Abstract
Background: The destruction of cartilage in patients with osteoarthritis is a consequence of an imbalance between matrix synthesis and degradation. The purpose of the present study was to determine the effects of electrical stimulation on these processes in full-thickness osteoarthritic adult human articular cartilage explants.
Methods: Full-thickness articular cartilage explants from osteoarthritic adult human knee joints were cultured in the absence or presence of interleukin-1ß (IL-1ß) and in the absence or presence of a specifically defined capacitively coupled electrical signal for seven or fourteen days. Total collagen and proteoglycan production were assessed by means of hydroxyproline and hexosamine analyses, respectively. Quantitative real-time polymerase chain reaction assays were used to measure mRNA expression levels of aggrecan, type-II collagen, collagenase-1 (MMP-1), collagenase-3 (MMP-13), stromelysin-1 (MMP-3), aggrecanase-1 (ADAM-TS4), and aggrecanase-2 (ADAM-TS5).
Results: Electrical stimulation of cultured explants for seven or fourteen days resulted in significant increases (p < 0.007) in proteoglycan and collagen production and a highly significant upregulation (p = 0.005) of aggrecan and type-II collagen mRNA expression. This occurred even in the presence of IL-1ß. In the absence of IL-1ß, the expression of metalloproteinases was at barely detectable levels in these explants. Treatment with IL-1ß led to the significant upregulation of metalloproteinase expression (p < 0.03), but simultaneous administration of the capacitively coupled electrical signal dramatically inhibited this stimulation.
Conclusions: The data show that, even in the presence of IL-1ß, a specific, defined capacitively coupled electrical signal can result in significant upregulation of cartilage matrix protein expression and production while simultaneously significantly attenuating the upregulation of metalloproteinase expression. These results support the contention that delivery of a specific, defined electrical field to articular cartilage could result in matrix preservation.
Clinical Relevance: Osteoarthritis ultimately results in the progressive destruction of articular cartilage through mechanisms involving metalloproteinase activity. The use of electrical stimulation to both increase matrix production and diminish matrix destruction has the promising potential to treat osteoarthritic patients in a noninvasive manner.
Human articular cartilage is a fascinating tissue. While it has the ability to transmit mechanical loads thousands of times—day after day—for many decades, it is a tissue without blood, nerve, or lymphatic supplies. The average chondrocyte seemingly resides alone in its lacuna in "splendid isolation," yet it has the ability—and sole responsibility—to respond to its environment and to preserve its surrounding matrix. This matrix is composed primarily of hydrated proteoglycan aggregates (containing aggrecan) providing compressive support embedded within a fibrous collagen network (containing type-II collagen) providing tensile strength. Both of these functional units are required to maintain the mechanical properties of hyaline cartilage1.
Although articular cartilage is relatively inert metabolically, the maintenance of its composition and function is nevertheless dependent on a critical balance between biosynthetic and degradative processes influenced by a variety of biological signals (e.g., growth factors and cytokines)1 and physical forces (e.g., joint loading and fluid flow)2-5. The physical forces are both mechanical and electrical. Because mechanical deformation during joint load-bearing produces internal electrical signals through streaming potentials2,4 and/or physicochemical changes6, it seems reasonable to suggest that the electrical fields that are generated would provide information to the chondrocyte. If so, perhaps externally applied electrical signals that mimic the endogenous electrical fields that are present in articular cartilage during weight-bearing may be able to help to maintain the integrity of the matrix by regulating the chondrocyte's anabolic and catabolic activities. Wang et al.7 provided a brief review of the effects of electrical stimulation on articular cartilage.
To this end, we recently reported that in vitro electrical stimulation can upregulate aggrecan and type-II collagen protein production and mRNA expression in fetal bovine articular chondrocytes7 and adult bovine articular cartilage explants8. On the catabolic side, it is generally recognized that interleukin-1 (IL-1) plays a major role in cartilage by downregulating collagen and aggrecan synthesis while upregulating metalloproteinase activities responsible for matrix degradation9.
