In-Vitro Mechanisms of Cell Proliferation Induction: A Novel Bioactive Treatment for Accelerating Wound Healing

Author(s): 
Frank R. George, PhD;1 Ronald J. Lukas, PhD;2 John Moffett, PhD;3 Mary C. Ritz, PhD1

Introduction

Over the past several years, much has been learned regarding the molecular and physiological bases of wound healing,1 as well as the causes of various chronic wounds, such as pressure ulcers. Recent cellular and molecular studies have substantially increased our understanding of the elegant cascade of signaling events necessary for the wound healing process.1–11 For example, several important biochemical mediators of cell migration and growth have been identified that are involved in tissue reformation.6,12,13 It is understood that, in many instances, these regulatory signals do not appear to be functioning properly in chronic, nonhealing wounds.14 There are distinct phases associated with the process of wound healing, and it is clear that fibroblasts and epithelial cells are two of several cell types critical to establishing and progressing through the wound healing process.1 For example, fibroblasts must proliferate and synthesize collagen to provide a strong matrix for vascularization and epithelial growth.

Growth factors have been considered candidate therapeutics for wound healing because they are synthesized by and stimulate cells required for tissue repair, they are deficient in chronic wounds, and there is evidence that pharmacological application enhances wound repair in a variety of animal models.14 Today, growth factors refer to an expanding class of molecules, sometimes with specificity for certain types of cells, that can have either pro- or antiproliferative effects under differing circumstances. Among the growth factors implicated in tissue repair are insulin-like growth factor (IGF),15 platelet-derived growth factor (PDGF),6 transforming growth factor-beta (TGF-b),16 and epidermal growth factor (EGF).17,18 These molecules and their receptors are the likely molecular substrates for tissue repair. Fibroblasts and endothelial cells and their surface growth factor receptors represent critical cellular targets for growth factors and related molecules associated with wound healing.1–3,8,15

Based on the hypothesis that defects in growth factor signaling contribute to the development and/or persistence of pressure ulcers, reinstitution or normalization of that signaling, whether by introducing new sources of growth factor molecules or by reinstituting appropriate receptor coupling to second messengers, should promote wound healing. However, the complexity and variability of clinical wounds have limited pharmacological approaches to accelerate wound healing, leaving dressings and nonpharmacological ancillary modalities to dominate a market associated with wound management. For example, while numerous studies have cited the in-vitro efficacy of growth factor-derived compounds in promoting cell proliferation,3,4,8,9,13–15 the use of these types of compounds in clinical trials of wound healing typically have not produced encouraging results. One notable exception is the current use of topically applied, platelet-derived growth factor (PDGF) in the treatment of diabetic foot ulcers.19 Although application of this growth factor has demonstrated efficacy in the healing of these and other chronic wounds, it requires a complicated treatment regimen to ensure effectiveness.20

As a result of this work, it has become apparent that focusing on a single growth factor or related compound or receptor site is not the most effective way to initiate and sustain the complex cascade of events needed to progress the wound healing process. Rather, what is required appears to be an appropriate sequential stimulation of multiple growth factor expression and secretion. In light of this realization, a novel bioactive technology has recently been developed based upon the concept of endogenously stimulating the cellular processes that initiate proliferation of fibro-blasts with subsequent introduction of granulation and epithelialization leading to wound closure.21–23

This novel biotechnological approach to the treatment of wounds is based upon the mitogenic (i.e., cell-cycle stimulating) properties of a specific spatial-temporal conformation of a low-level, confined, high-frequency, electromagnetic field.22 This cell proliferation induction (CPI) technology* has combined an understanding of wound physiology, cellular proliferation mechanisms, and market needs to produce an effective treatment within a low-cost, user friendly, and scientifically based product. Preliminary findings have shown that presenting this field as a train of rapid pulses at or near the cycle time for Ca+2 channels promotes the release of endogenous growth factors and increases the number of cells entering and progressing through the cell cycle.21 This, in turn, triggers the cascade of second messenger events necessary for cell growth and proliferation.23 The net effect of this “energy to molecule” transduction is observed as a significant increase in the rate of cell replication.21–23

The purpose of this report is to present findings from a series of in-vitro studies evaluating the cellular mechanisms involved in CPI. Specifically, this report shows dose-response, time-course effects of CPI on fibroblast and epithelial cell proliferation. In addition, data are presented suggesting that the proliferative effects of CPI treatment result from the rapid stimulation of growth factor secretion via Ca+2-mediated signaling pathways.

