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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 38  |  Issue : 2  |  Page : 65-72

Evaluation of the effectiveness of erbium yttrium–aluminum–garnet fractional laser, carbon dioxide therapy and platelet-rich plasma in treating striae distensae


Department of Dermatology and Andrology Shebin El-Kom Teaching Hospital, Shebeen El-Kom, Egypt

Date of Submission09-Sep-2017
Date of Acceptance21-Jan-2018
Date of Web Publication17-Aug-2018

Correspondence Address:
Enas A.M. Mahrous
Shebin El-Kom City, Menoufia Governorate
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ejdv.ejdv_37_17

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  Abstract 


Objective This study aims to compare the efficiency of erbium: yttrium–aluminum–garnet (Er: YAG) laser, carboxytherapy, and platelet-4rich plasma (PRP) in the treatment of striae distensae.
Background Striae distance or striae distensae are common dermal scars characterized by flattening and atrophy of the epidermis, and result in considerable aesthetic concern.
Patients and methods The study was conducted on 60 female patients with striae in the area between the diaphragm and the knee, whose ages ranged between 16 and 50 years. Cases were collected from the inpatient and outpatient dermatology clinics, Shebin El-Kom Teaching Hospital, in the period from October 2016 to April 2017. Detailed history, and full general and dermatological examination were done; photography and CO2 injection were performed on 20 patients (group A), while PRP injection was done to the resting 20 (group B), and 20 patients were treated by Er: YAG laser (group C). All the potential side effects were discussed with all patients’ group prior to their participation.
Results The mean age of the patients was 26.39±11.51 years. The most common sites affected by striae distensae were the abdomen, thighs and the hips. There was no significant difference regarding the patients’ skin type, duration of striae, and the result at the end of treatment sessions (P>0.05). However, there was a statistically significant difference (P<0.05) regarding the color of striae (P=0.036) and width of striae (P=0.023). Additionally, highly statistically significant (P=0.001) difference was obtained between the studied maneuvers regarding the type of pain and the modality of treatment and the improvement degree (P=0.00). The overall patient’s satisfaction after the end of the eighth session of carboxytherapy and the fourth session of PRP and the sixth session of Er: YAG laser was evaluated. The Er: YAG laser has an upper hand on carboxytherapy and PRP injection in the treatment of striae distensae regarding the results and patient’s satisfaction.
Conclusion The clinical improvement of striae distensae can be obtained with the use of the Er: YAG fractional laser. The results of this study demonstrated the safety and efficacy as well as low-incidence side effects of Er: YAG fractional laser in the treatment of striae distensae as compared with CO2 and PRP. So, more randomized, controlled studies are needed to further evaluate the efficacy of lasers for the treatment of striae distensae among different skin types and different ages.

Keywords: carbon dioxide, erbium: yttrium–aluminum–garnet fractional laser, platelet-rich plasma, striae distensae


How to cite this article:
Mahrous EA. Evaluation of the effectiveness of erbium yttrium–aluminum–garnet fractional laser, carbon dioxide therapy and platelet-rich plasma in treating striae distensae. Egypt J Dermatol Venerol 2018;38:65-72

How to cite this URL:
Mahrous EA. Evaluation of the effectiveness of erbium yttrium–aluminum–garnet fractional laser, carbon dioxide therapy and platelet-rich plasma in treating striae distensae. Egypt J Dermatol Venerol [serial online] 2018 [cited 2018 Dec 14];38:65-72. Available from: http://www.ejdv.eg.net/text.asp?2018/38/2/65/237276




  Introduction Top


Striae distensae, better known as stretch marks, are a common disfiguring skin disorder of significant cosmetic concern [1]. Recent or immature striae are defined as linear bands of smooth skin, which are erythematous or violaceous and maintain their characteristics for a short period, usually ∼6–10 months. In their later stage, they tend to become white, flat, or depressed, and are more difficult to treat. The histology of stretch marks is as that of a scar; the exact origin remains unknown, and the responsible factors for its development are poorly understood [2].

