Achilles tendons are commonly the site of surgical repair, especially following rupture (3,6), and often the site of corrective surgery in patients with neurological dysfunction. For example, some of the most commonly performed surgical procedures on patients following cerebral vascular accidents are tendon lengthening, release, and/or transfer (23). Standard treatment following surgical re-approximation of the tendon includes immobilization of the lower extremity with the tendon in a shortened position. In previous studies using a rabbit Achilles tendon model of tenotomy and repair, we showed that early removal of the immobilization cast promotes faster recovery from atrophy and speeds the rate of healing of the repaired tendon (11,14,16,19,29). Removal of plaster of Paris casts 5 d after surgical repair of rabbit Achilles tendons augmented the biomechanical strength of the tendons without re-rupture (16). Although a fair amount of muscle atrophy occurred in the soleus and plantaris muscles, recovery from atrophy began in the weight-bearing tendons days after the casts were removed, but recovery was delayed in the muscles of continuously casted lower extremities (29). These findings indicate that minimal immobilization and early functional loading accelerates tendon healing and minimizes muscle atrophy (38).
The benefits of early function must be weighed against the risk of re-rupturing the tendon. In our animal model, weight bearing on the repaired extremity within the first 5 d after surgical repair carried risk of re-rupturing the tendon. However, electrical stimulation of muscles in the casted limb provided mechanical stress that mimicked weight bearing with less risk of re-rupture (6,44). Thus, a safe amount of load could be imposed on repaired tendons during the initial days after repair. As the tissue healed, functional loads such as weight bearing could be applied safely to the repaired tendon (16). Electrical stimulation has been tested extensively in animal models and shown to improve the morphological profile and tension production in ankle muscles (6,34). In this report, we tested the use of electrical stimulation during the early phase of healing (1-5 d) following tenotomy to determine whether early electrical stimulation could produce positive effects without re-rupture.
Functional loading is not the only stimulus that has been shown to promote tendon healing. A low intensity laser beam, referred to as laser photostimulation, has been shown to enhance tissue healing (2,15,17,18,22,25,30,31,40). Although the precise mechanism(s) of action remains unclear (44), in vitro studies suggest that photostimulation promotes nucleic acid synthesis and cell division in cultures of human fibroblasts (31) and augments the pools of type I and type III procollagen mRNA in pig skin wounds (1). An increase in ATP production was found in rat liver mitochondria after photostimulation with a helium-neon (He-Ne) laser in vitro (37). These metabolic effects promote the healing strength of skin (25,26), and tendons (17).
Given our earlier results indicating that functional loading promotes tendon healing, and the positive effects of laser photostimulation on repaired tendons and other connective tissues, we hypothesized that the healing of repaired tendons would be accelerated further when treated with a combination of laser photostimulation and early mechanical loading using electrical stimulation. Thus, the purpose of this study was to compare the biomechanical, biochemical, and ultrastructural effects of a combination of laser photostimulation and electrical stimulation on rabbit Achilles tendons that were allowed to bear weight starting on day 5. Specifically, we measured and compared the tendons' maximum load, stress, strain, Young's modulus of elasticity, energy absorption, total collagen, collagen cross-links, mRNA, and ultrastructural morphometry.
Experimental tenotomy. The tendon surgery and animal care protocol was reviewed and approved by the institutional animal care and use committee. A total of 54 male New Zealand rabbits, ages 10-12 wk, were housed in standard 30.5 × 71 × 51 cm rabbit cages, with the temperature maintained at 22°C, and fed rabbit chow and water ad libitum. On the day of surgery each rabbit was weighed, anesthetized with intra-muscular injection of 3 mg·kg−1 body weight Xylazene, and 35 mg·kg−1 body weight Ketamine. Subsequently, the skin overlying the right Achilles tendon was shaved, scrubbed, and anaesthetized locally with 2 mg·kg−1 body weight lidocaine.
After anesthesia the right Achilles tendon of each rabbit was tenotomized and repaired as detailed in previous reports (12,16). A longitudinal incision was made lateral to the visible outline of the tendon. By blunt dissection, the tendon was isolated from adjoining tissue and transected approximately 1.5 cm above its calcaneal attachment. The severed ends were then approximated and sutured. Following skin closure, the surgical limb was immobilized using a custom designed premolded polyurethane splint (41). To promote rapid recovery from anesthesia, an injection of 0.2 mg·kg−1 body weight Yohimbine was given. Subsequently the rabbits were kept warm in an oxygen chamber and observed until they regained consciousness.
