Successful Treatment of Female Pattern Hair Loss with Injection of Autologous Adipose-derived Adult Stem Cells: A Controlled Clinical, Trichoscopic, and Immunohistochemical Trial

ABSTRACT

Background:

Hair follicles undergo growth, regression, and quiescence. It is suspected that adipocytes secrete factors that promote the activation of follicular dermal papilla cells, increase migration and proliferation in vitro, and increase the conversion of hair follicles from the telogen to anagen phase in vivo.

Objectives:

To evaluate the efficacy of adipose derived stem cells (ADSCs) injection for the treatment of female pattern hair loss (FPHL).

Patients and Methods:

This prospective randomized controlled trial included 33 patients divided into three groups according to Sinclair classification. ADSCs were extracted from autologous fat obtained from the lipoaspirate, cultured, and injected into the frontoparietal scalp. The patients were assessed clinically, trichoscopically, histopathologically, and immunohistochemically for hair cycle markers.

Results:

At weeks 12 and 24, there was an improvement in hair thickness and count, and an increased number of hairs per follicular unit. Histopathological and immunohistochemical assessments at week 12 showed a decrease in perifollicular inflammation, an increase in the hair cycle stimulatory marker (follistatin), and a decrease in hair cycle suppressor (DKK-1) immunostaining.

Conclusion:

The use of ADSCs in subjects with FPHL included in this study showed increased hair density and thickness, improved perifollicular inflammation, and enhanced hair cycle regeneration markers.

INTRODUCTION

Female pattern hair loss (FPHL) refers to the progressive thinning of scalp hair following a pattern distribution.[1] Hair follicle miniaturization and changes in hair cycle dynamics are the two main factors involved in the pathogenesis of FPHL.[2]

Various treatment modalities have been used for treatment.[3] However, the response to the available treatments is often inadequate. This hope lies in regenerative medicine, which uses the therapeutic potential of stem cell therapy.[4] Adipose-derived adult stem cells and conditioned media from ASCs have been reported to regenerate hair follicles in vitro[5] and are now being used to induce hair regeneration.[6] Treatment of FPHL has been attempted by intradermal injection of conditioned medium from ASCs.[7,8] However, the clinical injection of ASCs in patients for hair regrowth has been evaluated in a few studies.[9]

PATIENTS AND METHODS

This was a pilot prospective single-arm investigational study that included 33 patients selected clinically with Grades II, III, and IV (according to Sinclair’s scale) after exclusion of other causes of alopecia. Patients were selected from the outpatient clinic of the Dermatology Department of Ain Shams University Hospitals from December 2016 to June 2017. The protocol is registered in clinicaltrials.gov with Unique Protocol ID: FWA 0000175855.

Written informed consent was obtained after obtaining approval from the Research Ethics Committee of the Faculty of Medicine, Ain Shams University (FWA 000017585).

Patients were excluded from the study if they were <20 years or more than 50 years old, had co-existing chronic telogen effluvium, Grade V Sinclair’s FPHL, were taking any recent topical or systemic treatment that might affect hair shedding or regrowth for the past 6 months, or had clinical signs of hyperandrogenism. Pregnant or lactating females and those with a personal or family history of malignancy and immunosuppressed conditions were also excluded from the study.

Methods

Patients were divided into three groups (11 patients each) according to Sinclair’s scale: Group I included patients with Grade II, and Groups II and III included patients with Grades III and IV, respectively.

Procedure

Fat was harvested by liposuction from the lower abdomen under local anesthesia. Stem cell processing was performed at the Tissue Culture Laboratory of the Biochemistry Department, Faculty of Medicine at Ain Shams University. The stromal vascular fraction (SVF) was obtained after enzymatic digestion of lipoaspirate by collagenase. ADSCs were extracted from SVF and cultured on a DMEM media (13% fetal bovine serum, 1.5 penicillin-streptomycin, 0.05% fungizole) in 95% O₂ and 5% CO₂ atmospheric air in a CO₂ incubator for 2 weeks, the media was then changed twice every week. The cells were suspended in PBS to prepare for injection.

The cell count was standardized to 1 million cell/mL and 2 mL was injected intradermally in the frontoparietal region of the scalp. All the patients underwent a single session.

