The biomechanical integrity of bone in experimental diabetes.

Patients with diabetes mellitus incur a higher incidence of fractures compared to healthy individuals. This suggests that the structural integrity of the skeletal system may be compromised. To examine the biomechanical consequences of diabetes, we studied the structural integrity of the femur and tibia of rats with streptozotocin-induced diabetes. The induction of diabetes was confirmed by measuring blood glucose levels (>300 mg/dl). Seven-weeks following the establishment of diabetes, the animals were euthanized and the hind limbs removed. The femur and tibia of each hind limb were excised, and prepared for three point bending test on an Instron Materials Testing System. The results revealed a 37% decrease in maximum load (breaking strength) of the femur of diabetic rats when compared to controls. The diabetic femurs had 25% less deformation at maximum load compared to controls. Similarly, energy absorption capacity to yield point and toughness were reduced by 27 and 34%, respectively, in the diabetic femur. A 38% increase in the bending stiffness was observed in the femurs of diabetic rats. Similar results were obtained with the tibias of both groups. Measurement at the break point revealed that the bones of diabetic rats bore significantly less load, deformation and energy absorption capacity than controls. Overall, our findings warrant the conclusion that the diabetic state is associated with mechanical deterioration of bone, resulting in bones with inferior biomechanical integrity.

Laser photostimulation accelerates wound healing in diabetic rats.

In this study, we examined the hypothesis that laser photostimulation can facilitate healing of impaired wounds in experimental diabetes using a rat model. Diabetes was induced in male rats by streptozotocin injection and two 6 mm diameter circular wounds were created on either side of the spine. The left wound of each animal was treated with a 632.8 nm He:Ne laser at a dose of 1.0 J/cm2 for five days a week until the wounds closed (three weeks). Measurements of the biomechanical properties of the laser-treated wounds indicated there was a marginal increase in maximum load (16%), stress (16%), strain (27%), energy absorption (47%) and toughness (84%) compared to control wounds of diabetic rats. Biochemical assays revealed that the amount of total collagen was significantly increased in laser treated wounds (274 +/- 8.7 microg) over the control wounds (230 +/- 8.4 microg). Sequential extractions of collagen from healing wounds showed that laser treated wounds had significantly greater concentrations of neutral salt soluble (15%) and insoluble collagen (16%) than control wounds, suggesting accelerated collagen production in laser treated wounds. There was an appreciable decrease in pepsin soluble collagen (19%) in laser treated wounds over control wounds, indicating higher resistance to proteolytic digestion. In conclusion, the biomechanical and biochemical results collectively suggest that laser photostimulation promotes the tissue repair process by accelerating collagen production and promoting overall connective tissue stability in healing wounds of diabetic rats.

Matrix remodeling in healing rabbit Achilles tendon.

Biochemical, biomechanical and ultrastructural properties of the connective tissue matrix were investigated during the early remodeling phase of tissue repair in experimentally tenotomized and repaired rabbit Achilles tendons. Sterile surgical tenotomy was performed on the right Achilles tendons of 14 rabbits and allowed to heal for 15 days. The animals were euthanized and the Achilles tendons excised from both limbs. The left contralateral Achilles tendon of each rabbit was used as a control in the experiments. Prior to biochemical analysis, both intact and healing tendons were tested for their biomechanical integrity. The results revealed that the healing tendons had regained some of their physicochemical characteristics, but differed significantly from the intact left tendons. The healing tendons regained 48% tensile strength, 30% energy absorption, 20% tensile stress, and 14% Young's modulus of elasticity of intact tendons. In contrast, biochemical analysis showed that the healing tendons had 80% of the collagen and 60% of the collagen crosslinks (hydroxypyridinium) of normal tendons. Sequential extraction of collagen from the tissues yielded more soluble collagen in the healing tendons than intact tendons, suggesting either an increase in collagen synthesis and/or enhanced resorption of mature collagen in healing tendons compared to intact tendons. Electron microscopic studies revealed remarkable differences in the ultrastructure between intact and healing tendons. These observations could explain, in part, the connective tissue response to healing during the early phases of tissue remodeling.