Treatment. Rabbits were randomly assigned to two groups after surgery. The first group (designated MT for modality-treated) received 1.0 J·cm−2 He:Ne laser beam applied transcutaneously starting on day 1 and continuing for 14 d. The same group received mechanical stress with an interrupted galvanic current. The treatment protocol for application of the electrical stimulation was designed to induce maximal mechanical stress without re-rupture (44). Briefly, a calibrated "Z" tendon force transducer interfaced to a peak-hold meter was clamped to the repaired tendons. The triceps surae was electrically stimulated and the maximum force transmitted by the tendon without re-rupture was determined to be a current surged to yield a 3-s base to peak rise time and delivered at 120 pulses·s−1 at 40% duty cycle for 20 min. This stimulation protocol resulted in a maximal force across the tendon of 22 N (44). This protocol was used for all subsequent studies with the objective of mimicking the functional load of weight bearing during the early (days 1-5) postoperative period without re-rupture of the tendon. On the fifth postoperative day immobilization casts were removed from the surgical limbs of the treated and control groups of rabbits to permit functional loading via unrestricted weight-bearing activities.
Tendon excision. Two weeks after surgery, each rabbit was weighed, euthanized with an overdose of sodium pentabarbitol, and the surgical incision reopened. After separating the tendon from the surrounding tissue, sharp transverse cuts were made below the musculotendinous junction and above the calcaneal insertion of the tendon. For biochemical and molecular biology studies, the neotendon was excised manually under a dissecting microscope, bisected, snap frozen in liquid nitrogen, and stored at −70°C. For electron microscopy and computer morphometry, tendons were fixed in situ before excision, and only the site of the tenotomy and repair, the neotendon, was used as sample.
Total collagen content. Total collagen was determined by measuring the concentration of hydroxyproline in each tissue specimen using our well-established procedures (39). Briefly, the dried tissue specimen was homogenized in cold saline, hydrolyzed in alkali, and oxidized with chloramine-T. The chromophore was developed with the addition of Ehrlich's aldehyde, and the absorbance was measured at 550 nm. Concentrations of hydroxyproline in each tissue specimen were deduced from a standard calibration curve. The content of total collagen was calculated based on the fact that 14% of the total amino acids of collagen are hydroxyproline (33).
Measurement of collagen cross-links. The hydroxypyridinum cross-links in neotendon were determined using high performance liquid chromatography (HPLC; Shimadzu, Kyoto, Japan) with a binary gradient system control module managing a RF-10A spectrofluorometric detector, two pumps, and an auto injector as previously reported (20). Briefly, the C18 reverse phase column (Supelco Supelcosil, Bellefonte, PA; 25 cm × 4.6 mm with 5 mm LC-18 pore size) was equilibrated in 21% acetonitrile in water containing 0.01 M N-heptafluorobutyric acid. Aliquots of reconstituted sample were injected into the column using an automatic injector and the hydroxypyridinum cross-links were resolved using a linear gradient from 21% acetonitrile (containing 0.01 M N-heptafluorobutyric acid) to 25% acetonitrile in water. The concentration of hydroxypyridinium cross-links was measured by determining the area under the peak using pyridoxamine as a standard. Since pyridoxamine fluoresces three times greater than the hydroxypyridinum cross-links, the values obtained from the tendon samples were multiplied by three.
Assay of procollagen mRNA levels. RNA was isolated using a modification of the previously published procedure (9). Briefly, frozen neotendon samples (∼100 mg) were taken from storage and mounted on the specimen block of a cryostat with TBS Tissue Freeze Medium (Triangle Biomedical Sciences, Durham, NC). Sections (30 μm) were taken of the tendon and homogenized in 1.0 mL GT buffer (4.8 M guanidine thiocyanate, 25 mM sodium citrate, 1% sarcosyl). The following were added sequentially to the homogenate: 100 μL 2 M sodium acetate, pH 4.0, 1 mL water-saturated phenol, and 200 μL chloroform/isoamyl alcohol (49:1). The nucleic acids were isopropanol precipitated, centrifuged, resuspended in GT buffer, ethanol precipitated, washed, and resuspended in DEPC-treated dH2O. Densitometric absorbance readings at 260 and 280 nm were used to determine purity and concentration.