Two major classes of metalloproteinases have been linked to cartilage matrix catabolism: (1) the matrixins, including MMP-1 (collagenase-1), MMP-2 (gelatinase-A), MMP-3 (stromelysin-1), MMP-9 (gelatinase-B), and MMP-13 (collagenase-3), and (2) the adamalysins, including ADAM-TS4 (aggrecanase-1) and ADAM-TS5 (aggrecanase-2). The collective activities of these enzymes are such that all major structural components of cartilage are ultimately susceptible. As a result, these enzymes also have been implicated in the destructive activities associated with osteoarthritis10,11. Moreover, it has been proposed that the chondrocyte, under the influence of cytokines, is the most probable source for these enzymatic activities9,12,13.
A logical approach to ameliorating or preventing cartilage degradation would be to stimulate matrix production and/or to inhibit metalloproteinase activity. In spite of the presence of the naturally-occurring tissue inhibitors of metalloproteinases (TIMPs)11,14, it is apparent that the normal homeostatic balance between enzymatic and inhibitory activity is often altered in favor of the former. As a result, the search for therapeutic inhibitors of metalloproteinase activities is quite active15-18. In the present study, we investigated the effects of capacitively coupled electrical stimulation on collagen and proteoglycan production and expression and on the interleukin-induced expression of a number of metalloproteinases in human stage-II osteoarthritic articular cartilage explants.
Procurement of Cartilage Samples
The distal part of the femur and the proximal part of the tibia of patients with radiographic stage-II osteoarthritis (defined as definite osteophytes and absent or questionable narrowing of the joint space)19 were procured from patients who were undergoing total knee replacement at Penn Presbyterian Medical Center, Philadelphia, Pennsylvania. These samples were obtained anonymously in compliance with the Declaration of Helsinki and were approved by the Office of Regulatory Affairs of the University of Pennsylvania. Over the course of five months, tissue was received from eleven patients, including five men and six women with a median age (and standard deviation) of 63.3 ± 11.1 years (range, forty-six to eighty-four years). Immediately after removal, the tissue specimens were kept moist in sterile phosphate-buffered saline solution supplemented with antibiotics (penicillin [100 U/mL], streptomycin [100 µg/mL], and amphotericin B [0.025 µg/mL]; Invitrogen, Carlsbad, California) and were transported to the laboratory on ice. Full-thickness cartilage explants were removed from the cartilage-bone specimens with sterile 4 or 8-mm-diameter cork borers, were placed in sterile phosphate-buffered saline solution, and were processed further under sterile conditions. With the exception of the specimens that were used for histological analysis, all subchondral bone was removed from each explant by dissection. All explants (2 to 3-mm thick) were briefly (for less than one minute) washed once with 70% ethanol and three times with sterile phosphate-buffered saline solution.
Experimental Design
None of the cartilage was specifically chosen for individual studies. In fact, the experiments were planned first and then sufficient cartilage was obtained from the surgical specimens that were available that day in order to carry out the experimental protocol. In distributing the cartilage among groups in any single experiment, we were concerned about the large morphological variability among specimens taken from the same joint immediately following knee replacement surgery. Close examination of the joint surface of the excised 4 or 8-mm-diameter explants revealed great variation in the surface contours and imperfections even within the same explant, such that it was impossible to "match" explants with any degree of confidence that like was being compared with like. Therefore, we randomized the explant specimens between unstimulated (control) and stimulated groups in each experiment. Table I and the text below describe the overall experimental design in detail.
Four experiments studied matrix production (µg hexosamine and µg hydroxyproline per µg DNA) with use of tissue from four patients. Each experiment involved the use of tissue from one patient from which as many as fifty explants (4 mm in diameter) were randomly selected and divided into five groups of nine or ten each. The explants in two experiments were harvested after ten days of culture, and the explants in two experiments were harvested after seventeen days of culture. Each of the four experiments was run independently. Because there was sufficient matrix in each 4-mm explant for all of the biochemical analyses, each experimental group yielded nine or ten data points, and the results of two experiments at each stimulation time were pooled for a final total of nineteen or twenty data points for each group.
