Higher percentage of CD34+ stem cells and elevated efficacy in androgenetic alopecia treatment observed in CGF prepared from 640 nm laser‐pretreated blood: A preliminary study

Concentrated growth factor (CGF) injection has proven effective in treating androgenetic alopecia (AGA). The primary mechanism of CGF in treating AGA is thought to be the CD34+ stem cells and platelets‐associated growth factors being injected into the scalp. CGF efficacy in treating AGA may rely on the activation level of these stem cells and platelets. The 640 nm laser is a United States Food and Drug Administration approved AGA treatment that activates follicle stem cells. Therefore, we hypothesize that pretreating CGF with a 640 nm laser may further activate CD34+ stem cells and platelets, thereby improving the efficacy of CGF in treating AGA.

Methods: This study enrolled 10 patients (8 male, 2 female) with AGA aged 18-60 years who received CGF injections.The 640CGF group was pretreated with a 640 nm laser at an energy density of 4 J/cm 2 , with a 30 cm irradiation distance for 30 min.Half of the scalp was treated with 640CGF, whereas the other half was treated with N640CGF.
The injection was prepared by a doctor who did not know which blood tube had been pretreated.The treatment efficacy was evaluated using a trichoscope 1 month after injection.
Results: All 10 (100%) patients participated in the follow-up visit, and a higher quantity of new hairs was observed on the side injected with 640CGF than N640CGF (p = 0.019).Additionally, fewer malnourished hairs were observed on the 640CGF pretreated side (p = 0.015).No serious adverse events were reported.
Conclusions: A higher percentage of CD34+ stem cells and improved efficacy in AGA treatment could be observed with CGF prepared from 640 nm laser-pretreated blood.
Androgenetic alopecia (AGA) is the most common type of hair loss in clinics.2][3] Clinically, male AGA mainly shows "horseshoe-shaped" hair loss, whereas female AGA appears to have "Christmas tree-shaped" hair loss; they both greatly affect patients' quality of life, especially due to emotional distress, including low self-esteem, anxiety, depression, embarrassment, shame, decreased sense of value, excessive self-concern, etc. [4][5][6][7] Currently, a multitude of treatment modalities exist for AGA, including oral finasteride and topical minoxidil; however, with advancements in AGA knowledge and related technologies, new methods, such as low-level laser therapy (LLLT) 8 and concentrated growth factor (CGF) injections, 9 have emerged.In fact, the latter two treatment options were well accepted by patients who were reluctant to the traditional oral and topical treatment, for example, pregnant women or patients allergic to certain types of medicine.However, some limitations exist for these two treatments for AGA.For LLLT, the regularity of its application is closely associated with the treatment efficiency, 10 while for CGF, its concentration of growth factors could strongly affect the treatment outcomes. 11Thus, the possibilities of improving these specific treatments gained our attention.What might be the key to enhancing the LLLT and CGF treatment efficacy?
To answer this question, we reviewed the literature and focused on the connection of these two treatments.Approved by the United States Food and Drug Administration in 2007, the mechanism of how LLLT stimulates hair growth has been well observed.Studies [12][13][14] have shown that LLLT, mainly 640 nm laser, could improve hair density, thickness, and growth rate in patients with hair loss by stimulating hair follicle stem cells in the scalp, 15 which eventually results in elevating growth factors to promote the re-entry of dormant hair follicles into the growth phase, extending the duration of the growth phase, and increasing the proliferation rate of hair follicles in the active growth phase.
While growth factors and stem cells play a crucial role in LLLT, they are also critical in CGF treatment.CGF was first reported by Langer and Vacanti. 16They reported that CGF derived from patients' blood contains CD34+ stem cells and platelet-rich plasma, 17 which can stimulate cell proliferation and differentiation. 18The efficacy of CGF treatment strongly depends on the growth factors that platelets secrete during blood sample preparation and the activation rate of CD34+ stem cells, which are injected into the scalp and play a role in mimicking the hair follicle stem cells. 19According to the literature, a low-level laser can activate certain cells, including platelets and CD34+ stem cells 20,21  Thus, this pilot study aimed to evaluate whether 640 nm laser treatment could improve the efficacy of CGF treatment for AGA.We also prepared the mice experiment to evaluate the possible mechanism.

