Efficacy of autologous platelet‐rich plasma combined with a non‐cross‐linked hyaluronic acid compound in the treatment of female androgenetic alopecia: A retrospective, case‐series study

Female androgenetic alopecia (FAGA) is a condition that affects women and involves the gradual loss of terminal hair in specific areas of the scalp. The limited treatment options for FAGA necessitate the development of new strategies. This study aimed to evaluate the potential benefit of using a combination therapy composed of autologous platelet‐rich plasma (PRP) and a non‐cross‐linked hyaluronic acid (HA) compound in the treatment of FAGA.


| INTRODUC TI ON
Androgenetic alopecia (AGA) is a common medical condition that results in progressive hair loss due to the actions of androgen on hair follicles, typically beginning in adulthood. 1While the histopathological findings remain the same for both men and women, the clinical presentation and treatment options vary greatly. 2 Female AGA (FAGA) affects 40% of women over the age of 50 years. 3,4As a result, FAGA can negatively impact the quality of life and psychosocial well-being of these women.
AGA is characterized by follicular miniaturization and patterned hair loss, which are influenced by systemic androgen levels and genetic factors.The typical clinical manifestation of AGA is progressive hair loss in the frontotemporal region of the head, causing the hairline to recede.In two-thirds of patients, hair loss is primarily focused on the top of the head, while hair growth on the back and the sides remain normal, resulting in the "Mediterranean" pattern.Compared with men, women with AGA commonly experience diffuse thinning of hair on the crown of the head and the hairline.However, the front hairline's impact may vary as the expression levels of 5α-reductase and androgen receptors (ARs) in the hair follicles of women are usually half of those present in men.This variability could explain FAGA's inconsistent response to androgen inhibition therapy. 5trospective studies involving patients treated with both minoxidil and platelet-rich plasma (PRP) therapy reported that 86% of nonresponders were female. 6However, fewer clinical studies have evaluated FAGA treatment in comparison to male AGA. 7,8rrently, AGA treatment options primarily include systemic oral anti-androgen metabolizing drugs (e.g., finasteride and spironolactone), topical drugs to improve local microcirculation (e.g., fluridil and minoxidil), and hair transplantation. 9However, long-term systemic oral drugs have been associated with side effects, while patient compliance is poor with topical drugs and hair transplantation carries surgical risks and leads to continued hair shedding after surgery.Consequently, patients often turn to alternative therapies such as energy devices, PRP therapies, and drug combinations.Gupta et al.'s recent meta-analysis indicates that the therapeutic effect of PRP on hair density and diameter of male is significantly better than that of female AGA. 10 Further studies indicate that hyaluronic acid (HA) acts as an ideal scaffold for PRP, and that the combination of HA and PRP promotes tissue remodeling and wound healing by stimulating cell renewal. 11,12Due to the limitations of PRP treatment in FAGA and the demonstrated synergistic effect of PRP and HA in wound healing, we evaluated the efficacy of an autologous regenerative therapy comprising a combination of non-cross-linked HA compound and PRP in FAGA treatment in the study.

| PATIENTS AND ME THODS
This study was a retrospective, case-series study in patients with FAGA between September 2021 and December 2022.Nine patients with FAGA were enrolled and all of them provided the written informed consent prior to enrollment.Eligible individuals included female patients aged 20-65 years (the median was 45.7 years).
Enrolled patients need to be satisfied with the following diagnostic criteria: Type I-III according to the Ludwig classification.Hair microscopy revealed an uneven thickness of dry and increased vellus hairs.
Pregnant patients; those with chronic diseases like malignancy, inflammation, or any type of scalp infection; and anyone who has been on anticoagulant or antithrombotic therapy in the past year, with an intolerance to HA, or with permanent filler on any site of the skin were not suitable for treatment.In addition, patients with myasthenia gravis, multiple sclerosis, immune deficiency (immunosuppression), autoimmune disease, kidney disease, liver disease, epilepsy, or hypertension or who may have significant risk were excluded.The protocol of microinjection therapy included four sessions separated by a 4-week interval, and the follow-up period was 6 months.We mixed non-cross-linked HA compound and PRP into one syringe and transplanted it into affected areas of the scalp.The patients were assessed for the degree of hair loss before treatment and during each follow-up visit.In the pretreatment the Ludwig classification evaluation and hair microscope measurement of the nine patients, which were recorded as the baseline data.The data of baseline obtained from patients were compared with post-intervention results data.

| Preoperative anesthesia
Prior to treatment, patients received local anesthesia via multiplepoint-injection of lidocaine hydrochloride (5 mL, 0.1 g; Shanghai Pujin Linzhou Pharmaceutical Co., Ltd.).A 34-gauge-4 mm needle was used throughout the scalp for multiple injections (maximum 5 mL in total, 0.1 mL at each point with 1 cm apart).

