1. Dr. María Del Pilar Reyes Agüero, Aesthetic Medicine & Cosmetic Surgery. C. P. Ortiz Rubio 1325, Medardo González, 88550 Reynosa, Tamps., Mexico
  2. President of the WSCG World Society of Cosmetic Gynecology Medical Consultant and Educator, President of ICAM USA Inc International Consultants in Aesthetic Medicine, University of Buenos Aires, Dallas, Texas, USA


The bio-revitalization treatments for hair loss used in the present study focus on promoting hair growth by stimulating cell proliferation and differentiation during the hair growth cycle.

We use the terms bio-revitalization or bio-stimulation because these treatments help restore and promote normal physiological processes, leading to continuous renewal of scalp tissue and stimulating hair growth.

We present the treatment protocol and results obtained using a combination of PRP, carboxytherapy and a capillary solution of Alfatradiol 0.025 %.

Concerning these treatments :

  • Scientific studies on platelet-rich plasma (PRP) confirm that its benefits include increased wound healing of hard tissue and In addition, intradermal injections of PRP into the scalp have been shown to increase vascular structures around hair follicles and induce proliferation of dermal papilla cells.
  • Carboxytherapy involves the therapeutic use of carbon dioxide (CO2) in a sterile gaseous state. In the microcirculation, CO2 increases vascular tone and produces active microcirculatory vasodilation. Vasodilation induced by CO2 promotes the Bohr effect, resulting in increased tissue oxygenation and neo-angiogenesis. At the same time, the tissue stretching produced by CO2 induces subclinical inflammation that triggers tissue regeneration processes through activation of macrophages, fibroblasts and endothelial cells, stimulating neovascularization and restoration of the extracellular matrix.
  • Alfatradiol hair solution, 0.025 % is for use as a home care product, to be applied in the evening to a clean, dry scalp. Alfatradiol solution antagonizes the inhibitory effect of testosterone and dihydrotestosterone proliferation in anagen hair follicles. As a result, it reduces dermal synthesis of dihydrotestosterone by inhibiting the enzyme 5a reductase.

Our medical clinical study was conducted in Reynosa, Tamaulipas, Mexico, over the past 2 years.

  • We used a treatment protocol that included one session of intradermal PRP injections once a month and one session of deep intradermal carboxytherapy once a week over the entire balding area, plus Alfatradiol solution as daily maintenance at
  • Depending on the grade of alopecia, according to Hamilton's classification [43] Norwood [44Treatment duration for each patient was approximately 3 to 5 months, with a one-year follow-up.
  • Evaluation criteria were hair regrowth, using clinical, subjective and objective observation and before-and-after photographs. At the end of treatment cycles, patients showed a clinical improvement in the number of growing hairs.
  • At the end of follow-up, we achieved a high rate of patient satisfaction with the therapeutic outcome.
  • We believe this protocol is scientifically supported as a non-surgical treatment for androgenic alopecia.



The aim of the study is to evaluate the effects of a combination of biostimulation therapies using

  • PRP,
  • carboxytherapy
  • and Alfatradiol, a hair solution, on active hair growth.

Contrary to social belief, most men who suffer from male pattern baldness are unhappy with their situation and would do anything to change it.

Hair loss affects their quality of life, including social and professional relationships [...45].

