Fractional CO2 laser combined with 595-nm PDL inhibiting hypertrophic scars via measuring BMP-7 and Fas expression in rabbit models

Background and Objectives Although the treatment of hypertrophic scar (HS) remain challenging, fractional CO 2 laser (FCO 2 L) and 595-nm pulsed-dye laser (PDL) have proved clinical ecacy. Meanwhile, BMP-7 and Fas proteins are demonstrated to promote wound healing and inhibit scar formation, yet few reports on the effect of the two proteins on hypertrophic scarring are available, and their molecular mechanisms remain unclear. In current study, we attempted to observe the effect of combined use of FCO 2 L with 595-nm PDL in HS animal models through determining the expression of BMP-7 and Fas in scar inhibition. and simple and combined treatment group. Totally, 3 sessions of treatments were carried out once every 14 days. Then, the changes of broblasts and collagens in HSs and expression of BMP-7 and Fas proteins in the scar tissues were determined via histological and immunohistochemical studies, ELISA, CCK8 test, RT-PCR and Western blot assay.


Introduction
Hypertrophic scar (HS) represents a frequent complication in abnormal wound healing, and remains intractable in its treatment [1][2][3], because abnormal and excessive broblast growth and collagen deposition play critical roles in the process of HS formation [3][4][5][6]. HSs are characterized by signs of hard and red nodules, and symptoms of pain and itch, and treatment of them primarily rely on corticosteroid injection, laser therapy and surgery [4][5][6][7][8]. Fractional CO2 laser(FCO2L) and 595-nm pulsed dye laser (PDL) are selective phototherapeutics, and have been validated to be effective in therapy of HS [9][10][11][12][13][14], yet their underlying mechanisms remain unclear, and need validation through measurement of bone morphogenetic protein-7(BMP-7) and Fas gene, the two important proteins that affect the prognosis of this condition on molecular study basis.

Medicine and instruments
Upon the wounds developing into HS at day 28, FCO 2 L(Ultrapulse, Lumenis Ltd., Yokneam, Israel) was applied to the rabbits in FCO 2 L and combined treatment group, and 595-nm PDL(Vbeam2, Syneron & Candela., Boston, USA) was used to treat the animals in PDL and combined treatment group. Parameters were set according to previous studies [42][43][44] and the instructions for users supplied by the manufacturer. FCO 2 L: (Combo mode; square matrix: 6 mm; Deep:12.5 mJ, 15%; Super cial: 90 mJ,40%); 595-nm PDL: (Fluence:15J/cm2, square matrix: 7 mm, Pulse duration:10 ms). Treatment was repeated twice on each HS. Totally, three rounds of treatments were administered once every 14 days.
Histological study HS samples were obtained at day 70, and stained and measured with HE and Van Gieson(VG) for observation of the cellular proteins and collagen bers in tissues. Scar samples were initially xed and embedded in conventional protocol, and then sectioned into 5 µm in thickness. Staining was made in the middle of each section. The features of HS were examined, and hypertrophic index was measured (HI = A /B (A = vertical thickness from the highest point of HS to the surface of cartilage; B = vertical thickness from the normal skin to the surface of cartilage). Apart from that, NA (numerical densities of broblasts on area) and AA (area density of collagen bers on area) were calculated to determine the density of collagen bers in scar tissues.

Immunohistochemical study
Immunohistochemical study was performed to observe the expression of BMP-7 and Fas. The sections were dewaxed, rehydrated, graded and distilled, and then activated by endogenous peroxidase to trigger antigen repair. Moreover, streptavidin-peroxidase enzyme(CST, Mass., USA)and Mayer's hematoxylin (MHS32, Sigma, USA) were used to dye and re-stain the sections. Software Image-Pro plus for windows(version 6.0, Media Cybernetics, Inc. USA) was used to analyze the results.
ELISA study ELISA was conducted to measure Type I(SEA571Rb,Wuhan,China) and type III(SED087Rb,Wuhan, China) collagens. The scar samples were coated on the plastic porous plate and washed out, then the antibodies were added to bond the speci c antigens. The enzyme and substrates were colored by adding primary and secondary antibodies. Results were automatically estimated using enzyme-labeled instrument.