| Isolation of PRP
Patients were routinely disinfected before the treatment.9 mL of whole blood was collected from the antecubital vein of each patient into a PRP kit (ACR-C Classic; RegenLab), and immediately centrifuged in a high-speed centrifuge at 3000 rpm for 15 min.Once the erythrocytes were well separated, the upper platelet plasma was removed, resulting in 4.5 mL of PRP.The entire procedure was performed in a sterile environment.

| Injection of non-cross-linked HA compound and PRP
The main components of the non-cross-linked HA compound are sodium hyaluronate 5.00 mg/mL, proline 0.20 mg/mL, glycine 0.10 mg/ mL, alanine 0.10 mg/mL, l-carnosine 2.00 mg/mL and vitB 2 0.005 mg/ mL (named as Hearty® in the market, developed by Imeik Technology Development Co., Ltd.).This product was previously approved by the Chinese National Medical Products Administration for rejuvenation of neck wrinkles.We mixed Hearty® (2.5 mL) with PRP (3 mL) and injected into patients' scalp area, via multiple microinjection technique, in FAGA patients under sterilized conditions.The treatment protocol included four sessions separated by a 4-week interval.

| Assessment criteria
After four treatments of PRP and the non-cross-linked HA compound, hair loss improvements were assessed using a global assessment scale (4-point scale) by comparing posttreatment and pretreatment photos.The physicians used an SLR camera (EOS M6; Canon) to take photographs of the patients, including preinjection, 1-month, and 3month follow-up.Subjects maintained the same position, angle, and camera shooting parameters for each time.Hair microscope (dino-light AM7115MZT, ZKH) was also used to assess changes in hair density after combination therapy.The specific shooting method of the hair lens was to mark a 25 mm 2 area in the hair loss area, use the hair lens in this area, and use the computer-assisted software independently equipped by the hair lens to mark the hair for hair number statistics.
Patients were evaluated at each follow-up for symptomatic improvement in itching, grease and dandruff using a 4-point scoring system, as follows: −1 = symptoms are worse than before, 0 = no significant improvement in symptoms, +1 = symptoms are improved compared to before, and + 2 = symptoms are improved significantly compared to before.Furthermore, a score of ≥4 points (7-point scale) for patient satisfaction was considered indicative of good satisfaction.
According to Ludwig's grade criteria, Grade I cases had mild alopecia, Grade II cases had moderate alopecia, and Grade III cases had severe alopecia.Any adverse signs or symptoms, or adverse events related to the procedure are to be documented in detail, including redness, swelling, pain, itching, and bruising at the injection site.The safety of the combined treatment will be evaluated and the number and percentage of cases of each symptom will be counted.

| S TATIS TIC AL ANALYS IS
Data analysis was conducted using SPSS version 28.0 (IBM Corporation).Mean ± standard deviation values were used to present quantitative variables, such as baseline information.A ttest was performed to compare pretreatment and posttreatment physician's global assessment scores, with p < 0.05 indicating statistical significance.

| RE SULTS
Baseline statistics for the nine subjects who met the criteria were summarized in Table 1.The median age of patients was 45.67 ± 4.70 years.One female patient (11%) exhibited Ludwig scale type III and eight patients (89%) were in Ludwig scale II.None of the patients was lost to follow-up.

| Efficacy
According to the Ludwig scale, eight patients (88.89%) showed a onepoint improvement at 3-month compared to baseline data.The improvement rates subjectively evaluated by the study physician were 88.89% after 1 month and 100% after 3 months, respectively (Table 2).
Patients showed a significant increase in hair counts at all follow-up time points, which gradually increased occurring over the duration of treatment.After overall treatment for four times, the patients' hair count was 28.80 ± 1.24/cm 2 at 1 month and 33.04 ± 1.21 /cm 2 at 3 months after treatment, and there were significant differences in the hair count at 1 and 3 months relative to the baseline data (p < 0.001) (Table 3).The increase in the rate of hair density was 54.51% at 1-month treatment and 77.25% at 3-month treatment.Representative photographic images of FAGA patients are shown in Figures 1-6.Trichoscopy after 3month treatment of therapy showed a significant growth trend in hair density with excellent improvement in Figures 7-12.The hair density significantly increased after each treatment and target areas of alopecia diminished, indicating a significant attributed to improvement with PRP combined with a non-cross-linked HA compound.

| Patient satisfaction
Following treatment, nine FAGA patients reported that they showed good satisfaction with less scalp oil secretion, itching, and dandruff symptoms (Table 4).One month after treatment, 77.78% of FAGA patients were somewhat satisfied and 22.22% were satisfied; 3 months after treatment, 88.89% of FAGA patients were somewhat satisfied and 11.11% were satisfied for the appearance of hair.With the prolongation of treatment time, the patient satisfaction rate increased.