Mechanical stretching, normal growth, sudden weight gain or loss, pregnancy, and a high level of steroid hormones are all contributing factors to striae distensae formation [3]. Clinically, recent or immature striae are erythematous to violaceous pruritic linear bands of skin known as striae rubra. In the late stages, they become white, flat, and depressed closely resembling a scar, and are named striae alba. Histologically, a deep and superficial perivascular lymphocytic infiltrate with occasional eosinophils and dilated venules with edema of the upper dermis are characteristic of immature striae. In the terminal stages, there is thinning of epidermis due to blunting of rete ridges and a paucity of collagen and elastic fibers [4].

Several treatments have been proposed to improve the appearance of striae, yet no consistently effective modality is available to date. Topical retinoids, microdermabrasion, chemical peels, radiofrequency, skin needling, platelet-rich plasma (PRP), intense pulsed light, and ablative and no ablative lasers have all been used with variable results [2]. The effective treatment of striae distensae is preferably during the early and active stages. Lasers and light devices have recently become a good therapeutic option. It is also known that several lasers have proven to be efficient in the induction of new collagen formation [5].

The capacity for collagen stimulation, the consequent formation of new collagen, and the reorganization of the elastic fibers observed after treatment with diverse lasers and pulsed light enable a contraction of the striae and an improvement in the quality and texture of the skin in the treated areas [6]. Side effects such as prolonged healing and pigmentary alterations were related to ablative technologies, particularly in darker skin tones. The same occurs with pulsed dye laser and should be avoided for skin tones type IV–V because of the possibility of pigmentary alterations after treatment.

It is well established that some nonabrasive lasers have a strong attraction to vascular targets, which is an action related to an increase in dermal collagen. The first lasers utilized for this purpose were ablative, nonselective lasers: CO2 or erbium: yttrium–aluminum–garnet (Er: YAG) laser followed by different types of lasers described in the literature [7].

Another study [8] found the use of an ablative 2940-nm erbium: YAG fractional laser may provide an additional treatment approach particularly for patients suffering from later stage striae distensae; the risk of associated hyperpigmentation should not be underestimated. In general, striae distensae remain difficult to treat and any therapy and associated side effects should be critically discussed with the patient before therapy. Moreover, Elsaie et al. [3] have found that a satisfactory improvement in striae distensae lesions was seen through clinical and histological evaluation. Thus, long-pulsed (LP) Er: YAG laser is a safe and effective module of laser treatment for these common skin lesions.

Therefore, the aim of this study was to compare the efficiency of Er: YAG laser, carboxytherapy, and PRP in the treatment of striae distensae.


  Patients and methods Top


The current study was conducted on 60 female patients with striae in the area between the diaphragm and the knee, whose ages ranged between 16 and 50 years. Cases were collected from the inpatient and outpatient dermatology clinics, Shebin El-Kom Teaching Hospital, in the period from October 2016 to April 2017.

Ethical consideration

All patients in the study signed an informed written consent before the study initiation. Approval was obtained from the Ethics Committee of Menoufia University.

Inclusion criteria

Adult healthy women with striae in the area between the diaphragm and the knee, aged between 16 and 50 years and with skin type either 3 or 4, without associated dermatological or systemic diseases.

Exclusion criteria

Pregnancy and lactating women, history of keloids, uncontrolled diabetes, patients on retinoids or any vitamin A derivative for the last 6 months before sessions, any type of skin infections and cancer, any systemic diseases and bleeding disorders, any current use of regular systemic steroid or topical moderate to potent steroid at the site of strain, and on any immunosuppressive drugs.

Photography and CO2 injection were performed on 20 patients (group A), while PRP injection was done to the resting 20 (group B). All the potential side effects were discussed with all patients’ group prior to their participation.

Group A: they performed thorough intradermal injection of CO2 gas using a 30 G needle. Immediately after injection, the gas diffuses into the needle-surrounding tissue. The infusion velocity was 50–80 ml/min and the total volume was 40–60 ml/area. The session takes ∼10–20 min. The treated group received eight sessions with a 1-week interval between sessions. The CO2 gas is 1.5 times heavier than air, so it tends to go down with gravitation; depending on this fact the patients should receive the injection while they are in a sitting or supine position to allow good lymphatic return to the lungs [9].

Group B: They underwent a thorough intradermal injection of PRP. Each patient had four sessions with 2 weeks interval [10].