Methods and Study Design

Cells. Cells used in these studies included Rat-2 fibroblasts, SA-1 human primary fibroblasts (a gift from Dr. Joan Shapiro, St. Joseph’s Hospital, Phoenix, Arizona), TE671/RD muscle cells (ATCC#CRL-8805) (American Type Culture Collection, Rockville, Maryland), and SH-EP1 epithelial cells (Sloan Kettering Cancer Center, New York, New York).

Culture conditions. In-vitro studies of effects of CPI treatment were conducted using cells maintained in an incubator (Forma Scientific, Marietta, Ohio) at 37?C and 95-percent humidity in five-percent CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose pyruvate, 2mM glutamine, 3.7g/L sodium bicarbonate (GIBCO BRL, Bethesda, Maryland), 10-percent heat-inactive horse serum, five-percent fetal bovine serum (FBS) (15% total serum concentration) (Hyclone, Logan, Utah), 100 U/mL penicillin, 100 U/mL streptomycin, and 2mg/L amphotericin B (SIGMA, St. Louis, Missouri), unless otherwise indicated.

Growth experiments. Cells at approximately 70-percent confluence in 100mm dishes were harvested and prepared as suspensions of 1–40 x 103 cells/mL, as verified by hemocytometer counting. Cells were transferred to 96-well plates to achieve initial plating densities of between 1,000 and 40,000 cells per well. Cells were allowed to attach for 24 hours before each experiment. Just prior to the start of an experiment, cells were removed from their incubator and one of several CPI treatment conditions at ambient temperature (24±1 degrees C) was administered. Except where noted below, the CPI dose was 32mw/cm2 (measured at the position of the plated cells).21 Control cells were handled identically and administered a dose of 0mw/cm2 (sham treatment). Following CPI treatment, plates were returned to the incubator for 24 hours. Cells were then fixed by 10-minute exposure to one-percent glutaraldehyde, washed twice with phosphate-buffered saline (PBS), then stained for 10 minutes at room temperature using 0.5-percent crystal violet (SIGMA, St. Louis, Missouri) solution.24 After plates were rinsed and air dried, cells were either counted microscopically using an inverted microscope and phase contrast optics, or crystal violet was resolubilized using 100mL Sorensons solution24 and absorbance at 550nm was quantified.

Statistical analysis. Statistical analyses were conducted using PRISM software, with Analysis of Variance (ANOVA) and t-tests used where appropriate to determine alpha levels. Alpha level for statistical significance was set a priori at 0.05.

Experiment 1. Human primary fibroblasts and rat fibroblast and epithelial cells were plated under serial dilutions into 96-well trays in DMEM supplemented as described above. Samples in triplicate were CPI treated for 30 minutes at one of the following logarithmically incremented doses: 0 (Control), 3, 10, 32, 56, 100, or 178mw/cm2. For this and all subsequent studies, the duty cycle was 4.2 percent. Twenty-four hours after treatment, cell numbers were quantified by direct cell counting and by spectrophotometric analysis following crystal violet staining. Results were normalized to serially diluted controls.

Experiment 2. Human and rat immortalized and primary fibroblasts and epithelial cells were tested and analyzed as described above. Samples in triplicate were CPI treated for one of the following treatment times: 0 (Control), 5, 15, 30, or 60 minutes at 32mw/cm2. Twenty-four hours after treatment, cell numbers were quantified by direct cell counting and by spectrophotometric analysis following crystal violet staining. Results were normalized to serially diluted controls.