Laser photostimulation of collagen production in healing rabbit Achilles tendons.

BACKGROUND AND OBJECTIVE: Low energy laser photostimulation at certain wavelengths can enhance tissue repair by releasing growth factors from fibroblasts and stimulate the healing process. This study was designed to evaluate the influence of laser photostimulation on collagen production in experimentally tenotomized and repaired rabbit Achilles tendons. STUDY DESIGN/MATERIALS AND METHODS: A total of 24 male New Zealand rabbits, ages 10-12 weeks, were used. Following tenotomy and repair, the surgical hind limbs of the rabbits were immobilized in customized polyurethane casts. The experimental animals were treated with a 632.8 nm He:Ne laser daily at 1.0 J cm(-2) for 14 days. Control animals were sham treated with the laser head. On the fifth day after repair, the casts were removed to allow the animals to bear weight on the lower extremity. The animals were euthanized on the 15th postoperative day, then, the Achilles tendons were excised, processed and analyzed. RESULTS: Biochemical analyses of the tendons revealed a 26% increase in collagen concentration with laser photostimulation indicating a more rapid healing process in treated tendons compared to controls. Sequential extractions of collagen from regenerating tissues revealed that the laser photostimulated tendons had 32% and 33% greater concentrations of neutral salt soluble collagen and insoluble collagen, respectively, than control tendons suggesting an accelerated production of collagen with laser photostimulation. A significant decrease (9%) in pepsin soluble collagen was observed in laser-treated tendons compared to controls. There were no statistically significant differences recorded in the concentrations of hydroxypyridinium crosslinks and acid soluble collagen between treated and control tendons. CONCLUSION: This study of laser photostimulation on tendon healing in rabbits suggests that such therapy facilitates collagen production in a manner that enhances tendon healing.

Biochemistry and biomechanics of healing tendon: Part I. Effects of rigid plaster casts and functional casts.

PURPOSE: Traditional treatment of surgically repaired Achilles tendons includes complete immobilization of the joint in rigid casts for 6 to 8 wk. We tested the use of functional polyurethane casts as an alternative to rigid plaster casts after experimental tenotomy and repair of the rabbit Achilles tendon. METHODS: After repair the limbs of 15 experimental rabbits were immobilized in a functional polyurethane cast for 15 d, while those of 14 controls were immobilized in traditional rigid plaster casts for the same period. RESULTS: Functional casting resulted in a 60% increase in total collagen in the neotendon compared with that in rigid casting (P < 0.05). Mature collagen cross-links declined 8% in the tendons with functional casts. The biomechanical parameters of the tendons changed with functional casting, showing a 20% increase in maximum load and 21% increase in maximum stress. CONCLUSIONS: These changes were noted without any cases of tendon re-rupture in either type of cast. Thus, functional casting following surgery of Achilles tendons appears to improve healing without significant risks of re-rupture.

Biochemistry and biomechanics of healing tendon: Part II. Effects of combined laser therapy and electrical stimulation.

PURPOSE: In previous studies we demonstrated that early mechanical loading and laser photo-stimulation independently promoted tendon healing. Thus, we tested the hypothesis that a combination of laser phototherapy and mechanical load would further accelerate healing of experimentally tenotomized and repaired rabbit Achilles tendons. METHODS: Following surgical tenotomy and repair, the tendons of experimental and control rabbits were immobilized in polyurethane casts for 5 d. The repaired tendons of experimental rabbits received mechanical load via electrical stimulation-induced contraction of the triceps surae for 5 d. In addition, experimental tendons were treated with daily doses of 1 J.cm-2 low intensity helium-neon laser throughout the 14-d experimental period. RESULTS: The combination of laser photostimulation and mechanical load increased the maximal stress, maximal strain, and Young's modulus of elasticity of the tendons 30, 13, and 33%, respectively. However, MANOVA revealed no statistically significant differences in these biomechanical indices of repair of control and experimental tendons. Biochemical assays showed a 32% increase in collagen levels (P < 0.05) and an 11% decrease in mature cross-links in experimental tendons compared with that in controls (P > 0.05). Electron microscopy and computer morphometry revealed no significant differences in the morphometry of the collagen fibers and no visible differences in the ultrastructure of cellular and matrical components of control and experimental tendons. CONCLUSIONS: These findings indicate that the combination of laser photostimulation and early mechanical loading of tendons increased collagen production, with marginal biomechanical effects on repaired tendons.