Platelet-rich plasma preparation method

Twenty milliliters of venous blood was collected from the anticubital vein under complete aseptic conditions into tubes containing sodium citrate (10 : 1) as an anticoagulant. Then, the citrated whole blood was subjected to two centrifugation steps; initial centrifugation (soft spin) at 1419g for 7 min to separate the plasma and platelets from red and white blood cells. To avoid fragmentation of the platelets and the subsequent early release of the secreted proteins, with the corresponding negative impact on their bioactivity, low centrifugation speeds are recommended [10].

When the anticoagulated blood is centrifuged, three layers with different densities separate out: a lower layer, composed of red blood cells; a middle layer, composed of white blood cells and platelets; and an upper layer composed of the plasma. The resulting plasma supernatant, which contains the suspended platelets, was harvested to a second centrifugation step (hard spin) at 2522g for 5 min, leading to the separation of the plasma into two portions: PPP and PRP. Typically, the lower 2 ml of the plasma (10% of the initial volume of autologous blood) was yielded as PRP concentrate after centrifugation. Then, the PRP was activated by adding calcium chloride (0.1 cm of CaCl2 to each 1 cm of PRP) immediately before the injection. PRP was injected using an insulin syringe intradermally in the striae distensae with a space of 2 cm between different points of injections [10].

Postcare

The patients were advised to use a topical antibiotic ointment for 1 week. Avoid any tight clothes on the treated area, walking or light exercise, avoid exhausting exercise the same day of treatment, avoid any hot baths or saunas in the same day and drink plenty of fluids to help get out of the injected CO2 [9].

Photographic documentation

Photographic documentation was performed using the same digital camera Canon (PowerShot A2200 HD; Canon Inc., Ota, Tokyo, Japan, 14 mp, 4× optical zoom) set at a fixed distance from the patient before and after each treatment. Clinical improvement was analyzed according to the evaluation of the photographic material by two dermatologists blinded to the study group. In addition, each participant was asked to rate her overall satisfaction with the treatment 4 weeks after it was completed according to whether she was unsatisfied, slightly satisfied, satisfied, or very satisfied. The patients were also asked to report on any side effects of the treatment, including bleeding, oozing, posttreatment dyschromia, crust, and severe erythema.

Follow-up

All patients were seen in the following month after the last session to ensure the maintenance of results obtained and check for recurrence of striae.

The following score systems was used for the assessment of the degree of improvement after finishing the course as follows:
  1. Assessment of improvement of the striae by measurement of width of the widest striae (score from 0 to 4): 0, no improvement. 1, mild (<25%). 2, moderate (25–50%). 3, very good (51–75%). 4, excellent (>75%) [11].
  2. Improvement of color of the lesion (score from 0 to 4): 0 indicates no change in color. 1 indicates mild improvement in color (<25%). 2 indicates moderate improvement in color (25–50%). 3 indicates very good improvement in color (51–75%). 4 indicates excellent improvement in color (>75 or normal skin color) [12].
  3. Patient satisfaction (score from 0 to 2): upon final examination, the patients were asked about their overall satisfaction. 0, indicates no satisfaction. 1, indicates the patient was slightly satisfied. 2, indicates the patient was satisfied. 3, indicates the patient was very satisfied.
  4. Assessment of the degree of pain accompanying the used maneuver whether there was no pain, mild tolerated pain, or severe intolerable pain [13].


Group C: they were treated by fractionated 2940-nm Er: YAG laser after submitted to a test dose on their skin. Aiming to cool and protect the epidermis as well as to decrease the discomfort during laser treatment, an ice pack or cold air cooling was applied. No patients received any kind of local or topical anesthesia. A thin layer of cold, clear, water-based gel was applied to the treatment areas. After everyone in the room was given adequate eye protection, the lesions were treated with the 2940 nm LP Er: YAG laser (Twain, Italy) with a spot size of 3.5–7–8 mm, fluence fractional of 20 J/cm2, a delay of 15–20i ms, and a frequency of up to 6 Hz. In each session, each stria was treated over its entire length, avoiding overlapping pulses, as in the technique suggested by Alster and Greenberg [14] when using the pulsed dye laser in the treatment of striae or scars. The number of treatment sessions per patient was 3–6 with an interval of 18–21 days.

All patients were subjected to the following before treatment

Detailed history taking include

Age, sex, and occupation, onset, course, and duration of appearance of striae.