| Patient selection
This study was conducted from April 22, 2023, to June 13, 2023, and enrolled 10 patients aged 23-43 years diagnosed with AGA according to the literature, 22 of whom two (20.0%) were female.Each participant received CGF treatment without 640 nm laser pretreatment on one side of the scalp and 640 nm laser-pretreated CGF treatment on the other.All 10 (100.0%)participants were followed up 1 month after treatment.Participants were excluded if they met the following criteria: pregnancy or lactation, diagnosis of hypertension, hyperlipidemia, diabetes, malignancy, thyroid dysfunction, infectious disease, bleeding disorder, platelet dysfunction syndrome, platelet count <150 000/μL, history of drug allergy, anticoagulant therapy, history of AGA treatment (such as minoxidil, 5α reductase inhibitors, and LLLT) within the past 6 months, and any acute or chronic medical or laboratory abnormalities that increase the risk of participating in the study.Written informed consent was obtained from all participants.

| CGF preparation
We collected 36 mL of venous blood from the elbow vein.Half of the blood was irradiated with 640 nm laser (Nutralla, Suzhou) at 4 J/cm 2 for 30 min (Figure 1), and then all 36 mL blood was prepared with standard CGF extraction protocols (2700 r/min for 2 min, 2400 r/min for 4 min, 2700 r/min for 4 min, 3000 r/min for 3 min).

| Surgical procedure
Scalp disinfection and anesthesia were achieved using 5.0 g/L iodophor and 2% lidocaine, respectively.After anesthesia, 640 nm laser-pretreated CGF (640CGF) and 640 nm non-pretreated CGF (N640CGF) were injected randomly on the left and right scalp (10 cm above the central point of each brow bone, the injection range was 10 cm 2 ) of each patient by a doctor who did not participate in the CGF preparation.

| Treatment efficacy assessment
A follow-up visit was performed 1 month after treatment, and hair growth on the left and right sides of the scalp was obtained for each patient using a trichoscope (Beining, Nanjing) 23 according to the literature.The number of new hair growths and malnourished hairs was recorded under the trichoscope by two dermatologists who did not participate in the CGF preparation and administration.

| Statistical analysis
The number of newly grown and malnourished hairs under a trichoscope was compared, and the Mann-Whitney U-rank sum test was used to analyze the two datasets as neither group of data conformed to normality.Results were expressed as mean ± standard deviation (SD), and p < 0.05 was considered statistically significant.

| Mice experiment
The Institutional Animal Care and Use Committee of the Experimental Animal Welfare and Ethics Management Committee of Shanghai East Hospital (Shanghai, China) approved all animal experiments.Seven-week-old male C57BL/6 mice (obtained from JSJ experimental animal company, Shanghai, China) were anesthetized, and their blood was collected through retro orb sinus.The blood was separated into two groups (n = 3/group).CGF for Group A was pretreated with a 640 nm laser described in 2.2, whereas the CGF for Group B was prepared without a 640 nm laser.Then, the CGF samples of the two groups were collected, and CD34+ cells were detected by flow cytometry.

| Flow cytometry
The prepared CGF was treated with red blood cell lysis buffer (Shanghai Shenggong) and washed with phosphate-buffered saline.Then, the cells were stained with PerCP/Cyanine5.5 anti-mouse CD34 Antibody (Biolegend).Data were collected using a Cytoflex flow cytometer (Beckman) and were analyzed using Cytexpert software (Beckman).

| The side administered 640CGF exhibited better hair growth after 1 month
All 10 (100%) patients participated in the follow-up 1 month after the treatment.At the follow-up after 1 month of the treatment, more new hairs were reported on the 640CGF side than on the N640CGF side (p = 0.019), and fewer malnourished hairs were reported on the 640CGF side than on the N640CGF side (p = 0.015) (Table 1; Figure 2).No adverse events occurred in the 10 patients.

| The side administered pretreated CGF exhibited higher hair density after 5 months
The second follow-up was organized 5 months after the treatment for two patients whose hair growth was not apparent on both sides of the scalp during the first follow-up.The trichoscopic photographs showed that the side administered 640CGF had significantly higher hair density than the side administered N640CGF, and the difference in hair density between the two sides was more significant than that in the first follow-up (Figure 3).

| Patients subjectively perceive better hair growth on the side administered 640CGF
We had patients assess the hair growth on both sides and recorded their opinions; we found the side where patients evaluated better hair growth was highly consistent with the side injected with 640CGF (8 out of 10 patients) (Table 2).

