment because of our findings of lesion variability and limited lesion size.

The effect of laser energy on tissue is influenced by the wavelength of the laser in conjunction with the optical properties of the tissue, beam intensity, delivery time, and mechanical tension on the tissue.28,29 The delivery time and energy used in the present study are consistent with the induction of a photothermal process in irradiated tissue.30 The photon energy is absorbed by water and hemoglobin in the tendon tissue and converted to thermal energy that diffuses from the site of irradiation into surrounding tissue, which results in tissue vaporization and adjacent coagulation necrosis attributable to protein denaturation.

Clinical tendinopathy of the SDFT is believed to be secondary to degenerative changes that are enhanced with age and exercise.31–35 Although clinical disease is not typically characterized by coagulation necrosis, it was hypothesized that the diode laser–induced coagulation necrosis may mimic the inflammatory process of naturally developing tendinopathy, whereby damaged tendon must be removed by proliferative and remodeling healing responses. Similarities of the laser-induced lesions with naturally developing tendinopathy include visible reddened areas reported in natural degeneration,36 areas of increased cellularity,36 and more acellular areas, as described for aged SDFTs.24 The histologic features of naturally developing tendinopathy lesions in horses are dependent on chronicity. Nevertheless, fibroplasia, neovascularization, granulation tissue, foci of microhemorrhage, minimal inflammation, and cavitated regions of fibrin deposition have been identified in naturally developing and laser-induced tendon lesions.

The in vivo experiment was designed on the basis of results for the cadaver experiment. Cadaver lesions at the highest energy level (100 J) occupied approximately 20% of the mean forelimb SDFT CSA. This was believed to be a safe starting point for the in vivo experiment, considering the potential for lesion enlargement as a result of expected latent necrosis over time as has been reported for a surgical technique in which lesions continued to enlarge until approximately 35 days after induction.15

Differences in lesion CSA between the cadaver and in vivo experiments likely were related to differences in tissue properties, namely hydration, vascular perfusion, and mechanical stress. When tissue hydration and perfusion are reduced, photons are allowed to disburse through a larger volume of tissue before being absorbed by water or hemoglobin, respectively, which lowers the volumetric energy density and reduces ablation efficiency but increases the zone of collagen denaturation. The concentration of absorbers in tissue will influence the volume of tissue within which photons are absorbed and heat is generated. When a high concentration of absorbers is present, photons are confined to a small volume of tissue, which results in a more rapid increase in temperature per unit volume than when a low concentration of absorbers is present. Because both hemoglobin and water are absorbers of photons at a wavelength of 980 nm, differences in both tissue perfusion and hydration may influence similar tissues and result in different outcomes for exposure to the same laser settings. Additionally, when in vivo laser exposures exceed 3 seconds in duration, vascular circulation may dissipate some of the thermal energy, which will slightly decrease temperature per unit volume and thereby decrease ablation efficiency.37

When mechanical tension across a field of laser irradiation is increased, as in standing horses, the tension would be expected to improve ablation efficiency through separation of the weakened tissues. Each of these factors would seem to suggest that the application to tissues of live horses should have provided larger lesions than the application to cadaver tissues; however, the opposite results were observed. Thus, we speculate that the vascular circulation of the in vivo system provided some cooling effect on the tissue and reduced ablation efficiency or, more likely, that bleeding into the space around the fiber tip provided an absorptive shield of hemoglobin around the tip and substantially reduced irradiation of the tissue. Also, collagen shortening secondary to laser administration may have been greater in the unloaded cadaver tendon samples, which further increased the volume of tendon with thermal damage. The box or tailed-box shape seen in the in vivo experiment provided evidence of fascicle retraction following needle transection, whereas a spherical lesion was identified in the cadaveric experiment.

Compared with the surgically induced arthroscopic burr technique,11,16,20,23 the laser technique was minimally invasive and could be performed in standing horses. The arthroscopic burr technique yielded a mean ± SEM lesion volume of 1.86 ± 0.26 cm3 because it resulted in much longer lesions.20 It is possible that a greater diode laser energy setting (> 125 J) may result in a larger lesion, but it is also likely to cause charring, which may cause undesirable additional inflammation.