Combined ultrasound, electrical stimulation, and laser promote collagen synthesis with moderate changes in tendon biomechanics.

The biomechanical, biochemical, and ultrastructural effects of a multitherapeutic protocol were studied using regenerating rabbit Achilles tendons. The multitherapeutic protocol was composed of low-intensity Ga:As laser photostimulation, low intensity ultrasound, and electrical stimulation. Achilles tendons of 63 male New Zealand rabbits were tenotomized, sutured, immobilized, and subjected to the multitherapeutic protocol for five days, after which casts were removed and the therapy was continued for nine more days without electrical stimulation. The tendons were excised and compared with control tendons. Multitherapy treatment produced a 14% increase in maximal strength, a 42% increase in load-at-break, a 20% increase in maximal stress, a 45% increase in stress-at-break, a 21% increase in maximal strain, and a 14% increase in strain-at-break. Similarly, multitherapy treatment was associated with an increase in Young's modulus of elasticity of 31%, an increase in energy absorption at maximum load of 9%, and an increase in energy absorption at load-at-break of 11%. Biochemical analysis of the tendons showed an increase of 23% in the total amount of collagen in the multitherapy-treated tendons, with fewer mature crosslinks (decrease of 6%). Electron micrographs revealed no ultrastructural or morphologic changes in the tendon fibroblasts or in the extracellular matrix. The improvements measured in tendons receiving multitherapy were consistent but less remarkable compared with our earlier works with single modality protocols. The results warrant the hypothesis that the beneficial effects of ultrasound and laser photostimulation on tendon healing may counteract one another when applied simultaneously.

A simplified method for the analysis of hydroxyproline in biological tissues.

A critical study of the different steps involved in previous procedure for hydroxyproline assay allows the direct measurement of collagen content in tissue' homogenates without losing the advantages of the method. The procedure is based on alkaline hydrolysis of the tissue homogenate and subsequent determination of the free hydroxyproline in hydrolyzates. Chloramine-T was used to oxidize the free hydroxyproline for the production of a pyrrole. The addition of Ehrlich's reagent resulted in the formation of a chromophore that can be measured at 550 nm. Optimal assay conditions were determined using tissue homogenate and purified acid soluble collagen along with standard hydroxyproline. Critical parameters such as the amount of chloramine-T, sodium hydroxide, p-dimethylaminobenzaldehyde, pH of the reaction buffer, and length of oxidation time were examined to obtain satisfactory results. The method has been applied to samples of tissue homogenate and purified acid soluble collagen, with recovery of added hydroxyproline of 101 +/- 6.5 and 104 +/- 6.0 (SD) percent, respectively. The method is highly sensitive and reproducible when used to measure the imino acid in tissue homogenates. The modified hydroxyproline assay presented in this communication will be useful for routine measurement of collagen content in extracts of various tissue specimens. In addition, the modified method can be used for batch processing of column fractions to monitor the collagen concentrations during purification.

Immobilization-induced muscle atrophy is not reversed by lengthening the muscle.