| Higher Percentage of CD34+ cells was observed in CGF prepared from 640 nm laser-pretreated blood
The blood of mice was divided into two groups.Group A was pretreated with a 640 nm laser, while Group B was not treated with a 640 nm laser.Results showed that the percentage of CD34+ cells in Groups A and B was 55.78% ± 13.40% and 21.86% ± 4.60%, respectively.The percentage of CD34+ cells was significantly higher in Group A than in Group B (p = 0.0143) (Figure 4).To the best of our knowledge, we are the first to use the 640 nm laser-pretreated CGF for AGA treatment.Moreover, our pilot study showed that the combination of 640 nm laser and CGF is an effective way to enhance treatment efficacy.Our study showed that 640 nm laser-pretreated CGF had a higher therapeutic effect on AGA treatment.However, two participants (640CGF and N640CGF) showed no significant new hairs in the treated parts during the follow-up 1 month after the treatment, which might through photobiomodulation (PBM), which could eventually elevate the growth factors that the platelets secret and the CD34+ stem cells activity.Since platelets and CD34+ stem cells are the main sources of growth factors in CGF, LLLT pretreatment of venous blood in vitro could increase the concentration of growth factors by stimulating the activity of platelets and CD34+ stem cells in CGF.Thereby, improving the efficacy of CGF treatment for AGA.

F I G U R E 1
Abbreviations: NNHT: Number of New Hairs per Trichoscopic Photograph; NMHT: Number of Malnourished Hairs per Trichoscopic Photograph.

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be due to their more protracted period of hair loss, indicating a more deteriorated scalp micro-environment that might need more doses of CGF treatment and much longer time to get an accessible difference under trichoscope.Then, these two participants were involved in the second follow-up 5 months after the treatment.Their trichoscopic photographs showed that the hair growth was significantly better on the scalp injected with 640CGF than on the scalp injected with N640CGF.Five months after the treatment, this difference was mainly reflected in hair density.Therefore, the data of the second follow-up of these two participants also proved that 640CGF is more effective than N640CGF in promoting hair growth.Moreover, in the second follow-up, the difference in hair growth between the 640CGF and N640CGF groups was more significant than that in the first follow-up, which indicated that the enhanced ability of CD34+ cells to secrete growth factors activated by 640 nm laser has a long-lasting effect on hair growth.Higher concentrations of growth factors can promote faster, more efficient hair growth.Platelets could secret more growth factors, 24 like fibroblast and epidermal growth factors, under a lowlevel laser.Our experimental results may provide valuable insights into the efficacy of using a 640 nm laser to enhance the growth factors of platelets from CGF and improve the efficacy of CGF treatment for hair growth and tissue regeneration.The effect of LLLT on hair growth is related to its stimulation of hair follicle growth, and its use as a pretreatment method of venous blood may be related to the stimulation of CD34+ stem cells in CGF by 640 nm laser.The pre-stimulated CD34+ stem cells could be more activated, providing more growth factors for the hair follicles at the injection point.In conclusion, 640 nm laser-pretreated CGF showed improved efficiency in treating AGA.The possible mechanism might be the contribution of a 640 nm laser to stimulate the platelets and stem cells of CGF to secret more growth factors.Local injection of CGF pretreated with a 640 nm laser could be an effective treatment for AGA.Previous research on CGF was primarily focused on how to apply it to various diseases, that is, to broaden the application field of CGF.This study combined PBM with stem cells, starting from the CGF component itself, and used LLLT pretreatment to promote the role of CGF to a greater extent, providing ideas for more efficient treatment of AGA and other diseases.Our study provided new insight into the LLLT and CGF treatment for AGA and showed a novel pretreatment method for CGF, which could practically enhance the efficacy of CGF treatment in AGA.Although the sample size was small, we used autologous control in our study, which compensated for the inadequacy of the sample size.By programmatically scheduling the trichoscopy testing points (FigureS1), we ensured a relatively stable sampling location for patients' trichoscopy.The study was designed for 1 month because the patients were not willing to have the appearance of half side of the hair density significantly higher than the other side.One limitation of our study could be that we did not provide clinical photos, which obviously could not be solely ascribed to the patients' privacy exposure concerns; however, we TA B L E 2 The patients' and physician's judgments of the side administered pretreated CGF.The percentage of CD34+ cells in Groups A and B. Data were presented as mean ± SD, n = 3 samples/group.*p < 0.05, **p < 0.01, ***p < 0.001 compared with line indicated group.