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Author Response

Charles Godbout, PT, PhD, student

jerome.frenette{at}crchul.ulaval.ca

	Introduction

 
We welcome the opportunity to reply to Dr Spielholz’s comments. At first glance, we had the impression from Dr Spielholz’s comments that in vitro experiments are good and publishable if the results follow the mainstream of thoughts that are well accepted by the scientific or clinical community. Let’s start here with the raison d’être of in vitro experiments and wound closure model. In vitro experiments are used to investigate processes that are difficult or impossible to study in vivo. The main advantage of these studies is that we can control most of the parameters in order to obtain a very specific answer to a very specific question. Thus, in vitro experiments can provide useful insights to understand a process at the molecular, cellular, or tissue level. Obviously, and by definition, in vitro experiments do not mimic exactly the in vivo conditions. Regardless, all fields of medicine have progressed tremendously over the past century by using this logical and efficient approach that consists of doing research in a continuum that goes from in vitro experiments to animals and ultimately to human studies.

Getting back to our article, our question was: Can direct current (DC) promote cell migration in a model of wound closure in vitro? The answer to this question is "no." To help those who are not familiar with cells in culture, let’s look at the problem from a different angle. Cells in culture possess a contractile machinery (actin, myosin, calcium) that, when working in harmony and synchrony, allows cell locomotion. This contractile machinery is tightly regulated and, to some extent, is very similar to skeletal muscle. The next example may sound oversimple, but we believe that this explanation is the best to make our point clear to all readers. It is very easy to imagine how poorly a human being who is in good health would walk if we would apply constant electrical stimulation to the quadriceps femoris, hamstring, and gastrocnemius muscles during walking. The stride would be jerky, irregular, and inefficient. The same observation is actually seen with cells in motion. Direct current is probably perturbating and ultimately "freezing" the very complex mechanism responsible for cell migration in vitro.

Dr Spielholz does not agree that our model is one of wound closure. Again, let’s first define what a model and a wound are. A model is "something (as a similar object or a construct) used to help visualize or explore something else (as the living human body) that cannot be directly observed or experimented on."¹ A wound is defined as "an injury that causes a disruption of the normal continuity of a structure."² In our study, we used a construct (a cell layer) and created a disruption of the normal continuity of this layer to help visualize and explore cell migration during closure of that disruption. This is thus clearly a model of wound closure. Of course, we have never argued that our model recreates perfectly the complex process of healing at the cellular, molecular, and tissue levels. A detailed search by title/abstract of the National Center for Biology Information’s database (www.ncbi.nlm.nih.gov), using words such as "wound healing/closure in vitro" or "wound model in vitro," showed that a few hundred articles used a similar in vitro model to mimic wound healing or study cell migration. A lot of these articles were in very high impact journals. Have we all taken the wrong road? Probably not; we simply accept the limits of the wound closure model for what it is.

Another comment of Dr Spielholz concerned the decision of the Centers for Medicare and Medicaid Services (CMS) about the use of electrical stimulation for the treatment of chronic wounds.³ We would first like to mention that insurance coverage, lobbying, or economical considerations should not replace scientific judgment. In 2002, Medicare was finally willing to cover for electrical stimulation only for stage III or IV wounds as a second-line treatment after a contradictory decision. Why only as second-line treatment? Is it because it is not as effective as Dr Spielholz thinks, or simply because we do not know how to use it properly? Following the decision of the CMS, Ojingwa and Isseroff⁴ wrote a very good review on electrical stimulation and wisely suggested that the dermatologic community will need to develop treatment protocols and new therapeutic tools for the full advantage of patients. The CMS decision created new opportunities to perform additional investigations to increase our knowledge and refine our clinical practice. Additionally, the National Coordinating Centre for Health Technology Assessment in the United Kingdom mentioned that the evidence was generally insufficient to unequivocally conclude that electrical stimulation is beneficial for the treatment of chronic wounds.^{4, 5} The arguments of Dr Spielholz, unfortunately, are based on one misleading assumption that electrical stimulation is undoubtedly useful to heal wounds and that no other study, particularly in vitro, should be performed to improve our understanding of the complex mechanisms of healing under an electrical stimulation. The 2 questions that should be asked are: (1) Is the efficacy of electrical stimulation clearly established ("the study of things caused" or "the horse")? and (2) Do we really know what is happening during an electrical treatment ("the study of the causes of things" or "the cart")? We would personally answer "no" to those questions, and that is why additional studies are required to add new pieces of evidence. Furthermore, Ojingwa and Isseroff mentioned that more studies with larger sample sizes and rigorous study design are needed to be able to reach a firm conclusion regarding the utility of electrical stimulation in wound healing. Taken together, all of these observations strongly suggest that other in vitro and in vivo studies are essential for a clear comprehension of the effects of electrical stimulation, to define the mechanisms by which they are mediated, and to develop appropriate clinical protocols for wound treatment.