Experiment 3. This set of studies determined whether CPI treatment enhances cell proliferation through a specific cellular-based mechanism. This was done by testing whether cell culture medium removed from CPI-treated cells could stimulate proliferation in naïve cells that had not been CPI treated.

Cells were grown and maintained under the conditions described above. For these studies, two different groups of Rat-2 fibroblasts were tested according to the following procedure. For the first group, the CPI-treatment group, cells were treated using CPI for 30 minutes at 32mw/cm2. Subsequently, the CPI-conditioned cell culture medium was removed from the plates containing these treated cells and transferred onto naïve cells at one of the following time points: 0, 1, 3, or 7 hours after CPI treatment. After the conditioned medium was removed, the CPI-treated cells were rinsed, and fresh, untreated medium was then applied. For the second group of cells, the naïve group, cells were sham-treated for 30 minutes at 0mw/cm2. Subsequently, the unconditioned cell culture medium was removed from these sham-treated cells and replaced with the CPI-conditioned medium. In addition, some CPI-treated cells were maintained in their treated medium for the full time of the experiment to serve as positive controls. For negative controls, some naïve cells were maintained in their untreated medium, never being CPI-treated or exposed to medium from treated cells. After 24 hours, cell numbers were quantified by spectrophotometric analysis of crystal violet staining as described above.

Experiment 4. Growth factors are known to stimulate cell growth through a number of intercellular pathways. One growth factor pathway relies on a Ca+2-mediated set of events in order to function. To determine if Ca+2-signaling pathways are involved in the induction of cell growth by CPI treatment, Rat-2 fibroblasts maintained as described above were treated with CPI for 30 minutes at 32mw/cm2. Immediately following CPI treatment, the calcium chelating agent, ethylene-glycol-bis (_-aminoethlyether)N,N,N,N,-tetraacetic acid (EGTA) (SIGMA, St. Louis, Missouri), was added to the medium at a concentration of 1.0mM. EGTA was maintained in the medium for one, three, or five hours post-CPI treatment. The EGTA was then washed from the cells with phosphate-buffered saline (PBS) and the cells were allowed to grow for an additional 16 hours.

Results

In preliminary studies, we treated RAT-2 fibroblasts with CPI and monitored the effect using microscopy. Figures 1A and B show a photomicrograph of untreated (A) and CPI-treated (B) cells 24 hours post-treatment, illustrating the robust increase in cell number following a single 30-minute CPI treatment at 32mw/cm2.

Experiment 1. As shown in Figure 2A, a single 30-minute CPI treatment significantly increased fibroblast proliferation (p (p

Experiment 2. As seen in Figure 3, fibroblast proliferation also varied significantly as a function of treatment duration (p

Experiment 3. Figure 4A shows the effects of CPI-conditioned medium on proliferation of naïve cells as a function of the amount of time the naïve cells spent in the CPI-conditioned medium. Treating naïve cells with CPI-conditioned medium produced a significant increase in proliferation (p

Figure 4B shows that when the medium was removed from CPI-treated cells and replaced with fresh medium, the proliferation effect was directly related to the length of time the CPI-conditioned medium remained on the cells
(p

The CPI-treated and untreated cell groups show similar results in their CPI-induced cell growth kinetics. With shorter exposure to CPI-conditioned medium, modest proliferation effects were seen, and these effects became more robust as the exposure time to the CPI-conditioned medium increased. Taken together, the results in Figures 4A and B indicate that 1) an initial immediate partial effect is apparent; but 2) cells must remain in the CPI treatment-enriched media for several hours, as evidenced by the three- and seven-hour time points, to achieve maximum stimulation of cell growth. Together these results indicate that CPI-treated cells release factors into the media and that these factors must be present to signal cells to initiate increased growth and proliferation. The results also suggest that the peak proliferative response to CPI requires de novo protein synthesis, since the CPI effect required several hours to reach its maximum point.

