Evaluation of platelet‐rich plasma plus basic fibroblast growth factor combined with minoxidil in the treatment of androgenetic alopecia: A randomized controlled trial

Platelet‐rich plasma plus basic fibroblast growth factor (PRPF) has been confirmed to be a safe and valuable therapy for androgenetic alopecia (AGA). However, the efficacy of PRPF combined with minoxidil treatment remains unknown.


| INTRODUC TI ON
Androgenetic alopecia (AGA) is a common type of progressive hair loss. 1 Until recently, the main treatment for AGA has been medication such as topical minoxidil and oral finasteride, but these medications may have adverse effects. 2 Therefore, adjuvant therapy and improved treatment modalities are needed to achieve better outcomes. Platelet-rich plasma (PRP), an autogenous solution extracted from blood, centrifuged and filtered, and containing a high concentration of platelets and their bioactive growth factors (GFs), has been used to treat AGA in increasing numbers over the last decade. [2][3][4] Basic fibroblast growth factor (bFGF), also known as FGF2, is a member of FGF family. Previous studies have shown that bFGF increased the size of hair shaft and promote cell proliferation and cycle progression. 5 However, some studies have shown that compared with other GFs, the amount of bFGF in PRP was often less. 6,7 It is important to note that bFGF has been approved by the State Food and Drug Administration (SFDA) for many clinical use. 5,8 Therefore, adding bFGF to PRP should improve the efficacy of PRP. A previous study has demonstrated that PRP plus bFGF (PRPF) is effective in treating wrinkles. 9 In addition, our research found that PRPF had better efficacy in AGA treatment than PRP alone, which introduces a new method for AGA treatment. 10 Numerous studies and metaevidence-based medicine have shown that, compared with PRP/ minoxidil single treatment, the complex therapy has a higher improvement rate in AGA. 11,12 Regarding PRPF, it is necessary to use minoxidil in combination to achieve better results. However, there is a lack of relevant clinical studies. Therefore, we conducted a randomized controlled study to explore the clinical efficacy of PRPF monotherapy, minoxidil monotherapy, and their complex therapy, which aimed to provide a theoretical basis for finding effective and safe treatments in future clinical practice.

| Study design
This study was designed as a randomized controlled trial based on the principles of the Declaration of Helsinki. The study was approved by the local medical ethics committee, and informed consent for treatment was obtained from all patients before enrollment.

| Participants
This study was conducted in the Plastic and Aesthetic Department of Nanfang Hospital, Southern Medical University. The same clinician enrolled patients of AGA from April 2021 to December 2021.
Randomization assignments to any one of the three treatments were made sequentially according to a randomization table prepared by the research designers. All patients included in this study (n = 75) were randomly divided into three groups as follows: Group 1, 25 patients received monotherapy (direct intradermal PRPF injection); Group 2, 25 patients received classical therapy (topical minoxidil 5% was recommended twice daily); and Group 3, 25 patients received minoxidil combined with intradermal PRPF injection. The patients in Groups 1 and 2 received three PRPF treatment sessions at 1-month intervals. Detailed parameters of these three groups are shown in Table S1. A family history of AGA was recorded in all (75) of the patients included in this study. The exclusion criteria are introduced in Supplementary Methods S1. Except for the two designers of research, other researchers, including all outcome assessors were blinded to the treatment, and blinding was maintained until all data were analyzed.

| Treatment protocol
PRP was prepared first following the manufacturer's protocol of the

| Assessment criteria
Trichoscopy and global photographs were performed in four stages: T0, beginning of the study; T1, 1 month; T3, 3 months; and T6, 6 months. The 2 cm diameter circle centered on the marked tattoo was evaluated, and trichoscope photos were taken 3 days after hair shaving to assess hair growth rate and telogen hair ratio. This effect was reflected in the assessment of hair count, terminal hair count, vellus hair ratio, hair density, mean thickness, hair growth rate, telogen hair ratio, and global appearance. Patient satisfaction with the treatment and its side effects were also recorded, and the questionnaire was administered at T0 and T6. Please refer to Supplementary Methods S3 and S4 for patients' satisfaction scores and questionnaires.