Dr Spielholz questioned our conclusions by pointing to the findings of a recent in vivo investigation by Demir et al.⁶ Briefly, they investigated the effects of electrical stimulation and laser on wound healing in rats. A full-thickness incision was performed dorsally on rats and then sutured. The electrical stimulation treatment started within 2 hours after the surgical procedure and continued for up to 10 days. Histological and biochemical analyses were realized at 4 and 10 days following incision, and biomechanical measurements were obtained after 25 days. This study was published in a very low-ranked journal and presented some important flaws that make the authors’ conclusions at least questionable. First, of a few examples, there was no comparison with a control group (without injury and treatment). The control group was actually a sham one where the incision and suture were realized but no active treatment was applied. Second, histological staining is not acceptable by today’s standard to distinguish between subsets of leukocytes such as polymorphonuclear leukocytes or macrophages. Cells can now be labeled by specific antibodies. Third, the counting technique and the analysis of collagen arrangement were poorly defined. Fourth, the authors pledged that there is a difference in collagen density and arrangement, but no quantification or images were presented. Fifth, and most importantly, the authors overextrapolated by concluding "that electrical stimulation and laser treatment can be used in decubitus ulcers and chronic wound treatment, in combination with conventional therapies."⁶ They started their treatment on a sutured wound 2 hours after injury. Was this really a chronic wound? Was the protocol in agreement with the time course promoted by Dr Spielholz? Were the conclusions appropriate for the method used? Obviously, the answer is "no" for each question, and we wonder why these points have not been highlighted in Dr Spielholz’s commentary.

We feel that we need to clarify some points about the study by Sillman et al.⁷ In this study, the authors actually observed fibroblast migration, but they did not find a galvanotactic response. Dr Spielholz emphasized that the authors did not mention terms such as "wounds," "wound healing," or "wound closure." Why would they? As opposed to us, they used a model in culture where fibroblasts were plated at very low density with basically no contact between each other. Why would isolated cells deprived of any contact and communication be more physiologically relevant and closer to in vivo situations, as suggested by Dr Spielholz? In vivo, cells usually communicate with others even in relatively hypocellular tissue. For instance, in tendons, where cells are negligible constituents of the whole tissue, the tenoblasts and tenocytes keep contact and communication between them by cell processes and gap junctions.^{8, 9} Dr Spielholz criticized the comments we made on the study by Finkelstein et al,¹⁰ who used a similar model of wound in vitro to study the effect of DC on cell migration. Contrary to what Dr Spielholz mentioned, field strengths of 4.0 and 6.0 V/cm were used only on sparse cells and not on wounded cell layers, the latter being the central point of our discussion. The legend of Figure 4 in the article by Finkelstein et al stated that electrical fields greater than 2 V/cm were not used on cell layers because they caused cells to round up and motility was compromised. If they did not find any cell death in their study, it was simply because they never looked for it. Cells that round up go automatically into apoptosis, and cell death is the ineluctable exit. Changes in pH are probably what is killing the cells. Without constant circulation, we observed extreme acidic and basic pH near the anode and the cathode, respectively. Those electrochemical changes do not happen suddenly in fields greater than 2 V/cm. They are gradual, and, without any monitoring, one cannot assume that the conditions were appropriate even at 2 V/cm or below. This is why we mentioned that results of Finkelstein et al were weakened by the fact that electrical fields greater than 2.0 V/cm are deleterious. Interestingly, other authors¹¹ admitted their incapacity to maintain sufficient conditions in their model to allow cell survival in the long run.