Possible cause

Pregnancy, postpubertal, steroid intake, weight gain or loss, associated hormonal imbalance accompanied with virilization symptoms, or undefined cause. Last treatment taken for the striae, its date, duration, and end result.

General examination

To exclude any disease that may induce the striae or affect the quality of treatment in study patients.

Potential side effects of the injection (either CO2 or PRP) were discussed with the patients prior to participation.

Dermatological examination

To detect the type of striae, site, and severity according to the width of the majority of striae lines (mild≤0.5 cm, severe≥0.5 cm), the number of striae, duration in months or years, BMI (kg/m2), color assessment, appearance of striae, and side effects occurred to obtain a total measure.

Statistical analysis

Results were tabulated and statistically analyzed by a personal computer using Microsoft EXCEL 2016 and SPSS version 20 (SPSS Inc., Chicago, Illinois, USA) [15]. Statistical analysis was done using: descriptive, for example, percentage, mean and SD. Analytical that includes χ2. A value of P less than 0.05 was considered statistically significant.


  Results Top


In the present study, the age of the patients varied from 16 to 50 years with a mean age of 26.39±11.51 years. The number of the striae varied from 6 to 37 lines with a mean of 12.74±6.11. Regarding the possible etiological classification of striae distensae in the studied group, striae distensae was probably adolescence associated in 12 (20%) patients, 45% of patients were obese, 30% gave a positive family history, and 15% gave a history or a recent complain of virilization symptoms (acne vulgaris, hirsutism, or female pattern hair loss), which was followed by the appearance of striae distensae. The striae developed since less than a year in 63.3% patients, and 36.7% it developed since more than a year ([Table 1]).
Table 1 Demographic and clinical data in the studied cases

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Additionally, 35 (58.3%) patients have striae alba, while 24 (40%) patients show the presence of striae in the abdomen, and 31.7% have striae rubra. Also, 21.7% have striae in the buttocks, and 11.7% patients have in their flanks, 10% patients have striae in the thigh and legs. Moreover, 8.3% in the knee and popliteal fossa and 5% have striae in the groin. In majority (61.7%) of patients their striae lines width were less than 5 mm, while in 38.3% their striae lines width were more than or equal to 5 mm ([Table 1]).

Results of the current study has shown that there was a statistically highly significant difference (P=0.00) regarding the type of pain and the modality of treatment. Twenty-five (41.7%) patients had no pain during maneuver; 56% of them were treated by Er: YAG laser, followed by PRP (44%). In addition, 15 (25%) patients had tolerable pain, 60% of them were treated by PRP and 40% by Er: YAG. Moreover, 20 (100%) patients had severe pain; all of them treated by carboxytherapy (CT) maneuver ([Table 2]).
Table 2 Comparative study between carboxytherapy (CT), platelet-rich plasma, and erbium: yttrium–aluminum–garnet regarding pain and modality of treatment

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Additionally, a highly statistically significant (P=0.001) difference was obtained between the three studied maneuvers. The overall patient satisfaction after the end of the eighth session of carboxytherapy and the fourth session of PRP and the sixth session of Er: YAG laser. Thirteen (21.7%) patients were not satisfied, 11 of them were treated by PRP, and one of them was treated by CT and Er: YAG. Moreover, while 11 (18.3%) patients were slightly satisfied, four of them from group A and five from group B, and two from group C. Twenty-one (35%) patients were satisfied, nine of them from group A and four from group B and eight from group C. Also, 15 (25%) were very satisfied, six of them in group A and nine in group C ([Table 3]).
Table 3 Comparison between three studied maneuvers regarding patient’s satisfaction and modality of treatment

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By asking the patients about their skin response to sun exposure 36 (60%) were of skin type III, while 24 (40%) were of skin type IV (according to the Bolognian classification of skin typing) ([Table 4]).
Table 4 Improvement degree in skin type of the cases, duration and color of the striae, as well as width of the majority of striae lines

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There was no significant difference regarding the patients’ skin type, duration of striae, and the result at the end of treatment sessions (P>0.5). However, there was a statistically significant difference (P<0.5) regarding the color of striae (P=0.036), and width of striae (P=0.023), the result at the end of treatment sessions favoring striae alba (61.7%), and mild degree of striae (<5 mm) ([Table 4]).