| Statistical analysis
Repeated-measures analysis of variance (ANOVA) was used to determine the mean differences between hair parameters before and after treatment. The mean differences among the three groups were also examined using ANOVA. A post-hoc Bonferroni correction was applied to correct for the family wise error rate. The mean differences in the questionnaire results of the three groups were analyzed using the Wilcoxon rank test. Statistical analyses were performed using SPSS Statistics 23 and R software. For all tests, results with p < 0.05 were considered statistically significant.

| Hair density and hair thickness
As shown in Table 1 and Figure S1, the total mean hair count and density in all three groups increased significantly after 6 months treatment. At T6, the mean hair count increased by almost 31.28 hairs in PRPF group, 11.32 hairs in minoxidil group, and 41.96 hairs in complex therapy group (p < 0.05). The effects of PRPF monotherapy exceeded the effects of minoxidil treatment (p < 0.05). Both parameters of hair count and density suggested the effect of complex therapy exceeded both the effects of PRPF monotherapy (p < 0.05) and minoxidil treatment (p < 0.05). Nevertheless, the mean thickness showed no significant difference at T3 but declined in both the PRPF and complex therapy groups at T6.

| Terminal and vellus hair
Changes in terminal hair in the PRPF and complex therapy groups showed a sustained increase after treatment (p < 0.05). However, in the minoxidil group, the index of terminal hair began to increase 3 months after treatment (p < 0.05), and the vellus hair ratio in the PRPF group decreased at T1, T3, and T6 (p < 0.05). Nevertheless, the mean vellus hair ratio showed no significant change in the minoxidil and complex therapy groups (p > 0.05). It is worth noting that only significant improvements in terminal hair count were found in PRPF group versus minoxidil group (p < 0.05) and complex therapy group versus minoxidil group (p < 0.05).

| Telogen hair ratio and hair growth rate
The telogen hair ratio in all three observation groups decreased significantly, particularly in the complex therapy group. At the last visit, the mean ratio decreased by almost 11.53% in the PRPF group, 5.14% in the minoxidil group, and 14.41% in complex therapy group (p < 0.05). Through the comparative analyses, we found the effects of PRPF monotherapy exceeded the effects of minoxidil treatment (p < 0.05), and the effect of complex therapy exceeded both the effects of PRP monotherapy (p < 0.05) and minoxidil treatment (p < 0.05).
Regarding hair growth rate, this index in PRPF and complex therapy groups was augmented with significance in T1, T3, and T6. Hair growth rate had increased from 0.70 ± 0.01 mm/day at T0 to 0.83 ± 0.12 mm/day at T6 in the PRPF group (p < 0.05) and 0.72 ± 0.07 mm/day at T0 to 1.06 ± 0.12 mm/day at T6 in the complex therapy group (p < 0.05). However, there were no notable differences in hair growth rate in the minoxidil group. At T6, the hair growth rate of the complex therapy group was significantly higher than that of both PRPF therapy (p < 0.05) and minoxidil treatment (p < 0.05).

| Variation for photographs at baseline and 6 months follow-up
Representative images of patients in each group before and after treatment are shown in Figure 1. A significant improvement in hair density compared with the two single therapy groups was observed in the complex therapy group, as determined by TrichoScan ( Figure 2).

| Patient satisfaction and adverse effects
Most of the patients with AGA in the complex therapy group, with a mean score of 3.88, and the PRPF group, with a mean score of 3.2, were satisfied by the results at T6, and they were encouraged to continue treatment. Nevertheless, the mean patient satisfaction score in the minoxidil group was only 2.64. To better evaluate the subjective feelings of patients during treatment, we used a questionnaire survey before and after treatment. As shown in Table S3, significant improvements in questionnaire responses for complex therapy treatment were observed for 6 of 16 questions, and the degree of improvement was significantly better than that for PRPF/ minoxidil single treatment (e.g., impact of hair loss on social wellbeing, concern about bare spots, concern about continuation of hair loss, etc.). As for adverse effect, four patients (16%) in the minoxidil group experienced itchy scalp symptoms during treatment. Only three (12%) patients in the PRPF group and two (8%) patients in the complex therapy group experienced an adverse effect after PRPF injection, with minimal pain, redness, and pinpoint bleeding after instant PRPF injection. No other major side effects were reported during the treatment.