In clinical practice, repaired tendocalcaneus (Achilles tendon) ruptures are often protected in immobilization casts for 4 weeks in the fully plantar flexed position and for up to another 4 weeks after returning the ankle to joint neutral. Moderate to severe muscle atrophy occurs within 4 weeks of immobilization in plantar flexion, but it is not known if this atrophy is minimized or reversed following restoration of joint neutral position. We tested the hypothesis that the extent of atrophy could be reduced by returning the ankle to joint neutral after 4 weeks of immobilization. Eighteen rabbits were anesthetized, and their right hind-limbs were casted with the knee flexed 90 degrees and the ankle fully plantar flexed. Three animals each were studied after 3, 4, 6, or 8 weeks of immobilization. After 4 weeks of immobilization, the immobilization casts of the remaining six rabbits were modified to return the ankle to joint neutral for another 2 or 4 weeks. For muscle studies, the animals were anesthetized, and the soleus (SOL), plantaris (PLN), and gastrocnemius (GST) muscles were removed and weighed; the SOL and PLN were quick frozen and processed for histochemical fiber typing and fiber cross-sectional area measurement. All three muscles showed significantly reduced muscle weight to body weight ratios after 3 weeks of immobilization. SOL was the most affected, and GST was least affected. There was no significant further atrophy through 8 weeks of immobilization. The atrophy correlated with a significant reduction of mean fiber area (MFA) for Types I, IIo, and IIc fibers in SOL and PLN. In PLN, Type IIg fiber area was not significantly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscle atrophy continues after early cast removal following tendon repair.

We studied soleus (SOL), plantaris (PLN), and gastrocnemius (GST) muscles to determine whether early cast removal minimizes muscle atrophy or permits recovery from atrophy after tendon repair. After right tendocalcaneus (Achilles tendon) was transected and repaired, rabbit right hindlimbs were immobilized with the ankle plantar flexed and the knee flexed to 90 degrees. Rabbits were maintained in the cast and sacrificed at 5, 15, or 21 days postoperatively or the cast was removed on day 5 and the animals sacrificed at day 15 or 21. SOL, PLN, and GST muscles of both limbs were removed and weighed, and then histochemical analyses were performed on SOL and PLN muscles. Immobilization decreased SOL muscle wet weights, mean fiber cross-sectional area, and percentage of Type I fibers and increased the percentage of Type IIc fibers. Ten days after cast removal (i.e., postoperative day 15), SOL muscle atrophy and fiber composition did not differ significantly from continuously immobilized controls. However, 16 days after cast removal (i.e., postoperative day 21), SOL muscle fiber cross-sectional area and fiber composition were near normal, differing significantly from continuously casted controls. At each of the time intervals studied, PLN (containing many glycolytic fibers) did not atrophy as much as SOL (containing mainly oxidative fibers). Our results indicate that 1) early cast removal prevents atrophy of PLN glycolytic fibers, but not oxidative fibers in either PLN or SOL, and 2) early cast removal promotes recovery from atrophy of both oxidative and glycolytic fibers. In spite of the many differences between rabbits and humans, these findings suggest that, although early cast removal may not prevent oxidative muscle fiber atrophy after postoperative immobilization, it may facilitate recovery from atrophy.

Functional loading augments the initial tensile strength and energy absorption capacity of regenerating rabbit Achilles tendons.

The biomechanical effect of functional loading was studied in surgically tenotomized, repaired and immobilized right Achilles tendons of 34 rabbits. Beginning from the 5th day after surgery, loading was initiated by removing the immobilization casts of the animals to permit unrestricted weight-bearing to tolerance. At each of 12, 18 and 21 days after surgery, functionally loaded tendons and their corresponding nonloaded controls were excised and compared for differences in tensile strength, tensile stress and energy absorption capacity. Functional loading induced a twofold increase in the tensile strength (P less than 0.05) and energy absorption capacity (P less than 0.05) of the tendons, at both 12 and 18 days after surgery. No statistically significant differences were observed in the tensile strength and energy absorption capacity of the tendons at 21 days after surgery. Statistically significant differences in tensile stress were not observed at any time period throughout the study. These findings demonstrate that functional loading augments the tensile strength and energy absorption capacity of experimentally tenotomized tendons without promoting re-rupture, but only during the very early stages of healing. Even though the healing process of rabbit tendons may differ from healing of human tendons, these results suggest that similar beneficial effects may be attained if repaired human Achilles tendon ruptures are carefully loaded during the very early rather than later stages of healing.

