Preoperative and Postoperative Topical Tretinoin on High-Tension Excisional Wounds and Full-Thickness Skin Grafts in a Porcine Model: A Pilot Study
Clark C. Otley, MD,* Scott M. Gayner, MD,
†
Iftikhar Ahmed, MD,* Eric J. Moore, MD,
†
Randall K. Roenigk, MD,* and David A. Sherris, MD
†
Departments of
*
Dermatology and
†
Otorhinolaryngology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota
background.
Tretinoin induces neovascularization and theformation of collagen when applied topically.
objective.
The goal was to determine whether preoperativeand postoperative treatment with tretinoin enhances the heal-ing of high-tension, full-thickness excisional wounds and thesurvival of full-thickness skin grafts.
methods.
A blinded, randomized, placebo-controlled pilotstudy involved high-tension excisional wounds and full-thick-ness skin grafts treated perioperatively with tretinoin in a por-cine model.
results.
Perioperative treatment of high-tension excisionalsurgery sites with tretinoin appeared to have no consistent ben-eficial or adverse effects on wound healing or scar spreading. Inthe full-thickness skin graft model, a trend toward impairedwound healing was noted.
conclusion.
The collagen-inducing effects of topical tretinoindo not appear to enhance the healing of high-tension excisionalsurgery wounds in a porcine model. Tretinoin does not appearto improve the survival of full-thickness skin grafts and, in fact,a detrimental effect was apparent in our model.
TOPICAL TRETINOIN CREAM has been shown toactivate fibroblasts and increase collagen, glycosami-noglycan, and elastin formation.
1–3
Theoretically tretin-oin-induced collagen formation might enhance woundhealing and minimize scar spreading in high-tensionwounds, thereby leading to superior surgical results. Al-though tretinoin has been used preoperatively to acceler-ate reepithelialization of partial- and full-thicknesswounds that heal by second intention,
4–7
there are nodata on its effect on excisional surgery.
An infrequently recognized effect of tretinoin isenhancement of papillary dermal vascularization, asdocumented by light microscopy and laser Doppler ve-locimetry.
1,2,8
Theoretically retinoid-induced neovascu-larization could increase blood supply to skin grafts,enhancing survival and improving outcomes. The effectof tretinoin on skin flaps has been explored, but no re-search has been done with skin grafts.
4–7,9
The two as-pects of this experiment are pathophysiologically unre-lated, but are applicable to the same animal model.
Materials and Methods
Animal Model
The Yucatan hairless miniature pig has been demonstratedto have skin of high histologic similarity to human skin.
10
Therapeutic trials involving reconstructive surgery and woundhealing using this model have shown good results.
9,10
Thestudy was approved by the Institutional Animal Care andUse Committee and all procedures were performed in ac-cordance with the Mayo Clinic Animal Care informationmanual.
Treatment
Excisional Surgery Experiment.
Preoperatively the shouldersand hips of the animal were treated seven times per week for4 weeks with either 0.25 g of 0.1% tretinoin cream or pla-cebo base, which was randomly assigned by a coin flip. Tat-toos ensured accurate application to treatment sites. Proce-dures were performed with the animals anesthetized withketamine and xylazine. One Yucatan hairless minipig wasused for the excisional and grafting experiment, with surgi-cal sites separated by at least 15 cm.
Following pretreatment with tretinoin and placebo, four 3cm
3
2 cm fusiform full-thickness excisions of normal skin toinclude superficial subcutaneous fat were performed onmatched areas of high tension over the shoulders and hips bi-laterally. The long axis of the ellipses were oriented perpen-dicular to relaxed skin tension lines to maximize tension. Acontrol elliptical excision under minimal tension was per-formed on the midflank. Standard, sterile surgical procedure
Address correspondence and reprint requests to: Clark C. Otley, MD,Department of Dermatology, Mayo Clinic, 200 First St. SW, Rochester,MN 55905.
