Growth Factor for the Treatment of Skin Aging
dr.Ida Ayu Diah Purnama Sari, Sp.KK
Aging is a progressive accumulation of pathological changes in cells and tissues, including the skin, as well as decreased tissue ability to repair or replace itself and maintain its normal structure and function. In skin aging, barrier function, cell replacement, DNA repair, epidermis hydration, immune response, mechanical protection, sebum production, sensory perception, sweat production, temperature setting, and wound healing decreased.
There are various theories that are quite popular in the aging process but none of these theories can fully explain how the changes occur during the aging process. Essentially, the formation of reactive oxygen species (ROS) is the most important aspect of the skin aging process. Without an adequate defense system, free radicals will further cause oxidative stress and damage to the structure of proteins and DNA that are responsible for retaining the function of such cells. With increasing age, oxidative damage and free radicals are increasingly triggering the formation and accumulation of ROS, which leads to cellular damage and genetic mutation. In addition, accumulated advanced glycation end product (AGE) occurs on the skin. This AGE reacts with cellular and extracellular components causing inflammation, which further causes oxidative stress on those cells and eventually aging the cells.
There are two main processes of interrelated and overlapping skin aging, intrinsic and extrinsic aging.
Intrinsic aging is also known as natural aging process, which is an ongoing process that begins at the 20s age-group, caused by various factors from the physiological factors of the body itself such as genetic, hormonal and racial factors, as well as pathological factors such as diseases and malnutrition.
The shortening of the telomere on cell division can also be said to be one of the causes of the intrinsic aging to the skin. Telomere is a terminal part of a mammal chromosome consisting of hundreds of base-pairs short sequence of TTAGGG. These base-pairs cover the edges of the chromosomes to prevent fusion. During cell division, when the chromosome is splitting, the DNA polymerase enzyme cannot replicate the final base pair of chromosomes. Therefore, these terminal sequences will gradually disappear in the process of replication, resulting in shortening/shrinkage of the chromosome. When the telomere becomes too short, it will trigger the process of cell apoptosis. Because of this, the telomere is thought to play a role in the aging process. Telomerase is a reverse transcriptase enzyme found in stem cells that can replicate terminal-base pairs, but this enzyme is not found in most cells. Physiological aging process is more noticeable in women who enter the menopause period. At that time, ovarian function decreased, causing estrogen reduced, resulting in dryness and decreased skin elasticity so that it can cause aging of the skin. This suggests the influence of hormonal changes in aging skin.
Intrinsically aging skin will be accompanied by an expression line that is clearly visible but still maintain a geometric pattern of the skin. Under the microscope, intrinsically aging skin demonstrates epidermal atrophy, a flatter rete ridge and dermal atrophy. The collagen fibers increase in number with the increase in collagen III and collagen I. he histology of the skin that experiences intrinsic aging is summarized in table 1.
Table 1. Histology Overview of Skin Aging
|Flattening dermal-epidermal junction|
Decreased of thickness
Size and shape of cells vary
Diminish of the Langerhan cells
|Atrophy (Loss of dermis volume)|
Mast cell reduction
Reduced blood vessels
Capillary arch shortening
Peripheral nerve abnormalities
Change terminal hair into velus
Nail plate abnormalities
Reduced number of glands
Extrinsic aging is an aging process that occurs as a lifetime of exposure to ultraviolet radiation (UVR) so it is known as photoaging. Sun exposure can induce skin aging early and is often referred to as premature aging. The dominant extrinsic aging skin is found in sun-exposed areas such as the face, chest and extensor surface of the arm. Clinical findings from photoaging skin include wrinkles and pigmented lesions such as freckle, lentigines, hyperpigmentation patches as well as depigmentation lesions such as hipomelanosis gutata. Other signs of skin aging include loss of firmness and elasticity, purpura area on skin and benign lesions such as keratosis, telangiectasias and skin tag. Glogau develops a scale of photoaging to classify the clinical sun damage levels (table 2). Patients with significant exposure to sunlight on this scale may have a higher value than is appropriate for their age, so patients with minimal exposure to sunlight will have a lower value than their age.