The arthroscopic burr technique is a dynamic technique. A dynamic laser technique could be developed for introduction of the laser fiber parallel to the tendon fibers to create a longer lesion, although the lesion diameter is likely to be the same as for the static technique. A static laser technique was used in the present study to reduce potential lesion size variability attributable to laser fiber movement. The mean lesion percentage measured via ultrasonography and MRI in the present study at day 21 was approximately 13% and thus similar to results reported for the surgical technique (mean ± SEM, 16.7 ± 4.2%; range, 13% to 24%) 4 weeks after lesion induction and initiation of exercise.15 Investigators in previous studies25,38 have reported that clinical outcome of tendon injuries appears to be more highly associated with lesion severity as opposed to treatment choice, which suggests that the technique used to experimentally induce tendinopathy may need to emulate the size of severe naturally developing lesions to be of value in assessing the merit of treatments. Severe naturally developing lesions are considered to occupy approximately 40% of the tendon CSA.39

Further evaluation of the laser-tendon tissue interaction is required to provide information for potential modification of the laser source and application for the purpose of experimental induction of lesions. A larger area of cavitation in the tendon may be achieved by selection of a laser system that produces a wavelength with a higher absorption coefficient of water (eg, holmium yttrium-aluminum-garnet, erbium-doped yttrium-aluminum-garnet, or CO2 lasers) and by means of a delivery mode that provides enough energy to reach the vaporization threshold of water (heat of vaporization of water [2,500 J/cm3] divided by the absorption coefficient of water for the wavelength) within a single pulse that is equal to or shorter than the thermal relaxation time of the tissue (time necessary for energy to diffuse the optical penetration depth in the irradiated tissue) as determined for that wavelength. For the 980-nm wavelength, in which the absorption coefficient of water is 0.43/cm, the vaporization threshold would require an energy density of 5,800 J/cm2.40 In the present study, the energy density of a single pulse at the highest setting used was 293 J/cm2 (125 J/10 s = 12.5 J/s at 15 Hz = 0.83 J/pulse. Fiber diameter was 600 μm, so spot area was π times the square of the radius = 2.83 × 10−3; therefore, the energy density of a single pulse was 0.83 J/2.83 × 10−3 cm2 = 293 J/cm2).

Control lesions in the SDFT created by ultrasonographically guided placement of an 18-gauge needle were examined via ultrasonography, MRI, and histologic evaluation and ranged in size from 8% to 24% of the tendon CSA. The appearance of 2 tracks in 1 lesion during ultrasonography and MRI stressed the importance of using the smallest needle appropriate for intratendinous medications in standing horses and limiting needle redirection.

Interestingly, histologic examination, often referred to as the criterion-referenced standard for lesion verification, failed to identify an association between energy level and lesion size for the in vivo experiment. Histologic evaluation provided valuable information on the pathogenesis with regard to the qualitative effects of laser energy on the tissue matrix and cellular response, but the present study revealed the variation observed in data obtained from 2-D planar tissue sections. Histologic examination of lesions was limited by a finite number of tissue planes, which most likely failed to represent the entire lesion. Additionally, fixation in formalin and paraffin-associated tissue contracture may have confounded the results.

Ultrasonography and MRI enabled examination of lesions in 3-D planes, including the lesion track, and were strongly correlated with measurements of tendon and lesion size. Ultrasonography was useful to monitor lesion progression over the 21-day study period, whereby lesion age did not influence lesion size. These results are consistent with an ex vivo method in which mechanically induced lesions did not increase after cyclic loading, compared with the progression for collagenase-induced lesions.16

Considerable variation in the tendon response to lesion induction has been described.15 Two of the horses in another study32 did not have appreciable lesions after they were anesthetized and lesions were surgically induced with a 3.5-mm synovial resector. Furthermore, an exercise regimen in that study15 increased consistency of lesion appearance and enhanced lesion size by approximately 50%, which is consistent with the reported load enhancement in tendons of exercised horses.41 However, horse age and fitness limitations prevented exercise on a treadmill at a rapid gait in the present study. Also, it was believed that exercise may have introduced additional variation into the tendinopathy technique used in the present study.

In the present study, we evaluated a novel technique for induction of tendinopathy in the SDFT of horses. However, this technique resulted in small and variably shaped lesions; therefore, laser techniques require modification and evaluation before they can be used to induce tendinopathy in horses.

ABBREVIATIONS

CSA

Cross-sectional area

DACB

Distal to the accessory carpal bone

SDFT

Superficial digital flexor tendon

STIR

Short T1 inversion recovery

a.

