The Comparison between Microneedling and Fractional CO₂ Laser for Amniotic Membrane Stem Cell-Conditioned Medium and Vitamin C in Photoaging Treatment

Abstract

Context:

Photoaging correlates with ultraviolet radiation (UVR). In addition to direct and indirect DNA damage, UVR leads to a breakdown of collagen, resulting in enlarged pores, visible wrinkles, also uneven skin tones and pigmentations. Amniotic membrane stem cell (AMSC) can be a useful modality in cell regeneration. Vitamin C has been known as an agent of depigmentation, antioxidants, and collagen synthesis. A topical combination of amniotic membrane stem cell-conditioned medium (AMSC-CM) and vitamin C is expected to improve photoaging. Fractional CO₂ laser and microneedling served as transdermal drug delivery.

Aims:

To compare between microneedling and fractional CO₂ laser for the administration of AMSC-CM and vitamin C in photoaging treatment.

Methods and Material:

Sixty Indonesian women with photoaged skins participated in this experimental analytic, controlled, matching research and were divided into two groups. Thirty women received fractional CO₂ laser, and others received microneedling. All participants received a topical combination of AMSC-CM and vitamin C. Three treatment sessions were repeated at a four-week interval for fractional CO₂ laser and a two-week interval for microneedling. We evaluated the progression and side effects using a skin analyzer on weeks 0, 4, and 8 for the microneedling group and for the fractional CO₂ laser group on weeks 0, 4, and 12.

Results:

Assessment of wrinkles, UV spots, skin tone, and pore revealed a statistically significant effect of a topical combination of AMSC-CM and vitamin C after fractional CO₂ laser (p < 0.05).

Conclusions:

Combination of AMSC-CM and vitamin c using fractional CO₂ laser can improve photoaging and is better compared to microneedling.

Introduction

Intrinsic and extrinsic factors influence the skin aging process. Facial skin is frequently exposed to sunray and the accumulated damage can cause pigmentation and vascular changes, skin elasticity loss, and degradation of skin texture. Therefore, the skin would appear aged.[1]

The amnion membrane is a tissue in which cells have stem cell characteristics, differentiation abilities, and low immunogenicity. The amniotic membrane is acquired from a placenta obtained after the process of childbirth. The amniotic membrane can be used as cellular therapy and regenerative medication. Amniotic membrane stem cells (AMSCs) are better prospects for cell therapy and regenerative medicine compared with other adult mesenchymal stem cells because they are abundant and can be acquired easily and inexpensively.[2]

In the process of AMSC culture, there is a bioactive substance in the culture medium. The conditioned medium contains cytokine and a growth factor secreted when the AMSC is cultured. In regards to its skin healing properties, the growth factor has been proposed for skin rejuvenation. The use of growth factors for skin rejuvenation has now been much studied.[2,3,4]

Multiple methods are available for skin aging treatment, including (1) noninvasive conservative therapies such as the use of photoprotector substances to prevent photoaging, topical tretinoin as the gold standard, and an antioxidant such as vitamin C, (2) minimally invasive therapies such as chemical peeling, microdermabrasion, microneedling, botox injection, filler injection, and (3) laser therapies.[5]

Vitamin C is a water-soluble solution whose functions have been widely known as an agent of depigmentation, antioxidant, and collagen synthesis. It also functions as an antioxidant, neutralizing the reactive oxygen species (ROS), which is one of the pathomechanisms of photoaging.[6]

Several topical therapies, such as growth factor or stem cell, have a heavy molecular weight that it cannot penetrate the skin. The semiprolific (nonpolar) and small (< 500 Da) drugs can go through the stratum corneum. The big lipophilic and hydrophilic drugs such as AMSC-CM cannot go through normal intact skin.

