Low intensity ultrasound effects over osteopenic female rats bones

Ação do ultra-som de baixa intensidade sobre ossos de ratas osteopênicas

Abstracts

Several studies have already shown the beneficial effects of low intensity pulsed ultrasound on osteogenesis in fracture cases. However, few reports have related the ultrasound action in bone with some injury but without fracture. Thus, we induced a rat osteopenia model by ovariectomy and the proximal third of rat femur was stimulated by ultrasound (200mus burst of 1.5 MHz sine waves repeated at 1.0 kHz, 30mW/cm², SATA) for 20 min/day, during 20 days. After the treatment period, the body weight was significantly higher in the non-treated group than the treated one. No significant difference in bone mineral content was detected among the groups (p > 0.05). Also, no significant difference was noted in the mechanical properties of the femoral diaphysis. However, histologic investigations showed that the treated femur presented less microarchitectural deterioration than the non-treated group. Moreover, it was demonstrated that the treated group did show recent bone formation which was not there in the non-treated group. These results suggest that the low intensity ultrasound can interfere in a positive way on osteoporosis.

Ultrasonics; Bone diseases, Metabolic; Calcium; Phosphorus

Neste estudo, nós analisamos a ação do ultra-som pulsado de baixa intensidade (30mW/cm², freqüência de repetição de 1Khz e ciclo de trabalho de 20%) durante 20 dias consecutivos, por 20 minutos diários, como método não invasivo, em modelo de ratas osteopênicas. A estimulação ultra-sônica foi realizada na região proximal dos fêmures, bilateralmente. Apesar dos resultados quantitativos do conteúdo mineral ósseo (cálcio e fósforo) não terem demonstrado diferenças significativas (p >0,05) entre os grupos tratado e não tratado, as investigações histológicas mostraram que o fêmur tratado apresentou uma menor deterioração microarquitetural que o grupo não tratado, além da ocorrência de neoformação óssea, a qual não foi observada no grupo não tratado. Os resultados sugerem que o ultra-som pulsado de baixa intensidade pode interferir positivamente sobre a osteoporose.

Ultrasom; Osteopatias metabólicas; Cálcio; Fósforo

ORIGINAL ARTICLE

Low intensity ultrasound effects over osteopenic female rats bones

Daniela Cristina Leite de Carvalho^I; Alberto Cliquet Jr^II

^IPhysiotherapist. Master in Bioengineering. Doctorate Student at Post-Graduation Course in Surgery, UNICAMP

^IIFull Professor EESC, USP / FCM - UNICAMP

^{Correspondence}

SUMMARY

Several studies have already shown the beneficial effects of low intensity pulsed ultrasound on osteogenesis in fracture cases. However, few reports have related the ultrasound action in bone with some injury but without fracture. Thus, we induced a rat osteopenia model by ovariectomy and the proximal third of rat femur was stimulated by ultrasound (200ms burst of 1.5 MHz sine waves repeated at 1.0 kHz, 30mW/cm², SATA) for 20 min/day, during 20 days.

After the treatment period, the body weight was significantly higher in the non-treated group than the treated one. No significant difference in bone mineral content was detected among the groups (p > 0.05). Also, no significant difference was noted in the mechanical properties of the femoral diaphysis. However, histologic investigations showed that the treated femur presented less microarchitectural deterioration than the non-treated group. Moreover, it was demonstrated that the treated group did show recent bone formation which was not there in the non-treated group. These results suggest that the low intensity ultrasound can interfere in a positive way on osteoporosis.

Key words: Ultrasonics; Bone diseases, Metabolic;Calcium/analysis; Phosphorus/analysis

INTRODUCTION

Osteoporosis is a metabolic bone disorder affecting mainly the aged population, generally post-menopausal women due to lack of ovarian hormone. Bony mass reduction, mainly observed in cancellous bone, that is caused by increase in bony reabsorption by osteoclasts changes bones more fragile and subject to fractures⁽⁶⁾.

