Ageing is a natural and multidimensional process concerned with physiological, cellular and social changes of an organism over a period of time. Internal factors like genetics, hormones, metabolism and external factors like sun exposure, radiation and toxins contribute to ageing [1] . Some explanations for inevitable natural aging are cell damage by reactive oxygen species, mitochondrial dysfunction, and shorting of telomeres in successive cell division [2] . Aging in the sun exposed parts is typically characterized by wrinkling, laxity and roughness [3] [4] . Growth factor supplementation for preventing the aging of skin is an emerging concept. Stem cells release a host of growth factors into the conditioned medium (CM) used in culture [5] . Use of this conditioned medium in a suitable formulation could be used to prevent skin aging.

Cutisera^TM contains the CM obtained from adult human bone marrow derived mesenchymal stromal cells (BMMSCs) along with excipients formulated into a cosmetic serum. Human mesenchymal stromal cells (MSCs) are known to secrete various types of growth factors and cytokines (GF/CK) that possess important biological functions. These GFs/CKs present in the CM secreted by BMMSCs grown under specific culture conditions promote generation of healthy skin, hair growth and wound healing. CM reportedly contains more than 200 GFs/CKs. We have identified and qualified 30 GFs/CKs secreted by BMMSCs in our culture condition. Some of these factors have been implicated for their role in repair and regeneration of damaged tissue [6] [7] [8] . Using a novel production technology under Good Manufacturing Practice (GMP) conditions, homogenous CM is being consistently produced for developing the cosmetic product Cutisera^TM to derive benefits for skin care.

A total of 40 subjects were enrolled in the study. The subjects were equally distributed based on their skin type (normal, oily, dry and combination). Thirty eight subjects completed the study. Site application reaction by dermatological evaluation for product tolerance showed that one subject had mild dryness at baseline and day 30. One subject had mild acneiform eruption at day 15 and day 30. Frequency and severity of the noted reactions were neither clinically significant nor required any medical intervention. Site application reaction reported by the subjects based on self-evaluation for product tolerance is as follows: Mild itching, mild burning sensation, severe sweating (one subject each), Mild oiliness (two subjects), moderate oiliness (one subject) at day 15; Moderate and severe itching (one subject each), Mild oiliness (3 subjects) and moderate oiliness (one subject) at day 30. At day 60, no site application reaction was noted by the dermatologists or perceived by the subjects and the product was considered overall safe.

There was improvement in fine lines and wrinkles (dermatological assessment and Visioscan) (Figure 1), lateral crow’s feet (Figure 2), evenness of skin tone, Chromameter luminance (L* Value) at cheek and forehead (Figure 3), skin firmness/laxity (dermatological assessment and Cutometer) (Figure 4), hydration of skin (Figure 5), homogenecity of age spots (Figure 6), and visible pores (Figure 7). A good proportion of subjects felt improvement in several parameters (Figure 8). Representative images showing improvement are given in Figure 9, Figure 10, and Figure 11.

The study met its both primary and second objectives of efficacy and safety. Twice daily application of serum containing growth factors and cytokines for a period of 90 days in 38 healthy Indian females was safe and well tolerated. Application site reactions were not clinically significant and disappeared completely by day 60. Cutisera^TM application considerably improved tested anti-ageing parameters. The improvement was perceived starting from day 15 onwards (crow’s feet and hydration statistically significant at day 15) and continued over the course of study. The subjective assessments corroborated with dermatological

assessment and measurements by instruments. Impaired capacity of skin to synthesize collagen thereby loosing elasticity and turgor occurs as a part of chronological ageing and photoageing. Stem cells release a host of bioactive factors and the same are found in abundance in the conditioned medium used for culturing these cells. Because of the host of factors released by stem cells into the medium, the condition medium is being evaluated as an alternative to stem cell based therapy for a variety of indications including skin conditions [5] [9] [10] .

Natural extension of this application of therapy using conditioned medium is the treatment of aging. It is known that endogenous growth factors reduce with age [11] . Hence, administration of growth factors makes it logical for treatment

of skin aging. TGF beta causes increase in type I and type III collagen and plays a role in maintaining extracellular matrix [12] . Growth factors promote collagen synthesis, thus potentially benefit in rejuvenation [13] [14] [15] . Human growth factors are emerging as potential anti-aging agents [16] [17] [18] .