Transdermal drug delivery means drug delivery across an intact skin into the systemic circulation. It is a device which provides an alternative route for drug administration. Microneedling and laser-assisted drug delivery (LADD) are devices to enhance skin permeation to hydrophilic and macromolecules to widen the scope of drugs for transdermal drug delivery. Microneedling devices have been well developed for a controlled transdermal drug delivery in a minimally invasive and convenient manner to penetrate the stratum corneum and generate microchannels in order to deliver drugs into the target layer.[2,7,8]

Prakoeswa and colleagues reported a significant improvement in photoaging in the actions of microneedling and AMSC-CM. The study compared the normal saline and microneedling group. The AMSC-CM group showed better improvements in the pores, wrinkles, polarized spots, and UV spots parameters, whereas skin tones did not improve in either of the groups.[9]

LADD includes the selective control and eradication of epidermal and dermal layers to enable topical drug penetration and absorption, including drugs with a heavy molecular weight. The fractional CO₂ laser can serve as a LADD.[10]

This research aimed to examine the efficacy of transdermal drug delivery of a mixture of AMSC-CM and vitamin C after the microneedling compared to fractional CO₂ laser on photoaged skins using a Janus skin analyzer device.

Subjects and Methods

Research design and subject

This was a 14-week experimental analytic research using a controlled clinic test method, matching pair selection, and parallel design. This research involved 60 subjects at the dermatovenereology outpatient clinic of Dr. Soetomo General Hospital Surabaya. The Ethical Committee Board of Dr. Soetomo General Hospital Surabaya has approved this clinical study. All subjects gave signed informed consent prior to participation.

The 60 subjects passed the inclusion and exclusion criteria, and they were divided into microneedling group and fractional CO₂ laser group. Both groups received a mixture of AMSC-CM and vitamin C immediately after the procedure.

Preparation of a mixture of AMSC-CM and vitamin C

AMSC-CM was obtained from the Tissue Bank and Regenerative Medicine, Dr. Soetomo Teaching Hospital, Surabaya. The AMSC-CM was obtained legally per the international standards for tissue donor and stem cell culture.

The amniotic membrane isolation method uses the modified Soncinis protocol. The amniotic membrane was chopped with a knife into fine pieces with a size of about 20 mm³ then 0.25% trypsin was added and then centrifuged at 3000 rpm for 10 min at 4°C. The supernatant was discarded after the centrifugation process was complete. This process is done two times. The digested cell suspension was then added with phosphate-buffered saline (PBS) containing collagenase IV 0.75 mg/ml (Sigma-Aldrich, St. Louis, MO, USA) and Dnase 0.075 mg/ml (Takara Bio, Shiga, Japan) and incubated at a temperature of 37°C for 60 min. After filtering with a cell filter, centrifugation was carried out again for 5 min, and then the cells needed for culture were obtained.

The cells were cultured on collagen-coated plates/dishes using Dulbecco's Modified Eagle Medium (DMEM)/F12 (1:1) (Gibeo BRL, Gaithersburg, MD, USA),

The mixture contained 3 mL of AMSC-CM and 0.09 g of SAP (sodium ascorbyl phosphate). The mixing was conducted directly and at the Laboratory of Stem Cell Institute of Tropical Disease, Universitas Airlangga.

Procedure

We have conveyed all research procedures to the subjects before treatment. Before being treated with the AMSC-MP and vitamin E, the subjects received facial skincare (priming) with tretinoin 0,025% cream and sunscreen SPF 30 cream for two weeks.

The subjects underwent Janus 3D Facial Analysis System (Janus) facial examination as the objective baseline data before receiving the procedure. Five data measured in this research were wrinkles, polarized spots, UV spots, pores, and skin tones. The mixture of AMSC-CM and vitamin C was dosed 2 mL applied after microneedling and fractional CO₂ laser. The mixture was applied three times at 2-week interval in the microneedling group and at 4-week interval in the fractional CO₂ laser group. The laser fractional CO² (Fraxis Duo, Ilooda, Suwon, South Korea) parameter was energy 10 mJ/cm², 1 mm distance, single stack, single-pass, 0.34 ms pulse duration, and 120 μs spot size. For the microneedling group, we used Dermapen®, an automated handheld microneedling device with adjustable speed and depth, the parameter for the depth was 0.5 mm (periorbital and forehead area), 1 mm (cheeks and chin) and level 3 speed.[9] The evaluation using Janus was conducted on week 4 (observation 2) and 8 (observation 3) in microneedling group, and the evaluation for the fractional CO₂ laser group was conducted on week 4 (observation 2) and week 12 (observation 3). The whole procedures were performed by the researcher (medical doctor).