Considering this, treatments preventing and stabilizing this picture are important. However, treatments currently existing for osteoporosis are predominantly pharmacological, and in some cases contra-indicated or poorly tolerated by some patients. Thus, alternative noninvasive techniques are a good choice for controlling bony mass losses.

Based on biomechanical principles, where mechanical load induces bone deformation and consequently causes stimulus for a local bony response, already presented by WOLLF in 1870, the use of low intensity ultrasound is an alternative for bone formation stimulation, since ultrasound is a mechanical energy type generating local bone micro-deformations, similarly to those occurring under external forces⁽²⁾. Thus, therapy with ultrasound, by promoting beneficial effects over bony structure may be used in diseases challenging the ability to keeping bony mass, such as in osteoporosis. Many studies have already demonstrated the efficacy of low intensity ultrasound in accelerating healing of recent bone fractures, in cases of non-union and in pseudarthrosis^{(3,8,9,15,17)}. It was also demonstrated the beneficial effect of low intensity ultrasound in cases of patients with diseases impairing bony recovery, such as osteoporosis and diabetes^(10,16). However, there are few studies reporting effects of low intensity ultrasound in cases of osteoporosis without fractures.

Currently, the model of ovariectomized rat is gaining wide acceptance in the study of reflexes of lack of ovarian hormone on skeletal system, as well as in evaluation of bony responses to new treatments.

This study was divided into two parts. In the first it was evaluated the efficacy of a 30-day protocol for induction of osteopenia in femoral bone of female rats, after hysterectomy and bilateral ovariectomy. In the second, it was evaluated the efficiency of pulsed low intensity ultrasound (intensity of 30 mW/cm², frequency of 1.5 MHz, repetition frequency of 1 KHz, work cycle of 20%), over the proximal region of osteopenic femurs of female rats.

MATERIAL AND METHODS

For the first part were used 11 mature isogenic female Holstman rats (Rattus norvegicus albinus), with about 200 g of body mass. The animals were divided into two groups: control (5 animals) with non-ovarectomized animals and a group of 6 ovarectomized animals. The surgeries were performed under intramuscular anesthesia with Virbaxil 2% in a 0.04 ml/100 g of body mass and by Francotar in a 0.08 ml/100 g of body mass doses. Bilateral ovarectomy was performed in 6 female rats. After the surgery, the animals were feed ad libitum for 30 days. Body mass of the animals were evaluated in baseline and at the end of the experiment. In the 31st day the animals were sacrificed, their left femur removed, cleaned, washed in deionized water and kept in 10% formaldehyde solution for 48 hours. After this period the bones were standardized at 1.5 cm length from the great trochanter and calcined, dissolved in chloride acid at 37% and diluted in 100 ml. This phase allowed the evaluation of the bony mineral content. For quantitative analysis of calcium it was used a flame atomic absorption spectrophotometer and for phosphorus analysis a molecular absorption in an flow injection analysis (FIA) system spectrophotometer. The right femurs were decalcified by MORSE (Sodium citrate 20% and Formic acid 50% - equal parts) dehydrated and included in paraffin. For each piece seven serial longitudinal cuts were performed (6 mm thick) of proximal femur region by means of a microtome that were excluded. Sequentially were performed two serial cuts of 50 mm thick for each piece. After the cuts, these were de-paraffined, dried in stove and fixed to a support used for electronic microscopic evaluation. The cuts were gold metallized in order to facilitate electrical conduction. A LEO model 440 equipment was used.

The differences in body mass in both groups were evaluated with T test, with a 5% significance level.

In the second part of the study, 34 isogenic female Holstman rats (Rattus norvegicus albinus), with about 200 mg of body mass, were used. Se same surgical protocol of the first study part was performed. Due to the efficacy of the 30 days ovariectomy protocol for induction of osteopenia, the treatments started at the 31^st day after the surgery. The rats were divided into two groups: a group treated (25 animals) and a control group (9 animals). In the treated group, the rats underwent ultrasound with a spatial and temporal intensity of 30 mW/cm² in average. The equipment used was fixated to the support table, where the rats were immobilized. The head had normal size allowing the ultrasound application to be performed at the same time in both hind-feet involving a region from femoral head to distal third of femur. A gel was used in order to enable the ultrasound field conduction. The equipment was calibrated previously to its use in the experiment.