The combination growth factors in Cutisera^TM have shown benefit in skin aging in this study. The present study is similar to the study of growth factors administered along with a moisturizing cleanser and a sunscreen [19] . Clinical benefit in fine line and wrinkle was seen after three months of test product application compared to vehicle. Similar regime involving human epidermal

growth factor protein along with facial cleanser and sunscreen used for three months was effective in improving texture and appearance of facial skin [20] .

Local application is a predominant route for delivering drugs for dermatological purposes. It is known that the stratum corneum is impermeable to molecules with high molecular weight [21] . In spite of high molecular weight, topically applied growth factors and cytokines provide clinical benefit. One possible explanation is that they pass through hair follicles, sweat glands and microlesions in

the interfollicular stratum corneum [22] . It is likely that small quantity reaching the stratum corneum binds to specific receptors to produce downstream signaling and may be clinically beneficial. In consequence, keratinocytes produce growth factors and initiate the effects on skin layers including collagen formation and strengthening of extracellular matrix.

Skin biopsy findings can provide evidence for mechanism of action. In a pilot study by Fitzpatrick et al., application of mixture of growth factors on photodamaged skin showed clinical improvement and suggestion of collagen formation on histopathology [23] . Increase in epidermal thickness and increased fibroblast density in dermis in addition to clinical benefits of wrinkle reduction was seen in a study by Hussain et al. [24] . In another study, application of adipose derived stem cells conditioned medium increased the synthesis of type I and III procollagen and elastin genes [9] . However, the study failed to show clinical efficacy, probably due to one-time administration of conditioned medium. Thus implying that daily (or twice daily) application of growth factors may be ideal for skin health.

Growth factors have been used either alone or in combination with other agents which complement the antiaging activity. A regimen containing six products including growth factor serum, cleanser, skin brightener, moisturizer, retinol, and sunscreen was found to be safe and efficacious in photodamage over face [25] . Growth factor combination product with or without l-ascorbic acid has been evaluated and the benefits were attributable to TGF beta [26] . Growth factors have been combined with hyaluronic acid and evaluated as anti-aging therapies [27] [28] . Mixture of growth factors and hyaluronic acid has been studied in conjunction with laser for skin rejuvenation with positive outcome [29] .

Microneedling can facilitate entry of various agents in to the skin including growth factors. A novel method of combining microneedle fractional radiofrequency and conditioned medium in skin aging demonstrated clinical benefit and increase in dermal collagen content [30] . Subsequently, Lee et al. reported benefit of delivering growth factors through 0.25 mm microneedle for skin rejuvenation in a randomized, blinded study [31] .

Current study has few limitations: Firstly, it lacked a placebo group. Secondly, multiple dose levels might have identified minimal effective dose and the dose (if any) after which the response plateaus. Third, there was no wash out period before the final assessment which might have shown persistence of effect (if any) after cessation of application. Fourth, we did not conduct skin biopsy which might have provided insights into structural changes.

The current study establishes the efficacy of a test serum containing growth factors and cytokines as an anti-ageing and rejuvenating modality in people with all skin types without any major side effects. Such topical products can be successfully used as either an alternative or as an adjunct to invasive rejuvenation procedures such as laser resurfacing. Combining topical growth factors with invasive rejuvenation procedures may improve recovery time and provide a synergistic effect on skin rejuvenation.

This study was funded by Stempeutics Research Private Limited, Bangalore, India. Dr. Madhumita Panda and Dr. Jaideep Gogtay of Cipla Ltd. contributed in the protocol development.

Sachdev, M., Chu- llikana, A., Seetharam, R.N., Shilpakar, R., Sapna, R., Velugotla, K., Ritambhara, Majumdar, A.S. and Gupta, P.K. (2017) A Single Arm, Monocentric, Prospective, Open Label Study to Evaluate Anti-Ageing Efficacy of Human Growth Factors and Cytokines Containing Cosmetic Skin Serum in Indian Female Population. Journal of Cosmetics, Dermatological Sciences and Appli- cations, 7, 118-132. https://doi.org/10.4236/jcdsa.2017.71011