These experiments studied gene expression (mRNA levels) with use of quantitative polymerase chain reaction with use of tissue from five patients. In the first experiment, twenty-eight explants (8 mm in diameter) were chosen at random from a total of forty explants from one patient and were distributed into four groups of seven explants each as detailed below. In the second experiment, twenty-eight explants (8 mm in diameter) were selected at random from the common explant pool of forty explants from two patients and were distributed into four groups of seven explants each as described above. The third experiment was carried out exactly as described above for the second experiment. Each of the three experiments was run independently. At the end of each experiment, the seven explants in each of the four groups were combined into three pools (containing two, two, and three explants, respectively) because there was not enough RNA in a single explant to be analyzed accurately. Thus, each experimental group yielded three data points, and the results of the three experiments were pooled for a final total of nine data points for each group.
An eighth experiment was performed to determine cell viability within representative explants. Sixty explants (4 mm in diameter) from one patient were randomly divided into five groups of twelve explants each. For analysis of the explants within each group, they were randomly divided into three groups of four explants each, giving three data points per group.
Last, a ninth study was performed in which six full-thickness cartilage explants (4 mm in diameter) were randomly selected from a pool of sixty explants (the remaining specimens were part of another study) and were submitted for histological evaluation.
Histological Analysis
Fresh osteoarthritic cartilage explants (4 mm in diameter) containing subchondral bone were obtained from both knee joints from one patient. All specimens from this patient were first photographed, and then six explants were randomly chosen, fixed in formalin, embedded in paraffin, decalcified in Immunocal (Decal Chemical, Tallman, New York) for forty-eight hours at room temperature, rinsed in 70% ethanol, and sectioned at 6 µm. Sections were subsequently stained with either hematoxylin and eosin or safranin O20,21. Representative sections were photographed.
Cartilage Explant Culture
For each tissue-culture experiment, fresh osteoarthritic cartilage from one or two patients was cut into multiple full-thickness explants (either 4 or 8 mm in diameter) and the subchondral bone was removed. The explants were randomly transferred into individual sterile modified Cooper dishes22 and were incubated for three days in Dulbecco's Modified Eagle Medium (Invitrogen) supplemented with 1% insulin-transferrin-selenium-G (Invitrogen), 1% antibiotics, and 50 µg/mL L-ascorbate (Sigma-Aldrich, St. Louis, Missouri) with the medium changed daily. Because all of the experiments involved the use of fresh tissue, it was impractical to obtain histopathological data on representative specimens used for in vitro experiments. Each electrical stimulation experiment used the cartilage freshly harvested from the joint or joints of one or two patients and contained either fifty to sixty 4-mm explants (matrix production experiments) or thirty-five to forty 8-mm explants (mRNA expression experiments).
For the matrix production experiments, 4-mm-diameter explants were randomly divided into five groups: (1) Day 3, (2) Unstimulated, (3) Stimulated, (4) Unstimulated + IL-1ß, and (5) Stimulated + IL-1ß (Fig. 1, A). On Day 3, interleukin treatment (100 ng/mL human recombinant IL-1ß [R&D Systems, Minneapolis, Minnesota]) and/or electrical stimulation were initiated for treated explant groups; untreated explant groups received neither. One untreated group was harvested on Day 3, and the remaining four groups were divided as shown in the figures and were harvested after either seven more days or fourteen more days. Each experiment was performed twice. In each of these experiments, the n value equals the total number of explants cultured and individually analyzed. This experimental design is identical to that described previously8.
For the mRNA expression experiments, 8-mm-diameter explants were randomly divided into four groups containing seven explants each: (1) Unstimulated, (2) Stimulated, (3) Unstimulated + IL-1ß, and (4) Stimulated + IL-1ß (Fig. 1). After three days of incubation, the groups were treated as shown in the figures and were harvested after seven more days. To ensure that sufficient amounts of total RNA could be isolated, multiple explants were combined and treated as described in detail below. This experiment was performed three separate times. Thus, data were obtained from three pairs of explants from three separate experiments, for a total of nine data points. The experimental design is identical to that described previously8.