Effects of exercise and food restriction on rat skeletal muscles.

Studies were undertaken to compare the effects of exercise and food restriction on body weight (BW), muscle weight (MW), muscle fiber size, and proportion of muscle fiber types. 20 male Fischer 344 rats were randomly assigned to four equal groups: ad libitum-fed control (AC), ad libitum-fed exercise (AE), food restricted control (RC) and food restricted exercise (RE). From 6 weeks of age, RC and RE rats received 60% of the daily food intake of AC and AE rats, respectively. At 7 months of age, AE and RE rats began 40-50 min of daily treadmill exercise. Running speed increased from 1.2 to 1.6 miles/hour and the grade increased to 15% during the first 2 weeks of training. After 10 weeks of training, rats were weighed, sacrificed, and the soleus (SOL), plantaris (PLN) and extensor digitorum longus (EDL) muscles were removed at in situ rest length, weighed, and quick-frozen. Standard histochemical assays were performed, and muscle fiber cross-sectional area was determined planimetrically. Training had little effect on MW or BW, but food restriction greatly reduced BW. This resulted in greater MW/BW ratio in RC and RE than AC and AE rats, respectively. Exercise also increased SOL muscle fiber area in ad libitum-fed but not food restricted rats resulting in smaller fibers in SOL of RE than AE. No changes in percentage of SOL fiber types occurred with food restriction or exercise. In PLN, the percentage of fast-twitch oxidative fibers of AE and RE was greater than in AC and RC, but there was no effect of food restriction or exercise on fiber area.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural morphometry of matrical changes induced by exercise and food restriction in the rat calcaneal tendon.

The ultrastructural morphometry of collagen fibril populations in 24 calcaneal tendons obtained from 12 Fischer 344 rats were studied to elucidate matrical changes induced by food restriction and/or endurance exercise. Rats were randomly assigned to four equal groups: ad libitum control (AC), ad libitum exercise (AE), restricted diet control (RC) and restricted diet exercise (RE) groups. Beginning from 6 weeks of age, animals in the two food restriction groups were fed 60% of the mean food consumption of ad libitum fed rats. Then, starting from 6-7 months of age, the rats in the two exercise groups performed 40-50 min of treadmill running at 1.2-1.6 miles h-1 every day for a total of 10 weeks. Endurance training did not significantly alter body weight, but food restriction with or without exercise resulted in a significant loss of body weight. In ad libitum fed controls, food restriction alone did not significantly alter the mean collagen fibril CSA, but predisposed a preponderance of small-sized collagen fibrils. Endurance training per se induced a significant (32%) increase in mean fibril CSA (P less than 0.05), but this adaptive response to exercise was prevented by food restriction, as indicated by a 33% decline in fibril CSA (P less than 0.05). These findings demonstrate that dietary restriction modifies the adaptation of tendon collagen morphometry in response to endurance training, and that weight loss is better achieved with food restriction than endurance exercise.

Ultrastructural morphometry of membrane-bound intracytoplasmic collagen fibrils in tendon fibroblasts exposed to He:Ne laser beam.

Collagen fibrils are not found in fibroblast cytoplasm except in certain pathological conditions or in the presence of drugs and other agents that accelerate collagen turnover. Because low energy laser photostimulation is both a non-pathogenic and non-chemical accelerator of collagen synthesis, its effects were studied on four groups of calcaneal tendons from 18 rabbits (1) to test the hypothesis that vacuolar fibrils are not produced exclusively by diseases and chemical agents, and (2) to compare the morphometry of matrical and vacuolar fibrils. The right calcaneal tendons of nine rabbits were surgically tenotomized and repaired; six of these were transcutaneously irradiated with He:Ne laser everyday. The right calcaneal tendon of six of the remaining nine rabbits were similarly irradiated with laser, but without prior tenotomy and repair. 21 days later, all tendons were fixed in situ and processed for electron microscopy. Fibril-bearing vacuoles were found only in fibroblasts of tenotomized laser-irradiated tendons. Similar vacuoles were not seen in non-tenotomized laser-irradiated tendons nor in non-irradiated tendons whether tenotomized or not. Mann-Whitney U tests revealed no statistically significant differences in the cross-sectional areas or diameters of matrical and vacuolar fibrils. These findings suggest (a) that matrical and vacuolar fibrils have a common origin, and (b) that vacuolar fibrils can be induced by a non-pathologic, non-chemical accelerator of collagen synthesis.

