Effect of extracorporeal shock wave therapy on keratinocytes derived from human hypertrophic scars

Hypertrophic scars represent a common complication in burn patients. In addition to cosmetic defects, they may cause serious sensory abnormalities such as pain and itching, severe dysfunction depending on the site, and emotional disorders such as anxiety and depression. The present study aimed to identify the molecular mechanisms underlying the use of extracorporeal shock wave therapy in keratinocytes. Keratinocytes derived from hypertrophic scar tissue were cultured and expression of proliferation markers (keratin 5 and 14), activation markers (keratin 6 and 17), differentiation markers (keratin 1, 10, and involucrin), apoptosis factors (Bax, Bcl2, and Caspase 14), and proliferation/differentiation regulators (p21 and p27) was investigated to compared with that of those in keratinocytes derived from normal skin tissue. Scar-derived keratinocytes were treated with extracorporeal shock waves under 1000 impulses at 0.1, 0.2, and 0.3 mJ/mm2. Shock waves altered the molecular pattern of proliferation, activation, differentiation, and apoptosis, as well as proliferation/ differentiation regulators, including Bax, Bcl2, ASK1, p21, p27, and Notch1. In summary, we show that extracorporeal shock wave therapy regulates the proliferation and differentiation of keratinocytes derived from hypertrophic scar to maintain normal epidermal integrity.

The p21 and p27 are cell cycle regulator, to control proliferation to differentiation in keratinocyte, both were significantly higher at both mRNA and protein levels in HTSKs than that of those in HNKs (p21-mRNA:  TGG ATG GTG CTT ATA TGA C  GAC AAC TCT GCT TGG TAG   KRT5  GTG GAA GAC TTC AAG AAC AA  TAG GCA GCA TCT ACA TCC   KRT6A  TGA AGA AGG ATG TGG ATG  ATC ATA CAA GGC TCT CAG   KRT10  GAT TCT CAA CCT AAC AAC TG  GCT ACC TCA TTC TCA TAC T   KRT14  GCT GAG ATC AAA GAC TAC A  AGA AGG ACA TTG GCA TTG   KRT17  ATC CTG CTG GAT GTG AAG ACGC TCC ACA ATG GTA CGC ACC TGAC   IVL  GGA CTG CCT GAG CAA GAA TGTG TAA GCT GCT GCT CTG GGT TT   NOTCH1  AGC CTC AAC GGG TAC AAG  TTG ACA CAA GGG TTG GAT TC   CASP14  CCT GTT GTC ACC TTG CTA T  GTC CTT GCC TCT GTC TTA C   BAX  CCT TTT GCT TCA GGG TTT CA  CCA TGT TAC TGT CCA GTT CG   BCL2  TGC GGC CTC TGT TTG ATT T  AGG CAT GTT GAC TTC ACT TGT   ACTB  AGA GCT ACG AGC TGC CTG AC  AGC ACT GTG TTG GCG TAC AG   Table 2. Western blot primary antibodies.  (Fig. 4).
These results indicate that HTSKs are exhibiting an activated, over-differentiated, and augmented anti-apoptosis phenotype.