CCK8 test
Cell counting kit-8(CCK8) was employed to investigate the viability of broblasts and the content of Col-I and III in the samples. Fibroblasts from HSs were inoculated in 96-well plates(2 × 10 4 cells/ml) by cell suspension (100 ul/well)for 24 h.CCK8( HY-K0301,MCE,USA) was dissolved and added into each well at 0, 24, 48, 72 and 96 h, respectively. Then, ELISA was performed to observe the absorbance value of Col-I and II at 450 nm.
Real time-PCR TRIzo™ Reagent(Invitrogen™, Hudson estuary, USA) was used to isolate RNA from the HSs, and transcribed to cDNA by PrimeScript™ Double Strand cDNA Synthesis Kit(Takara, Shiga, Japan). And Real-Time PCR System®(Applied Biosystems, Life Technologies) was performed at 95 °C for 5 min, 60℃ and 72℃ for 30 s, and 40 cycles in 4℃ for 20 min. The Primer Information(5'to3') of this two proteins was:BMP-7(F:ACCTCCAGGGCAAGCACAACT, R:CGTCAGTGAGGAAGCGGCTATCT),Fas(F:TGAACCATCACCGACTTCCTCCTA,R:GCCACTCTTGGACACCAGCAT). Gel UV analyzer (Applied Biosystems, Life Technologies) was used to photograph according to the manufacturer's protocols, The formula of 2-(ΔΔCt) was used to analyze relative gene expression in the samples.

Western blot assay
Western blot assay was carried out to examine the expression of BMP-7 and Fas. The sections were initially digested, lysed and transmitted to a polyvinylidene di uoride membrane, and immunoblotted in buffer overnight. Primary and secondary antibodies were applied to incubate the reactive protein. Mouse monoclonal anti-BMP-7(ab99359,abcam,UK), anti-Fas(ab56032, abcam,UK) antibodies and control protein (ACTIN, CST,Mass., USA) were used to measure BMP-7 expression, and the results were analyzed using Image J software.

Statistical analyses
The results were analyzed by SPSS 19.0 software(SPSS Inc., Chicago, IL). Means(M) ± standard deviation(SD) was used to express the databases and A value of *, P < 0.05,**,P < 0.01 or ***,P < 0.001 were accepted as statistical signi cance.

FCO 2 L combined with 595-nm PDL reduced HSs
The HSs became attened and shrunk in rabbits treated by combined FCO 2 L with 595-nm PDL compared to the therapy with simple FCO 2 L or 595-nm PDL (Fig. 1).The scar thickness was lower in combined treatment group(0.61 ± 0.05) than in other groups(1.10 ± 0.08;1.19 ± 0.10) (Fig. 2a)(***P < 0.001). Fibroblasts were more regular, and collagen bers were less in the treatment groups, especially in the combined treatment group (Fig. 1). Furthermore, HI, AA and NA were all reduced most in rabbits treated by FCO 2 (Fig. 2b-d), with signi cant difference among groups(***P < 0.001).