The follow-up visits were scheduled flexibly if any subject experiences side effects, such as itchiness, red face, or other conditions that require a doctor's examination. In the event of severe cases, such as infection and complications related to the procedure, the research would be dismissed. The subjects were advised to protect their facial skins from direct sun exposure for optimal results.

Statistical analysis

The data were analysed using the comparative statistic test specifically using the SPSS software version 21 (SPSS Inc., Chicago, IL, USA). The level of significance was P < 0.05.

Results

This research involved 60 subjects with photoaged skins who passed the inclusion and exclusion criteria. All research subjects were willing to take part in the study by signing information for consent, informed consent, and medical agreement sheet.

The microneedling group (control) demonstrated a significant result on wrinkles, UV spots, polarized spots, and pores on the second observation when compared with the baseline data (observation 1) [Table 1].

Table 1.

Intragroup of the control group (A mixture of AMSC-CM and vitamin C after microneedling as LADD)

Observation	Wrinkles	UV Spots	Polarized Spots	Skin Tones	Pores
1–2 (week 0)	0.044*	0.015*	0.052	0.297	0.018*
2–3 (week 4)	0.046*	0.009*	0.036*	0.319	0.029*
1–3 (week 8)	0.983	0.848	0.876	0.963	0.848

The fractional CO₂ laser group (treatment) also demonstrated a significant improvement in wrinkles, polarized spots, and pores. We found an immediate significant improvement in wrinkles during the second observation on the fourth week after treatment. Furthermore, we also found a significant improvement in polarized spots during the third observation [Table 2].

Table 2.

Intragroup of treatment group (a mixture of AMSC-CM and vitamin C after fractional CO₂ laser as LADD)

Observation	Wrinkles	UV Spots	Polarized Spots	Skin Tones	Pores
1–2 (week 0)	0.000*	0.099	0.000*	0.176	0.007*
2–3 (week 4)	0.001*	0.604	0.134	0.264	0.050
1–3 (week 12)	0.152	0.255	0.027*	0.812	0.440

Table 3 shows the comparison of the control group and treatment group's results. Wrinkle, skin tone, and pore of the treatment group appeared significantly improved in the second and third observations. A significant difference in UV spot observation was found at the second observation.

Table 3.

A comparison between the treatment group and control group

Observation	Wrinkles			UV spots			Polarized spots			Skin tones			Pores
	Microneedling AMSC-CM-VC	CO2 fractional laser AMSC-CM-VC	P	Microneedling AMSC-CM-VC	CO2 fractional laser AMSC-CM-VC	P	Microneedling AMSC-CM-VC	CO2 fractional laser AMSC-CM-VC	P	Microneedling AMSC-CM-VC	CO2 fractional laser AMSC-CM-VC	P	Microneedling AMSC-CM-VC	CO2 fractional laser AMSC-CM-VC	P
1-2	−1.56 (5.22)	−0.85 (4.09)	0.15	−1.48 (3.89)	0.50 (6.02)	0.33	−1.21 (3.55)	0.46 (5.30)	0.20	−1.03 (8.01)	−0.43 (3.40)	0.30	−1.05 (4.75)	−0.65 (4.26)	0.10
2-3	−1.55 (6.89)	−0.23 (3.34)	0.00*	−1.58 (3.87)	1.41 (7.15)	0,04*	−1.30 (4.62)	1.90 (5.10)	0.89	−0.98 (8.02)	−0.31 (3.91)	0.00*	−0.95 (3.77)	−0.23 (4.42)	0.02*
1–3	−2.08 (6.18)	−1.13 (3.71)	0.00*	−1.97 (4.17)	0.66 (6.23)	0.21	−1.62 (4.21)	0.62 (5.27)	0.12	−1.37 (8.34)	−0.57 (3.78)	0.00*	−1.40 (4.13)	−0.86 (4.29)	0.01*

Discussion

This research compared fractional CO₂ laser and microneedling in administering a mixture of AMSC-CM and vitamin C (AMSC-CM-VC). Microneedling is a modality therapy that inducts collagen using micro-sized needles, creating small holes invisible to the naked eyes. We used an automatic microneedling instrument with controllable speed and depth.[7,10,11] We use fractional CO₂ laser (Fraxis Duo, Illoda, Suwon, South Korea) as the LADD with a parameter of 10 mJ/cm², 1.0 mm distance, single stack, single pass, and 120 μ spot size. The parameter was expected to provide optimal LADD results. The subjects' skin faces were cleaned and smeared with 10% lidocaine topical anaesthesia for comfort during the procedure.

