Abstract
Background In addition to systemic treatment, osteoporosis may be treated topically by incorporating calcium and phosphate into the bone.
Objective This article describes the use of a recently developed, novel iontophoretic apparatus suitable for local ion delivery into bones. In this study, in vivo experiments were performed to compare the effects of local electrotherapy and those of systemic hormone replacement on bone.
Design In this study, local iontophoresis was carried out in ovariectomized and control rats. Bone density, biomechanical, and elemental studies were performed.
Methods Forty 12-week-old Sprague-Dawley rats received an ovariectomy (OVX) or were sham-operated (sham). Twenty-one weeks later, tibias of subgroups of sham-operated and OVX animals were subjected to serial local iontophoresis (IOP) treatments, received systemic subcutaneous 17β-estradiol (E2), or were treated with the combination of IOP and E2. Changes in bone density were detected by quantitative ultrasound densitometry and expressed as amplitude-dependent speed of sound (AD-SoS). Biomechanical studies and elemental analysis were performed at the end of the experiments.
Results Osteopenia developed 21 weeks after OVX in the proximal tibial regions; the mean difference estimate (95% confidence intervals) of AD-SoS values between the sham-operated and OVX animals was 188.7 (140.4–237.1). Serial iontophoretic treatment resulted in an increase in bone density in both sham-operated and OVX animals (sham+IOP versus sham: 121.4 [73.01–169.7]; OVX+IOP versus OVX: 241.6 [193.2–289.9]). Similar changes in AD-SoS were detected after 17β-estradiol (E2) treatment; however, even greater changes occurred after OVX+E2+IOP versus OVX+E2 (123.4 [75.1–171.8]). Similar improvements also were evident regarding the biomechanical features of the tibias.
Limitations A limitation of this study was the relatively small number of rats.
Conclusions The efficacy of local IOP using calcium- and phosphate-donating microparticles is comparable to that of estrogen therapy as evidenced by steadily increasing bone density, restoration of the calcium and phosphate balance, and improvement in the biomechanical properties of the bone.
Osteoporosis affects more than 750 million people worldwide. Without effective therapy and patient follow-ups, osteoporotic fractures may occur and lead to increased mortality, especially among elderly people.1 In Hungary, osteoporosis-related fractures are detected in approximately 50,000 patients per year.2 The systemic treatment of osteoporosis using bisphosphonates, strontium ranelate, teriparatide, raloxifene, and other compounds has been widely utilized and characterized.3 Hormone supplementation with estrogen therapy also has been used,4 but its application has been shown to carry higher potential risk5 than those of selective estrogen receptor modulators, bisphosphonates, or calcitonin.6 For decades, topical iontophoresis (IOP) and calcium ionostasis have been used to treat fractures, incomplete bone fusion, and osteoporosis.7–10 Various compounds, including calcium, phosphorous, magnesium, vitamin D, parathormone 1-34, and calcitonin, have been administered locally using IOP.11–14
We recently set up a new iontophoretic apparatus with calcium- and phosphate-donating microparticles and recently presented preliminary in vitro and in vivo data on the use of this technique for the topical treatment of osteoporosis. Our in vitro studies using porcine tissues demonstrated that both calcium and phosphate ions were absorbed into the bone. Preliminary in vivo assessments revealed that IOP increased amplitude-dependent speed of sound (AD-SoS), as determined by ultrasonography, in ovariectomized rats.10
In this article, we present in vivo observations on the effects of topical IOP with calcium- and phosphate-donating particles versus systemic estrogen supplementation.15,16 The effects were assessed via determination of bone density by ultrasound, biomechanical markers, and analytical composition of rat tibias.
Materials and Method
Animals
The experiments were initiated in female Sprague-Dawley rats with an average body weight of 200 g at 12 weeks of age. The animals were housed in an environmentally controlled room with a 12-hour light-dark cycle. They were supplied with commercial rat chow (Charles River, Wilmington, Massachusetts) and tap water ad libitum.
All animal procedures used in this experiment were approved by the Animal Welfare Committee of the University of Szeged and were performed in accordance with National Institutes of Health guidelines.17,18
Surgical Procedure of Ovariectomy (OVX)
Rats were randomly assigned to main groups of sham-operated (sham) animals or animals that received an ovariectomy (OVX) at the age of 12 weeks. In brief, the animals were anesthetized with a combination of ketamine and xylazine (25 mg/kg and 75 mg/kg intraperitoneally, respectively), and a median laparotomy was performed under sterile conditions. The connection of the Fallopian tubes was cut between hemostats, the ovaries were removed, and the stumps were ligated. Thereafter, the abdomen was filled with warm sterile physiological saline, and the abdominal wall was closed in 2 layers. Sham-operated rats underwent the same procedures except for the manipulation of the internal genital organs.