Effect of ESWT on expression of proliferation and differentiation regulators in
In the literature on the ESWT, no agreement on how to achieve maximum potential. There are several in vitro studies has been used with 100-300 pulses at 0.03-0.13 mJ/mm 2 in mesenchymal cell line 14 , 1000 impulses at 0.14 mJ/ mm 2 in human tenocytes 25 , and 0.06-0.50 mJ/mm 2 in human osteoblasts 26 . Recent we applied ESWT with 4 Hz frequency, 0.05-0.30 mJ/mm 2 energy flux density, and 1000 to 2000 impulses/cm 2 per treatment according to patient's tolerance to pain. This treatment improved scar thickness, vascularity, and functionality, when treat to HTS of hand without obvious side effects 24 . Moreover, in our previous study, that ESWT with 1000 impulses/cm 2 at 0.03, 0.1, and 0.3 mJ/mm 2 not only altered the expression of fibrosis-related molecules in fibroblast derived from HTS, but did not affected the cells viability. Therefore, in this study, we selected the parameter with 1000 impulses/cm 2 at 0.1, 0.2, and 0.3 mJ/mm 2 , applied to keratinocytes, and determined the molecular changes of cells.
At present, the fundamental epidermal pathology of HTS is still unclear. In previous studies, determined the expression of keratin 5 and 14, both are proliferation marker, and keratin 6, 16, and 17, they are activation marker by immunohistochemistry in HTS tissues that provided evidence that HTSKs were activated, but there was no difference in proliferation compared with site matched normal skin tissues 27 . Moreover, differentiation marker, filaggrin and involucrin were abnormally detected in HTS 27,28 . The increased expression of involucrin also found in other hyperproliferative disease, such as psoriasis 29 . The authors considered that abnormal expression of involucrin possibly affect stratum corneum formation 28 . In our results, the characteristic of HTSKs is similar to those of above previous study that is activated keratinocytes highly expressed keratin 6 and 17, and involucrin expression was also increased. Activated keratinocytes may play an important role in the development of HTS through the epidermal-mesenchymal interaction 17 . In HTSKs, the anti-apoptotic factor Bcl2 expression at mRNA and protein levels increased, while pro-apoptotic factor Bax expression was unchanged, compared to HNKs that suggest up-regulated anti-apoptosis in HTSKs. This results also reflect the low level of apoptosis is a cause of HTS formation 30 . The differentiation marker, keratin 1 and involucrin expression were increased in HTSKs, suggest differentiation of HTSKs was increased than HNKs. Moreover, the cell cycle regulator, p21 and p27 expression were also up-regulated in HTSKs. Study revelated the keratinocyte proliferation and differentiation were tightly regulated by the p21 and p27 expression. Moreover, over-expression of p21 and p27 induced keratinocyte differentiation and inhibited proliferation 31 . On the contrary, down-regulation of p21 and p27 expression resulted inhibition of differentiation with decrease of keratin-1 expression 32 . Therefore, increased expression of keratin 1 and involucrin may be related to up-regulated p21 and p27 expression in HTSKs.
We found the proliferation marker, keratin 14 expression was decreased at 24 h and increased at 72 h, while activation marker keratin 6 expression was increased at 24 h and decreased at 72 h. Moreover, early time differentiation marker keratin 1 expression was decreased at 24 h and increased at 72 h, and later differentiation marker, involucrin was increased at 24 h and decreased at 72 h. These results indicate that ESWT coordinately regulate HTSKs proliferation, activation, and differentiation, rather than blindly increase or decrease. Whereas, expression of keratin 6, and keratin 1 and involucrin in HNKs at 24 h after ESWT was increased and decreased at 72 h. These results suggest that ESWT exhibit different effects depending on physiological phenotypes. To maintain normal epidermal integrity, the proliferation-differentiation-death cycle must operate seamlessly. The several apoptosis-related factors physiologically involved in keratinocyte proliferation and differentiation 33 , including Bax, Bcl2, ASK1, and Caspase 14 etc. In normal epidermal development, Bcl-2 is highly expressed in basal proliferating keratinocytes and absent from suprabasal keratinocytes 34 . Bax expression has been reported to be stronger in suprabasal than in basal keratinocytes 35 . Over-expression of ASK1 in keratinocytes doses not induce apoptosis, but induce cell differentiation along with increasing the expression of involucrin 36 . Caspase-14 is uniquely expressed in skin tissues unlike other caspases type expressed ubiquitously 37 and its accumulation is  www.nature.com/scientificreports/ associated with keratinocyte differentiation and stratum corneum formation 38 . In our results, Bcl2 expression was correlated with Bax expression increased at 24 h decreased at 72 h after ESWT. Moreover, ASK1 expression was increased at 24 h and 72 h after ESWT. Caspase 14 expression was increased just at 72 h after treatment. These results are further supported the effect of shock wave therapy regulate HTSKs proliferation and differentiation.
To sum up the above, that over-or down-expression of apoptosis-related factors reflects ESWT treatment induces the proliferation or differentiation phenotype keratinocyte. Overall, the shock wave therapy altered the expression of proliferation and differentiation-related molecular in HTSKs. The increased or decreased expression of molecular in HTSKs 24 h or 72 h after treatment was close to that of those in the normal cell; for instance, keratin 1 expression at 24 h, involucrin, bcl2, p21, and p27 expression at 72 h vs. that of those in HNKs. The consequence its dynamic regulation may be partially involved in pathological phenotype restore to normal cell physiological state contribute to maintain normal epidermal homeostasis.
Notch1, p21, and p27 are also involved in regulating the proliferation and differentiation of keratinocytes [39][40][41][42] . Specifically, activation of Notch1 upregulation of p21 to induce differentiation associated cell arrest, whereas p27 activation promotes the differentiation of keratinocytes [40][41][42] . In our results, expression of Notch1, p21, and p27 was increased at 24 h and decreased at 72 h after ESWT. Their expression pattern was consistent with that expression of keratin 14 and involucrin. Therefore, the effect of ESWT on the proliferation and differentiation of HTSKs possibly through regulate expression of Notch1, p21, and p27.
In summary, present study demonstrated that extracorporeal shock wave dynamically regulates proliferation, activation, and differentiation of keratinocytes originated from hypertrophic scar tissue (Table 3). This evidence partially supports observed in clinical beneficial effects of shock wave therapy for post-burn hypertrophic scar. Future study will be carried out in a three-dimensional culture model to fully explore the mechanism of action and to provide more physiologically relevant information.