Evaluation of results

Patients were evaluated by four methods: clinically, trichoscopically, histopathologically, and immunohistochemically.

Clinical evaluation

Each patient was examined and photographed using a digital camera before and at 12 and 24 weeks after the procedure. The clinical classification was recorded by digital photography of the frontal and vertex scalp areas showing the central hairline using a 12-megapixel camera (Iphone 7). The images were standardized for light, angle, and position.

Trichoscopic evaluation

The patients were trichoscopically evaluated using a Dermlite DLIII dermoscope. Trichoscopic images were obtained using the same camera mounted on the Dermlite DLIII dermoscope (3Gen Inc., California, USA) at 20-fold. An area in the frontal scalp was photographed, measured from the bony prominence 12 cm from the tip of the nose.

The trichoscopic images of each patient were analyzed using simple morphometric software (ImageJ, National Institutes of Health, Bethesda, Maryland, USA). It permits clicking of the vellus and terminal hairs on the trichoscopic image, and a software-aided count is displayed by two blinded investigators. As for the thickness, it gives the mean hair diameter by measuring 30 hairs (of different shaft thickness) cross-sectionally in the trichoscopic image with 75% magnification. Total, terminal, and vellus hair counts were calculated for each patient at baseline and at 12 and 24 weeks postinjection.

Histopathological evaluation

Two 4 mm deep punch biopsies were taken from the frontal area. Biopsies were obtained at baseline and 12 weeks after the injection. One biopsy specimen was horizontally sectioned for Hematoxylin and Eosin staining to assess anagen follicles, and the other was vertically sectioned to assess perifollicular inflammation and fibrosis. The quantity of inflammatory infiltrates and width of perifollicular fibrosis in the lower infundibulum and isthmus were assessed. An average of five readings at the lower isthmus under ×200 was obtained. Perifollicular fibrosis was scored from 0 to 3 as negative, mild, moderate, and marked based on the width of the condensed collagen in the lower infundibulum and isthmus. Perifollicular infiltrate was scored from 0 to 3 as negative, mild, moderate, and marked.[10]

Immunohistochemical evaluation

Tissue sections for immunohistochemistry were obtained from vertically positioned biopsies in paraffin blocks, cut to 4-μm thickness, and mounted on positively charged slides. Immunostaining for Follistatin and DKK-1 was performed using a Ventana Benchmark Autostainer (Ventana Medical Systems [F. Hoffmann-La Roche]), which automated the processes of deparaffinization, cell conditioning (standard conditioning for 80 min), antibody application (application of 100 μ of Follistatin and DKK-1 purified rabbit anti-human polyclonal antibodies, Enzo Biochem, Inc.[NYSE: ENZ] after dilution 1:100, put into incubation temperature at 42°C for 32 min), application of 3,3’-diaminobenzidine (DAB), counter stain with hematoxylin for 8 min, and postcounter staining with bluing reagent for 4 min. Following staining, slides were washed, dehydrated in ascending alcohol grades, cleared in xylene, covered with slips, and immunostained using an Olympus light microscope (C × 34). The staining intensity for both markers was used using the i score (0 = very mild, 1 = mild, 2 = moderate, and 3 = marked). Although immunohistochemical staining intensity is difficult to grade objectively, it has the advantage of protein localization.[11] Scoring was performed by an independent observer blinded to the case-control status of the slides.

Assessment of side effects

Patients were examined immediately after the treatment session to evaluate postprocedural pain, which was rated using a numerical rating scale. It is a 0- to 10-point scale that correlates to no pain at zero, mild pain from to 1 to 4, moderate pain from to 5 to 7 and severe pain from to 8 to 10. Each patient was asked to rate their pain intensity from 0 to 10. Patients were also asked and examined for other complications, such as edema, infection, ecchymosis, vasovagal attack, and skin necrosis.

RESULTS

Simple descriptive statistics (arithmetic mean and standard deviation) were used to summarize the normal quantitative data, and frequencies were used for qualitative data. Bivariate relationships were displayed in cross-tabulations, and a comparison of proportions was performed using Chi-square and Fisher’s exact tests where appropriate. The paired t-test, one-way ANOVA, and posthook tests were used to compare normally distributed quantitative data.