Quantitative Ultrasound Bone Densitometry (QUS)
Bone density measurements were performed at the tibia and the tail using a DBM-Sonic 1200 (IGEA, Carpi, Italy) ultrasonic bone densitometry device under ketamine-xylazine anesthesia (25 mg/kg and 75 mg/kg intraperitoneally, respectively). The values were expressed as changes in the average AD-SoS values.19,20 After calibration, the AD-SoS values of the soft tissues (muscle and skin) were determined, and the system deducted these values from the bone density. The AD-SoS values were calculated by a computer software program, and the average of 5 measured values was used at each time point of measurement. Measurements were conducted every second week between the age of 23 and 33 weeks. Twenty-one weeks after OVX (at the age of 33 weeks), statistically significant density alterations were observed in the proximal tibia; thus, this location was used for QUS measurements at the age of 36, 39, and 45 weeks (Fig. 1).
The time sequence of surgical interventions, treatments, and measurements. Vertical black boxes indicate a measurement with ultrasonic densitometry (QUS). OVX=ovariectomy, sham=sham operation, IOP=calcium-phosphate iontophoresis, E2=17β-estradiol treatment.
Local Calcium and Phosphate IOP
At the age of 33 weeks, the proximal portions of the tibias of 6 rats in the sham group and 6 rats in the OVX group were subjected to serial local iontophoretic treatment (sham+IOP and OVX+IOP, respectively) using a 3-electrode iontophoretic apparatus and calcium- and phosphate-donating microparticles as described previously10 (Fig. 1). In brief, we used silicate and hydrotalcite layer lattices as a calcium and phosphate source. Iontophoresis was performed using a 3-electrode instrument supplied by a one-phase network.10,18 In this apparatus, the voltage/current of the anode and cathode is separately adjustable.
Iontophoretic treatments were conducted at 33 weeks of age (day 0) and repeated 4 times (on days 2, 4, 11, and 18) around the left tibia of the animals under ketamine and xylazine anesthesia (25 mg/kg and 75 mg/kg intraperitoneally, respectively). This 30-minute treatment was carried out in the sham group, the OVX group, and a group of OVX animals that received chronic 17β-estradiol (E2) therapy (sham+IOP, OVX+IOP, and OVX+E2+IOP) (Fig. 1). During the treatment, the positive and negative electrodes were applied onto the skin at the 2 sides of the treated limb, and the third reference electrode was placed proximally to the other 2 electrodes. The region of interest for topical treatment (where bone loss was detected by bone densitometry) was marked on the skin. The instrument was constructed to comply with strict safety requirements.
Estrogen Therapy
In order to directly compare the effects of calcium-phosphate IOP with those of estrogen, E2 therapy was initiated in further subgroups of the OVX animals and was continued until the end of the experiments, at the age of 45 weeks. These effects were elicited by daily subcutaneous injections of 20 μg/kg 17β-E2 (Sigma-Aldrich Co, St Louis, Missouri) dissolved in 100% ethanol and diluted in corn oil, administered 5 days a week, at the time of the initiation of the IOP treatment (OVX+E2).21 The E2 treatment also was conducted in combination with IOP treatment (OVX+E2+IOP). The remaining 2 OVX subgroups (OVX and OVX+IOP) received vehicle treatment (combination of 100% ethanol and corn oil) only.
Experimental Design
Twelve-week-old rats were randomly assigned to 2 major categories of sham-operated animals (n=16) and OVX animals (n=24) and were further allocated to subgroups in a randomized fashion. Within the major categories, animals in subgroups 1 and 2 were sham-operated or ovariectomized and were not subjected to any further interventions other than QUS measurements (sham [n=10] and OVX [n=6]) (Fig. 1). Other sham-operated and OVX animals were subjected to serial local iontophoretic treatment (subgroup 3 [sham+IOP, n=6] and subgroup 4 [OVX+IOP, n=6], respectively) at the age of 33 weeks. At the same time, chronic E2 therapy was initiated in a further subgroup of the OVX animals (subgroup 5 [OVX+E2, n=6]). In some of the OVX animals, this intervention also was combined with IOP (subgroup 6 [OVX+E2+IOP, n=6]). Ultrasonic bone density changes were compared at the age of 33, 36, 39, and 45 weeks. At the end of the experimental protocol, the animals were overanesthetized (with a single overdose of pentobarbital, intraperitonially, 100 mg/kg), and their tibias were removed and subjected to biomechanical tests and elemental analysis.