To determine cell viability, chondrocytes were isolated from representative explants with use of sequential enzymic digestion with testicular hyaluronidase, trypsin, and bacterial collagenase as described previously7, stained with Trypan Blue, and counted with a hemocytometer.
Electrical Stimulation
For capacitively coupled electrical stimulation in vitro, electrodes are placed in contact with glass coverslips on opposite sides of the explant and a uniform field is generated between the electrodes. The term capacitively is used because the arrangement of electrodes and targeted cells/tissue resembles a capacitor (an electrical element used to temporarily store a charge) with the coverslip/gel + culture medium + explant + coverslip/gel constituting a continuous inter-electrode dielectric medium. Stimulation occurs by means of a transfer of electrical energy from the capacitor plate to the targeted tissue (coupling) through the induced electrical field23.
To construct the electrical stimulation apparatus, Cooper dishes were modified by cutting 33-mm holes in the top and bottom and covering the holes with 35-mm quartz coverslips that were glued to the plastic edges. An explant was placed on the bottom coverslip, and a circuit was established by completely filling the dish with tissue culture medium to eliminate air bubbles, placing the dishes on a common stainless steel electrode plate, attaching 33-mm stainless steel electrodes to each top coverslip with use of conductive paste, and then connecting both the top and bottom electrodes to a power source (Fig. 2). More details can be found in our previous report22.
In the experiments described, a capacitively coupled 20 mV/cm electrical field at a frequency of 60 kHz—a thirty-minute continuous stimulation (100% duty cycle) followed by a pulsed (one hour on and five hours off, four times per day) 50% duty cycle (one minute on, one minute off)—was delivered to the cultured explants beginning on Day 3 as described in detail previously7,22; a schematic description of the signal protocol is given in Fig. 1, C. Incubation was continued for either seven or fourteen days of stimulation, with the medium being changed every three or four days, at which time IL-1ß and vitamin C were replenished in the appropriate cultures. Untreated explant groups were treated identically, except that electrical stimulation was not applied. At the end of the experiment, each explant was harvested and frozen either for biochemical analysis or for RNA analysis as described below.
Biochemical Analyses
Explants were harvested after seven days (Day 10) or fourteen days (Day 17) of treatment, were washed with phosphate-buffered saline solution, and were frozen at -80°C until the time of analysis. All explants were thawed at 4°C, were cut into smaller pieces with a blade, and were papain-digested (Sigma-Aldrich)24. The digest (representing the whole explant) was analyzed for total DNA (as an index of cell number)25, hydroxyproline (as a measure of total collagen)26, and hexosamine (as a measure of total proteoglycan)27. All analytical data for each explant were expressed as a ratio to the DNA content of that explant.
Total RNA Isolation from Articular Cartilage Explants
At the appropriate times, explants were collected and immediately stored in RNAlater Solution (Ambion, Austin, Texas) at -80°C. Two explants from the same group were combined, cut into smaller pieces with a blade, and pulverized in liquid nitrogen with an equal volume of white quartz sand (Sigma-Aldrich) with use of a ceramic mortar and pestle. The resultant powder was transferred into a nuclease-free 60-mm-diameter Petri dish, and 2 mL of TRIzol Reagent (Life Technologies, Rockville, Maryland) was added. Total RNA was then isolated with use of the TRIzol Reagent method combined with a column-purification step (RNeasy Total RNA Mini Kit; QIAGEN, Valencia, California)28 according to the manufacturer's instructions and was stored at -80°C. This procedure resulted in DNA-free RNA suitable for amplification by quantitative real-time polymerase chain reaction (qRT-PCR) with high reproducibility and efficiency29. The typical yield of total RNA/explant was 15 to 20 µg.