| Safety and tolerability
No serious adverse event occurred in any patient.Three patients had injection-related reactions (pruritus, erythema, or edema), which resolved within 72 h.There was no adverse event occurred during the follow-up period, indicating PRP combined with HA is safe and well tolerated in the treatment of FAGA.tion. 3 Given this complexity, a simple and effective treatment approach that can be easily adhered to and widely adopted is needed.9) 7( 9) 0( 9 Moreover, patients reported high levels of satisfaction with no adverse events occurring during the follow-up period.This suggests that the safety of this treatment contributed to the high satisfaction levels reported. To our knowledge, this study is the first to use the combination of PRP and a non-cross-linked HA compound in treating patients with FAGA.Autologous plasma contains fibrinogen, which has a strong affinity for HA when converted to fibrin.This interaction forms a three-dimensional scaffold that increases the stability, biocompatibility, and viscoelasticity of PRP.It prolongs the action time of PRP, improves its storage modulus, and regulates the release of growth factors.Previous clinical studies have demonstrated the efficacy of the PRP and HA combination in managing moderate and severe osteoarthritis. 13Additionally, mesotherapy studies support the compatibility, efficacy, and safety of combining treatments. 14 who did not respond to topical Minoxidil®. 24Micrograft or PRP are equally effective in males and females with AGA, although the effectiveness in females was not as significant as in males.TGFβ reduced bleeding, sped up healing time, and stimulated angiogenesis, cell growth, and morphogenesis in would healing, which processes also required to improve hair regrowth.Furthermore, the stimulation of Wnt/β-catenin signaling is implicated in both wound healing and hair regrowth. 25PRP is considered a biological stimulator for the reasons previously mentioned.In contrast, HA is a biopolymer found in the extracellular matrix of various tissues, such as cartilage, skin, bone, and brain tissue.To investigate the synergistic effect of PRP and HA in the treatment of FAGA, we used a non-cross-linked HA compound complex with l-carnosine, trace amino acids, and vitamins.These ingredients can also be used as supplemental nutrients to promote hair follicle growth and improve FAGA treatment.HA is an essential component of the extracellular matrix with unique hydrodynamic, viscoelasticity, and strain properties.7][28] Additionally, oligomeric HA stimulates the secretion of VEGF, which triggers the formation of new blood vessels. 29We thus speculated that HA could reduce scalp inflammation in patients with FAGA, improve scalp blood supply, and promote hair follicle growth. 30Non-cross-linked HA is preferable for superficial scalp injections compared to cross-linked HA, as it degrades more slowly and does not promote the formation of nodules on the superficial skin layer. 31l-carnosine is a natural dipeptide consisting of B-alanine and L-histidine that can reduce free radical and advanced glycation end product (AGEs) levels.Narda et al. demonstrated that applying a carnosine-containing cream to human skin grafts significantly decreased the levels of AGEs in the dermis, epidermis, and reticular layers. 32l-carnosine may create a more favorable environment for hair follicle tissue repair by maintaining the activity of growth factors and adjusting the arrangement of elastic fibers.Our study showed a significant increase in increase in scalp hair number compared to baseline after 3 months of follow-up, supporting the beneficial role of l-carnosine in protecting hair follicles from additional oxidative damage posttreatment.Additionally, the low doses of proline, glycine, and alanine provide nutrients for newly formed hair, while VitB 2 regulates cell activity during hair follicle repair. 33Gentile et al. conducted a halfhead controlled trial to evaluate the impact of PRP injections on the scalp of 5 females and 18 males with AGA.After 3 months, a 31% increase in hair density was observed in the treated area, compared to a 1% increase in the control area. 34In our study, we observed an increase in hair density rate of 54.51% after 1-month treatment and 77.25% after 3-month treatment.Combination therapy has demonstrated greater effectiveness than PRP treatment alone.
Our study revealed that the combination of a non-cross-linked HA compound with PRP injection into the skin of patients with FAGA was an effective alternative therapy, which significantly reduced hair

TA B L E 1
Demographic and clinical characteristics.treatment (root/cm 2 ) 18.64 ± 1.32 AGA is typically attributed to abnormal hair follicles sensitivity to androgens due to increased expression of ARs in hair follicles.This occurs through heightened activity of 5α-reductase-2, leading to increased dihydrotestosterone (DHT) expression.DHT then binds to ARs, causing hair follicle miniaturization or even atrophy and resulting in hair loss.Molecular biology studies have shown that activating hair follicle stem cells requires the combined action of multiple cytokines and growth factors to facilitate hair regenera-