  • Androgenetic alopecia or male pattern baldness accounts for over 95 % of hair loss in men.
  • By age thirty-five, two-thirds of American men will experience some degree of noticeable hair loss, and by age fifty, around 85 % of men have significantly thinning hair.
  • For around twenty-five percent of men suffering from male pattern baldness, the process begins before the age of twenty-one.
  • Dihydrotestosterone (DHT) is the specific hormone responsible for male and female baldness due to changes in its metabolism [...45].
In normal hair loss, fewer than 100 hairs fall out each day and are replaced by thick new hair.
  • In the evolution of male and female pattern baldness, the new hair is fine and sparse (intermediate hair or miniaturized hair). Male and female pattern baldness generally progresses in a definitive pattern.
  • Medical treatments for hair loss, such as minoxidil lotion, oral finasteride 1 mg or oral cyproterone acetate, can slow the development of baldness [45].
Research into biostimulation treatments confirms the chain of events that lead to the recovery of tissue physiology, and to an improvement in the condition of connective tissue, i.e. the epidermis and dermis [...].46].
Thanks to biostimulation treatments, free radicals and oxidative stress are reduced, there is a recovery of glycation and protein methylation and there is vasodilatation of the micro-vessels, so the inflammatory process is diminished so that fibroblasts and their anabolic functions are activated, and in particular the production of type III collagen, elastin and hyaluronic acid from their precursors is enhanced [48].
  • In the present study, we focused on restorative treatments, defined as procedures aimed at restoring skin metabolism and function (return to normal).
  • We used a combination of procedures, PRP injections and carboxytherapy, an excellent example of biostimulation results as restorative treatments.


The benefits of platelet-rich plasma (PRP) include increased healing of hard and soft tissue wounds.

  • Furthermore, the role of PRP in the treatment of hair loss has been confirmed in recent reports [...1, 2, 3, 4Rinaldi described the use of PRP in alopecia areata [4].
  • Uebel et al. showed that pretreatment of follicular units with PRP prior to transplantation resulted in improved hair growth and density [3].
  • Activated autologous PRP has been reported to induce dermal papilla cell proliferation by upregulating fibroblast growth factor 7 (FGF-7) and catenin b as well as ERK protein kinases (extracellular signal-related kinase) and Akt [2].
  • Angiogenesis associated with anagenesis has been suggested as an important factor in active hair growth [...5This is due to secretion of vascular endothelial growth factor (VEGF) by keratinocytes in the outer root sheath and fibroblasts in the dermal papilla [...].5, 6, 7].
  • Increased VEGF secretion influences the growth of normal and pathological dermal structures [8]. Tobin et al. have reported that hair follicle mesenchyme exhibits significant plasticity associated with the hair cycle. Modulation of these cellular exchanges is likely to be crucial during the clinically significant hair follicle transformations that occur during androgenetic alopecia [ ].9].


Injecting PRP intradermally or subcutaneously into areas of baldness has been shown to improve cutaneous ischemic conditions and increase vascular structures around hair follicles [...].1, 10]. Many current treatment modalities for hair loss have been shown to modulate angiogenesis and improve blood flow [...11].

  • The anti-apoptotic effects of PRP have been suggested as one of the main factors in stimulating hair growth [...2, 23].
  • PRP-induced activation of anti-apoptotic regulators, such as Bcl-2 protein and Akt signaling, prolongs dermal papilla cell survival during the hair cycle [2, 23].
  • Furthermore, upregulation of FGF-7/b-catenin signaling pathways with PRP treatment is suggested to stimulate hair growth by inducing follicular stem cell differentiation as well as prolonging the anagen phase of the hair growth cycle [.2, 24].


The data we report demonstrate the clinical efficacy of PRP treatment. What's more, patient satisfaction confirms the quality of the results.


Consequently, alfatradiol reduces cutaneous synthesis of dihydrotestosterone, inhibiting the enzyme 5 a reductase. With topical dermal administration, effective concentrations of alfatradiol are obtained in the skin, and only a negligible amount is absorbed [27].


  • Carbon dioxide (CO2) is an odorless, colorless gas first discovered by Van Helmont in 1648.
  • The clinical use of CO2 is not new: in Buenos Aires, Argentina, and in Clermont Ferrand, France, injections of CO2 have been used to treat peripheral arteriopathies of the lower limbs, particularly those that are obliterating [29].
  • When CO2 is administered subcutaneously, it immediately diffuses to the skin and muscle microcirculation.
  • At the vascular level, CO2 produces microcirculatory vasodilation through its direct activity on the smooth muscle cells of arterioles [30]. In addition, it promotes the Bohr effect, a mechanism leading to higher tissue oxygenation (PO2 is increased) and neo-angiogenesis [ .31, 41, 42].