Tabel 2. Photoaging Glogau Classification
|Type I||Type II||Type III||Type IV|
|No wrinkles||Wrinkles in motion||Wrinkles at rest||Only wrinkles|
|Usually in the 20-30 age group||Usually in the 30-40 age group||Usually in the 50 and above age group||Usually in the 60 and above age group|
|Mild photoaging||Moderate photoaging||Advanced photoaging||Severe photoaging|
|Mild pigment changes||Senile lentigines||Dyschromia, telangiectasias||Yellow-gray skin colour|
|No keratosis||Keratosis palpable but not visible||Visible keratosis||Prior skin malignancies|
|Minimal wrinkles||Parallel smile lines begin to appear||Visible wrinkles||Wrinkles throughout-no normal skin|
Histopathology alterations in the skin experiencing photoaging can be easily distinguished and characterized by elastosis. Skin experiencing photoaging is also characterized by epidermal atrophy and discoloration changes in the fibers of elastin and collagen. In the skin experiencing heavy photoaging, the collagen fibers are fragmented and thickened. Elastin fibers also appear fragmented. Changes in the fibers of elastin and collagen deteriorate with ultraviolet exposure (UV).
Skin Aging Treatment Modalities
In principle, anti-aging skin therapy is prioritizing prevention factors. Prevention is done by avoiding exposure to sunlight such as the use of sunscreen with adequate protection power and adjusted to the skin condition. When photoaging occurs, any action taken to overcome or correct the disorder is considered a treatment. Such treatment includes the use of chemical peeling, techniques such as micro-dermabrasion, and the use of ablative and non-ablative laser beams. Recently the role of growth factor as a therapy on aging skin was discovered. Anti-aging skin therapy with growth factor can be both topical and injectable.
Growth factor is a polypeptide or protein that plays a role in the regulation of various cell processes. Together with cytokines, this substance helps to regulate differentiation, chemotaksis and adhesion, activation, apoptosis, survival, and cell transformation. Growth factors are capable to metabolize activity in a specific micro-network environment at very low concentrations.
Growth factor is a key regulator of wound healing, and the topical use has been widely prescribed. Clinical trials indicate the administration of topical growth factor can shorten the healing time and increase the speed of the wound closure. This growth factor includes Epidermal Growth Factor (EGF), Fibroblast Growth Factor-2 (FGF-2), Transforming Growth Factor-beta (TGF-β), and Platelet-Derived Growth Factor (PDGF).
In recent years, topical and injection growth factor emerged as an appealing modality of anti-aging skin therapy, with increasing interest in skin rejuvenation.Growth factor can be applied topically or injected in platelet-rich plasma autologous (PRP). To understand the scientific reasons regarding this therapy is necessary insight into the pathophysiology of aging skin, and how it can be handled at a cellular level.
Mechanical, protective and restorative skin properties decrease as they age. Daily exposure to environmental stressors, extrinsic factors, including UV and cigarette smoke, enhances oxidative stress. The result is tissue damage, due to decreased antioxidants accompanied by increased production of ROS, also known as free radicals. Some biochemical pathways that are triggered by the ROS often result in the suppression of transforming growth factor-β receptor II (TGF-β-R2), an excess expression of the metalloproteinase matrix (MMP) which is a cholestylase, and an increase in inflammation through the nuclear factorkappa β pathway. UV radiation also causes direct damage to the structural protein of the skin. This extrinsic factor increases intrinsic damage, which is related to the decrease of progressive antioxidant capacity, which is age-related accompanied by increased ROS production from oxidative metabolism of cells in the skin. Intrinsic deterioration contributes to the excess ROS and biochemical effects. The analysis of aging cells shows the shortening of progressive telomere, which is also related to tissue damage.
Picture 1. Biochemical pathway of skin aging.
The intrinsic and extracellular aging of skin cause the breakdown of collagen and elastin tissues in the dermis. It is clinically manifested as xerosis, loss of elasticity, atrophy, and dyschromia. A safe, non-invasive treatment searches to restore or inhibit this change is still a challenge. Studies on the healing of chronic and acute wounds have provided insight into the pathophysiology of photoaging. There are some parallels between the pathways involved in wound healing and that which are necessary for the regeneration of skin aging. Therefore, an understanding of the wound healing process can improve the understanding of skin aging process and the intervention that can overcome both.
Growth factor consists of a large group of protein regulators that attach to surface receptor of the cell and serve as chemical messengers. Through this interaction, the growth factor mediates inter-and intracellular signaling pathways that control the growth, proliferation and differentiation of cells. Unlike hormones, growth factor activity is limited to the vicinity of its production. In the skin, the growth factor is synthesized by fibroblasts, keratinocytes, platelet, lymphocytes and mast cells. The growth factor specifics regulate important cell activities, including mitogenesis, angiogenesis, chemotaxis, extracellular matrix (ECM) formation and growth factor control.