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was used. The wounds were sutured under tension with 4-0vicryl interrupted dermal or subcutaneous vertical mattresssutures and 4-0 nylon interrupted epidermal sutures. Thewounds were dressed with antibiotic ointment and Telfa, andepidermal sutures were removed after 14 days.
Treatment with tretinoin and placebo cream was reinsti-tuted 5 weeks postoperatively, delayed so as not to irritate afreshly sutured wound and yet reinstituted so as to enhanceneocollagen formation and vascularization during the contrac-tion phase of wound healing. Punch biopsies (6 mm) were per-formed on days 24 and 84 postoperatively from matched sitesalong the excision scar (ie, anterior, middle, and posterior sec-tion). Biopsy sites were not sutured to avoid extra tension.
Skin Graft Experiment.
Four treatment sites were randomlyassigned by coin flip to be pretreated with either 0.25 g of0.1% tretinoin cream or placebo base as above. Anesthesia,operative, and postoperative procedures are outlined above.After 4 weeks of pretreatment, four 2 cm
3
2 cm circularfull-thickness excisions of normal skin were performed onmatched areas of the back. The excised tissue was defattedand the full-thickness skin grafts were replaced and suturedwith 4-0 nylon interrupted epidermal sutures. Cream appli-cation resumed after 5 weeks. One full-thickness 6 mmpunch biopsy specimen from the midback was processed forhistopathology to serve as a pretreatment control. At 10, 24,and 84 days postoperatively, 6 mm punch biopsy specimenswere taken from identical portions of the graft sites (i.e., 12-,6-, and 3-o’clock positions) and processed as above.
Clinical and Histologic Evaluation.
Blinded clinical and pho-tographic assessments were performed on postoperative days7, 14, 60, and 143. Variables for the excisional experimentincluded the width of incision, the presence and extent ofdehiscence and crusting, and a global subjective qualitygrade from 1 to 5 (1
5
worst, 5
5
best). Skin grafts were as-sessed for percentage graft survival, necrosis, and sloughing;presence of crusting and dehiscence; incision width; and anoverall subjective qualitative grade from 1 to 5. The resultswere assessed by three blinded investigators.
Staining of biopsy specimens was performed with hema-toxylin and eosin and Giemsa. Staining with inducible nitric
oxide synthetase to highlight vascular structures was unreli-able. Blinded histologic evaluation was done by a board-certi-fied dermatopathologist who assessed epidermal and dermalthickness, collagen bundle diameter, and scar width for theexcisional specimens and depth of necrosis, dermal thickness,vascular concentration and dilation, and scar width for graftspecimens. Subjective qualitative impressions were recorded.
Results
Excisional Surgery Experiment
After 4 weeks of cream application, a retinoid derma-titis was apparent at the tretinoin-treated sites. Post-operatively, blinded clinical assessments revealed nosignificant differences in all of the assessed variablesbetween tretinoin-treated sites and placebo-treated ex-cisional sites (Figure 1). There were imperfections atall high-tension surgical sites, but they were equivalentat tretinoin- and placebo-treated sites. In contrast, thelow-tension control site healed perfectly at all timepoints (Figure 1). At the final end point of 143 days,all excisional sites, including the tretinoin and placebosites and the low-tension control site, had similar clin-ical appearances.
Likewise, the results of our blinded histologic as-sessments revealed no significant differences in mostof the assessed variables between tretinoin-treated andplacebo-treated excisional sites, including dermal thick-ness, vascular concentration, vascular dilation, scarwidth, and qualitative scar features (Figure 2). The onlyvariable for which there was a significant difference be-tween tretinoin- and placebo-treated sites was dermalthickness at day 24, when the average tretinoin thick-ness was 8.2 mm, whereas placebo thickness was 5.6mm. However, by day 84 there was no difference. Atretinoin effect was evident after 4 weeks of tretinoinpreoperatively as increased epidermal thickness com-pared with the placebo site. However, once surgery wasperformed, this difference was not apparent. In contrastto the high-tension sites, the low-tension control site
Figure 1. A) Placebo-treated excision site with healing imperfections similar to tretinoin site on postoperative day 24. B) Tretinoin-treatedsite excision with healing imperfections similar to placebo site on postoperative day 24. C) Low-tension control excision site with perfecthealing on postoperative day 24.