| DISCUSS ION
Owing to the limited effective treatment of AGA, PRP is gradually becoming an effective alternative therapy. Many clinical studies have shown that PRP is beneficial for treating hair loss. 13,14 However, there has been no unified standard for the preparation process of PRP. Due to the multifold enrichment of platelets that secrete GFs after PRP injection, the heterogeneity of GFs may lead to inconsistent clinical responses. 15,16 Various GFs in PRP have been found to regulate the growth cycles and morphological changes in hair follicles (HFs), 14,17 and studies have shown that bFGF can promote hair growth. 18 Recently, researchers have shown that the combination of bFGF and 5% minoxidil improved treatment efficiency and patient satisfaction in the TA B L E 1 Relevant change of hair growth parameters at 1 month, 3 months, and 6 months. treatment of male patients with AGA. 18 Additionally, our team conducted a randomized controlled clinical study, which confirmed that PRPF was more effective than PRP in promoting hair growth. 10 Our study, in conjunction with previous studies, lay a foundation for the clinical application of PRPF in AGA.

Time
It has already confirmed that PRP combined with minoxidil treatment enhances the functions of HFs, and it is currently the recommended treatment for AGA. 18 Our team further confirmed that PRPF was more effective than PRP in promoting hair growth. 10 Therefore, we assessed the efficacy of PRPF and minoxidil complex therapy in AGA. The results again showed better improvement in treatment efficiency and patient satisfaction with PRPF than those with minoxidil. It also demonstrated the high clinical efficacy of complex therapy and its advantages over single treatment with PRPF or minoxidil. PRPF complex therapy dramatically increased hair count, promoted hair regrowth, and improved hair loss, with a statistically higher efficacy than treatment with PRPF or minoxidil alone.
PRPF complex therapy showed better efficacy than monotherapy in this study, which emphasized the importance of combination  18 Consistently, PRPF acts synergistically with topical minoxidil to promote hair growth in AGA in our study.
Up to now, the mechanism by which minoxidil promotes hair growth has not yet been fully elucidated. Studies have shown that minoxidil, by activating potassium channels, can increase blood flow to the scalp 22 and promote the transition of HFs to the active anagen phase. 23 Minoxidil requires sulfotransferase catalysis to form minoxidil sulfate, 24 and it works to promote HF growth by increasing blood flow and allowing more nutrients to be delivered to the HF, thus stimulating the hair growth cycle. 24,25 Platelets have exhibit minoxidil sulfotransferase activity. 26 PRPF not only provides a large number of GFs to promote hair growth but might also promote the production of active metabolite of minoxidil. Meanwhile, by increasing the blood flow to the scalp, minoxidil provides HF with more oxygen and adenosine triphosphate (ATP) for hair growth. PRPF and minoxidil potentiate each other's actions when used in combination. Moreover, complex therapy allows AGA patients to achieve more significant results in a shorter treatment period, which may increase patient confidence and compliance during treatment. To date, AGA treatment has been recommended as hybrid therapy, and the combination of PRPF and topical minoxidil can be considered a promising treatment strategy.
There are still several parts in this study that need to be improved in the future. Firstly, sample size needs to be increased and the follow-up time needs to be extended. In addition, considering the heterogeneity in GFs and resultant yield of PRP, GFs quantification analysis of PRPF need to be performed in our further study.

| CON CLUS ION
Significant increase in hair count, hair density and hair growth rate were observed after PRPF therapy than minoxidil treatment.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors have declared that no conflict of interest exists.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C S S TATEM ENT
All procedures in this study were reviewed and approved by research ethics board at Nanfang Hospital, Southern Medical University (approval code: NFEC-2019-212), and participants provided informed consent explaining therapeutic approaches in this study, their benefits, and possible adverse effects.