Zedler S, Schaer T, Ebling A, et al. Evaluation of a novel model of equine superficial digital flexor tendonitis in regenerative medicine research (abstr). Vet Surg 2008;37:E34.

b.

SMA 600-μm bare fiber assay, flat tip, Biolitec, East Longmeadow, Mass.

c.

DiodeVET-25, B&W Tek Inc, Newark, Del.

d.

Sharpie pen, Sanford Corp, Oak Brook, Ill.

e.

Olympus BX41 microscope, Olympus Corp, Center Valley, Pa.

f.

Olympus DP25 camera, Olympus Corp, Center Valley, Pa.

g.

Olympus DP2-BSW software, Olympus Corp, Center Valley, Pa.

h.

Euthasol, Virbac Animal Health, Fort Worth, Tex.

i.

Dormosedan, Pfizer Animal Health, Exton, Pa.

j.

Torbugesic, Pfizer Animal Health, Exton, Pa.

k.

Carbocaine-V, Pfizer Animal Health, Exton, Pa.

l.

Biosound Technos, Biosound Esaote, Indianapolis, Ind.

m.

GE Signa 1.5-T horizon HiSpeed LX 9.1 MR system, GE Healthcare Inc, Waukesha, Wis.

n.

GE Healthcare South Asia Ltd, Bangalore, India.

o.

OsiriX, version 2.7, Open Source. Available at: www.homepage.mac.com/rossetantoine/osirix/Index2.html. Accessed Mar 3, 2009.

p.

30-inch Apple Cinema HD display, Apple, Cupertino, Calif.

q.

Proc Mixed, SAS, version 9.1.2, SAS Institute Inc, Cary, NC.

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    • Export Citation
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    van Schie HTBakker EMCherdchutham W, et al. Monitoring of the repair process of surgically created lesions in equine superficial digital flexor tendons by use of computerized ultrasonography. Am J Vet Res 2009; 70:3748.

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    • Search Google Scholar
    • Export Citation
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    Watkins JPAuer JAMorgan SJ, et al. Healing of surgically created defects in the equine superficial digital flexor tendon: effects of pulsing electromagnetic field therapy on collagen-type transformation and tissue morphologic reorganization. Am J Vet Res 1985; 46:20972103.

    • Search Google Scholar
    • Export Citation
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    Schramme MKerekes ZHunter S, et al. Mr imaging features of surgically induced core lesions in the equine superficial digital flexor tendon. Vet Radiol Ultrasound 2010; 51:280287.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Guest DJSmith MRAllen WR. Equine embryonic stem-like cells and mesenchymal stromal cells have different survival rates and migration patterns following their injection into damaged superficial digital flexor tendon. Equine Vet J 2010; 42:636642.

    • Crossref
    • Search Google Scholar
    • Export Citation
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    Guest DJSmith MRAllen WR. Monitoring the fate of autologous and allogeneic mesenchymal progenitor cells injected into the superficial digital flexor tendon of horses: preliminary study. Equine Vet J 2008; 40:178181.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23.

    Bosch Gvan Schie HTde Groot MW, et al. Effects of platelet-rich plasma on the quality of repair of mechanically induced core lesions in equine superficial digital flexor tendons: a placebo-controlled experimental study. J Orthop Res 2010; 28:211217.

    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
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Contributor Notes

Supported by the Alamo Pintado Medical Foundation.

The authors thank Dr. Neil Willits for assistance with the statistical analysis, John Doval for assistance with the figures, and Dr. G. M. Peavy for technical assistance with the diode laser.

Address correspondence to Dr. Vidal (mavidal@ucdavis.edu).
  • Figure 1—

    Photomicrographs obtained by use of bright-field (A) and polarized light (B) microscopy of transverse sections of the SDFT from an equine cadaver after induction of lesions with a diode laser at 75 J. In panel A, the central spherical cavitated core is surrounded by a variably thick hypereosinophilic rim of coagulated tissue. In panel B, the demarcation between the region of coagulated tissue and unaffected tendon is most readily identifiable by use of polarized light. H&E stain; bar = 1 mm.