Experiment 4. To determine if calcium mediates the CPI effect, we treated cells with EGTA, a specific calcium ion chelator. The results suggest an important role for Ca+2 in mediating the CPI pro-proliferation effect. As shown in Figure 5, CPI-treated cells without EGTA showed a significant increase in proliferation relative to controls. Conversely, in the presence of 1.0mM EGTA, proliferation was significantly reduced relative to controls as a function of EGTA incubation time. These results indicate that CPI induces cell growth via a Ca+2 mediated process.

Taken together, the results of Experiments 3 and 4 suggest that CPI stimulates cell growth by inducing growth factors that in turn stimulate cell replication through a Ca+2 mediated intracellular pathway.

Discussion

This paper presents results from in-vitro studies using CPI, a bioactive stimulus that induces proliferation in cell types critical to wound healing processes. Recent clinical findings25 show that this technology can significantly enhance wound healing to closure. The present in-vitro results showing a cellular basis for this clinical effect contribute towards defining the conditions under which CPI treatment can be most effective.

The results of Experiments 1 and 2 establish that CPI-induced proliferation of fibroblasts and epithelial cells varies as a function of the treatment dose as well as the duration of treatment. These findings served as the basis for establishing dose- and treatment-time parameters for clinical studies as described elsewhere.25 In addition, the large windows of efficacy seen between doses of 32 to 178mw/cm2 and treatment durations of 15 to 60 minutes are consistent with a large therapeutic index for clinical use.

The mechanisms of wound healing are known to have distinct phases and involve several cell types, including keratinocytes, macrophages, fibroblasts, epithelial cells, and myofibroblasts. Initiation of the wound healing process involves fibroblasts and keratinocytes. These cells are thought to be stimulated to proliferate by a complex set of signals of which the most important appear to be a number of growth factors. Growth factors initiate cell proliferation by binding to their receptors and stimulating a series of internal pathways. These signal transduction pathways often utilize calcium as a key mediator.

The results from Experiments 3 and 4 provide evidence that CPI increases cell proliferation in fibroblasts by inducing the de novo synthesis of growth factors via stimulation of a Ca+2-mediated process. An important feature of CPI treatment is that it stimulates the proliferation of multiple cell types critical to the wound healing progress. This suggests that several different types of growth factors are being induced. The identification of the specific growth factors involved needs to be determined. Also, CPI may be inducing other gene products involved in the regulation of the cell cycle. It has previously been shown that other forms of magnetic fields can induce expression of c-myc, c-fos, c-jun, and PKC mRNA.26 Other genes that may be induced need to be determined by further studies. It has also been shown that in lymphocytes Ca+2 fluxes can be induced by certain electromagnetic fields,27 consistent with the data we have presented. Further, reports suggest that certain types of electromagnetic fields may activate macrophages, increase cell proliferation of fibroblasts, induce collagen synthesis, and increase the expression of receptors on human fibroblasts.26–31 Whether these same types of effects would be seen with keratinocytes and whether stimulation of keratinocyte activity is an important mediator of CPI effects in vivo remains the topic for future investigations.

CPI treatment has many features of conventional pharmaceuticals, such as dose and time dependence, in its pro-proliferative effects. These findings are consistent with induction of specific molecular event(s) during CPI treatment leading to a cumulative increase in cell numbers in treated, experimental samples relative to sham-treated control samples. Based upon these findings, we hypothesize that CPI treatment may have significant clinical efficacy in terms of accelerating wound closure by stimulating the release of novel growth factors that produce their effects through Ca+2-dependent messenger systems. While determining the specific nature of the growth factors and genes involved in this process and their precise relationship to the wound healing process requires future studies, this hypothesis is consistent with both the present findings and current understanding of wound physiology and cell-cycle mechanisms. The ability of CPI to induce proliferation in fibroblasts and epithelial cells suggests that this biotechnology may be of significant value in the treatment of chronic wounds.

*Provant® Wound Closure System (Regenesis Biomedical Inc., Scottsdale, Arizona)

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