G. Kesava Reddy

Laser photostimulation of collagen production in healing rabbit Achilles tendons

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.

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 632.8nm He:Ne laser at a dose of 1.0J/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 μg) over the control wounds (230 ± 8.4 μg). 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.

Effect of mulberry (Morus indica L.) therapy on plasma and erythrocyte membrane lipids in patients with type 2 diabetes

BACKGROUND Mulberry (Morus indica L.) is non-toxic natural therapeutic agent shown to possess hypoglycemic, hypotensive, and diuretic properties. METHODS The hypoglycemic effect of the mulberry leaves was evaluated by comparing the anti-diabetic activity of the standard drug, glibenclamide. A total of 24 type 2 diabetic patents were divided randomly into two treatment groups: the mulberry agent and glibenclamide, for 30 days. Serum and erythrocyte membrane lipid profiles of the patients were analyzed before and after the treatments. RESULTS Patients with mulberry therapy significantly improved their glycemic control vs. glibenclamide treatment. The results from pre- and post-treatment analysis of blood plasma and urine samples showed that the mulberry therapy significantly decreased the concentration of serum total cholesterol (12%, p<0.01), triglycerides (16%, p<0.01), plasma free fatty acids (12%, p<0.01), LDL-cholesterol (23%, p<0.01), VLDL-cholesterol (17%, p<0.01), plasma peroxides (25%, p<0.01), urinary peroxides (55%, p<0.01), while increasing HDL-cholesterol (18%, p<0.01). Although the patients with glibenclamide treatment showed marginal improvement in glycemic control, the changes in the lipid profile were not statistically significant except for triglycerides (10%, p<0.05), plasma peroxides (15%, p<0.05), and urinary peroxides (19%, p<0.05). Both treatments displayed no apparent effect on the concentrations of the glycosylated hemoglobin (Hb A(1)c) in diabetic patients. However, the fasting blood glucose concentrations of diabetic patients were significantly reduced by the mulberry therapy. CONCLUSIONS Mulberry therapy exhibits potential hypoglycemic and hypolipidemic effects in diabetic patients.

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.

Comparison of the photostimulatory effects of visible He-Ne and infrared Ga-As lasers on healing impaired diabetic rat wounds

In this study, the ability of photostimulation to promote healing of impaired wounds was investigated using a Ga‐As laser in rats with experimental diabetes and the results were compared with previously reported findings of the effects of a He‐Ne laser on the repair of healing‐impaired diabetic rat wounds 1 .

Glucose-mediated in vitro glycation modulates biomechanical integrity of the soft tissues but not hard tissues

Glycation induced crosslinking of connective tissue collagen is thought to be involved in the pathogenesis of various disorders associated with diabetes and aging. Although the formation of the glycation related collagen crosslinks appears to be universal for all tissues, currently it is unknown whether differences exist between soft and hard tissue biomechanics in response to glucose‐mediated in vitro glycation. In this study, the impact of non‐enzymatic glycation was investigated on tendons and bones, using them as models for soft and hard tissue respectively. Achilles tendons from rabbits, and femur and tibia from rats, were subjected to in vitro glycation with glucose. Sixty days following glycation, the matrix integrity of the tendons and bones was evaluated and compared with the respective non‐glycated tissue (n = 10 in each group). The results revealed that the impact of glycation was significant on the tendon but not on the bone. Measurements of the biomechanical stability of glycated tendons indicated a significant increase in maximum load (21%), Youngs modulus of elasticity (72%), energy to yield (35%) and toughness (68%) compared to the non‐glycated tendons. No significant differences were found in breaking strength, bending stiffness, energy to yield and toughness between glycated and non‐glycated femurs or tibias. The deformation of both soft and hard tissue was unaffected by the glycation. Measurements at ultimate tissue failure (break point) revealed that glycated tendons bore significantly higher load and energy absorption than non‐glycated tendons. In contrast, the deformation of the glycated tendons at break point was considerably reduced as compared to control tendons. However, glycation had no significant effects on the hard tissue biomechanical properties at break point. The results of this study demonstrate that in vitro glycation influences the biomechanical properties of soft tissue but not hard tissue.

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.

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 Youngs 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.

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.

High-dose inhaled nitric oxide and hyperoxia increases lung collagen accumulation in piglets

Nitric oxide (NO), a pro-oxidant gas, is used with hyperoxia (O2) to treat neonatal pulmonary hypertension and recently bronchopulmonary dysplasia, but great concerns remain regarding NO’s potential toxicity. Based on reports that exposure to oxidant gases results in pulmonary extracellular matrix injury associated with elevated lavage fluid levels of extracellular matrix components, we hypothesized that inhaled NO with or without hyperoxia will have the same effect. We measured alveolar septal width, lung collagen content, lavage fluid hydroxyproline, hyaluronan and laminin levels in neonatal piglets after 5 days’ exposure to room air (RA), RA + 50 ppm NO (RA + NO), O2 (FiO2 > 0.96) or O2 + NO. Matrix metalloproteinase (MMP) activity and MMP-2 mRNA were also measured. In recovery experiments, we measured lung collagen content in piglets exposed to RA + NO or O2 + NO and then allowed to recover for 3 days. The results show that lung collagen increased 4-fold in the RA + NO piglets, the O2 and O2 + NO groups had only a 2-fold elevation relative to RA controls. Unlike the RA + NO piglets, the O2 and O2 + NO groups had more than 20-fold elevation in lung lavage fluid hydroxyproline compared to the RA group. O2 and O2 + NO also had increased lung MMP activity, extravascular water, and lavage fluid proteins. MMP-2 mRNA levels were unchanged. After 3 days’ recovery in room air, the RA + NO groups’ lung collagen had declined from 4-fold to 2-fold above the RA group values. The O2 + NO group did not decline. Alveolar septal width increased significantly only in the O2 and O2 + NO groups. We conclude that 5 days’ exposure to NO does not result in pulmonary matrix degradation but instead significantly increases lung collagen content. This effect appears potentially reversible. In contrast, hyperoxia exposure with or without NO results in pulmonary matrix degradation and increased lung collagen content. The observation that NO increased lung collagen content represents a new finding and suggests NO could potentially induce pulmonary fibrosis.