Formaldehyde gel electrophoresis was performed as previously reported (10). Briefly, total RNA (20 μg) was electrophoresed through a 1% SeaKem LE agarose gel at 5 V·cm−1. The RNA was blotted to an MSI Magnagraph (Micron Separations, Inc., Westborough, MA) nylon membrane as described (8) and dried in an 80°C oven for 30 min. Slot blots were performed using BioRad (Hercules, CA) BioDot SF blotting system. E. coli carrying the following plasmids were kindly donated by Jeanne C. Myers: HF677 (10), HF32 (32), and E6 (28). Plasmid pHcGAP (46) was obtained from American Type Culture Collection (Rockville, MD) (ATCC # 57090). Plasmids HF677, HF32, E6, and pHcGAP carry partial sequences for the following genes: procollagen alpha 1, type (I); procollagen alpha 2, type (I); procollagen alpha 1, type (III); and glyceraldehyde-3-phosphate dehydrogenase, respectively. Using a Rad-Free nonradioactive labeling system (Schleicher & Schuell Inc., Keene, NH), plasmids were linearized, labeled, hybridized to the RNA blots, and detected by chemiluminescence.
Transmission electron microscopy. The tendons of five rabbits in each group were excised for ultrastructural and morphometric analyses (35,36). The tendons were fixed in situ, and representative specimens of neotendons were taken and fixed for 2 h in 2.5% glutaraldehyde (pH 7.4), buffer washed, and postfixed for another 2 h in 1% aqueous solution of osmium tetroxide (pH 7.4). Each specimen was washed with dH20 and dehydrated in graded alcohol before final dehydration in propylene oxide. After gradual infiltration with a mixture of propylene oxide and resin, each specimen was embedded in EMBED 812 resin and kept in an oven at 60°C for 65 h. Each resin-embedded specimen was trimmed and sectioned transversely at 700-800 Å to obtain representative silver/silver grey sections that were mounted on a grid. Finally, grid mounted sections were stained with both uranyl acetate and lead citrate for 5 min each before they were studied under the electron microscope. From each grid electron micrographs of several fields of collagen fibrils were taken at a uniform magnification of 140,000 X, so that at least 2,500-4,000 fibrils per group were obtained. Our previous studies (13,14) suggest that this range of fibril count is necessary for reliable morphometric analyses.
Ultrastructural study and morphometric analysis. Before morphometric measurements, the ultrastructure of the two groups of tendons were compared using the following common criteria: a) presence or absence of fibrils, b) orientation and organization of fibrils, c) cell types present, and d) morphological appearance of 1) endoplasmic reticulum, 2) mitochondria, 3) lysosomes, 4) ribosomes, 5) Golgi complexes, 6) nuclear chromatin, and 7) nucleoli (13). The descriptive information was used to complement the morphometric data, derived by measuring the profile of the highly magnified collagen fibrils as detailed in previous reports (13,14).
Briefly, each electron micrograph depicting perfect cross-sectional profiles of the collagen fibrils was placed on a calibrated Jandel Scientific digitizing tablet (Sausalito, CA) interfaced to a desk top computer loaded with SIGMA-Scan (Corte Madera CA), a software developed for morphometric measurements. An electronic pen was used to trace the visible outline of each collagen fibril. Simultaneously, the cross-sectional area of each fibril was computed and stored in the computer database.
Biomechanical analysis. The cross-sectional area of each tendon was measured with a caliper customized as described by An et al. (4). A serrated pneumatic action clamp was used to attach each tendon to the cross heads of a microprocessor controlled Instron materials testing system model 8511 (Instron Inc., Canton, MA) interfaced to a desktop computer. The gauge length of the measurements (distance between the clamps on the tendon) was 15 to 18 mm. Using a 500-N load cell each clamped tendon was pulled to rupture at a cross head speed of 250 mm·min−1. In preliminary experiments we found no significant difference in the biomechanical results when the tendons were pulled at either 250 mm·min−1 or the 500 mm·min−1 used in our previous studies (N = 16). The biomechanical parameters of each tendon, including strength, stress, strain, Young's modulus of elasticity, and energy absorbed were computed from the load/deformation curve automatically and stored for subsequent statistical analysis.