1 F I G U R E 2 F I G U R E 3 F I G U R E 4 F I G U R E 6 F I G U R E 7 F I G U R E 8 F I G U R E 9 F I G U R E 1 0
Photograph taken of a 52-year-old female androgenetic alopecia patient before treatment.Photograph taken of a 52-year-old female androgenetic alopecia patient at 1-month posttreatment.Photograph taken of a 52-year-old female androgenetic alopecia patient at 3-month posttreatment.Photograph taken of a 62-year-old female androgenetic alopecia patient before treatment.In this study, we evaluated the efficacy of PRP and a non-crosslinked HA compound for treating FAGA.Our findings indicated that this combination treatment significantly improved hair growth and density, as evaluated by the study physician, relative to baseline at 1 month posttreatment follow-up.Furthermore, hair count remained higher than the baseline level during the 3-month follow-up period (p < 0.001), suggesting that this treatment's clinical efficacy could last beyond 3 months.Patients reported high satisfaction levels F I G U R E 5 Photograph taken of a 62-year-old female androgenetic alopecia patient at 1-month posttreatment.Photograph taken of a 62-year-old female androgenetic alopecia patient at 3-month posttreatment.Microscopic hair image of a 52-year-old female androgenetic alopecia patient before treatment.Microscopic hair image of a 52-year-old female androgenetic alopecia patient at 1-month posttreatment.Microscopic hair image of a 52-year-old female androgenetic alopecia patient at 3-month posttreatment.Microscopic hair image of a 62-year-old female androgenetic alopecia patient before treatment.during the last follow-up visit.Mild pruritus, erythema, or edema occurred following the injection of the PRP and non-cross-linked HA compound combination treatment but only lasted up to 72 h.
Our study results indicate that the combination of PRP and HA was safe and well tolerated in treating FAGA.In both in vivo and in vitro studies, the use of PRP, human follicle stem cells, and adipose-derived stem cells has shown promising results in promoting hair regrowth and wound healing, likely due to their anti-inflammatory and immunomodulatory properties that favor hair regeneration and wound healing[15][16][17] PRP contains various signaling proteins and major growth factors (e.g., PDGF, VEGF, TGFβ).The beneficial properties of these factors have been exploited in various contexts, including venous ulcer treatment, burn healing, and skin rejuvenation.18,19The growth factors released after PRP injection can penetrate deeply into all layers of the skin and stimulate the proliferation of dermal papilla cells in the affected area through the activation of the Bcl-2 protein (an anti-apoptotic regulator) and Akt signaling.These growth factors also induce hair follicle stem cells to turn into mature hair follicle cells to repair and improve the condition of hair follicles.20Moreover, PRP-associated growth factors improve the health of the perifollicular vascular plexus by increasing VEGF and PDGF levels, promoting angiogenesis, providing oxygen and nutrients, improving microcirculation in the scalp, and ultimately promoting hair regeneration.21Additionally, PRP promotes the release of anti-inflammatory mediators (e.g., the interleukin 1 receptor antagonist, soluble tumor necrosis factor receptor I, IL-4, IL-10, and IL-13) while suppressing inflammatory responses.22As reported by Fukuoka et al. infusion of adipose-derived stem cell medium intradermally into 22 patients with AGA resulted in an increment of 15.6 ± 4.2 hairs in female patients, which is consistent with our findings.23A systematic review analyzing RCTs and observational studies demonstrated the relative effectiveness and safety of PRP in the treatment of FAGA, particularly in those patients

F I G U R E 1 2 Follow
HA and PRP work together to promote tissue remodeling and recovery, stimulate cell renewal, and improve wound healing.HA regulates and hydrates the cellular microenvironment and induces cell migration, proliferation, cell-substrate adhesion, and intercellular adhesion through binding to cell surface receptors. 11Therefore, HA provides an effective scaffold that works cooperatively with PRP to aid wound healing and hair regeneration by promoting cell proliferation, differentiation, and neoangiogenesis.F I G U R E 11 Microscopic hair image of a 62-year-old female androgenetic alopecia patient at 1-month posttreatment.Microscopic hair image of a 62-year-old female androgenetic alopecia patient at 3-month posttreatment.TA B L E 4 Patient satisfaction.
loss and improved the patients' quality of life.Nevertheless, some limitations of our study include a sample size and short follow-up duration.Therefore, it is essential to conduct randomized controlled trials involve larger cohorts and standardized study design protocols, which objectively evaluate the extent of hair loss, number and interval of treatment sessions, platelet count, activation method, and long-term follow-up outcomes.Future randomized controlled trials will need to verify the superior effectiveness of the combined PRP and HA formulation compared to PRP alone or other FAGA treatment methods.In conclusion, our study demonstrates the effectiveness and safety of combining PRP with a non-cross-linked HA compound in treating FAGA.The encouraging results of our study underscore the significance of conducting further randomized controlled trials with large sample sizes to acquire high-quality clinical evidence on the efficacy of this therapeutic combination.