  • Figure 2—

    Arithmetic mean ± SEM size of lesions in the SDFT of 4 equine cadavers (white circles) and 5 live horses (black triangles) after laser induction of lesions. Linear relationships for energy settings and histologic size of lesion area were significantly (P < 0.001) different between equine cadavers (y = [0.1508•x] − 1.366; R2 = 0.8) and live horses (y = [0.0264•x] + 3.301; R2 = 0.2). Cadaver histologic lesion areas differed significantly (P < 0.001) among energy levels. In the live horses, there were significant (P < 0.05) differences in lesion size between needle-induced (control) lesions (0 J) and lesions induced with 87.5 or 125 J.

  • Figure 3—

    Photograph of the forelimb of a preliminary horse (A) and the corresponding sagittal image for T1-weighted fast spin-echo MRI (B) at day 21 after laser induction of lesions in the SDFT at proximal (25 J; arrow) and distal (75 J; arrowhead) locations. Peritendinous edema is visible grossly in the photograph and on the corresponding sagittal image at each lesion site. Notice the characteristic box-shaped lesion on the sagittal image.

  • Figure 4—

    Transverse T1-weighted fast spin-echo MRI (left column) and corresponding transverse (middle column) and sagittal (right column) ultrasonographic images of lesions in an SDFT of each of 5 horses during the in vivo experiment. Treatments to induce lesions and age of lesions are as follows: A, 21-day-old needle-induced (control) lesion; B, 21-day-old lesion induced via a laser at 75 J; C, 21-day-old lesion induced via a laser at 75 J; D, 14-day-old lesion induced via a laser at 125 J; and E, 21-day-old lesion induced via a laser at 125 J. The lesions were categorized as a track lesion (A, D), track and core lesion (B, E), and double track lesion (C). In the middle column, A1 represents the cross-sectional area of the SDFT and A2 represents the cross-sectional area of the lesion, respectively.

  • Figure 5—

    Photomicrographs of sagittal images obtained by use of bright-field (A and C) and polarized (B and D) microscopy of 21-day-old lesions in the SDFT of horses during the in vivo experiment. Lesions were induced with a needle (A and B) and laser at 87.5 J (C and D). The laser-induced lesion (C and D) has prominent eosinophilic coagulation of necrotic material (white arrows) flanking a central rectangular region of granulation tissue (asterisk). Flanking coagulation was not identified in the lesion induced by insertion of the needle alone (A and B). H&E stain; bar = 1 mm.

  • 1.

    Patterson-Kane JCFirth EC. The pathobiology of exercise-induced superficial digital flexor tendon injury in Thoroughbred racehorses. Vet J 2009; 181:7989.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Smith RK. Mesenchymal stem cell therapy for equine tendinopathy. Disabil Rehabil 2008; 30:17521758.

  • 3.

    Dyson SJ. Medical management of superficial digital flexor tendonitis: a comparative study in 219 horses (1992–2000). Equine Vet J 2004; 36:415419.

    • Search Google Scholar
    • Export Citation
  • 4.

    O'Meara BBladon BParkin TD, et al. An investigation of the relationship between race performance and superficial digital flexor tendonitis in the Thoroughbred racehorse. Equine Vet J 2010; 42:322326.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Schnabel LVLynch MEvan der Meulen MC, et al. Mesenchymal stem cells and insulin-like growth factor-I gene-enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons. J Orthop Res 2009; 27:13921398.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    Nixon AJDahlgren LAHaupt JL, et al. Effect of adipose-derived nucleated cell fractions on tendon repair in horses with collagenase-induced tendinitis. Am J Vet Res 2008; 69:928937.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Dahlgren LAMohammed HONixon AJ. Expression of insulin-like growth factor binding proteins in healing tendon lesions. J Orthop Res 2006; 24:183192.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Dahlgren LAvan der Meulen MCBertram JE, et al. Insulin-like growth factor-I improves cellular and molecular aspects of healing in a collagenase-induced model of flexor tendinitis. J Orthop Res 2002; 20:910919.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Donnelly BPNixon AJHaupt JL, et al. Nucleotide structure of equine platelet-derived growth factor-A and -B and expression in horses with induced acute tendinitis (Erratum published in Am J Vet Res 2006;67:1627). Am J Vet Res 2006; 67:12181225.

    • Search Google Scholar
    • Export Citation
  • 10.