Results in [Table 5] shows that striae distensae showed characteristic improvement in appearance after the end of the eighth sessions of carboxytherapy and the fourth session of PRP, and sixth sessions of Er: YAG laser. Seven (11.7%) patients showed no improvement, one of them was treated by CT and six by PRP, while 15 (25%) patients have mild improvement (25%, grade I), four of them were treated by CT and 10 by PRP and one of them was treated by Er: YAG laser. Also, nine (15%) patients had moderate improvement (25–50%, grade II), three of them were treated by CT and PRP and Er: YAG (three for each). Furthermore, 15 (25%) patients had a marked improvement (50–75%, grade III), one treated by PRP and seven by CT and Er: YAG (seven for each). Moreover, 14 (23.3%) showed excellent improvement, five of them were treated by CT and nine by Er: YAG ([Table 5]).
Table 5 Degree of improvement at the end of treatment sessions

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  Discussion Top


In the current study, the age of patients ranged from 16 to 50 years old, which is the age of many causes of striae such as pregnancy, weight loss and growth spurt [16]. The age of the patient was not a factor in decreasing the number of striae, which contrasts with the study of Pinheiro et al. [17] that compares the effect of carboxytherapy with radiofrequency on skin rejuvenation and claimed that the younger patients respond better than the older.

Additionally, in the present study, the most common sites affected by striae distensae were the abdomen, thighs and the hips. Cho et al. [18] reported that the most common sites of striae distensae are the abdomen, buttocks, and less common on the outer aspect of arms. Also, Osman et al. [19] reported that striae distensae mostly affect the abdomen and breasts in pregnant women.

Furthermore, our results show that there was noticed improvement in both types of striae, but it was obvious in alba than rubra. In contrast to the previous study on the effect of infrared laser at 1450 nm on stretch marks that showed no improvement in striae alba [20], the present study is in agreement with Koutna [9] who reported that the age of the scar was not important for the level of improvement reached by carboxytherapy, that is, old scars reacted similar to the younger ones. So, in cases of striae alba, it does not matter whether the striae lines were old or recent.

In the present study, the efficacy of treatment has not been affected by the skin type, while other treatments like pulsed dye laser (PDL), nonablative 1, 450 nm diode laser, and fractional photothermally caused post-inflammatory hyperpigmentation in skin type IV [19].

In the current study, we noticed that the narrower the lesion, the better the outcome. This may be due to the local effect of injected CO2 gas on collagen and elastin synthesis and increase in the local blood flow so that the skin appears healthier as the improvement in the color of striae indicates better improvement in pigmentation and vascularization after therapy [17]. That also went with Koutna [9] who detected that in the wider scar tissue, there was no change in appearance after carboxytherapy and scar tissue reacted well only if not too wide.

Regarding the pain in the present study, severe untolerated pain was caused by carboxytherapy sessions which was referred to the local effect of the gas in tissues and its physical properties which is in agreement with Koutna [9] who found that the whole area looks healthier and better and often slightly tighten, which is highly appreciated by the patients, despite severe discomfort during the gas administration, but at the same time in contrast to Zenker [21] who stated that ‘more often than not, the patient will not necessarily experience any pain with this treatment as the stretch marks already represent ruptured tissues; this means that the gas can diffuse more easily and is therefore less painful. That pain can be lowered by using a topical anesthetic lotion such as xylocaine lotion. However, Koutna [9] reported that the local anesthetic creams initiate vasoconstriction, we did not notice that erythema was not affected by the anesthetic lotion.

In the present study, it was obvious that the Er: YAG laser has an upper hand over carboxytherapy and PRP injection in the treatment of striae distensae regarding the results and patient’s satisfaction. Also, most of high satisfactory results (satisfaction score 2 or 3) went for group C (Er: YAG laser), while most of poor satisfactory results (satisfaction score 0 or 1) results went for group B (PRP injection). After treatment of group C, 17 (85%) patients had high satisfactory results and three (15%) had poor satisfactory results, while in group B only four (20%) patients had high satisfactory results and 16 (80%) had poor satisfactory results.

That is referred most probably to the usage of Er: YAG laser as a monotherapy without adding any of the methods of skin ablation and rejuvenation such as microneedling, microdermabrasion, fractional laser, or RF needling. So, we suggest that induction of skin micro-opening may be mandatory for better results with Er: YAG laser in the stimulation of nucleogenesis and skin remodeling. In the present study, there were some limitations such as the absence of histopathological examination due to participants’ refusal, although it has a good clinical improvement in striae alba and good patient satisfaction.

