Could -79 °C Spray-Type Cryotherapy Be an Effective Monotherapy for the Treatment of Keloid?

Abstract

Cryotherapy has been regarded as an effective modality for the treatment of keloids, and the spray-type device is one of the novel cryotherapeutic units. However, the biological mechanisms and therapeutic effects of this technique are incompletely studied. We evaluated the clinical efficacy of our cryotherapy protocol with molecular and pathologic evidence for the treatment of keloids. We evenly split each of ten keloid lesions into a non-treated (C-) and treated (C+) area; the C+ area was subjected to two freeze-thaw cycles of spray-type cryotherapy using -79 °C spray-type CryoPen™. This treatment was repeated after an interval of two weeks. The proliferation and migration abilities of the fibroblasts isolated from the dermis under the cryotherapy-treated or untreated keloid tissues (at least 5 mm deep) were compared and pathologic findings of the full layer were evaluated. Molecular analysis revealed that the number of dermal fibroblasts was significantly higher in C+ group as compared with C- group. The dermal fibroblasts from C+ group showed more than two-fold increase in the migration ability as compared with the fibroblasts from C- group. The expression of matrix metallopeptidase 9 was increased by more than two-fold and a significant increase in transforming growth factor beta 1 expression and Smad2/3 phosphorylation level was observed in C+ group. C+ group showed more extensive lymphoplasmacytic infiltration with thicker fibrosis and occasional "proliferating core collagen" as compared with C- group. Thus, -79 °C spray-type cryotherapy is ineffective as a monotherapy and should be used in combination with intralesional corticosteroids or botulinum toxin A for favourable outcomes in the treatment of thick keloids.

Keywords: cryotherapy; fibrosis; keloid; matrix metallopeptidase 9; transforming growth factor β 1.

Figure 1

Schematic illustration of our tissue sampling for pathologic and molecular study. (A) The dermal keloid (3 mm thick and at least 5 mm deep) from the epidermis was used for primary cell culture, (B) while the adjacent full layer from the epidermis to reticular dermis was used for pathologic examination.

Figure 2

Clinical cases of keloids treated with −79 °C spray type CryoPen™. (A) A 56-year-old female patient with anterior chest keloid was presented with recurrent central ulceration, pruritus, and intermittent pain; (B) Split therapy was performed such that the right half was treated with cryotherapy, while the remaining left half was left untreated. The therapeutic effect was noticed 1 week following four sessions of cryotherapy in terms of improvement in the texture, softness, and redness accompanied with a slight shrinkage of the right treated half; (C) Three weeks after the four sessions of cryotherapy, the lesion reverted to its original status unless it was subjected to repeated cryotherapy; some skin erosion was observed in the treated half (small white arrow). Keloid excision and full thickness skin grafting from inguinal area were planned; (D) A 21-year-old female patient was presented with spontaneous keloids of the upper arm and axilla; (E) Split therapy was performed such that the lower half was treated with cryotherapy, while the upper half was left untreated. The therapeutic effect was evaluated after four sessions of cryotherapy in terms of reduction in the volume or shrinkage of keloid. Cryotherapy was ineffective and caused skin erosion at the site of cryotherapy; no volume reduction or shrinkage (small white arrow) was observed; (F) Post-treatment appearance of keloid at 3 weeks after final cryotherapy. The erosion was improved with conservative wound management. Keloid excision and primary closure were planned.

Figure 2

Clinical cases of keloids treated with −79 °C spray type CryoPen™. (A) A 56-year-old female patient with anterior chest keloid was presented with recurrent central ulceration, pruritus, and intermittent pain; (B) Split therapy was performed such that the right half was treated with cryotherapy, while the remaining left half was left untreated. The therapeutic effect was noticed 1 week following four sessions of cryotherapy in terms of improvement in the texture, softness, and redness accompanied with a slight shrinkage of the right treated half; (C) Three weeks after the four sessions of cryotherapy, the lesion reverted to its original status unless it was subjected to repeated cryotherapy; some skin erosion was observed in the treated half (small white arrow). Keloid excision and full thickness skin grafting from inguinal area were planned; (D) A 21-year-old female patient was presented with spontaneous keloids of the upper arm and axilla; (E) Split therapy was performed such that the lower half was treated with cryotherapy, while the upper half was left untreated. The therapeutic effect was evaluated after four sessions of cryotherapy in terms of reduction in the volume or shrinkage of keloid. Cryotherapy was ineffective and caused skin erosion at the site of cryotherapy; no volume reduction or shrinkage (small white arrow) was observed; (F) Post-treatment appearance of keloid at 3 weeks after final cryotherapy. The erosion was improved with conservative wound management. Keloid excision and primary closure were planned.

Figure 3

Cryotherapy increases the migration and proliferative activity of keloid dermal fibroblasts. (a) The comparison of the proliferative activity of the dermal fibroblasts from keloids before and after cryotherapy. Cell proliferation was promoted in the fibroblast treated with cryotherapy. The cell number was estimated at 48 h after cell seeding; (b,c) The migration activity of dermal fibroblasts was increased following cryotherapy. The migration of the cells was analysed by the transwell assay (b) and wound-healing assay (c); (d) qRT-PCR experiments were performed to evaluate the mRNA levels of fibronectin, MMP2 (Matrix metalloproteinase 2), MMP9, PAI-1 (Plasminogen activator inhibitor-1), TGF-β1, and Col2a1 (Collagen Type II Alpha 1) in fibroblasts derived from the dermis under the cryotherapy-treated (+) or untreated (−) keloids. MMP9 and TGF-β1 were significantly increased in the fibroblast treated with cryotherapy as compared with controls; (e) Immunoblot analysis of phosphorylated Smad2/3. Total Smad2/3 and GAPDH were used as the loading controls. Error bars represent the standard error from three repeated experiments. * p < 0.05, ** p < 0.01, and *** p < 0.005 (Student’s t-test).

Figure 4

Histologic findings of keloids pretreated and untreated with cryotherapy. (a) Cryotherapy-treated keloid showed extensive lymphoplasmacytic infiltration characterised by thicker fibrosis and occasional “proliferating core collagen” (black arrow) in the deeper part (H&E stain, ×12.5); (b) The untreated keloid showed less inflammation and less surface-parallel fibrosis without core collagen (H&E stain, ×12.5).