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Research Reports |
nspielholz{at}adelphia.net
 | Introduction |
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The literature contains conflicting reports about whether fibroblasts on glass slides migrate in a direct current (DC) field or not. Like these authors, previous researchers1 also reported they do not, but other researchers2–5 have reported they do. Workers in this area will have to sort this out. My disagreements with this article are quite different. First, and most importantly, I will address various aspects of the authors’ claim that "The model of wound closure [used in this study] is well accepted to mimic wound healing in vitro." I argue that this is not a model of "wound healing," and I will show this by comparing the findings of this study with the findings of some in vivo studies.6–9 Indeed, the authors’ concluding statement, "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," far exceeds the method used. Second, the claim that this in vitro preparation is "well accepted" as a model of wound closure and wound healing might surprise other investigators.1, 2, 10, 11 Third, the description of "weaknesses" inherent in a study that did report fibroblast migration in vitro using a DC field2 is itself flawed.
| The Model Is Not One of a Wound or Wound Healing |
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 TopIntroduction The Model Is Not... Contrasting the Authors'...How the Authors Explain...References |
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I claim: None of the above. First, this "model of wound healing" ignores the fact that, following real wounding, fibroblasts are not among the first cells to enter a wound. Fibroblasts only appear days later, after other cell types (and their secretions) have modified the wound bed.12 None of these other cells or their secretions are present in this model. Scratching a confluent layer of fibroblasts and then seeing whether cells migrate into it over the next 8 hours does not make this a "model of wound closure." Migration, yes; clinical wound closure, no. Second, because there is no tissue from epidermis to periosteum consisting solely of a layer of confluent fibroblasts, this "model of healing" recedes further from mimicking reality.
I am not arguing against this in vitro preparation. When used and interpreted appropriately, it is indeed a unique method for studying how controlled perturbations of artificial environments may affect cell migration. And, although it is true that this preparation is sometimes referred to colloquially as a "wound" (as in "Migration and proliferation of diploid human fibroblasts following ‘wounding’ of confluent monolayers"10(p235) or "‘wounding’ confluent cell monolayers exposes a cell-free stripe"2(p1533)), this term is obviously used metaphorically (and more about this later).
Because this preparation does not model healing of an acute wound, it is even further removed from the "chronic wounds" that are approved for treatment with electrical stimulation.13 And to cap the irrelevance to clinical practice, electrical stimulation clinically is not instituted until a stage III or IV wound, regardless of etiology, has shown no healing for at least 30 days.13 How much further from real wound healing and clinical applicability can this model get?
In addition to claiming that this in vitro preparation mimics wound healing, Godbout and Frenette also declare, "Although human or animal studies may provide very important information, in vitro studies are essential to evaluate the effects of external stimulations at the cellular or molecular level." Thus, in vivo studies "may provide very important information" but "in vitro studies are essential to evaluate...." This clearly puts the cart before the horse. Hughlings Jackson warned against such thinking when he advised, "The study of the causes of things must be preceded by study of things caused."14(p15) If this in vitro study had been done prior to observing that electrical stimulation was beneficial, it could have been concluded that electrical stimulation was useless for "wound closure," and the clinical investigations showing benefit might not have been done.
We can test the authors’ claims that this is a "model of wound healing" and that it is "essential to evaluate the effects of external stimulations." Let’s compare the findings of this in vitro study with those of a recent in vivo study using 124 rats.9 Following full-thickness wounding, 2 different treatments (DC stimulation or laser therapy) were administered daily for 10 days, and treated rats were compared with sham-treated controls. Histological, biochemical, and biomechanical outcome measures were performed 4, 10, and 25 days after wounding. Consult the article for quantitative details, but here are some quotations concerning the findings:
Both treatment modalities had positive effects on the proliferation phase, increasing the fibroblast number and hydroxyproline level, and stimulating the synthesis and organization of collagen compared with their control groups.9(p149)On the 4th and 10th days, the collagen density and arrangement were significantly better in the ES [electrical stimulation] and laser treatment groups than in their controls.9(p149)
The difference in fibroblast number between the ES and the ES sham groups ... on the 4th day indicates the beneficial effect of ES treatment on the early proliferative phase, and this finding correlates with the literature.9(p151)
Tissue hydroxyproline level is accepted as an important parameter in the evaluation of collagen metabolism ... we too found a significantly increased level of hydroxyproline in the ES group compared to the sham ES group on the 4th and 10th days.9(p152)
Both ES and laser treatment increased the wound breaking strength significantly compared with their control groups....9(p152)
Which is more "essential" to determining whether DC promotes healing or not, the in vivo preparation or the in vitro preparation?