The treatment of erythematous striae using the 1064-nm-LP Er: YAG laser has demonstrated a clinical improvement of such lesions, probably due to the laser’s affinity for the vascular target present in the striae, the LP Er: YAG laser induces the formation of new collagen; this leads to an improvement in the atrophy of the skin and consequently improves the appearance of immature striae. This improvement was evident for both patients and doctors. The full clearance of the lesions is very rare and seems to be occasionally obtained in some isolated areas of recent striae. Hence, it is vital to start treatment early [14].

While, Sanchez et al. [22] stated that the disadvantages of PRP therapy stem from the fact that it is a readily available, autologous blood product. These features mean that if the platelets are minimally manipulated, PRP is not classified as a drug by the FDA and, accordingly, not subjected to federal regulation. PRP preparation and administration protocols are not specifically defined. As a result, and because of the numerous variables involved in PRP use, clinical and experimental methodologies are extremely inconsistent, making it difficult to draw conclusions about the true efficacy of PRP and best practices for its use.

Another study, Goldman et al. [23], concluded that the clinical improvement of immature striae can be obtained with the use of the 1064-nm-LP Er: YAG laser. The low incidence of side effects makes this laser a good alternative in the treatment of these common skin lesions. Also, Gungor et al. [24] treated 20 women, compared the efficacy of 2940 nm erbium: YAG laser and1064-nm-LP Er: YAG laser in the treatment of striae distensae; 17 striae alba had poor response; and three striae rubra had moderate response to both lasers. So, there is a need for systematic treatment to improve the effect of 2940 nm Er: YAG AFL in striae alba and to overcome the problems associated with ablative skin resurfacing procedures. While, Gauglitz et al. [8] have found the use of an ablative 2940-nm erbium: YAG fractional laser may provide an additional treatment approach particularly for patients suffering from later stage striae distensae, the risk of associated hyperpigmentation should not be underestimated. In general, striae distensae remain difficult to treat and any therapy and associated side effects should be critically discussed with the patient before therapy. Moreover, Elsaie et al. [3], have found a satisfactory improvement in striae distensae lesions through clinical and histological evaluation. Thus, the LP Er: YAG laser is a safe and effective module of laser treatment for these common skin lesions.Additionally, the Er: YAG laser treatment enhances wound contraction, the remodeling of collagen fibers by thermal necrosis, activation of the release of basic fibroblast growth factor, and inhibition of transforming growth factor β1 [19]. Moreover, Ye et al. [25] found a significant increase in the expression of tissue inhibitors of metalloproteinases and a decrease in the expression of matrix metalloproteinases after the laser treatment. Other studies eluded that the Er: YAG could increase the antioxidant capacity as well as the expression of heat shock proteins 70 that might contribute to further collagenases.

There are inconsistent results described in the literature regarding the management of the striae distensae by different laser systems. Yang and Lee [26] showed the effectiveness of the CO2 laser, and Suh et al. [27] demonstrated the efficacy of the pulsed dye laser. However, Nehal et al. [28] showed no clinical or histological improvement of alba type of striae distensae when pulsed dye laser was used. Furthermore, Jimenez et al. [29] have shown that the pulsed dye laser was effective for rubra lesions but not alba lesions. In another study conducted with a diode laser on 11 patients with striae distensae, Tay et al. [20] reported no clinical improvement on photographic evaluation. Also, very recently, El-Ramly et al. [10] showed a significant histological improvement when using the LP Er: YAG for treating striae distensae. Monteiro et al. [30] demonstrate excellent patient and plastic surgeon satisfaction of striae rubrae after treatment using the 1550 fractionated Er: YAG laser.


  Conclusion Top


The clinical improvement of striae distensae can be obtained with the use of the Er: YAG fractional laser. The results of this study demonstrated the safety and efficacy as well as low-incidence side effects of Er: YAG fractional laser in the treatment of striae distensae as compared with CO2 and PRP. So, more randomized, controlled studies are needed to further evaluate the efficacy of lasers for the treatment of striae distensae among different skin types and different ages.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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