Bone Healing Effects of Diode Laser (808 nm) on a Rat Tibial Fracture Model

Materials and Methods

Animals and experimental design. A total of 48 male rats (Sprague-Dawley), 8 weeks old and weighing approximately 250-300 g, were randomly divided into two groups containing 24 animals each, and killed 1, 3, 4 and 8 weeks after surgery. The groups included: group 1 (control) – sham control, animals not exposed to laser; and group 2 (laser group) – animals treated with low level laser. All control animals were submitted to the same procedures of handling as were the laser-irradiated animals. Upon arrival at the Laboratory Animal Research Center of Chungbuk National University, the animals were housed in an environmentally controlled animal facility for 7 days for acclimatization. The rats were kept in standard cages, 2-3 in each, with 12-hour light-dark intervals. The room temperature and the humidity were maintained at 20±2°C and 50±20%, respectively. All rats were fed pellet-type of commercial diet (Purina®, St. Louis, MO, USA) and tap water was available ad libitum from bottles. Care was taken to avoid unnecessary stress and discomfort of the animals throughout the experimental period. The animals were treated in accordance with the “Guide for Care and Use of Laboratory Animals” of Chungbuk National University. The study was conducted as an animal trial of 8 weeks duration with a blind controlled design.

Surgical procedures. At the beginning of the experiment, all animals underwent surgery in order to produce cortical bone defects and fractures of the right tibia. General anesthesia was obtained by intraperitoneal administration using a mixture of 2% xylazine hydrochloride (Rompun®; Bayer, Pittsburgh, PA, USA) 13 mg/kg and 125 mg of zolazepam hydrochloride with 125 mg of tiletamine hydrochloride (Zoletil®; Virbac Lab., Carros, France) in the recommended dose of 20 mg/kg. Under general anesthesia, the right leg of the animals was shaved, thereby exposing the tibia. The surgery was performed under an aseptic condition.

A 2.5-cm incision was made in the anterolateral direction of the right tibia. The incision penetrated the epidermis, dermis, and fascial layers. Lateral reflection of these tissues exposed the underlying periosteum. An additional medial-anterior incision was made through the periosteum. The periosteum was elevated and was retained by Debakey forceps. A cortical bone defect was made on the lateral aspect at one-third along the tibia which at the level of the tibial tuberosity using a 3-mm in diameter round burr. A transverse fracture was created at the center of the round-shaped cortical bone defect with an orthopedic saw. A Kirschner wire (1.0 mm in diameter) was inserted into the medullary canal with a small Jacobs handchuck using a retrograde method. The fascial and superficial tissue layers were repositioned and were sutured with 4-0 polyglycolic acid (Surgifit; AILEE, Busan, Korea) and 4-0 mono non-absorbable suture (Nylon; AILEE). As antibiotics and analgesics, 10 mg/kg of enrofloxacin (Baytril®; Bayer, Pittsburgh, PA, USA) and 0.2 mg/kg of meloxicam (Metacam®; Boehringer Ingelheim Vetmedica, St. Joseph, MO, USA), respectively, were injected daily through a subcutaneous route for three days.

Laser treatment. The animals were randomly divided into two equal groups (1 and 2). Groups 1 and 2 were treated in a sham and experimental setting, respectively, differing only in the use of a diode laser device. A diode laser device of 808-nm wavelength (DVL-20 Diode Laser System®; Asuka Medical Inc., Kyoto, Japan) was used in this study. This system operates in the near-infrared spectrum at a continuous wavelength of 808 nm and with an output power of 1 W. Laser irradiation was performed under general anesthesia using isoflurane (3%) chamber induction. A light probe with a diameter of 0.4 mm delivered the laser beam, and the irradiation was administered by placing the probe in light contact with the area to be treated. Treatment started after three days and was carried out every other day. The treatment points were administered as follows: anterior, posterior, medial, and lateral aspect of the surgical site. A total of four points were irradiated. The treatment time per point was 0.02 seconds, giving an energy density of 15.38 J/cm².

Body weights. Under general anesthesia, the body weight of each animal in each group was measured using an automatic electronic balance (Precisa Balance Series XB®; Precisa Instrument Ltd., Dietikon, Switzerland). In order to reduce errors originating from feeding, all animals were fasted with water allowed for 18 hours before sacrifice.

Radiological evaluation. The animals were radiographed at the end of the experimental periods (1, 3 and 4 weeks, respectively). The rats were euthanized by an intracardiac injection of 2 ml potassium chloride after general anesthesia using intraperitoneal administration of 13 mg/kg xylazine (Rompun®; Bayer, Pittsburgh, PA, USA) and 20 mg/kg of tiletamine/zolazepam (Zoletil®; Virbac Lab.). Immediately after the euthanasia, radiographs (KODAK DirectView CR500 system; Eastman Kodak Company Health Group, New York, NY, USA) in two planes, were captured from every tibia that had been operated on. Thereafter, differences between the control and the laser groups were quantified by applying a scoring system for bone defect healing evaluation (Table I). The scoring system was previously reported in the study of Santic et al. (16).

Histological evaluation. Histological evaluation was performed at 1, 3 and 4 weeks after surgery, respectively. Every right tibia was removed and soft tissues including skins and muscles were eliminated from the tibia. The Kirschner wire was removed from the tibia. Tissue samples (proximal half of each right tibia including the fractured and defected areas) were fixed in 10% formalin for 48 hours, decalcified, embedded in paraffin blocks, and cut longitudinally into 5-μm thick sections with a microtome (from medial aspect to lateral aspect of the tibia). For a microscopic descriptive analysis of each group, slides were prepared by using hematoxylin and eosin (H&E) dyes. Bone healing evaluation was performed using a microscope connected to an image analyzer. All measurements were performed using a magnifying objective (×40). For the comparison of the differences between the two groups, objectively, a semiquantitative scoring system, which was modified from that of Santics et al. (16), was applied (Table II). The evaluation was carried out by three independent observers. The maximum quality of bone healing was evaluated at eight points.

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Figure 1.

Representative radiological images of tibial bones after artificial fracture. The laser-treated group exhibited earlier new bone formation than did the control. The presented figures are representative images of each group. A, C, E, G, I, and K: Right lateral view of each tibia, B, D, F, H, J, and L: The Craniocaudal view of each tibia.

Blood chemistry and complete blood count. At 4 weeks post surgery, complete blood count (CBC) (Cell-Dyn® 3700; Abbott Diagnostics, North Chicago, IL, USA), and blood chemistry (HITACHI 7020 Autonomic Analyzer; Hitachi High-Technologies Corporation, Tokyo, Japan) including alkaline phosphatase (ALP), calcium, and phosphate, were performed.

Bending test. A three-point bending test was performed at 8 weeks, for the measurement of the mechanical strength of regenerated bone tissue. The bending test was performed with a computer-controlled tensile test system (MultiTest1-i®; Mecmesin Limited, West Sussex, UK) fitted with a calibrated load-cell of 1000 N. The cross-speed range was set to 5 mm/min. The callus area of each tibia was tested and the maximum bone tolerance force before the point of fracture was measured.

Statistical analysis. All statistical analyses were performed with the SPSS software (Windows version 12.0, SPSS, Chicago, IL, USA). Statistical analyses were performed by using the Student's t-test to compare the data from the laser treated and control groups. Measurement of all the data was made blindly with no clue to whether the animal being evaluated was treated or untreated. All data were presented as the mean±standard deviation (SD). The results were considered to be significant when the level of probability (p-value) was 0.05 or less.