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healed with a thinner epidermis and a narrower scar,which might be expected (Figure 2). At our final endpoint of 84 days, all high-tension surgical sites, includ-ing the tretinoin and placebo sites, had similar histo-logic appearances.
Skin Graft Experiment
Postoperatively the tretinoin-treated graft sites showedclinical evidence of impaired healing manifest by a de-creased percentage of graft survival, increased percentageof graft necrosis and slough, and lower overall subjectiveclinical quality grades (Figure 3). The adverse effectswere clearly apparent and maximal at the 7-day evalua-tion, gradually decreasing until the final evaluation atday 143. The final overall quality scores at day 143 werehigher for the placebo-treated sites. At day 143, the tret-inoin-treated grafts were significantly contracted relativeto the placebo-treated sites owing to extensive graft lossfrom ischemic necrosis. Table 1 shows the relative scoresof clinical evaluations at various times.
The histologic findings were comparable betweentreatment and control graft sites for most variables, in-cluding dermal thickness, vascular concentration, vas-cular dilation, scar width, and qualitative scar features.The one exception was an early trend toward increasedthickness of superficial necrosis at the tretinoin sites.The average depths of necrosis at days 10, 24, and 84for the tretinoin-treated sites and placebo-treated sites,respectively, were 1.1 and 0.9, 1.9 and 1.2, and 0 and0 mm (Figure 4).
Comment
Full-thickness wounds on high-tension areas such asthe back tend to heal with an increased incidence ofunsightly spread scars, despite optimal suturing mate-rials and technique. It is unreasonable to expect pa-tients to submit to prolonged inactivity to minimizescar spreading after surgery. A pharmacologic methodto enhance scar formation and reduce scar spreadingin high-tension areas would be clinically beneficial.
Theoretically tretinoin might enhance wound heal-ing and minimize scar spreading via the formation ofcollagen, glycosaminoglycans, and elastin.
1–3
This the-ory has never been explored. In our model, preopera-tive and postoperative topical application of tretinoinwas not associated with any significant beneficial ordetrimental effect. Histologic examination confirmedthe similar outcomes in placebo- and tretinoin-treatedsites. In contrast, the low-tension control demonstratedsuperior healing clinically and histologically, showingthat tension was a more important variable than wasmedication.
Several mechanisms might explain the lack of benefitfrom tretinoin. Tretinoin may simply be ineffective forthis purpose. Other explanations might include interfer-ence by the cellular and soluble mediator responseevoked by tretinoin. Inadequate duration of treatmentand the pilot nature of the study may have compro-mised the outcome. We performed a pilot study beforeembarking on a larger trial to define any obvious posi-tive or negative effects without excessive use of animals.
Figure 2. A) Placebo-treated excision site with scar similar to tretinoin site on postoperative day 84. B) Tretinoin-treated excision site withscar similar to placebo site on postoperative day 84. C) Low-tension control excision site with narrower scar on postoperative day 84. (He-matoxylin and eosin; magnification 23.)
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One of the drawbacks of skin grafts is the potentialfor ischemic necrosis, which can compromise out-come. Enhanced graft survival via tretinoin-inducedneovascularization is an appealing, albeit theoretical,solution.
1,2,8
This concept has never been explored. Inour model, preoperative and postoperative topical ap-plication of tretinoin was not associated with im-proved survival or outcome. Rather, graft sites treatedwith tretinoin exhibited a rather pronounced trend to-ward impaired wound healing, as evidenced by in-creased epidermal sloughing and necrosis, decreasedarea of graft survival, and lower scores on subjectiveglobal quality ratings. Histologic examination con-firmed the greater degree of necrosis with tretinoin.