Quantitative Real-Time Polymerase Chain Reaction
Reverse transcription followed by quantitative real-time polymerase chain reaction was performed as described previously7 with use of a two-step quantitative real-time polymerase chain reaction method (with reverse transcription and amplification in separate vessels) based on SYBR Green I detection chemistry30 in a Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, California). Oligonucleotide primers for human aggrecan, collagen type II, MMP-1, MMP-3, MMP-13, ADAM-TS4, ADAM-TS5, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, reference gene) were designed from published sequences with use of PrimerExpress (Applied Biosystems) software. To further ensure that the polymerase chain reaction signal was generated from cDNA (as opposed to genomic DNA), primer pairs were placed in different exons or across an intron/exon boundary wherever possible. All primer pairs had melting temperatures of 58°C to 60°C and yielded products of <140 base pairs; efficiencies of quantitative real-time polymerase chain reaction amplification were determined in a dilution series set of experiments under the same conditions as described by Ginzinger31. To verify that the expected products with respect to size and homogeneity were obtained, melting profiles of polymerase chain reaction products were acquired and aliquots were analyzed by means of electrophoresis on 2% agarose gels. Sequences of primer pairs and calculated reaction efficiencies are given in Table II.
Relative quantification of mRNA expression for each of the target genes was accomplished by normalizing to a housekeeping gene as described previously; GAPDH was chosen because it did not vary significantly with electrical stimulation in these cells7. For each experiment, corrected ?CT values (?CT [control - stimulated]) were obtained for pooled pairs of control (unstimulated) samples and experimental (stimulated) samples whose RNA was isolated, reverse-transcribed, and analyzed in duplicate or triplicate at the same time; for comparison among experiments, values representing relative gene expression were expressed as the ratio of stimulated/control.
Statistical Methods
For the data represented in Figures 4 and 5, analysis of variance was used to determine the effect of electricity on the levels of either hexosamine or hydroxyproline production at Day 10 or Day 17 in either the absence or presence of IL-1ß. For the data represented in Figure 6, analysis of variance was carried out to measure the effect of electricity on the levels of aggrecan or type-II collagen mRNA expression in either the absence or presence of IL-1ß. For the data represented in Figure 7, analysis of variance was performed to compare the control group with the control + IL-1ß group as well as the control + IL-1ß group with the control + IL-1ß + electricity group with regard to MMP-1, MMP-3, MMP-13, ADAM-TS4, and ADAM-TS5 mRNA expression. Since either two or three experiments are represented for each outcome measured, the analysis of variance included an "experiment" effect in addition to the treatment effect of interest. The data were analyzed on the basis of the assumption of a randomized block design (with patients serving as blocks) and the presence of stimulation as the treatment. The primary focus of the analysis was to compare a stimulated group with an appropriate control group in the absence or presence of IL-1ß; thus, separate analysis of variance models were fit for each comparison of interest. Each analysis of variance included a patient (i.e., block) effect as well as the treatment effect of interest. As each analysis of variance was fit to assess the difference between two levels (treatment and control), there was no need to perform post hoc testing. Logarithmic transformations of the response variables were performed where needed to meet analysis of variance normality assumptions, which were carefully checked with residual diagnostics. The Day-3 groups were not included in any of the analysis of variance models. The level of significance was set at p = 0.05.
Histological Analysis of Human Cartilage Explants
Visual inspection of the articular cartilage on removal at the time of total knee arthroplasty revealed no discernable "normal" cartilage. That is, there was no white, smooth, glistening cartilage anywhere on either the tibial or femoral side of the joint. A photograph of a typical articular cartilage explant is shown in Fig. 3, A. The normal smooth surface characteristic of articular cartilage is replaced with a surface containing fissures, ridges, and numerous other imperfections, signifying a loss of integrity. At low microscopic power (Fig. 3, B and C), the surface imperfections in a cartilage section are manifested by the presence of numerous vertical clefts. The matrix also shows evidence of disorganization by the presence of eosinophilic, safranin O-deficient "whorls" just beneath the surface. The latter can be seen at higher power (Fig. 3, E) to consist of chondrocytes scattered in a safranin O-poor matrix, signifying the loss of proteoglycan. A higher-power view of a cleft is shown in Fig. 3, F. As shown in Fig. 3, B, this cleft penetrates approximately 25% of the cartilage thickness. Surface irregularities, the presence of vertical clefts, and loss of safranin-O staining are characteristic changes seen in osteoarthritic articular cartilage32.