Biomechanical Tests
At the end of the experiments, the left tibia of each rat was break tested in a 3-point bending procedure using a servohydraulic instrument (Instron 4302, Instron, Hungary).22,23 The soft tissues and periosteum were carefully removed from the tibia. Bones were immersed in Tris-buffered Ringer solution (pH 7.4) until further use. For the break test procedure, tibias were placed with their posterior side facing downward between the edges of the instrument (the gap being 6 mm in length), and the examined region included the proximal end and the mid-diaphysis of the tibia. The middle of this area then was pressed downward with a constant speed of 1.5 mm/s until the bone was broken. Load and displacement values were continuously recorded during this process. The maximum load (Fmax) (in newtons) was defined as the force resulting in breakage of the tibia. Stiffness (in newtons per millimeter) was defined as tangent of the angle between the linear region of the load-displacement curve and the x-axis.24
Elemental Analysis
At the end of the experiments, calcium and phosphate elemental analyses were performed on the removed tibias. All reagents used in elemental analysis were of analytical reagent grade unless otherwise stated. All experiments were carried out at room temperature. Triple deionized water produced by a Millipore Milli-Q RG apparatus (Millipore, Billerica, Massachusetts) was used to prepare all solutions. We used 65% nitric acid and 30% hydrogen peroxide (Spektrum 3D Ltd, Debrecen, Hungary) for the wet digestion of the samples. A Milestone MLS-1200 Mega MDR apparatus (Gemini BV, Appeldoorn, the Netherlands) was used for microwave digestion. Merck 1,000 mg/L calcium and 1,000 mg/L phosphate standards (Merck Chemicals, Darmstadt, Germany) were used for the calibration of flame atomic absorption spectrometry (AAS) and spectrophotometer instruments, respectively. For the assessment of phosphate mass concentration, L-(+)-ascorbic acid (Spektrum 3D Ltd) and phosphate reagent (sulfamic acid ACS reagent [Sigma-Aldrich], ammonium molybdate tetrahydrate [Sigma-Aldrich], antimony [III] chloride ACS reagent [Sigma-Aldrich], and L-(+)-tartaric acid ACS reagent [Fluka Chemical Corp, Milwaukee, Wisconsin]) were used.25 Flame AAS and spectrophotometry were performed by a Unicam SP 1900 spectrometer (Pye Unicam, Cambridge, United Kingdom) and a Hach DR 2000 UV-Vis direct reading spectrophotometer (Hach Lange GmbH, Dusseldorf, Germany), respectively.
All samples were prepared for elemental analysis by the following method. The wet digestion of the samples under atmospheric pressure was not complete; therefore, it was followed by fast microwave-assisted digestion. During the latter procedure, 0.5 g of solid samples, 3 mL of concentrated nitric acid, and 0.5 mL of hydrogen peroxide were placed into the Teflon (E. I. du Pont de Nemours and Company, Wilmington, Delaware) vessels and microwave digested for 5 minutes at 300 W followed by 5 minutes at 600 W. Sample solutions were filled with triple deionized water after digestion.
Calcium content of the digested sample solutions was assessed by flame AAS at 423.5 nm. Samples were analyzed in triplicates. The phosphate content of the samples was analyzed by spectrophotometry at 890 nm. Before measurements, 0.25 mL of ascorbic acid (10%) and 1 mL of phosphate reagent containing ammonium molibdenate were added to 25 mL of each digested sample. To avoid potential bias, biomechanical and elemental analyses were performed in a blinded fashion using coded samples.
Data Analysis
All data are expressed as means ± standard error of the mean. Data analysis was performed with SigmaStat statistical software (Jandel Corp, San Rafael, California) and with SPSS version 17.0 software (SPSS Inc, Chicago, Illinois). Changes in AD-SoS values between and within groups (over time) were analyzed by 2-way repeated-measures analysis of variance. For the other parameters, differences between groups were analyzed by 2-way analysis of variance. Pair-wise comparisons were performed based on estimated marginal means; P values were corrected by the Bonferroni method. Differences between values at P<.05 were considered statistically significant.
Role of the Funding Source
This work was supported by research grant T 048541 from the Hungarian Scientific Research Fund (OTKA) (Dr Szekanecz), research grants TAMOP 4.2.2-08/1-2008-0013 and TAMOP 4.2.1/B-09/1/KONV-2010-0005 (Dr Szabó), and research grant 315/10 from the Medical Research Council (ETT) (Dr Szekanecz).