Some explanations for the adverse effects of tretin-oin on grafts are similar to those for high-tension exci-sional surgery, including a true-negative effect, inter-ference from the retinoid inflammation, or small studysize. Explanations uniquely applicable to the grafts in-clude the possibility that tretinoin neovascularizationselectively induced arterioles, with resultant venoussludging due to inadequate venous drainage; con-versely, if venules were selectively enhanced, then noincrease in oxygenation might result.
Whether the adverse effects of tretinoin on skingraft survival in a porcine model are relevant to recon-structive surgery in humans is unknown. Tretinoin is acommonly used medication in the population under-
Table 1.
Average Blinded Clinical Scores of Graft Qualities
Figure 3. A) Placebo-treated graft with normal healing on postoperative day 7. B) Tretinoin-treated graft with impaired healing and necro-sis on postoperative day 7. C) Placebo-treated graft with hypopigmentation and intact graft tissue on postoperative day 143. D) Tretinoin-treated graft with hyperpigmentation and extensive contractural graft loss on postoperative day 143.
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going frequent reconstructive surgery; no apparent ad-verse effect on skin grafts has been noted by the authors.
Acknowledgment
The authors thank Christopher D.Marrs, RN, for expert technical assistance throughoutthe study.
The results were presented at the annual meeting ofthe International Society for Dermatologic Surgery,Amsterdam, The Netherlands, September 27, 1997.The research was supported by an intramural grantfrom the Mayo Foundation.
References
1. Kligman AM, Grove GL, Hirose R, Leyden JJ. Topical tretinoin forphotoaged skin. J Am Acad Dermatol 1986;15:836–59.
2. Kligman AM, Dogadkina D, Lavker RM. Effects of topical tretin-oin on non-sun-exposed protected skin of the elderly. J Am AcadDermatol 1993;29:25–33.
3. Griffiths CE, Russman AN, Majmudar G, Singer RS, Hamilton TA,Voorhees JJ. Restoration of collagen formation in photodamagedhuman skin by tretinoin (retinoic acid). N Engl J Med 1993;329:530–35.
4. Hung VC, Lee JY, Zitelli JA, Hebda PA. Topical tretinoin and epi-thelial wound healing. Arch Dermatol 1989;125:65–9.
5. Mandy SH. Tretinoin in the preoperative and postoperative man-agement of dermabrasion. J Am Acad Dermatol 1986;15:878–9.
6. Popp C, Kligman AM, Stoudemayer TJ. Pretreatment of photoagedforearm skin with topical tretinoin accelerates healing of full-thick-ness wounds. Br J Dermatol 1995;132:46–53.
7. Hevia O, Nemeth AJ, Taylor JR. Tretinoin accelerates healing aftertrichloroacetic acid chemical peel. Arch Dermatol 1991;127:678–82.
8. Grove GL, Grove MJ, Zerweck CR, et al. Determination of topicaltretinoin effects on cutaneous microcirculation in photoaged skinby laser Doppler velocimetry. J Cutan Aging Cosmet Dermatol1988;1:27–32.
9. Canady JW, Thompson SA. The effects of tretinoin on random skinflap survival in the swine model. Ann Plast Surg 1994;32:180–85.
10. Brodland DG, Cullimore KC, Roenigk RK, Gibson LE. Depths ofchemexfoliation induced by various concentrations and applicationtechniques of trichloroacetic acid in a porcine model. J DermatolSurg Oncol 1989;15:967–71.
Commentary
The allure of improving the natural healing process of the skinis a basic motive for all dermatologic surgeons to study woundhealing. This article studies the effect of an important topicalmedication which is well known to alter the baseline metabolicactivity of skin and enhance some of its reparative functions.The stated premise of the study is the search for a pharmaco-logic method to enhance the tensile strength in scar formation,to reduce scar spreading in a high tension area, and to enhancegraft survival through neovascularization of the graft skin. Itwas hoped that tretinoin could accomplish these theoretical en-hancements in the healing process and augment the clinical out-come.