Effects of Ethanol Washing and Culture Period on Chondrocyte Number and Viability
Analyses showed that neither ethanol rinsing nor seven or fourteen-day culture with or without electrical stimulation had any significant effect on cell viability or normalized cell number (Table III).
Effect of a Seven-Day Electrical Stimulation Regimen on Matrix Production
Exposure of adult human articular cartilage explants to seven days of electrical stimulation resulted in a 1.8-fold increase (p < 0.0001) in proteoglycan (hexosamine) content (Fig. 4, A) and 1.7-fold increase (p = 0.0003) in collagen (hydroxyproline) content (Fig. 4, B) in comparison with unstimulated Day-10 controls. To simulate a disease state, treatment of explants with 100 ng/mL of IL-1ß for seven days resulted in a 21% loss of proteoglycan and a 25% loss of collagen as compared with untreated controls, but simultaneous application of electrical stimulation led to 1.3-fold (p = 0.1310, not significant) and 1.6-fold (p = 0.0003) increases, respectively, as compared with the interleukin-treated controls (Fig. 4, A and B). The effects of stimulation over this period can be more clearly appreciated if the pretreatment (Day-3) values for hexosamine and hydroxyproline content are subtracted from the Day-17 values: the increase in hexosamine is then on the order of nineteenfold, and that for hydroxyproline on the order of fortyfold. The interleukin-treated samples cannot be compared in this way because the unstimulated values are less than pretreatment values.
Effect of a Fourteen-Day Electrical Stimulation Regimen on Matrix Production
Extending the electrical stimulation experimental protocol to fourteen days resulted in nearly identical results, showing the maintenance of significantly elevated matrix production for hexosamine and hydroxyproline both in the absence (p = 0.0062 and 0.0016, respectively) and presence (p = 0.0209 and 0.0251, respectively) of interleukin (Fig. 5).
Effect of a Seven-Day Electrical Stimulation Regimen on Matrix Protein mRNA Expression
With use of an experimental protocol identical to that described above, it was shown that electrical stimulation also markedly upregulated the expression of both aggrecan mRNA (16.1-fold, p < 0.0001) and type-II collagen mRNA (10.5-fold, p < 0.0001) in the absence of IL-1ß. Although the response in interleukin-treated samples was attenuated, it was still highly significant for both aggrecan and type-II collagen mRNAs (p < 0.0001 and p = 0.0050, respectively) (Fig. 6). In these experiments, IL-1ß treatment appeared to have minimal effect on either aggrecan or type-II collagen mRNA levels in unstimulated samples. Similar results were obtained with use of adult bovine cartilage explants8.
Relative mRNA Expression of Metalloproteinases in Untreated Adult Human Articular Cartilage Explants
Quantitative polymerase chain reaction analysis of metalloproteinase expression showed that very low levels of MMP-1, MMP-3, MMP-13, ADAM-TS4, and ADAM-TS5 were detectable after seven days of culture. There were relatively large variations among experiments in the expression of all of the metalloproteinases studied (data not shown); but, for comparison, GAPDH, aggrecan, and type-II collagen mRNAs were expressed at levels from fifty to 350 times higher than metalloproteinases in these explants.
Effect of a Seven-Day Electrical Stimulation Regimen on Metalloproteinase mRNA Expression
With use of an experimental protocol identical to that described above, it was shown that, in the absence of IL-1ß, electrical stimulation had no appreciable effect on either MMP or ADAM-TS mRNA expression (Fig. 7). In contrast, incubation in the presence of 100 ng/mL IL-1ß resulted in highly significant (p < 0.0001 to p = 0.0284) increases in mRNA levels of both classes of these degradative enzymes, but simultaneous application of electrical stimulation resulted in highly significant (fourfold to fourteenfold) inhibition of this upregulation (p = 0.0023 to 0.0109), except in the case of ADAM-TS5, the expression of which was dramatically but not significantly reduced (p = 0.1446).