The patients’ ages ranged from 20 to 50 years, with a mean age of 32 ± 8 years. Disease duration ranged from 1 to 10 years with a mean duration of 5 ± 2 years. Twelve (36.4%) of the patients denied the presence of any family history.

Clinical evaluation

The patients’ photographs were assessed by two blinded investigators. At week 12, twenty-five patients (75.75%) showed clinical improvement in terms of improvement of scalp coverage (hair volume) [Figures 1 and 2].

Figure 1.

A 40-year-old patient. (a) Sinclair IV at week 0, (b) Improvement of hair density at week 12 after treatment, (c) Further improvement (Sinclair III) at week 24

Figure 2.

A 20-year-old patient. (a) Sinclair III at week 0, (b) Improvement of hair fullness at week 12 after treatment, (c) Further improvement (Sinclair II) at week 24

At week 24, 56% of the improved patients maintained the improvement, whereas 40% showed slight worsening but better than the baseline, and only one (4%) patient showed worsening, returning to the same baseline state.

Trichoscopic evaluation

The mean terminal hair thickness was significantly higher than that at week 24 (P < 0.001). The mean vellus hair thickness increased significantly at weeks 12 and 24 (P < 0.001) [Table 1].

Table 1.

Terminal and vellus hair thicknesses at baseline, and at weeks 12 and 24 after injection (frontal and occipital scalp)

Terminal hair thickness frontal			Vellus hair thickness frontal			Terminal hair thickness occipital			Vellus hair thickness occipital
T0	T12	P	T0	T12	P	T0	T12	P	T0	T12	P
0.078±0.011	0.123±0.161	0.126	0.029±0.005	0.038±0.005	<0.001	0.082±0.010	0.164±0.233	0.053	0.034±0.003	0.050±0.070	0.201
T0	T24	P	T0	T24	P	T0	T24	P	T0	T24	P
0.078±0.011	0.101±0.0173	<0.001	0.029±0.005	0.039±0.005	<0.001	0.082±0.010	0.118±0.140	0.150	0.034±0.003	0.040±0.003	<0.001

T0 – Baseline trichoscopic assessment; T12 – Trichoscopic measurements at week 12; T24 – week 24

The mean total and terminal hair counts increased significantly at weeks 12 and 24 postinjection (P < 0.001). However, the mean vellus hair count decreased at weeks 12 and 24. However, the decrease was not statistically significant [Table 2].

Table 2.

Comparison between terminal and vellus hair counts at baseline, and weeks 12 and 24 after injection (frontal and occipital scalp)

Terminal hair count frontal			Vellus hair count frontal			Terminal hair count occipital			Vellus hair count occipital
T0	T12	P	T0	T12	P	T0	T12	P	T0	T12	P
25.83±9.06	50.82±14.25	<0.001	27.09±7.86	27.54±7.51	0.743	42.54±11.25	70.08±16.61	<0.001	24.45±5.61	24.45±5.46	1.000
T0	T24	P	T0	T24	P	T0	T24	P	T0	T24	P
25.83±9.06	62.37±13.98	<0.001	27.09±7.86	25.35±6.05	0.139	42.54±11.25	85.44±19.92	<0.001	24.45±5.61	25.74±3.75	0.256

T0 – Baseline trichoscopic assessment; T12 – Trichoscopic measurements at week 12; T24 – week 24

Single, double, and triple hair follicular units:

Follicular units (FUs) with single, double, or triple hair were counted. They constituted 38.39 ± 16.54, 44.27 ± 11.19, and 17.33 ± 6.65% of FUs, respectively, at baseline trichoscopic assessment [Table 3]. Predominance of double hair per FU was observed in patients with Grades II and III Sinclair classification (100% and 72.7% of patients, respectively). Single hair per FU was observed in all patients with Grade IV FPHL.

Table 3.