The animals in control group did not underwent ultrasound treatment, however suffered the same interference stress can cause to animals. For this, they were immobilized for 20 minutes during 20 consecutive days, that is, the same as with the treated animals.

Animals were sacrificed 1 day after the treatment ended, and their left femurs removed and submitted to a three-point flexion mechanical assay in order to determine their mechanical properties. A MTS TestStart II equipment was used for the mechanical assay. The bones were laid over their extremities over two supports with 18 mm distance. The speed of load application was 5 mm/min using a 100 Kgf load cell. For each load increase applied to the bone, it was retrieved the deformation caused, allowing to plot a load versus deformation curve. Through this curve, it was possible to evaluate mechanical properties such as maximal deformation, rigidity, resiliency and tenacity (Figure 1). The parameters were calculated by means of the Mathematics 2.2 software. After the flexion assay, the femurs were standardized at 1.5 cm length from the great trochanter and prepared for a quantitative analysis of calcium and phosphorus. In each pot were placed two bones, except in some cases in it was necessary to put three bones. The right femurs were taken apart for common light microscopy and electronic microscopy. After preparation, the pieces were cut longitudinally (6 mm thick) from proximal femur region by means of a microtome, and the flakes stained with Masson Tricromion.

Sequentially, serial cuts were performed at 50 ìm thick, and prepared to be analyzed under electronic microscopy. The body mass were weighted from baseline to the end of the experiment.

In order to compare the female rats body mass it was used the T test; for comparing mechanical variables and bony mineral contents (calcium and phosphorus) between the groups, the variance analysis (ANOVA). Significance level was 5%.

RESULTS

First study part: at the time of the sacrifice it was observed a significant increase in body mass of ovarectomized female rats as shown in (Table 1).

By means of a quantitative analysis of calcium and phosphorus of the femur of animals from both experimental groups, it was observed that rats from control group had a higher amount of calcium and phosphorus (average: calcium, 180.59 g/kg; phosphorus 90 g/kg) in comparison to the group of ovarectomized rats (average: calcium, 140.94 g/kg and phosphorus, 73.49 g/kg). There was a 21.956% reduction in calcium and 18.34% in phosphorus in the ovarectomized rats in comparison to the control ones (Figure 2).

By means of electronic microscopy it was possible to compare the structural differences between femur of control and ovarectomized groups ( Figures 3) shows the differences in cancellous structures between the groups. Through the comparison it is possible to observe that the bony trabeculae in ovarectomized female rats are more spaced and with reduced conectivities in comparison to the normal rats group, characterizing a larger bony reabsorption by osteoclasts in trabecullary plates.

Second part of the study: (Table 2) presents the mean and SD values of the body mass measurements of female rats in control and treated groups, in baseline and at the end of the trial. Statistically significant differences (p= 0.005) in body mass gain between both groups were observed, larger in the control animals.

By means of the flexion assay it was possible to plot curves load versus deformation (Figures 4 and 5), so obtaining and comparing the values of mechanical properties of femoral shafts from different experimental groups. Table 3 shows the mean values and SD of mechanical properties of female rats in both groups: control and treated. It is observed that there were no statistically significant differences when mechanical variables were compared (maximum load p = 0,5603; maximum deformation p=0.1551; rigidity p=0.0546; resiliency p=0.5835; tenacity p=0.2926).

The quantitative calcium and phosphorus analysis showed that the group of ovarectomized female rats that underwent low intensity ultrasound treatment reached non-statistically significant values in comparison to those found in control animals (calcium, p=0.734; phosphorus, p=0.396). (Table 4) shows calcium and phosphorus values in both groups.