The most important findings of this study were that application of a specific and defined capacitively coupled signal7 that was on for only 4.5 hours per day over a one to two-week period8 to cultures of human osteoarthritic articular cartilage plugs led to (1) significantly increased collagen and proteoglycan protein production, (2) significant upregulation of type-II collagen and aggrecan mRNA expression in the absence or presence of IL-1ß in most cases, and (3) significant downregulation of the interleukin-induced mRNA expression of MMP-1, MMP-3, MMP-13, and ADAM-TS4 but not of ADAM-TS5. The overall effect is a concerted increase in matrix anabolism with a concomitant decrease in matrix catabolism.
Employing dose-response experiments for the past ten years, our laboratory has been systematically defining the parameters (e.g., frequency, amplitude, duration, duty cycle, and effective stimulation period) of capacitively coupled electrical fields in micromass cultures of fetal7 and adult (data not presented) bovine articular cartilage chondrocytes and adult bovine articular cartilage explants8. Those investigations led to the adoption of a one-hour stimulation period every six hours with use of a 60-kHz, 20-mV/cm, 50% duty cycle (one minute on/one minute off) signal to stimulate cartilage matrix production7. Other studies with micromass cultures of fetal bovine chondrocytes showed that just a thirty-minute stimulation every twenty-four hours using a 60-kHz, 20-mV/cm, 100% duty cycle (continuous) signal resulted in a marked downregulation of interleukin-induced MMP-1 expression in these cultures (data not presented). These field parameters were combined into the compound signal schematically depicted in Figure 1, C with the intention of increasing cartilage matrix production and decreasing cartilage matrix destruction. The data presented here offer proof of that concept.
Total proteoglycan and collagen production were significantly increased by approximately 1.8-fold and 1.7-fold, respectively, after seven days of electrical stimulation. Matrix production decreased in the presence of IL-1ß, but the concurrent application of electrical stimulation partially prevented this loss (1.3-fold and 1.6-fold for hexosamine and hydroxyproline, respectively) (Fig. 4). Stimulation for fourteen days gave similar results in both the absence and the presence of IL-1ß (Fig. 5). It is important to note that, compared with untreated samples, electrically stimulated samples showed that matrix production was increased over the two-week stimulation period in the absence of interleukin and was at least maintained in the presence of interleukin.
Subsequent analysis of aggrecan and type-II collagen mRNA expression levels in these cultures showed a marked upregulation (by 16.1-fold and 10.5-fold, respectively) after seven days of electrical stimulation. Although IL-1ß had little effect on mRNA expression of these targets, electrical stimulation under these inhibitory conditions still produced a significant upregulation compared with unstimulated samples (Fig. 6). Similar results were obtained in a previous study involving cultures of normal adult bovine articular cartilage explants8.
The expression levels of a variety of metalloproteinases known to be involved in cartilage degradation33,34 were also investigated in our human explant cultures as these enzymes are potential therapeutic targets in a number of musculoskeletal disorders35. In the absence of interleukin, mRNA levels of all of the enzymes that were investigated were very low in our samples and were so highly variable that there were no differences in expression among them (data not shown). Similar observations in human cartilage have been made by others36-40. Nevertheless, incubation in the presence of IL-1ß for twenty-four hours resulted in a marked upregulation in the expression of all of the metalloproteinases that were studied. This increase was also quite variable among different cartilage samples as well as among the individual enzymes, with the overall levels of induction from highest to lowest being MMP-1, MMP-13, MMP-3, ADAM-TS4, and ADAM-TS5. Concurrent electrical stimulation, however, resulted in a fourfold to fourteenfold inhibition of metalloproteinase expression (Fig. 7). Although the ADAM-TS enzymes responded to both IL-1ß and electricity, the effects were not as dramatic as those seen with the MMPs.