Predominance of single, double and triple hair follicular units at baseline, and at weeks 12 and 24 after injection (frontal scalp)

Single hair/follicular unit frontal			Double hair/follicular unit frontal			Triple hair/follicular unit frontal
T0	T12	P	T0	T12	P	T0	T12	P
38.39±16.54	32.15±14.84	<0.001	44.27±11.19	48.21±8.62	<0.001	17.33±6.65	19.69±7.36	<0.001
T0	T24	P	T0	T24	P	T0	T24	P
38.39±16.54	29.30±14.08	<0.001	44.27±11.19	50.03±7.56	<0.001	17.33±6.65	20.72±7.98	<0.001

T0 – Baseline trichoscopic assessment, T12 – Trichoscopic measurements at week 12; T24 – week 24

Single hair FUs decreased significantly, whereas double and triple hair per FU increased significantly at weeks 12 and 24 (P < 0.001).

Histopathological evaluation

Miniatured hair follicles

Before treatment, miniaturization of hair follicles was observed in 13 (39.4%) patients. Majority of patients showed reversal of miniaturization after treatment; (93.9%) showed no miniaturization of hair follicles, with an increased number of terminals compared to vellus follicles. However, this difference was not statistically significant (P = 0.070), probably because of the small sample size. Including a larger sample size may enhance the significance of the results.

Perifollicular inflammation and fibrosis

Regarding perifollicular inflammation, only six patients (18.2%) showed no perifollicular inflammation in their baseline scalp biopsies, 15 (45.5%) patients had mild inflammation, 11 patients (33.3%) had moderate inflammation, and only 1 (3%) patient had marked perifollicular inflammation. At 12 weeks after ADSCs injection, statistically nonsignificant (P = 0.873) improvement in perifollicular inflammation was found, resolved in 27 (81.8%) of patients.

In the context of perifollicular fibrosis at 12 weeks after injection, 9 (27.3%) patients had no perifollicular fibrosis, increasing from 18.2% at baseline assessment (P = 0.719). However, most of the patients, 24 (72.7%), had hyalinization of perifollicular fibrous tissue at week 12 after injection, representing a form of collagen remodeling of perifollicular fibrous tissue.

Terminal: vellus follicle ratio

At baseline scalp biopsy, the mean terminal-to-vellus ratio was 1.40:1 ± 0.675, which increased significantly to 2.42:1 ± 1.36 (P ≤ 0.001). Terminal: vellus ratio improvement differed between groups, being significant only in patients with Grades II and III (P = 0.007, 0.014, respectively) and nonsignificant in patients with Grade IV (P = 0.066) [Table 4].

Table 4.

Correlation of mean histopathological terminal: Vellus ratio in scalp biopsies at week 0 and week 12 after adipose-derived adult stem cells injection with Sinclair’s grade of female pattern hair loss

Grade	Mean±SD		P
	Week 0	Week 12
All patients	1.40:1+0.675	2.42:1+1.361	<0.001
Grade II	1.80:1±0.919	3.09:1±1.514	0.007
Grade III	1.18:1±0.405	2.10:1±0.994	0.014
Grade IV	1.22:1±0.441	2.00:1±1.333	0.066

SD – Standard deviation

All histopathological findings reflected follicular stimulation and translated clinically into enhanced trichoscopic findings in terms of increased hair count and thickness.

Immunohistochemical evaluation

Immunohistochemical staining of follistatin (hair cycle stimulatory marker): [Figure 3]

Figure 3.

Medium Power (×40). Mild immunohistochemical staining of Follistatin (interfollicular epidermal keratinocytes, perifollicular dermis and external root sheath). Staining intensity for both markers was used using i score (0 = very mild, 1 = mild, 2 = moderate, and 3 = marked). Scoring was carried out by an independent observer blinded to the case–control status of the slides (No photos of control slides are available) (Yellow arrows point to positive Follistatin immunostaining).

Follistatin immunohistochemical staining was observed in all biopsies along the interfollicular epidermal keratinocytes, in the external root sheath of hair follicles, and in the dermal extrafollicular environment. At baseline assessment, 30 patients had mild staining (90.9%), whereas only three had moderate (9.1%) and none of the patients had marked staining. Twelve weeks after treatment, the majority of patients (21 patients) showed moderate staining, but none showed marked staining. However, this correlation was not statistically significant (P = 0.272), probably due to the small sample size.

Immunohistochemical staining of DKK-1 (hair cycle inhibitory marker): [Figure 4]

Figure 4.