Figures 6-AB and 6-CD compare the femoral head region between the control and treated groups. Masson Tricromium staining allows studying the bony age. Mature bone is stained in blue and immature ones in red. It is possible to observe that, in control group, bony matrix is predominantly formed by mature bone, without indication of recent bony formation. In the treated group, there are neo-bony formation zones, indicating active osteoblasts. The increase in the osteoblastic activity was subjectively observed, without the use of morphometric methods.

Images obtained by electronic microscopy show structural differences of the femoral head region between control and treated group. It is possible to observe structural differences between the experimental groups, with a larger loss of trabecullar bone in control group (Figures 7 and 8).

DISCUSSION

Ovarectomized female rats is an useful model due to presenting biological mechanisms that are similar to those occurring in osteoporotic women^(11,14). This study showed that the ovarian hormone deficiency for 30 days leads to bony loss in mature female rats, and a body mass increase that is statistically significant in comparison to the control group (p < 0.05), supporting the literature reports showing that ovariectomy causes metabolic changes responsible for a pronounced increase in food intake and body mass (as a consequence of increase of body fat)^(5,18,19). The body mass increase is higher in ovarectomized rats in comparison to intact female rats in similar food-intake conditions^(5,13).

By means of electronic microscopy analysis it was possible to observe structural differences between the femurs of control and ovarectomized groups. The findings support the results of studies that also found trabecullar bone loss in consequence of lack of ovarian hormone^(1,6,7,18).

After the analysis of the efficiency of the ovariectomy protocol, it was studied the effect of low intensity ultrasound on osteopenic bones. Our results showed an increase in body mass that was statistically significant in the control group (p = 0.005) in comparison to the treated group, establishing a correlation between ultrasound and a lower body mass increase. However, there were not found in the literature works correlating ultrasound to body mass in ovarectomized animals.

Flexion test results had no statistically significant differences (p > 0.05) in relation to the several studied mechanic variables. The treatment did not interfere in femoral shaft mechanic properties. Ultrasound may have not stimulated an increase in the bony mineral content, and consequently in femoral shaft bony endurance, due to a lower change in bony metabolism in regions with predominance of cortical bone, making them less sensitive to ultrasound stimuli.

Quantitative results of mineral content did not show a significant increase of calcium and phosphorus in osteopenic bone of female rats treated with low intensity ultrasound in comparison to non-treated control female rats. Nevertheless this quantitative analysis failed to show statistically significant differences (p > 0.05) in comparison of the groups, qualitative findings pointed to structural differences between the groups.

The histological analysis (stained with Masson Tricromium) showed bony neoformation in the treated group which was not observed in the control group.

By means of electronic microscopy it could be noticed that in the group treated with low intensity ultrasound, bony micro-architecture appears to be more preserved than the one in the control group, with less bony trabecullar loss, making it to appear as more dense.

From the quantitative and qualitative obtained data, it can be presumed that treatment with low intensity ultrasound in osteopenic bone lead to an improvement in the bony trabecullate. Qualitative results showed a slight improvement of the architecture of treated bone. However, this increase and/or preservation of the mineral content was not quantitatively observed. Thus, it can be supposed that the quantitative analysis did not demonstrate any difference due to the presence of large quantities of new formed not yet mineralized bone ^{4, 12}. Perhaps the duration of the low intensity ultrasound treatment was not sufficient for allowing the total mineralization of the stimulated regions, preventing these changes to be quantitatively observed.

AKNOWLEGEMENTS

To FAPESP (#96/12198-2) and to CAPES for financial support.

Trabalho recebido em 29/09/2002

Aprovado em 27/11/2002

Work performed at Escola de Engenharia de São Carlos - (USP) at the Post-Graduate Course in Interunidades Bioengenharia and at the Ambulatory of the Orthopedics and Traumatology Department of Faculdade de Ciencias Médicas - UNICAMP.

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Publication Dates

• Publication in this collection
  16 Apr 2003
• Date of issue
  Jan 2003

History

• Received
  29 Sept 2002
• Accepted
  27 Nov 2002