" Pharmacodynamics of carboxytherapy

-   Active vasodilation [32].
  • Direct action on arteriolar smooth muscle fibers
  • Sympatholytic activity
- Increase in arterioarteriolar sphygmata.
  • Increased oxidative phenomena, with the resulting hydrolysis of triglycerides into fatty acids and glycerol in adipose tissue.
  • Stimulates neo-collagenases [33, 34].
- Hemorheological activity.
  • Improves erythrocyte deformation.
- Tissue stretching during CO2 :
  • induces subclinical inflammation,
  • which triggers tissue repair and regeneration processes,
  • stimulating macrophages, fibroblasts and endothelial cells,
  • leading to neovascularization and matrix remodeling

" Carboxytherapy unit

The CO2 is equipped with a control panel and a handpiece. The electronic control allows various parameters to be selected, such as slow flow rate, gas volume and dose percentage. The CO2 cartridge is intended for medical use only. CO2 arrives in sterile conditions.

Carboxytherapy device.



  • Men aged 25 to 49.
  • Opting for the first time for non-surgical treatment of baldness. No previous surgery. Between 2 and 5a in the Hamilton Norwood ranking.


  • Patients who had received topical (such as minoxidil, prostaglandin, or analogues, retinoids and corticosteroids) or systemic treatments for baldness (such as finasteride, dutasteride and anti-androgens) in the previous 12 months were excluded.
  • Patients with a propensity for keloids and immunocompromised patients were also excluded.

Informed consent

  • All patients gave written informed consent prior to participation in the study, which was conducted in accordance with the Declaration of Helsinki..


Obtaining PRP

  • The process begins with the collection of a 10cc (1 tube) or 20cc (2 tubes) autologous blood sample in a tube with separating gel and anticoagulant.
  • Alfatradiol 0.025 % Hair solution: for home care, apply in the evening to a clean, dry scalp.

After centrifugation, approximately 5.5 mL of non-activated autologous PRP is obtained per tube (centrifugation: 3,200 rpm for 5 minutes).


  • 39 male patients with various grades of androgenetic alopecia or male pattern baldness received intradermal PRP, using a mesotherapy technique, 0.05 mL injected every 1 to 1.5 cm, every 4 weeks.
  • Immediately after the PRP, they received CO2 intradermally, 5 cc into each injection site, using a 30G needle, with a distance of 2 to 3 cm between them, over the entire bald area.
  • The carboxytherapy technique was then repeated, once or twice a week, using the same dose, 5 cc at each injection site, with a distance of 2 to 3 cm between them, and over the entire bald area.
  • The duration of treatment in each patient was approximately 3 to 5 months, depending on the degree of alopecia according to the Hamilton Norwood [44].
  • The follow-up was carried out over a one-year period.

Evaluation criteria

  • All patients were evaluated in four stages:
    • T0, start of study.
    • T1 after 12
    • T2, after 6
    • T3, after 12
  • The results of the treatment on hair growth were measured using before and after photographs (different stages), the doctor's clinical observation and patient satisfaction.


Histological evaluation.

  • Incisional punch biopsies (3 mm diameter) of the scalp were obtained at baseline and two months after the last treatment and fixed in buffered formalin.
  • Histological analysis was performed on serial hematoxylin-eosin-stained kerosene sections, assessing epidermal thickness and the number of follicles per mm2.
  • For skin biopsies, all samples were embedded in the correct orientation and cut perpendicular to the surface.

Before treatment.

The sections show a thinned epidermis with little prominence, covered by a thin epithelium. At dermal level, follicular structures are minimal, connective tissue fibers are sparse and perivascular inflammatory mini-changes are observed.

After treatment.

Sections show an epidermis with mild hyperkeratosis and irregular acantosis. Numerous pilosebaceous appendages of normal character are observed on a vascularized connective-type dermal stroma.

Evaluating before and after photography



Androgenetic alopecia       

3 PRP sessions    

12 carboxytherapy sessions     

3-month treatment.






after five sessions of PRP and 20 sessions of carboxytherapy.


Once PRP is injected into the dermal layer, platelets are activated. They expand and growth factors are released from the Alpha granules.