The participating subjects have passed the inclusion and exclusion criteria. We conducted a matching pair selection per the Glogau criteria and Janus analysis. The subjects received priming for 2 weeks, which was stopped 3 days before the scheduled treatment. We used 0.025% tretinoin cream for priming to minimize the side effects, accelerate the healing, and condition the skins.

All research subjects were women experiencing photoaging. The gender uniformity aimed to minimize dropouts of the research subjects as it is believed that women concerned about their skin more than men. Therefore, the level of compromise was expected to be higher than men. Visitation data of photoaging patients at the cosmetics unit of Outpatient Clinic of Dermatovenereology RSUD Dr. Soetomo showed that the visitors were predominantly females. There were 711 female patients, and 24 male patients opted for skin rejuvenation in 2018.

UV radiation can cause wrinkles through the decrease of stretch strength, elasticity, and degradation in structural components that support the extracellular dermal matrix. UV radiation can increase the ROS, increasing the AP-1 and NF-kB and lowering the TGF-β. Furthermore, there will be a decrease in the production of collagen and an increase in collagen destruction.[12] The AMSC-CM consists of several growth factors that can stimulate the proliferation and migration of dermal fibroblasts, epidermal keratinocytes, and increase the collagen synthesis from the fibroblasts.[13,14]

Seo et al.[13] reported an improvement in skin roughness after subjects underwent a photoaging treatment using the metabolite product of stem cell and microneedling. They also reported an increase in dermal collagen with a minimum side effect. Lee et al.[2] compared between metabolite products of stem cell after microneedling and microneedling only, and they reported a significant result in wrinkles repair. Subjects of the research were treated five times at 2-week interval. They used the Visiometer analysis, which examines the skin roughness and depth of the wrinkles. Prakoeswa and colleagues have reported that AMSC CM and microneedling had better results compared to the placebo for photoaging treatment.[9]

Research by Zhou et al.[14] on atrophic acne scar and skin rejuvenation assessed the efficacy and side effects of PM-ADSC (product metabolite of adipose-derived stem cell) after fractional CO₂ laser compared to the DMEM. The research involved 29 individual subjects at 8-W static mode and 25% spot density. The result showed a meaningful satisfaction, increase in hydration, elasticity, decrease in TEWL, skin roughness, and index of melanin within 12 weeks. The histological analysis also showed that the PM-ADSC increases the dermal collagen density and elastin density. Split-face research on the efficacy and safety of AMSC-CM after Er-Yag laser showed a more significant repair in pores compared to normal saline.[15] Rahmadewi has also published a study on AMSC metabolite product and vitamin E after laser fractional CO₂ and reported improved pores in photoaging patients.[16]

We used vitamin C in consideration of its function in stimulating collagen synthesis, improving wrinkles through its role as a co-factor of lysyl and prolyl hydroxylase.[17,18,19] This was in line with research by Crisan et al.[20] which reported a significant increase in collagen synthesis, as evidenced in the results of ultrasonography evaluation. Figures 1-4, shows baseline and after treatments using Janus 3D Facial Analysis System.

Figure 1.

Baseline of microneedling + AMSC CM VC control group

Figure 4.

After three sessions of fractional laser CO2 + AMSC CM VC group

Figure 2.

After three sessions of microneedling + AMSC CM VC control group

Figure 3.

Baseline of fractional laser CO2 + AMSC CM VC group

In conclusion, the fractional CO₂ laser as LADD provided better results in transdermal drug delivery compared to microneedle-facilitated delivery. Also, a combination of AMSC-CM and vitamin C has a promising role in photoaging treatment. However, more studies are needed in the future.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.