    McCarrel TFortier L. Temporal growth factor release from platelet-rich plasma, trehalose lyophilized platelets, and bone marrow aspirate and their effect on tendon and ligament gene expression. J Orthop Res 2009; 27:10331042.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Bosch GRene van Weeren PBarneveld A, et al. Computerised analysis of standardised ultrasonographic images to monitor the repair of surgically created core lesions in equine superficial digital flexor tendons following treatment with intratendinous platelet rich plasma or placebo. Vet J 2011; 187:9298.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Mitchell RD. Treatment of tendon and ligament injuries with UBM powder, in Proceedings. 14th Am Coll Vet Surg Symp 2004;190193.

  • 13.

    Moraes JRFacco GGMoraes FR, et al. Effects of glycosaminoglycan polysulphate on the organisation of collagen fibres in experimentally induced tendonitis in horses. Vet Rec 2009; 165:203205.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Wallis TWBaxter GMWerpy NM, et al. Acellular urinary bladder matrix in a collagenase model of superficial digital flexor tendonitis in horses. J Equine Vet Sci 2010; 30:365370.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Schramme MHunter SCampbell N, et al. A surgical tendonitis model in horses: technique, clinical, ultrasonographic and histological characterisation. Vet Comp Orthop Traumatol 2010; 23:231239.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16.

    Bosch GLameris MCvan den Belt AJ, et al. The propagation of induced tendon lesions in the equine superficial digital flexor tendon: an ex vivo study. Equine Vet J 2010; 42:407411.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Williams IFMcCullagh KGGoodship AE, et al. Studies on the pathogenesis of equine tendonitis following collagenase injury. Res Vet Sci 1984; 36:326338.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    van Schie HTBakker EMCherdchutham W, et al. Monitoring of the repair process of surgically created lesions in equine superficial digital flexor tendons by use of computerized ultrasonography. Am J Vet Res 2009; 70:3748.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Watkins JPAuer JAMorgan SJ, et al. Healing of surgically created defects in the equine superficial digital flexor tendon: effects of pulsing electromagnetic field therapy on collagen-type transformation and tissue morphologic reorganization. Am J Vet Res 1985; 46:20972103.

    • Search Google Scholar
    • Export Citation
  • 20.

    Schramme MKerekes ZHunter S, et al. Mr imaging features of surgically induced core lesions in the equine superficial digital flexor tendon. Vet Radiol Ultrasound 2010; 51:280287.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Guest DJSmith MRAllen WR. Equine embryonic stem-like cells and mesenchymal stromal cells have different survival rates and migration patterns following their injection into damaged superficial digital flexor tendon. Equine Vet J 2010; 42:636642.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Guest DJSmith MRAllen WR. Monitoring the fate of autologous and allogeneic mesenchymal progenitor cells injected into the superficial digital flexor tendon of horses: preliminary study. Equine Vet J 2008; 40:178181.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23.

    Bosch Gvan Schie HTde Groot MW, et al. Effects of platelet-rich plasma on the quality of repair of mechanically induced core lesions in equine superficial digital flexor tendons: a placebo-controlled experimental study. J Orthop Res 2010; 28:211217.

    • Search Google Scholar
    • Export Citation
  • 24.

    Webbon PM. A histological study of macroscopically normal equine digital flexor tendons. Equine Vet J 1978; 10:253259.

  • 25.

    Marr CMLove SBoyd JS, et al. Factors affecting the clinical outcome of injuries to the superficial digital flexor tendon in National Hunt and point-to-point racehorses. Vet Rec 1993; 132:476479.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Berger NEeg PH. Fundamentals of laser-tissue interactions. In: Berger NEeg PH, eds. Veterinary laser surgery: a practical guide. Ames, Iowa: Blackwell Publishing, 2006:2942.

    • Search Google Scholar
    • Export Citation
  • 27.

    Kester WO. Definition and classification of lameness. In: Guide for veterinary services and judging of equestrian events. 4th ed. Lexington, Ky: American Association of Equine Practitioners, 1991;19.

    • Search Google Scholar
    • Export Citation
  • 28.

    Peavy GM. Lasers and laser-tissue interactions. Vet Clin North Am Small Anim Pract 2002; 32:517534.

  • 29.

    Peavy GMWilder-Smith P. Laser surgery. In: Verstraete FJMLommer MJ, eds. Oral and maxillofacial surgery in dogs and cats. Philadelphia: Elsevier, 2011:7988.

    • Search Google Scholar
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