Histomorphometrical analysis on the effects of two therapeutic ultrasound intensities on fracture healing in aged rats

Introduction: Experimental studies conducted in young animals show that therapeutic ultrasound (TUS) has been successfully used to shorten the healing time of bone fractures. However, they were not found in the literature, studies comparing the effect of different intensities of UST in aged animals. Objective: To test the efficacy of intensity 1.0 W/cm2 and of 0.5 W/cm2 in the consolidation of experimental fracture of the tibia from aged Wistar rats. Materials and methods: Three groups of 15 month old rats were submitted to a midshaft osteotomy of the tibia and then, the hind member was immobilized with a metal splint and plaster of Paris, wrapping the knee and ankle joint. One group (L), received ultrasound at 0.5 W/cm2; the other group (I), were exposed to ultrasound at 1.0 W/cm2. One control group (C), did not receive the ultrasound. Fifteen animals (five from each group) were euthanatized at the end of the first week and fifteen (five from each group) at the end of the third week. The progress of the fracture healing was performed for each group by morphometric analysis of histological sections of the fracture region. Results and conclusion:


Introduction:
Experimental studies conducted in young animals show that therapeutic ultrasound (TUS) has been successfully used to shorten the healing time of bone fractures.However, they were not found in the literature, studies comparing the effect of different intensities of UST in aged animals.Objective: To test the efficacy of intensity 1.0 W/cm² and of 0.5 W/cm² in the consolidation of experimental fracture of the tibia from aged Wistar rats.Materials and methods: Three groups of 15 month old rats were submitted to a midshaft osteotomy of the tibia and then, the hind member was immobilized with a metal splint and plaster of Paris, wrapping the knee and ankle joint.One group (L), received ultrasound at 0.5 W/cm²; the other group (I), were exposed to ultrasound at 1.0 W/cm².One control group (C), did not receive the ultrasound.Fifteen animals (five from each group) were euthanatized at the end of the first week and fifteen (five from each group) at the end of the third week.The progress of the fracture healing was performed for each group by morphometric analysis of histological sections of the fracture region.Results and conclusion:

Introduction
One of the most common kinds of fractures, both in the young and elderly is that of the middle third of the tibia (1).In the repair process of such kind of fracture (stable bone fracture), after the removal of the blood clot by cells of the connective tissue, there is an initial proliferation of osteogenic cells (2), that is followed by the appearance of minuscule bone and cartilaginous strands and fibrous tissue in the area.As a result, immature bone tissue is formed, and then the lamellated bone tissue appear constituting the bone callus (3).This entire process generally takes between 10 and 13 weeks in humans and 3 weeks in young rats (4).
Several works have been carried out searching for methods to abbreviate the time for fracture consolidation (4,5).Methods that have been used to accelerate bone healing include electrical stimulation, therapeutic ultrasound (TUS) and precocious mobilization, among others.Low-intensity pulsed ultrasound has been shown to accelerate fracture healing in both animal models and clinical trials (2,6,7,8,9,10,11).The effectiveness of this resource has been studied using different degrees of intensity.The values that showed positive results are that of low intensity: 0.5 W/cm² and 0.3 W/ cm² (7,12).The use of high intensities such as those of 1.5 W/cm² proved to be detrimental to the bone tissue.However, investigations showing the effects of values between 0.2 and 1.5 W/cm² are scarce and have been carried out in young animals (12,13).In this study, the time for fracture consolidation of the middle third of the tibia using two different intensities (0.5 W/cm² and 1.0 W/cm²) of pulsating ultrasound were compared by histomorphometric method in aged rats.