Membrane-bound intracellular collagen fibrils in fibroblasts and myofibroblasts of regenerating rabbit calcaneal tendons.

The ultrastructures of 33 rabbit calcaneal tendons were studied to determine (1) whether vacuolar fibrils are present in three regions of tendons undergoing normal healing after tenotomy and repair, and (2) to stimulate collagen synthesis via functional loading, and hence determine the effect of loading on the presence of vacuolar fibrils in healing tendons. In all the loaded tendons, electron microscopy revealed membrane-bound collagen fibril equivalents in sections of neotendon obtained from the site of tenotomy, and in sections of tendon segments proximal and distal to the site of surgery. Similar vacuolar fibrils were visualized in sections of the proximal and distal segments of the non-loaded regenerating tendons, and also in sections of neotendons formed at the site of tenotomy after 12 and 15 days of healing without functional loading. No such fibrils were visualized in the non-tenotomized normal control tendons. These findings indicate that chemical agents and disease are not necessary to induce the appearance of intracytoplasmic fibrils in vivo and that functional loading augments the presence of fibril-bearing vacuoles in regenerating tendons.

Connective tissue plasticity: ultrastructural, biomechanical, and morphometric effects of physical factors on intact and regenerating tendons.

Supported in part by a grant from the Foundation for Physical Therapy and a merit grant from Veterans Affairs Rehabilitation Research and Development Program. Proceedings from the 1991 Eugene Michels Research Forum, Combined Sections Meeting, Orlando, FL, January-February, 1991. Special thanks to A. Joseph Threlkeld, University of Kentucky Medical Center, Lexington, KY, for helping acquire the presentation material for publication. J Orthop Sports Phys Ther 1991;14(5):198-212.

The biomechanical effects of low-intensity ultrasound on healing tendons.

The effect of low-intensity ultrasound on the healing strength of tendons was studied in experimentally tenotomized, repaired and immobilized right tendo calcaneus (Achilles tendon) of 24 rabbits. Ten tendons were sonicated in continuous waves at a space-averaged intensity of 0.5 W cm-2 for 5 min every day. The remaining 14 tendons were mock-sonicated as controls. After nine consecutive treatments, the tendons were excised under anesthesia and compared for differences in tensile strength, tensile stress and energy absorption capacity. Sonication at 0.5 W cm-2 induced a significant increase in the tensile strength (p less than .02), tensile stress (p less than .005) and energy absorption capacity (p less than .001) of the tendons. These findings suggest that high-intensity sonication may not be necessary to augment the healing strength of tendons and that sonication at similarly low intensities may enhance the healing process of surgically repaired human tendo calcaneus.

The effects of therapeutic ultrasound on tendon healing. A biomechanical study.

The effect of 1 MHz therapeutic ultrasound on the healing strength of tendons was studied in the tenotomized, repaired and immobilized right Achilles tendons of 26 rabbits. Twelve tendons were sonicated daily in continuous waves at a spatial averaged intensity of 1 W cm(2) [corrected] for 5 min. After nine consecutive treatments, the tendons were excised under anesthesia and compared biomechanically. Exposure to ultrasound induced a significant increase in both the tensile strength and the energy absorption capacity of the tendons. Although healing in rabbits may not translate directly to healing in humans, these findings suggest that surgically repaired human Achilles tendons may heal faster if ultrasound is applied during the early stages of healing.