Data analyses. Multivariate analyses of variance (MANOVA) were used to compare the biomechanical and biochemical characteristics of the two groups of tendons. Because the cross-sectional area of collagen fibrils is not normally distributed, the Wilcoxon test was used to compare the cross-sectional area of the two groups of tendons.
The surgical procedure was tolerated well by both groups of rabbits with minimal weight loss ranging from 0 to 0.24 kg per animal. No statistically significant differences were found in the body weight changes per group. Similarly, the mean cross-sectional area of the tenotomized site of the modality-treated tendons (61.5 129 ± 9.1 mm (2) did not differ significantly from the 66.2 ± 5.5 mm2 value obtained from control tendons subjected to weight bearing alone (P > 0.05). There was no re-rupturing of tendons following surgery in either group.
Biochemical analysis. Total collagen production increased with daily modality treatments, 380 ± 41 μg·mg−1 dry tissue weight compared with 288 ± 27 μg·mg−1 dry tissue weight for controls (N = 9/group; P < 0.003; Fig. 1). The number of mature cross-links in specimens obtained from the same tendons used for the collagen assay decreased with modality treatment (MT) from 323 ± 20 pM·mg−1 dry tissue in controls to 290 ± 29 pM·mg−1 dry tissue (Fig. 1, N = 9/group; P > 0.05). When the cross-links were normalized per mole of collagen, it became apparent that there were significantly fewer mature cross-links per collagen molecule in the modality treated tendons (2.60 ± 0.02 pM·mole−1 collagen) than in controls (4.02 ± 0.04 pM·mole−1 collagen, P < 0.01). This finding is consistent with work from other labs showing that type III collagen is produced first during tendon healing. Type III collagen lacks disulfide cross-links and has fewer mature cross-links than type I collagen. To verify that type III collagen had increased by the fifteenth day after injury, we measured the procollagen mRNA levels using collagen-specific probes. Although we failed to measure a difference in type I collagen levels in the two groups (MT vs control), there was an increase in the type III collagen levels in the tendons treated with modalities.
Ultrastructure and morphometry. Electron microscopy of specimens obtained from each group (N = 6/group) revealed no obvious differences in the ultrastructural appearance of both groups of tendons. There was an abundance of collagen fibrils and a correspondingly large number of fibroblasts that were characterized by well-developed euchromatic nuclei, massive cytoplasm with an extensive array of rough endoplasmic reticulum, a few stacks of Golgi apparatus, numerous ribosomes, vesicles, and other transitional elements (Figs. 2A and B). Viewed in cross section, the collagen fibrils of both groups of tendons were aligned longitudinally along the long axis of the tendon and aggregated into clusters reminiscent of collagen bundles (Figs. 2C and D). The distribution profile of the cross-sectional area of each collagen fibril was similar for both control and treated tendons (Fig. 2). This profile represents the analysis of approximately 5000 individual fibrils per group (MT = 5115, control = 4984). The mean value for the crosssectional area of each fibril was 1844 ± 490 nm2 for samples from the MT group and 1851 ± 493 nm2 for controls, indicating that the increase in type III collagen in the MT group had not changed the structure of the collagen fibrils.
Biomechanical analysis. When pulled to rupture all tendons failed at the site of the tenotomy and repair, indicating that this site was the weakest portion of the tendon. Modality treatment resulted in modest, statistically insignificant increases in the biomechanical characteristics of the tendons with 15 d of healing. The results are summarized in Table 1, illustrating small changes in the maximum load. Stress measurements were calculated based on the load values divided by the cross-sectional area of the tendon, thus providing a measurement of the tensile strength of the unit fibers (7). Maximum stress increased 30% with modality treatment, but this change was not statistically significant. Strain values were calculated as the percent change in the length of the tissue during the pull to rupture. This percent change in length of the tendon is based on the displacement of the whole tendon, not just the neotendon. Maximum strain was not statistically different with modality treatment. Young's modulus of elasticity increased 33% with modality treatment; however, the change was not statistically significant. The energy absorbed by the tendons did not differ between groups. In summary, the biomechanical data revealed little or no change with treatment.