Surgical proceeding
In this work, 30 fifteen-mo-old Wistar male rats were used.All the animals were submitted to peridural anesthetics (Xylocayne 2% and Epinefrine, 1:4000) (Ariston LAB, São Paulo, Brazil).After preparation of the right hindlimb, flexion position was given.A two cm linear incision was made on the medial aspect of the limb and the tibia was exposed.The tibia was transversally and incompletely divided in its middle third, using an appropriate saw.Then, the skin was sutured and bone tissue were counted and then the V v bone was calculated by the formula: the hind limb was immobilized by a metal splint and plaster of Paris, wrapping the knee and ankle joints.Animals were divided into three groups with 10 rats each: Control group (C); Low intensity TUS group (L) and Intermediary intensity TUS group (I).C Group was submitted only to the immobilization.L Group received 1.0 MHz TUS (Sonopulse III Ibramed, Amparo, São Paulo, Brazil) in pulsed mode at 0.5 W/cm² for 5 minutes daily, 5 times a week; 5 animals received TUS for 1 week and 5 for 3 weeks.I Group received 1.0 MHz TUS in pulsed mode at 1.0 W/ cm², for 5 minutes daily, 5 times a week; 5 animals received TUS for 1 week and 5 for 3 weeks.The application of the TUS was performed through a water balloon.A thin layer of gel was placed on the area of the lesion and on top of the balloon.Then, the balloon was placed over the wound surface area, and the TUS probe was placed over it.The rats were allowed to move freely in their cages.At the end of the first week 15 rats, being 5 from each group were killed and at the end of the 3 rd week the last 15 rats, being 5 from each group were also killed.The segment of the tibia with the fracture trace was collected and processed for examination.All procedures to which the animals were submitted received the approval of the Commission of Ethics in the Use of Animals of the Faculdade de Medicina Veterinária of Universidade de São Paulo, which follows the International Guiding Principles for Biomedical Research Involving Animals, under protocol nº 734/2005.

Histomorphometry
From each animal, the segment of the tibia containing the fracture trace was removed, fixed in formaldehyde in 0.07 M phosphate buffer (pH 7.0) and decalcified with 10% EDTA (Sigma-Aldrich, St. Louis, MO, USA) and 4% formaldehyde in 0.01 M phosphate buffer (pH 7.4) at 4 °C.The blocks were washed in the phosphate buffer and dehydrated, cleared and embedded in Paraplast.Sections 7 micrometers (μm) thick were performed parallel to the long axis of the tibia.Every third section of a total of 10 sections was then stained with hematoxylin and eosin (HE) and analysed in a digital image analysis system (KS-400, Carl Zeiss, São Paulo, Brazil).
To estimate the volume density (%) of neoformed bone (V v bone), a test-system with 132 points was superimposed to each image over the monitor screen in the video-microscopic system.The points hitting Vv bone = Pp (bone)

PT
Where: PP(bone) are the number of points hitting the bone, and PT are the total number of points (54) of the test-system (14) (Figure 1).The results are expressed as means ± SEM.

Statistical analysis
Data were compared by analysis of variance (ANOVA), and Tukey's test for multiple comparisons, as appropriate.The significance level was set at P < 0.05.