Dr Spielholz complained that the duration of our protocol lasted for only 8 hours but, in the case of Finkelstein et al, a 90-minute duration of stimulation was considered adequate. In our case, preliminary experiments indicated that fibroblasts in control conditions (no electrical stimulation) would close the wound in less than 8 hours. A prolongation of the stimulation protocol, therefore, was superfluous and unnecessary. Coming back to the article by Finkelstein et al, it is true that stimulation of NIH-3T3 fibroblasts with DC at 2.0 V/cm for 1.5 hours induced significant migration toward the cathode when compared with control cells and that cathodal-facing cells migrated faster than anodal-facing cells at 0.6 and 2.0 V/cm. However, as discussed in our article, when Finkelstein et al performed another set of experiments that included an additional 2.5 hours of incubation before the exposition of injured cell layers to DC, there was no increase of cathodal-directed migration and anodal-directed migration was retarded. In brief, DC could increase cathodal-directed migration immediately after wounding, but, if there is a delay of only 2.5 hours, the electrical stimulation is no longer beneficial. Does it sound like our results? Can we conclude that there is a window of opportunity of about only 2 hours where electrical stimulation can be applied?

One major disagreement of Dr Spielholz relates to the following sentence in our article: "We hope that these results will pave the way to determining the best time to use DC and refine the clinical decision-making process of physical therapists using electrotherapy." We did not mean that we should transfer our results directly to clinical practice, as Dr Spielholz seems to interpret our statement. Our statement means that we hope to stimulate other research in vitro or in vivo, and physical therapists need to be more critical about electrical stimulation. We like to believe that our results were all collected very meticulously and conscientiously and that a good report always raises more questions than answers. We think that we clearly explained the nature of this in vitro model and defined the implications of our results. We hope that readers will be more conscious about the use of electrical stimulation and that they will understand that electrotherapy is not ‘‘black or white,’’ but mainly composed of gray zone. Nevertheless, the beauty of science is that everything is true until someone challenges it, and the possibility to challenge ideas should not be limited. The new emerging technologies such as fluorescent staining of live cells should provide valuable information as to whether or not electrical stimulation inhibits movement of migrating cells in vivo. Audi alteram partem ...

	References

Top Introduction References

 

1. Merriam-Webster’s Medical Dictionary, 2002. Available at: http://dictionary.reference.com/search?q=model. Accessed October 11, 2005.
2. Hollister Web site, Wound Care Glossary. Available at: www.hollister.com/us/wound/resource/glossary.html. Accessed October 11, 2005.
3. Centers for Medicare and Medicaid Services. Coverage and Billing Requirements for Electrical Stimulation for the Treatment of Wounds, November 8, 2002. Available at: http://www.cms.hhs.gov/manuals/pm_trans/AB02161.pdf. Accessed October 5, 2005.
4. Ojingwa JC, Isseroff RR. Electrical stimulation of wound healing. J Invest Dermatol 2003;121 :1 –12.[ISI][Medline]
5. Cullum N, Nelson EA, Flemming K, Sheldon T. Systematic reviews of wound care management: (5) beds; (6) compression; (7) laser therapy, therapeutic ultrasound, electrotherapy and electromagnetic therapy. Health Technol Assess 2001;5 :1 –221.[Medline]
6. Demir H, Balay H, Kirnap M. A comparative study of the effects of electrical stimulation and laser treatment on experimental wound healing in rats. J Rehabil Res Dev 2004;41 :147 –154.[ISI][Medline]
7. Sillman AL, Quang DM, Farboud B, et al. Human dermal fibroblasts do not exhibit directional migration on collagen I in direct-current electric fields of physiological strength. Exp Dermatol 2003;12 :396 –402.[ISI][Medline]
8. McNeilly CM, Banes AJ, Benjamin M, Ralphs JR. Tendon cells in vivo form a three dimensional network of cell processes linked by gap junctions. J Anat 1996;189(pt 3) :593 –600.
9. Ralphs JR, Benjamin M, Waggett AD, et al. Regional differences in cell shape and gap junction expression in rat Achilles tendon: relation to fibrocartilage differentiation. J Anat 1998;193(pt 2) :215 –222.
10. Finkelstein E, Chang W, Chao PH, et al. Roles of microtubules, cell polarity and adhesion in electric-field-mediated motility of 3T3 fibroblasts. J Cell Sci 2004;117(pt 8) :1533 –1545.
11. Obedencio GP, Nuccitelli R, Isseroff RR. Involucrin-positive keratinocytes demonstrate decreased migration speed but sustained directional migration in a DC electric field. J Invest Dermatol 1999;113 :851 –855.[ISI][Medline]

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