When the skin is injured, the growth factor accumulates on the wound area and interacts synergistically to initiate and coordinate wound healing. These growth factors can reverse the effects of colagenase, increase collagen levels and reduce tissue inflammation. Clinical studies show that growth factor applications derived from animals or humans, administered topically or by injecting growth factor autologous, can also increase skin collagen synthesis and this is related to the reduced signs of skin aging process such as fine lines and wrinkles.
Wound healing of the skin is governed by complex interactions between growth factors. The success of wound healing requires a balance between inflammatory development and resolution. It involves a lot of growth factors, including Platelet-Derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF), Transforming Growth Factor-β (TGF-β), Epidermal Growth Factor (EGF), Granulocyte Colony-Stimulating Growth Factor (G-CSF), Keratinocyte Growth Factor (KGF), Interleukin 6 (IL-6), Interleukin 8 (IL-8), and Hepatocyte Growth Factor (HGF). The relevant growth factor for wound healing induces dermis remodeling with the stimulation of new glycosaminoglican, elastin and collagen synthesis, and by triggering angiogenesis.
Picture 2. Important growth factor during the three main stages of wound healing.
There is a prominent similarity between these events and effective events overcoming the effects of the intrinsic and extrinsic skin aging. It has been hypothesized that skin aging is in line with a considerable wound that transcends skin repair mechanisms, which weaken with age. The purpose of a topical or injectable factor is to fill the depletion of the growth factor on the skin itself and to regulate the cell activity responsible for skin remodeling, thus slowing down or even reversing the manifestation of skin aging. This thought can be expanded to lesions of iatrogenic skin, such as during laser procedures and other skin rejuvenation, that there is a hypothesis that the injection and topical growth factor can also facilitate healing in this situation and may even increase the outcome.
Mechanism Action of Growth Factor
Topical and injectable growth factors have the potential to modulate complex mobile interactions that ultimately results in an increased regulation of collagen synthesis and a lower regulation of collagen degradation. The signal from the presence of cytokines after the application of topical or injection growth factor can be an overview of reflection of interactions that occur during wound healing. If topical growth factor successfully penetrates the stratum korneum, it can bind to the specific receptors of the keratinocytes and initiate a signal cascade. Once tied to the growth factor receptors, the growth factor is secreted by the keratinocytes itself, stimulating fibroblasts to synthesize the growth factor that gives effect to dermis. The growth factor derived from fibroblasts also stimulates the proliferation of keratinocytes. Research has been conducted to show minimal penetration intact in the stratum corneum by a hydrophilic molecule that has a molecular weight greater than 500 Da. Growth factor is a hydrophilic molecule with a molecular weight more than 15.000 Da. Therefore, it is unlikely that the Growth factor could penetrate the intact epidermis in sufficient quantities to exhibit significant effects clinically.
One of the routes that can be reached by topical growth factor to epidermal keratinocytes receptors is through hair follicles and sweat glands. Another consideration is that aging skin tends to begin to experience impaired barrier function, and it allows for better penetration. Once the growth factor has crossed the stratum corneum, their interactions with specific receptors in keratinocytes can initiate cytokines that trigger cascade that affect fibroblasts and other cells in the dermis. The results of the coloragenesis and remodeling of ECM have been observed histologically, and can be correlated with visible clinical outcomes. PRP and its derivatives are injected, containing growth factor potentially causing the effects of similar mechanisms. Direct intradermal or subdermal injection can accelerate or enhance the clinical effect.
Picture 3. Mechanism action of growth factor.
The main function of platelets is to control blood loss after a vascular injury. Interactions between platelets and plasma proteins lead to the formation of fibrin clot. This clot is a reservoir of growth factor, which is disposed to plasma of the alpha platelet granules when activated and destroyed during wound healing and tissue regeneration. The rational for platelet rich plasma (PRP) is concentrating and providing direct growth factor to the target tissues, such as aging skin; or injured muscles, tendons, or cartilage. Usually, the concentration of platelets in the PRP can reach 5 to 10 times the normal concentration of platelet in the blood. PRP injection allows direct transmission of growth factor to the dermis. After passing the stratum korneum, the efficacy does not depend on the TEWL penetration of the active substance.
PRP techniques are prepared using the blood of the patient, which is taken shortly before the therapy. This PRP therapy is prepared with different centrifugation techniques, using a double-spin method. At high speeds, cellular materials are formed with different specific gravity. Previous patients are advised not to consume number of medications such as aspirin, and other non-steroidal anti-inflammatory drugs drug types for two weeks.