High Power (×100). Marked immunohistochemical staining of DKK-1 (follicular keratinocytes). Staining intensity for both markers was used using i score (0 = very mild, 1 = mild, 2 = moderate, and 3 = marked). Scoring was carried out by an independent observer blinded to the case–control status of the slides (No photos of control slides are available) (Yellow arrows point to positive DKK-1 immunostaining)

DKK-1 staining was observed along the follicular keratinocytes. At baseline assessment, majority of patients, 31 patients (93.9%), showed marked immunohistochemical staining for DKK-1. Twelve weeks after treatment, only 2 (6.1%) patients had marked staining, whereas the majority had moderate staining; 22 (66.7%) patients. The correlation between baseline and DKK-1 staining after treatment was not statistically significant due to the small sample size (P = 0.587).

Side effects and complications

Following liposuction, all patients experienced mild pain and bruising in the lower abdomen that lasted for < 1 week. No serious side effects (e.g. damage to deeper structures such as nerves, blood vessels, muscles, and abdominal organs, cardiac and pulmonary complications, fluid accumulation, and infection) have been reported in our patients. Following ADSCs injection, 15 (45.5%) patients experienced scalp pain only, whereas 18 (54.5%) patients experienced low-grade fever and lymphadenopathy (submandibular and postauricular) in association with scalp pain. These symptoms lasted for 3–5 days and were self-limiting in all the patients. No side effects or complications were reported at the site of the scalp biopsy.

DISCUSSION

To the best of our knowledge, this is the first randomized clinical trial to evaluate the efficacy and safety of ADSCs in patients with FPHL, proving hair regenerative effects at four levels: clinical, trichoscopic, histopathological, and immunohistochemical.

Although the molecular mechanisms underlying the hair regenerative effect of ADSCs are well known, the efficacy of ADSCs in hair regeneration has not been previously demonstrated in a randomized clinical trial.

Trichoscopically, our results demonstrated that ADSCs significantly increased the total and terminal hair count and density and decreased the vellus hair count. Moreover, ADSCs increased double- and triple-hair FUs while decreasing single-hair FUs and increasing the terminal: vellus ratio. These results reflect the recovery of hair follicle miniaturization.

Our study is unique in demonstrating the beneficial effects of ADSCs on hair follicles histopathologically, with a decrease in perifollicular inflammation and fibrosis and remodeling of perifollicular fibrous tissue (perifollicular hyalinization), reflecting a disease-modifying effect on FPHL, not just a hair regeneration effect.

Although the effect of ADSCs on the hair follicle macroenvironment and hair follicle dynamics has been discussed in a few previous studies, this is the first clinical trial to combine assessment of ADSCs on two opposing hair cycle stimulators and inhibitors, Follistatin and DKK-1, respectively, immunohistochemically with protein localization of both markers before and after treatment with reversal of their staining intensity: increased follistatin and decreasing DKK-1 after ADSCs injection.

In androgenetic alopecia, a reduction in the number of hairs per FU precedes the development of baldness.[12] This may reflect a hierarchy of androgen sensitivity in FUs, leading to the selective miniaturization of secondary terminal hairs within each FU. Loss of terminal hair leads to a reduction in hair density that precedes the diffuse loss of volume over the crown and the appearance of a bald scalp. Loss of (secondary) hair can be recognized on trichoscopy as a reduced ratio of double and triple (terminal) hair FU on the mid-frontal scalp.[13] This is thought to be reversed by ADSCs treatment by stimulating hair follicle stem cells to restore the normal hair cycle, reversing miniaturization.[14]

Improvements in hair density and thickness can be explained by inducing anagen re-entry and conversion of hair follicles into terminal follicles and terminalization. Another proposed mechanism is follicular activation, particularly dermal papilla stimulation through paracrine effects of growth factors and activation of epidermal cellular proliferation pathways such as Wnt/β-catenin, Akt, and Erk signaling pathways promoting adipocyte differentiation and hair growth. Activation of the Wnt pathway is associated with a dramatic and premature increase in the number of adipocytes, synchronizing adipocyte differentiation with the hair growth cycle.[15] Moreover, activation of the Akt and Erk signaling pathways enhances the survival and proliferation of dermal papillae cells by 130%.[16] Increased follistatin[17] and decreased DKK-1[18] levels are associated with the activation of the Wnt/β-catenin signaling pathway, highlighting hair cycle activation.