The most important PRP growth factors for hair growth are :

  • Platelet-derived growth factor (PDGF)

  • Vascular endothelial growth factor (VEGF)

  • Epidermal growth factor (EGF)

  • Insulin-like growth factor 1 (IGF-1)

  • Fibroblast growth factor (FGF)

  • Nerve growth factor (NGF).

  • PDGF stimulates the growth of dermal mesenchyme. PDGF signals are involved in both epidermal-hair follicle interactions and those required for hair canal formation and dermal mesenchyme growth, respectively [35].
  • VEGF belongs to a family of powerful growth factors that act on mitotic and endothelial cells in blood vessels, increasing vascular permeability. Hair follicle growth depends on the vessels and capillaries that form the vascular plexus of the dermal papilla.  Rinaldi et al [28] have shown, using confocal microscopy, that stimulation of VEGF via adenosine receptors induced significant changes in the mean diameter of perifollicular scalp vessels compared with placebo treatment, and a lengthening of the anagen phase of transplanted follicles. Other studies have shown that stimulation of adenosine receptors contributes to hair growth by directly stimulating VEGF production by the dermal papilla [36].
  • EGF stimulates mitosis of epithelial cells and fibroblasts, and improves the proportion of hair in the anagen phase. EGF inhibits entry into the catagen phase, favoring the anagen phase [37].
  • IGF slows down apoptosis. IGF-1 acts as a mitotic signal in different cell lines and protects cells from apoptosis. IGF-1 is anti-apoptotic and able to prevent cell death [38].
  • The FGF stimulates proliferation and differentiation of keratinocytes and endothelial cells [ ].39].
  • NGF strongly stimulates hair growth and slows down apoptosis. NGF has a modulating effect on hair, depending on the receptor with which it interacts: at the level of the outer root sheath, the NGF TrkA complex stimulates keratinocyte proliferation, while NGFj p75NTR promotes apoptosis, follicle regression and inhibits hair growth [...].39].

During the anagen-catagen transition, increased neuro-immune communication leads to perifollicular neurogenic inflammation, apoptosis in hair follicles and premature development of the catagen phase.

  • A review of the literature on key candidates in cutaneous stress responses reveals that not only noradrenaline, the dominant signaling molecule in the sympathetic stress response, but also NGF, act as stress mediators. This is certainly one of the best ways to explain the correlation between stress and hair loss [40].

Giusti et al. demonstrated that the optimal platelet concentration for induction of angiogenesis in human endothelial cells was 1,500,000 platelets/μl, while excessively high platelet concentrations were suggested to diminish angiogenic potential [26].

  • When administered subcutaneously, CO2 causes vasodilation in the subcutaneous microcirculation. This is evidenced by an increase in flow velocity and the opening of "virtual" capillaries otherwise closed under paraphysiological conditions.
  • The mechanism of action of CO2 is visible and measurable by micro-angiology (video capillaroscopy with optical probe and laser Doppler fluxometry). Video capillaroscopy with optical probe shows active capillary and venous vasodilation, and an increase in the relationship between microvascular tissues. Laser Doppler fluxometry quantifies vasodilation (perfusion units) through an increase in arteriolar sphygmata (flow motion) and blood velocity in the microcirculation [47].
  • The action of CO2 produces a Bohr effect that increases PO2 and neo-collagenases.

[41] [42].


The combination of PRP, CO2 and a capillary solution of Alfatradiol 0.025 % showed excellent clinical results in the treatment of different grades of androgenic hair loss.

In future studies, it will be interesting to assess the impact of different factors such as the use of derma-roller for collagen induction therapy and biostimulation precursors.

Another element will be to find out how the protocol works in other hair loss pathologies, such as alopecia areata and telogen effluvium.

Our protocol may be a very good proposition as a non-invasive treatment for androgenic baldness. Until now, the range of non-invasive treatments has been mediocre.

The proposed treatment does not replace the hair transplant technique, but it can satisfy patients looking for non-surgical interventions, or it can be used to delay surgery and improve results.

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Aesthetic health based on scientific evidence

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