Results
Bone Density Changes
As shown by the AD-SoS values, a decrease in bone density in the proximal region of the tibia was detected 21 weeks after OVX (at the age of 33 weeks) (Fig. 2), and the differences in AD-SoS values between sham-operated and OVX animals persisted until the end of the observation period (Fig. 3). Serial IOP treatment caused sustained elevations in this parameter after the third iontophoretic treatment in all of the treated groups, lasting during the rest of the examination period (Fig. 3). The E2 therapy resulted in a moderate increase in bone mineral density, which reached significance by 3 weeks (Fig. 2). The efficacy of E2 in increasing AD-SoS, however, could be greatly potentiated when OVX+E2 was combined with IOP. As a result, by the end of the examination period, bone mineral density was significantly lower in the OVX group than in any of the other groups. Furthermore, IOP brought about higher density values in all of the experimental groups (sham+IOP, OVX+IOP, and OVX+E2+IOP).
The time course of changes in bone density in the proximal tibia as measured by amplitude-dependent speed of sound (AD-SoS) in sham-operated (sham) and ovariectomized (OVX) rats and in those treated with topical calcium-phosphate iontophoresis (IOP) (sham+IOP and OVX+IOP), with 17β-estradiol-treated (E2) (OVX+E2), or with the combination of these treatments (OVX+E2+IOP). Serial IOP treatments were started at the age of 33 weeks (day 0) and are marked by arrows. Data are presented as mean±standard error of the mean. Two-way repeated-measures analysis of variance was used. Pair-wise comparisons were performed based on estimated marginal means; P values were corrected by Bonferroni method. *P<.05 vs sham, #P<.05 vs corresponding group without IOP.
The effects of iontophoresis (IOP) and 17β-estradiol treatment (E2) and their combination on the tibial bone density values measured ultrasonically by amplitude-dependent speed of sound (AD-SoS) in ovariectomized (OVX) and sham-operated (sham) rats measured at the end of the experimental protocol (45 weeks of age). Two-way analysis of variance was used. Pair-wise comparisons were performed based on estimated marginal means; P values were corrected by Bonferroni method. *P<.05 vs OVX (between groups without IOP), #P<.05 vs corresponding group without IOP.
Biomechanical Changes
Ovariectomy caused a significant reduction of Fmax values of the tibias, but these changes were not observed if OVX animals were treated with E2 (OVX+E2) (Fig. 4). Iontophoresis caused increases in this parameter in all groups examined. As a result, Fmax values significantly increased above the levels of the sham-operated and OVX+E2 animals. Likewise, Fmax values were restored to the levels of sham-operated animals if OVX animals were treated with IOP.
The effects of iontophoresis (IOP) and 17β-estradiol treatment (E2) and their combination on the maximum load of the tibias in ovariectomized (OVX) and sham-operated (sham) rats at the end of the experimental protocol (45 weeks of age). Two-way analysis of variance was used. Pair-wise comparisons were performed based on estimated marginal means; P values were corrected by Bonferroni method. *P<.05 vs OVX (between groups without IOP), #P<.05 vs corresponding group without IOP.
Similarly, untreated OVX animals had significantly lower stiffness values than sham-operated rats (Fig. 5). This loss of stiffness, however, could not be evidenced in the E2-treated OVX group. Iontophoresis caused complete restoration of this parameter in the OVX animals.
The effects of iontophoresis (IOP) and 17β-estradiol treatment (E2) and their combination on the bone stiffness of the tibias in ovariectomized (OVX) and sham-operated (sham) rats at the end of the experimental protocol (45 weeks of age). Two-way analysis of variance was used. Pair-wise comparisons were performed based on estimated marginal means; P values were corrected by Bonferroni method. *P<.05 vs OVX (between groups without IOP), #P<.05 vs corresponding group without IOP.
Changes in Bone Mineral Content
At the end of the experimental protocol, calcium mass concentration and concentration values were significantly lower in the OVX animals than in the sham-operated animals (Table). These measures of OVX-induced bone loss were prevented by E2 treatment (see OVX+E2). In response to IOP, calcium concentrations remained significantly elevated not only in the osteopenic OVX group but also in sham-operated and E2-treated animals even 10 weeks after the last IOP treatment.
Effect of Serial Local Iontophoresis (IOP) and 17β-Estradiol (E2) Treatment or Their Combination on the Analytical Calcium and Phosphate Content of Rat Tibias in Sham-Operated (Sham) and Ovariectomized (OVX) Animalsa
Phosphate concentrations, however, did not show significant changes in response to OVX. Calcium-phosphate IOP resulted in considerable elevations in this parameter only when sham and OVX+E2 treatments were combined with IOP.