One last point concerning the conclusion based on this "model": Godbout and Frenette claim that because DC did not facilitate fibroblast migration into the scratch, "periodic DC does not promote wound closure." But even if nonmigration of fibroblasts is correct (which other studies2–5 contradict), the conclusion ignores the contributions of the other cells involved with healing (eg, keratinocytes, macrophages) that are influenced by electrical fields.15–17 Therefore, the blanket statement that "DC does not promote wound closure" because one cell type did not migrate into a scratch is an inappropriate generalization.
| Contrasting the Authors’ Interpretation of Their Findings of Nonmigration With the Interpretations of Other Authors Who Also Reported Nonmigration |
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TopIntroductionThe Model Is Not...Contrasting the Authors'...How the Authors Explain...References |
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| How the Authors Explain the "Weaknesses" of Other Reports That Fibroblasts Do Migrate in an Electrical Field |
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TopIntroductionThe Model Is Not...Contrasting the Authors'...How the Authors Explain...References |
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This article attempts to explain why the migratory findings of Finkelstein at al2 are suspect. The authors state, "The significance of Finkelstein and colleagues’ results was weakened by the fact that EFs [electrical fields] greater than 2.0 V/cm are deleterious for cells and that pH measurements and culture medium perfusion were not performed."
I do not understand, if Finkelstein et al2 were careless with controlling the pH and other aspects of their perfusion medium, how such "errors" would cause cells to migrate in a DC field, which Godbout and Frenette did not find with their "correct" pH and perfusion medium. In fact, this "criticism" actually contradicts the 13 references given in this article addressing the importance of pH, because if Finkelstein et al erred, not only should they not have found migration, they should have found cell death. But they did not; they found migration. So is this a valid criticism of the study by Finkelstein et al?
Now back to the authors’ criticism that "The significance of Finkelstein and colleagues’ results was weakened by the fact that EFs greater than 2.0 V/cm are deleterious for cells...." Finkelstein et al,2 studied the effects of DC fields of different strengths (0, 0.6, 2.0, 4.0, and 6.0 V/cm) on both "sparse" cell populations and "confluent layer" populations. For readers who are not familiar with cell culture, in confluent layers, cells are so numerous that they touch one another all around, resulting in "contact inhibition of locomotion."18 Conversely, in sparse cell populations, cells are farther apart and freer to move (clearly more like the situation in vivo). What did Finkelstein et al report? To quote them: "When applied to monolayers and sparse cells, EFs elicited intriguingly different responses. First, dose dependence differed markedly. For sparse cells, EFs <2 V·cm–1 produced no effects, 2–6 V·cm–1 linearly increased speed and directionality, and EFs 
10 V·cm–1 were poorly tolerated. By contrast, for wound-facing monolayer cells, EFs of only 0.6–2 V·cm–1 yielded directional motility; these EFs yielded no time lag before cells commenced cathode-facing migration and no change in final speed. EFs 2 V·cm–1 were deleterious."2(p1542)
This fuller description contrasts with the authors’ criticism that the work of Finkelstein et al2 was "weakened by the fact that EFs greater than 2.0 V/cm are deleterious." Obviously, the field strengths of 2 to 6 V/cm to which the sparse cultures were exposed were not "deleterious," because these fibroblasts migrated, while fibroblasts in the confluent cultures that migrated did so to field strengths not greater than 2 V/cm. How does the comment that "field strengths greater than 2.0 V/cm are deleterious" bear on what Finkelstein et al reported?
In summary, my major disagreement with this article is not that it reports nonmigration of fibroblasts in a DC field, but that it states a clinical conclusion based on an in vitro model that does not mimic wound closure or wound healing. Generalizations that reach far beyond the methods are not only inappropriate but also may be misleading. Furthermore, although in vitro studies can reveal much about mechanisms of action, the claimed "essentiality" of them over in vivo studies is backward. Last, the 2 suggested "weaknesses" of a study that showed that fibroblasts do migrate under a DC field do not themselves hold up to scrutiny.
| References |
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TopIntroductionThe Model Is Not...Contrasting the Authors'...How the Authors Explain...References |
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