Discussion
Hypertrophic scar commonly results from abnormal wound healing with a complex pathologic process, and poor understanding of its etiology and pathology makes it di cult to completely remove the scars and achieve better prognosis of this condition [1][2][3]. Development and reoccurrence of HS are associated with irregular and disorder broblasts and overproduction of collagens in the skin, which potentially trigger certain clinical symptoms and manifestations, including high and red nodules, pain and itch at the lesion [3,4]. Untreated HSs can also bring about some physiological and psychological disturbance from cosmetic dis gurement and even economic burden for the victims [2]. Clinically, lasers of diverse parameters are recognized as effective and safe treatment options for HSs, especially fractional CO2 laser and 595-nm PDL [5][6][7][8][9][10][11][12][13][14]. The primary mechanisms of the two laser systems may be involved in some inter-cell factors and pathways in skin resurfacing, such as BMP-7 and Fas expression [29-33, 45, 46]. Nonetheless, few evidences on how the two proteins work in HS remodeling following combined use of FCO2L with 595-nmPDL, for which we attempted to observe and verify the mechanisms in rabbit models.
In our study, attened, shrunk and softened HSs were seen in the animals models after 3 sessions of therapy, and the improvement was better at the lesions treated by FCO2L plus 595-nm PDL than simple use either of the laser modality. Still, irregular and disordered broblasts and collagens were suppressed after treatment, especially in rabbits in the combined treatment group via veri cation by ELISA, CCK8 test, HE and VG staining. Further immunohistochemical study, Western blot analysis and RT-PCR indicated evidently up-regulated expression of BMP-7 and Fas at the HSs after combined treatment. These ndings demonstrate that combined use of FCO2L with 595-nm PDL can generate expression of BMP-7 and Fas proteins in HS tissues.
BMP7 is a member of TGFβsuperfamily and has been veri ed to reduce brosis in various tissues [16][17][18][19][20][21][22][23][24][25][47][48][49]. Previous clinical observations indicated that BMP-7 can suppress brosis in renal and myocardial through reducing epithelial-mesenchymal transition and counteracting TGF-β1 signaling pathway [21,22]. Moreover, BMP-7 participates in the process of wound healing, inhibits dermal papilla cells, thus limiting the initiation of proliferative broblasts in hypertrophic scars [15,16,24,25]. Guo J and Lin Q [25] had created HSs in mouse models to investigate the inhibitory effects of BMP7 on scar formation, and found that BMP-7 promoted wound healing, induced broblast apoptosis and inhibited scar formation by suppressing collagen deposition and expression of brotic proteins in scar tissues. The inhibitory effects of BMP-7 may be attributed to activated BMP-7/Smad1/5/8 signaling pathway, which is consistent with another observation [24]. By the ndings aforementioned, we hypothesized that the e cacy in our protocol that combined use of FCO2L with 595-nm PDL has achieved should be the result of boosted BMP7 expression.
We also found that FCO2L plus 595-nm PDL pro le could result in increase of Fas expression in hypertrophic scar samples. This gene is of important role in the process of cell apoptosis by Fas-associated death domain (FADD) and activation-induced cell death (AICD) [26][27][28], and signi cantly elevated in broblasts during HS formation and wound repair through apoptotic signal-way transaction and reducing redundant brosis [29][30][31], on which basis many clinical and pre-clinical measures have been developed to target the treatment of HSs [29][30][31][32][33][34]50]. Le X and Wu WW[50 investigated the expression of Fas ligand and FADD in HS models, and found that FADD can check cell proliferation and promotes apoptosis of hypertrophic scar broblasts by enhancing FasL expression. Additional clinical study also con rmed imbalance of proliferation and apoptosis of cells in HS tissues due to the sensitivity of Fas expression, and concluded that Fas sensitivity enhancement should be a promising therapeutic target in HSs [34]. Besides, Li XY[32] and Liu YB [33] reported that TSG-6 and tunicamycin could trigger Fas/FasL signaling pathway and activate the apoptosis of broblasts in hypertrophic scars, which is consistent with our ndings.
Nevertheless, simple use of either FCO2L or PDL failed to achieve satisfactory prognosis in treating HSs, and there remains necessary to expand evidence and develop optimal therapeutic options, for which combined use of FCO2L and 595-nm PDL was attempted in early-stage of HSs, and the results showed that the clinical scores (VAS, VSS and POSAS) as well as disordered collagens and bers were signi cantly improved after treatment [11,13,37,39,41,[55][56][57]. This may be involved in the role of 595-nm PDL that is capable of promoting BMP-7 and Fas expression, which is con rmed in our study.

Limitations and prospects
Current study was performed merely in rabbit models, and need further investigation on diverse animal models.
In addition, veri cation of long-lasting bene ts and scar recurrences is necessary in clinical settings because of the hard follow-up in animal models.

Conclusions
Combined use of FCO 2 L with 595-nm PDL can inhibit HSs in rabbit models, which may be associated with boosted BMP-7 and Fas protein expression through regulating the signaling pathway of the two proteins that are responsible for scar formation. Our ndings may lay a foundation for an emerging option in the treatment of HS in clinic.

Con ict of Interest
The authors declare that they have no competing interests in this study.