Growth factors comprise activating signals originating from the mesenchymal dermal compartment (including mature adipocytes) and act on hair follicle stem cells, which are implicated in the anagen phase. Moreover, the Wnt/β-catenin signaling-dependent secreted factors from keratinocytes promote adipocyte differentiation.[19] This is known as mesenchymal-epithelial reciprocal interaction.[20]

Growth factors induce anagen re-entry in telogen hair follicles,[21] prolong the duration of the anagen phase, increase the rates of proliferation in active anagen hair follicles, and prevent premature catagen development.[22]

These effects are continuous and progressive even 6 months after injection of ADSCs due to progressive proliferation of dermal papillae cells and continuous secretion of growth factors from dermal mesenchymal cells including adipocytes.[13]

Finally, Adipose-derived adult stem cells (ADSCs) show promising regenerative benefits for treating FPHL compared to traditional therapies such as minoxidil and antiandrogens. ADSCs promote hair follicle development and overall scalp environment without the continuous application required by minoxidil, and they avoid the potential side effects of hormonal imbalances associated with antiandrogens. ADSCs work through a more natural mechanism that not only fosters hair growth but also improves tissue repair and regeneration without the systemic side effects often associated with pharmacological treatments. This novel therapy holds the potential for more sustainable and holistic management of FPHL.

CONCLUSION AND RECOMMENDATIONS

In this study, it has been proven that ADSCs are an effective modality for improving FPHL. However, complete resolution of the condition did not occur, possibly because of the progressive nature of the disease. Halting disease progression is considered successful in managing this multifactorial progressive disease. Better results were achieved with better FPHL grades. No serious adverse effects or complications were observed. We recommend conducting studies with larger sample sizes and longer follow-up durations (>24 weeks) to evaluate the onset of posttreatment regression. ADSCs treatment is costly and requires a special setting; therefore, we recommend comparing ADSCs with other modalities to explore newer regenerative treatment options. It is also advisable to conduct future studies comparing various methods of preparing adipose-derived adult stem cells (ADSCs), such as in vivo mechanical versus enzymatic techniques, and to investigate how different doses of stem cells impact treatment outcomes.

Statement of ethics

This study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki. The study protocol was approved by the ethical committee of the Faculty of Medicine, Ain Shams University (reference number: FWA 000017585). All participants provided written informed consent prior to participating in the study. The participants were informed of the potential benefits and risks associated with the procedure, the use of their images in the publication, and they were free to withdraw from the study at any time. The study was conducted by experienced dermatologists with utmost care and consideration of participants’ welfare, and every effort was made to ensure their safety and comfort throughout the study.

Author contributions

(Author 1; Dr Hoda Moneib) conceived and designed the study, supervised all clinical steps, performed data analysis, statistical expertise, collected and analyzed data and provided critical revisions to the manuscript. (Author 2; Dr. Ghada Fathy) contributed to the study design, recruited participants, collected and analyzed data, supervised clinical steps, and provided critical revisions to the manuscript. (Author 3; Dr. Naglaa Samir) contributed to the study design, supervised biopsy cuts, and preparation of the slides for histopathology and immunohistochemistry, interpreted the slides, and generated and analyzed data for both histopathology and immunohistochemistry. (Author 4; Dr. Nashwa Nagy) performed the extraction of ADSCs from the lipoaspirate, cell culture, and prepared the ADSCs suspension ready for injection. (Author 5; Dr. Mahy El-Bassiouny) contributed to the study design, wrote the manuscript, performed liposuction, injected the ADSCs, and performed the clinical and trichoscopic follow-up visits. All authors have read and approved the final manuscript.

Data availability

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding authors.

Capsule summary

Several clinical trials have assessed the effectiveness of ADSC injections for patterned hair loss, employing various methods for processing lipoaspirate and primarily relying on clinical evaluations. In this study, however, an objective assessment was conducted, incorporating comprehensive trichoscopic measurements along with histopathological and immunohistochemical evaluations of follicular regeneration.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.