Equipment commonly used is the Digital Bench Top centrifugation machine, Tarson Pasteur Pipette, Tarson Tube 14 ml, spuit 10 cc, insuline syringe, solution sodium citrate 3,8% and solution calcium chloride 10%. There is no standardization in the preparation and application of PRP. The preparation for concentrating platelet from autologous blood should be through double centrifugation technique as red blood cells will interfere with PRP formation if not removed first. Platelet rich plasma can be applied topically around the target area or by intradermal injection. After application, PRP will trigger and strengthen signal to start tissue healing.
The first step is to take 10 ml of blood samples from the arm veins, to be inserted into the tubes that have been labeled with the patient’s identity (name and age), mixed with the anticoagulant Acid Citrate Dextrose (ACD) with a ratio of 10 : 1,5. In room temperature (22 – 260 C) samples were centrifuged for 10 minutes at 2000 rpm, with the aim of separating the plasma from red blood cells and other blood components.
Furthermore, the formed plasma was transferred to another tube and centrifuged for 10 minutes at 3000 rpm, with the aim to precipitated platelet. The low platelet plasma is then extracted and disposed, so there are approximately 3-6 cc of PRP available. The normal count of platelet counts is 200.000/ µL roughly, after being PRP the amount increases by 5 times or at least one million platelet/ µL.
Picture 6. PRP preparation.
To stimulate platelet degranulation as well as release of growth factor, in general, platelets are activated ex vivo first by adding calcium chloride or thrombin with a ratio of 1:10 and rotated firmly. Degranulation will take place rapidly, therefore PRP preparations should be applied within 10 minutes after platelet is activated. During the PRP preparation process, patients can be administered topical/local anesthesia if necessary. Apply antiseptic on the target area before PRP application. The quality of the PRP is determined by the ability and experience of the individual conducting plasma separation. To obtain the effect of platelet degranulation and activation of certain number of growth factors, the bottom of the plasma must be activated. Products can be applied in the form of injection or gel. An activated product must be used within 10 minutes after the formation of the gel. The patient must also signed the informed consent which explains the procedures, contraindications, effectivity and possible side effects.
A study was conducted on 23 patients (an average of 47 years old, between 29-70 age group) for 3 months. Injection with platelet rich plasma is done once a month. The evaluated parameters are the nasolabial fold, the skin elasticity assessment, texture and homogeneity of the skin, wrinkles around the eyes and wrinkles around the neck. Evaluation is performed every month after injection. Each patient receives 4 ml of PRP which has been activated. PRP concentrations are injected within 7 minutes.
In this study, no serious long-term side effects have been reported. Mild and temporary side effects that have been reported are bruises and petechiae (3% patients), burning in 3 minutes (70% patients), and minimal erythema at the injection site (80% of patients). The results of several studies were evaluated after one session of therapy (using dermoscopy and digital camera), as well as based on patient satisfaction. Based on this clinical study, the use of PRP can be concluded with significant improvement, and the score varies between good and very good.
Another study showed the use of PRP in the facial area. This study used 408 patients (387 women and 21 males). Clinical effects of skin rejuvenation and side effects were evaluated in 3-month intervals. The results gained vary based on the age of the participants. In young people (less than 35 years old), rapid results showed, and therapy aimed to rejuvenate and to inhibit aging of the skin. For this age group, therapy every 12 to 24 months is very effective. Patients over 45 years of age required a second therapy of 9-12 months after the second injection, and could be combined with other therapies. Patients aged 50-60 years needed second therapy after 6 months, third therapy after 15 months from the first therapy. Patients over 60 years old needed second therapy after 3 months, third therapy after 9 months and fourth therapy after 1.5 years first therapy.
Aging process occurs in all of the body’s organs, including the skin. Skin health is an overall reflection of internal health. Skin aging is a complex biological process and is influenced by intrinsic and extrinsic factors. Intrinsic skin aging occurs in line with the increase in age due to the body’s response to sustain vital life. Intrinsic aging is influenced by age, genetic, racial and hormonal factors. Meanwhile, extrinsic skin aging is influenced by environmental factors especially chronic sun exposure. It is often also called photoaging.
Various strategies can be done to inhibit the aging process of the skin, one of them by administering therapy with growth factor. Growth factor is a polypeptide or protein that plays a role in the regulation of various cell processes. Together with cytokines, this substance helps regulate differentiation, chemotaksis and adhesion, activation, apoptosis, survival, and cell transformation. The analysis of biochemical and structural changes that occur as the person ages has triggered the observation that skin aging has several connections with widespread acute and chronic skin injuries. Topical and injectable growth factors (or called platelet rich plasma) have the potential to cope with skin aging through cell regeneration stimulation. Number of studies show that the growth factor applied topically or by injectable can stimulate positive epidermal changes, and this is related to clinical improvement in the science of skin’s anti-aging.