The above observations become important when one considers the therapeutic use of electrical stimulation for osteoarthritic patients. It is generally agreed that metalloproteinase activity is a vital step in the pathogenesis of osteoarthritis, yet there is still no effective way to locally inhibit enzyme activity or expression. Although there are numerous matrix metalloproteinase activity inhibitors in the offing, clinical trials have been somewhat disappointing. Problems include the systemic effect of these drugs as well as their potency, which can lower enzyme activity below basal levels16,41. An alternative approach, however, is to inhibit metalloproteinases at the gene transcription level42. Rather than employing systemic inhibitors, our approach has been to focus on the target area with use of capacitively coupled electrical fields. Our in vitro results demonstrate that mRNA levels of all of the metalloproteinases monitored were dramatically and significantly downregulated under electrical stimulation to values only slightly greater than basal levels (Fig. 6). Moreover, it is noteworthy that electrical stimulation in the absence of IL-1ß had no detectable effect on metalloproteinase expression (Fig. 7), suggesting that this modality might not affect enzyme levels in noninflammatory tissues.
The mechanism or mechanisms whereby capacitively coupled fields both stimulate matrix production and inhibit matrix destruction are not yet known. Nevertheless, the observed effects are generally consistent with an inhibition of the interleukin signal transduction pathways, which can proceed via the mitogen-activated protein kinase (MAPK) pathway and/or the nuclear factor-?B (NF-?B) pathway to elicit the production of transcription factors that inhibit their target genes42. Interference in interleukin signal transduction via these pathways could be responsible for the observed results43. On the other hand, previous studies of bone cells from our laboratory showed that capacitively coupled signal transduction proceeded via calcium influx through voltage-gated channels, resulting in subsequent increases in cytosolic calcium and cytoskeletal calmodulin and leading to production of prostaglandin E2 and transforming growth factor beta-1 (TGF-ß1)44-46. As TGF-ß can locally counteract the effects of interleukin in cartilage47, this relationship is also worthy of future studies involving capacitively coupled fields. A similar conclusion was reached with use of pulsed electromagnetic fields in a guinea-pig model of osteoarthritis48. Future long-term in vitro studies will compare the effects of electrical stimulation on normal human articular cartilage and will monitor histological changes occurring during treatment of osteoarthritic specimens.
It is interesting to note that Wang et al.49 found that the electrical potential at the surface of bovine articular cartilage after relaxation of a 10% compression load was 2 mV at zero seconds. If one adds dimensionality to Wang's calculations, the electrical field at the surface of the cartilage under the above conditions is 20 mV/cm (S.R. Pollack, personal communication), the same optimal field that we determined experimentally in the research described herein.
To date, we are aware of only three clinical trials that have evaluated the role of electromagnetic fields for the treatment of osteoarthritis50-52; none of those studies employed capacitive coupling. The main outcome measures were assessments of patient pain and mobility as well as physician evaluations. Those studies showed small to moderate clinical benefits, but no biochemical or genetic information was obtained. Our approach is to first use in vitro experiments to determine the most effective electrical signal parameters that result in reproducible, beneficial biological outcomes. If substantiated by an appropriate animal study, such findings could have profound clinical implications when this technology is transferred to patients with degenerative arthritis. Among the advantages of capacitive coupling are that it is noninvasive, it is easily adaptable to a variety of anatomic sites, it delivers effective stimulatory fields to a defined tissue area with relatively low applied voltages (~1 to 10 V), and it has modest power requirements, permitting portable units23. 
Note: The authors thank Craig Israelite, MD, (Department of Orthopaedic Surgery) for providing cartilage specimens, Tim Baradet, PhD, (Penn Center for Musculoskeletal Disorders core facility supported by NIH/NIAMS AR050950) for performing the histological procedures on cartilage explants, and Amy Praestgaard, MS, (Department of Biostatistics and Epidemiology) for performing the statistical analyses. This work was supported by a Sponsored Research Agreement between BioniCare Medical Technologies, Inc. (Sparks, Maryland) and the University of Pennsylvania.
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