Histomorphometric analysis
Qualitatively, at the first week, the C group has an empty space, corresponding to the area of the lesion, showing that the consolidation process has not yet begun (Figure 2A).At third week, proliferation of osteogenic cells can be observed in the lesion area.In the deep part of the lesion, there are recently formed bony trabeculae.Much more osseous tissue was observed in the callus (Figure 2D).In L Group (Figure 2B), at the first week, osteogenic cells fill in the lesion space, indicating that the formation of bone tissue will be initiated.In the third week, newly formed bone fill almost entirely the space of the lesion (Figure 2E).At the first week of the I Group (Figure 2C), newly formed immature bony trabeculae fill the region of the fracture and osteogenic cells can be observed in the lesion space indicating that beginning of primary ossification takes place in the space of lesion.At the third week, bone trabeculae (arrows) can be seen filling almost all space of the fracture (Figure 2F).In conclusion, on day 21 st (Figures 2D, 2E, 2F), fractures treated with US at 1.0 W/cm² (Figure 2F) healed more rapidly than did that treated with 0.5 W/cm² (Figure 2E) or the controls (Figure 2D).
Quantitatively, significant differences on the volume density (%) of newly formed bone tissue were observed among the groups at the 1 st and 3 rd week There is disagreement among authors, related to the best intensity of TUS to be applied in the case of fractures (18).Several authors (5,12) obtained favorable results in fractures of rats treated with 0.1 W/cm² for two minutes every 2 days or with 0.2 W/cm 2 for ten minutes over five consecutive days (13).According to others, intensities from 0.3 W/ cm² to 0.5 W/cm 2 are the most efficient (19).The present study showed that fractures treated with ultrasound at 1.0 W/cm 2 healed significantly faster than that treated with ultrasound at 0.5 W/cm 2 and the control.The fracture healing has been accelerated in TUS 1.0 W/cm 2 group than in the 0.5 W/cm 2 group, both at the first week but mainly at the 3 rd week (p < 0.05).
In the present study, tibia was choose due the easy surgical access to this bone, because of its superficial location and because the fracture in the middle third of the tibia is one of the most frequent (1,20).
(p < 0.05).The percent of the new bone tissue was higher in I (7%) than in the C (0%) and L (3%) group at the first week (P < 0.05).At the third week, the percent of the newly formed bone tissue was higher in L (25%) than in the C (9%) group and in I (60%) than in L and C group (P < 0.05) (Figure 3).

Discussion
The consolidation time for the bone fractures that frequently occur both in sports and in aged sedentary people obliges the individuals to remain distant from their normal activities (2,15,16).Application of TUS is among the resources suggested with the objective of recovering the individuals in the shortest possible time.This resource have been used by several authors obtaining positive effects (2,6,7,8,10,17), although some have not obtained favorable results in humans with its application (16).Assessment of the results of TUS application for the study of fracture healing with experimental animals has generally been done through X-ray or bone densitometry (5,13,16,19,21,22).In the present work, results were obtained through histological and morphometric analysis of the fracture area.Only a few authors have used histological and quantitative methods for this purpose (4,5,19) and none used histological methods associated with morphometric methods.
The pulsed TUS mode was used because it presents less thermal effect and because it has a proved action on the inflammatory reaction (23).However, some care must be taken when the TUS is used, as for example, to avoid direct contact of the head of the equipment with the surgical wound.In this study we used the application with the water balloon, because of the impossibility of immersion of the fractured area in the water, due to the limb immobilization through the plaster of Paris.
The exact mechanism of action of the ultrasound device is unknown.It has been theorized that the ultrasound pressure waves may mediate biologic activity directly by mechanical deformation of the cell membrane or the extracellular matrix or indirectly by an electrical effect caused by cell deformation (24,25).Low intensity pulsed ultrasound in isolated cell systems produced significant multifunctional effects of direct relevance to bone formation and resorption such as increasing calcium uptake and modulating adenylate cyclase activity, transforming growth factor beta synthesis, bone morphogenic protein effects, and parathyroid hormone response (11,26).Furthermore, ultrasound stimulation increases the mechanical properties of the healing fracture callus by stimulating earlier synthesis of extracellular matrix proteins in cartilage, possibly altering chondrocyte maturation and endochondral bone formation (17,27).Others studies suggested that TUS acts on some cellular reactions involved in each phase of the healing process such as inflammatory reaction, angiogenesis, chondrogenesis, intramembranous ossification, endochondral ossification, and bone remodeling (11,19).In summary, the application of ultrasonic waves of 1.0 W/cm 2 promoted a more rapid healing of experimental bone fractures than 0.5 W/cm 2 in Wistar rats.

Figure 1 -Figure 2 -Figure 3 -
Figure 1 -Test-system with 132-points (yellow points) superimposed at a histological section from the fracture trace, used to quantify the newly formed bone (white arrows) in the four groups of rats Note: Black arrows show nuclei of osteogenic cells.Bar: 100 micrometers μm.Source: Research data.