In the present investigation we examined the therapeutic effects of laser photostimulation in combination with early electrical stimulation on the tissue repair process of surgically tenotomized rabbit Achilles tendons. Our previous studies concerning the effects of low-intensity laser photostimulation on regenerating Achilles tendons showed an increase in tensile strength, Young's modulus of elasticity, and energy absorption capacity of the photostimulated tendons (17). Similar results have been observed in tendons following treatment with either electrical stimulation (21) or functional loading and physical activity (16,43). Thus, it was anticipated that the combination of laser photostimulation and early mechanical stress through electrical stimulation would have greater beneficial effects, i.e., quicken the repair process of tenotomized tendons. However, our findings indicate that a combination of these modalities produced marginal effects on the biomechanical characteristics of the regenerating tendons (Table 1). Although there was a trend toward increased values with treatment in several of the parameters measured (e.g. maximum load, maximum stress, maximum strain, Young's modulus of elasticity), no dramatic changes were noted.
The trend toward enhanced healing in modality-treated tendons was manifested more in the biochemical results. The outcome of the biochemical analysis indicated that collagen production was appreciably enhanced by a combination of laser phototherapy and early electrical stimulation. However, trifunctional hydroxypyridinium cross-links of collagen in the modality-treated group were decreased, based on dry weight of the tendon samples. Relative to the molar amounts of collagen, the quantities of mature cross-links decreased significantly in the modality-treated group. This finding coincided with an increase in type III procollagen mRNA. Thus, modality treatment appears to have enhanced tendon healing. It is possible that at 15 d after tendon repair modality treatment produces changes at the molecular level that are yet to develop into gross tissue changes such as increased tendon strength.
The failure to measure an increase in tendon strength following modality treatment was a surprise, since we have previously reported significant increases in this parameter with functional loading via weight bearing (100% increase in strength) (19) or with laser photostimulation (40% increase) (17). A comparison of any differences in our procedures between earlier investigations and this study reveals two possible explanations for the discrepancy. First, slight variations in animal health, nutrition, and age could explain the lack of beneficial results from the treatment in our present study. However, the animals were allowed food ad libitum as in previous studies. A notable difference is that the animals used in this study were 2-2.5 months younger than the animals used in previous studies. However, preliminary work investigating the biomechanical load capacity of nontenotomized tendons showed no difference in strength resulting from age differences within this range (results not shown).
A second explanation for the lack of change with modality treatment is that our earlier works, showing an improvement with treatment, were completed using plaster of Paris casts for immobilization (15,17). For this study we used functional polyurethane casts identical in design and appearance to the rigid plaster casts used in previous reports (41). Since both experimental and control groups for this study were immobilized in functional casts, it is possible that no improvement was noted because the casts increased healing optimally so that treatment with modalities had no further effect. While separate studies showed that functional casting is advantageous compared with rigid plaster of Paris immobilization, functional casting was not shown to be solely responsible for optimal healing (41). Our studies of single modality treatment with laser photostimulation or electrical stimulation showed greater magnitudes of improvement (for example increased maximum load, stress, and energy absorption) than the increases noted with functional casting alone (41).
It is possible that combining laser and electrical stimulation may have negated any beneficial effect of the individual modality alone. We have shown (13,15,16,18) that laser photostimulation applied alone to healing tendons improves biomechanical properties; others have shown that electrical stimulation improves the stress, Young's modulus of elasticity, and strain in healing skin (24). Furthermore, biochemical changes have been reported with the use of these individual modalities. For example, electrical stimulation (both AC and DC) caused significant increases in the collagen content around the incision line of skin wounds in rats (5). Few studies have examined the effect of combined noninvasive modalities applied to healing tissue. It is important to stress that in our protocol, electrical stimulation and laser photostimulation both were applied daily during the early healing phase. Since the exact molecular changes involved in cellular signaling initiated by laser photostimulation and electrical stimulation have not been identified, it is possible that the stimulatory effects of one could be offset by the effects of a second modality applied simultaneously. Further animal studies are underway to explore the interactive effects of these modalities during tendon healing.
The clinically important finding of the results summarized here is that combining daily laser therapy and electrical stimulation did not enhance the healing of ruptured tendons, in contrast to earlier publications when each modality was applied separately. It is possible that the therapeutic modalities tested here (electrical stimulation and laser photostimulation) work best individually and may actually dampen the beneficial effects when combined. The clinically important finding of the results is that combination therapies should be used cautiously in human cases of Achilles tendon repair.
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