In one sense, it is disappointing that this was essentially anegative study. On the other hand, negative studies are vital toour understanding and force us to look further for ways to pos-itively affect wound healing. While a pilot animal study cannotbe casually considered correlative to humans, these resultsshould call to question any assumption that may be made byextrapolations from other wound healing studies. Based on
other studies, tretinoin appears to stimulate the repair of someof the chronic ultraviolet light-induced damage, has been re-ported to speed the healing of full-thickness wounds, and en-hances the healing of the partial-thickness wounds of resurfac-ing, when used preoperatively. Until recently, these beneficialeffects have been largely unchallenged. There are studies whichhave reported enhancement of pretreated wounds and impair-ment of reepithelialization by continued use of tretinoin in par-tial-thickness wounds.
1
The conclusions of this study would indicate that there is nobenefit from topical tretinoin preoperatively and postopera-tively on high-tension wounds and that there is a trend towardsa less favorable healing of full-thickness skin grafts treated pre-operatively and postoperatively with tretinoin. When one ex-amines these results and tries to develop explanations, severalpossibilities come to mind. First, it could be theorized that theexpected changes induced by preoperative treatment with tret-inoin have no effect on the subsequent formation of scar in theensuing hiatus of 5 weeks when the tretinoin is not applied. It
Figure 4. A) Placebo-treated graft with lesser depth of necrosis onpostoperative day 10. B) Tretinoin-treated graft with greaterdepth of necrosis on postoperative day 10. (Hematoxylin andeosin; magnification 23.)
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may be that the outcome of the scar is most significantly deter-mined by the healing process within the first month after injury.A sutured wound would be expected to be in the remodelingphase of wound healing by the 5th week and beyond which iseither not significantly affected by tretinoin or is a phase ofwound healing in which the clinical results are not determined.
Another issue regarding the lack of benefit to high tensionsutured wounds has to do with whether or not the beneficial ef-fects of tretinoin exist beyond the superficial dermis. A fullthickness wound may not be significantly affected by the papil-lary dermal vascularization induced by tretinoin since the ma-jority of the injury exists in the deeper portion of the dermis.
Regarding the trend towards a worse outcome of the treatedfull thickness skin grafts, several potential explanations exist.Tretinoin’s effect on the cells of actinic keratoses is reported topromote the maturity of the cells which are in developmentalarrest. Healing skin has many characteristics in common withneoplasm and this reversal of neoplastic characteristics couldinhibit beneficial metabolic activities, thereby hampering thewound healing process. Another consideration for the tendencytowards worse results in tretinoin-treated full-thickness skingraft is the issue of metabolic requirements of the graft skin.Empirically, we know that the lower the metabolic requirementfor a graft, the higher the likelihood of complete survival of thegraft is. This is evidenced by the increased take of a split-thick-ness skin graft over a full-thickness skin graft in a poorly vascu-
larized wound. If the pretreated graft skin has neovasculariza-tion and an increase in collagen formation and has a generallyincreased metabolic activity, it may be at increased risk for fail-ure based on higher metabolic requirements. One may expect agreater degree of graft necrosis in tretinoin treated graft skin.
Although it is disappointing that this study did not show tre-tinoin to be an asset in the healing of high-tension suturedwounds or full-thickness skin grafts, it has secondarily raised is-sues of potential clinical import. Should the dermatologic sur-geon avoid harvesting grafts from donor sites treated preopera-tively with tretinoin? When is it safe to reinstitute tretinoinfollowing reconstruction? Is wound tension the prime determi-nant of postoperative spreading of scar? If so, are there anyother ways to positively affect the cosmetic wounds other thanmechanical tension reducing tactics such as periostial suspen-sion sutures and complete subcutaneous and dermal suturing ofthe wound edges? Only continued thoughtful and carefully con-trolled studies such as this can clarify these questions.
David G. Brodland, MD
Rochester, Minnesota
References
1. Hung VC, Lee JY, Zitelli JA, Hebda PA. Topical tretinoin and epi-thelial wound healing. Arch Derm 1989;125:56–69.