Discussion
Osteoporosis affects millions of people, especially elderly women. Several systemically administered pharmacotherapeutic modalities have become available to treat this disease.1,2 Local osteoporosis therapy may be useful to treat local forms of bone loss (eg, fractures or complex regional pain syndrome).4–6 The use of IOP and calcium-donating microparticles enables the delivery of calcium and phosphate ions to the underlying bone.4–7,10,11 “Calcium ionostasis” was developed in Hungary in the 1950s.4–6
We have recently developed a new iontophoretic apparatus that uses calcium-donating microparticles, including silicate and hydrotalcite layer lattices, for the topical treatment of bone loss.7,12 Our preliminary in vitro studies using porcine tissues revealed that calcium and phosphate ions indeed penetrate the skin and the underlying bone. Furthermore, initial in vivo assessments suggested that IOP increased AD-SoS in OVX rats.7
The current study presents in vivo evidence supporting the efficacy of IOP and calcium-donating microparticles in increasing bone calcium and phosphate contents, increasing AD-SoS values, and improving the biomechanical properties of osteoporotic rat tibias. We compared the effects of IOP on sham-operated and ovariectomized rats, as well as the effect of estrogen therapy and the combination of electrotherapy and estrogen on OVX rat bones. The data showed that iontophoretic treatment increased the AD-SoS values in both sham-operated and osteopenic OVX rats. Five repeated treatments of IOP appeared to be steadily effective, as marked by a long-lasting restoration of bone density values (observed at day 90, 72 days after the last treatment). The OVX-induced osteopenia could be effectively reversed by E2 treatment. The data also show that IOP and estrogen monotherapy appeared to be similarly effective and that their combination provides the highest efficacy. The same conclusion can be drawn from the biomechanical parameters. As such, OVX resulted in significantly decreased Fmax and bone stiffness compared with the sham intervention. Iontophoresis or 17β-estradiol monotherapy was able to restore Fmax and stiffness lost after OVX. Likewise, the combination of the 2 therapeutic modalities was even more effective in normalizing the biomechanical properties of OVX rat tibias.
Elemental analysis provides further support for these findings. Ovariectomy caused a significant decrease in calcium content and mass concentration, but only a moderate reduction in phosphate content. Again, IOP increases these ion contents not only in OVX animals but also in sham-operated animals, and hormone therapy appears to be less effective than the combination of hormone therapy and electrotherapy in restoring bone mineral content.
The limitation of the proposed method mainly lay in the topical application of the IOP, which restricts it to areas where the electrodes can have a relatively close proximity to the bone and to each other. Potential species dependence of efficacy and imitations derived from the depth of penetration provided by the applied method also would limit extrapolation of the results to humans. Treatment of the most affected areas of osteopenia (ie, the vertebrae) may require further technical modifications. For these reasons, this novel approach may have therapeutic potential for local forms of osteopenia such as Sudeck atrophy. Other limitations of the present study and experimental model were the relatively small number of experimental animals included in the experiments and the limited clinical relevance (or even the risks of) estrogen supplementation as a basis of comparison for the effects of IOP in women with osteoporosis.
In conclusion, by means of this local new 3-electrode iontophoretic apparatus and the use of various calcium- and phosphate-donating microparticles, bone mineral density and biomechanical properties of the osteopenic bone can effectively be improved. The efficacy of this approach is comparable to that of chronic estrogen therapy, and the combination of these approaches further improves the effectiveness. Hence, topical IOP may represent an effective novel treatment modality in the management of local forms of osteopenia.
Footnotes
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Dr Pap and Professor Szekanecz provided concept/idea/research design. Dr Gomez, Dr Szabó, Dr Pap, and Professor Szekanecz provided writing. Dr Gomez, Dr Szabó, Dr Pap Jr, and Professor Szekanecz provided data collection. Dr Gomez, Dr Szabó, and Dr Boda provided data analysis. Professor Szekanecz provided project management. Dr Gomez, Dr Szabó, Dr Pap Jr, Dr Pap, and Professor Szekanecz provided participants. Dr Pap and Professor Szekanecz provided consultation (including review of manuscript before submission).
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This work was supported by research grant T 048541 from the Hungarian Scientific Research Fund (OTKA) (Dr Szekanecz), research grants TAMOP 4.2.2-08/1-2008-0013 and TAMOP 4.2.1/B-09/1/KONV-2010-0005 (Dr Szabó), and research grant 315/10 from the Medical Research Council (ETT) (Dr Szekanecz).
- Received November 19, 2010.
- Accepted September 26, 2011.
- © 2012 American Physical Therapy Association
References
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