Short-Term Efficacy of Autologous Cellular Micrografts in Male and Female Androgenetic Alopecia: A Retrospective Cohort Study

Introduction

Androgenetic alopecia (AGA) or pattern hair loss (PHL) is the most common cause of hair loss. It affects approximately 50% of men and women by the age of 50, and its prevalence increases with advancing age.^1,2 As the name implies, AGA has a clear genetic predisposition mediating an excessive response to androgens.³

The treatments for AGA are limited. Topical minoxidil is the only FDA-approved treatment in females, and topical minoxidil and oral finasteride 1 mg are the only FDA-approved treatments in males. Minoxidil efficacy is limited, and studies suggest that only one-third of the patients experience a cosmetic benefit or moderate terminal hair regrowth after 1 year of use.^4–7 Scalp irritation, increased seborrhea, erythema and hypertrichosis are commonly reported after topical minoxidil use.^8–10 Oral finasteride 1 mg is FDA-approved for male but not female AGA, due to its teratogenicity. Decreased libido, erectile dysfunction and ejaculatory problems have been reported in around 3–5% of men using finasteride.^11–13 In addition, both minoxidil and finasteride require a long-term commitment by the patient on a daily basis, most likely for an individual’s life.

Furthermore, in the advanced stages of PHL, the above treatments are less effective and hair transplantation would be the only option for some of these patients. Hair transplantation also comes with limitations. Many balding males have a limited donor supply, which is not sufficient to cover the bald area. Also, many females with PHL have a weak donor area and are not candidates for hair transplantation.^14–16

The previous observations highlight the importance of finding effective new treatments, which ensure better patient compliance and have limited side effects. Since AGA is characterized by defects in and loss of hair progenitor cells, while hair follicle stem cells (HFSCs) remain viable,¹⁷ transplantation of multipotent stem cells has become a well-accepted treatment option. Autologous cellular micrografts (ACM) is a method that obtains autologous mature stem cells from scalp biopsies of a patient using a preparation system for mechanical disintegration and filtering of solid tissues.

The advocated mechanisms of action of ACM in AGA include the enhancement of hair follicle regeneration by transplantation of mature multipotent stem cells, besides the reactivation of existing stem cells and progenitor cells of miniaturized follicles, by restoring hair growth signaling via the injection of growth factors.^17,18 Yet, these mechanisms have not been established. On the other hand, given the novelty of the treatment method, further data is needed to establish the clinical efficacy in AGA. Yet, the few published reports provide promising results, showing an increase in hair density and thickness in a high percentage of patients.^22,37,38 However, these studies comprise limitations that impede the generalizability of the findings, notably due to limited sample size.

In this paper, we report the short-term clinical efficacy of a single application of ACM in the treatment of male and female AGA. We also proposed a pixel-based method to portray the treatment efficacy and cosmetic benefit in hair loss, in a standardized visual fashion.

Materials and Methods

Design and Setting

A retrospective cohort study was carried out at the author’s dermatology clinic in Jeddah, Saudi Arabia, from September 2019 to August 2020.

Population

The study involved 18–65-year-old consecutive males and females with clinically diagnosed and dermoscopy-confirmed AGA, who received a single session of ACM (Regenera Activa^®). Only females classified Sinclair 2–4 and males classified Hamilton-Norwood 2–4 were included. Patients using any other topical or systemic medication for hair loss during the 6 months prior to inclusion or during the study period were excluded, as well as those using medical devices such as low-level laser therapy (LLT) or procedures such as platelet-rich plasma (PRP) or micro-needling for hair loss during the same periods. Other exclusion criteria included: cancer within 2 years; pregnancy or breastfeeding; immunosuppression; hemoglobin below 12 mg/dL; presence of other causes of alopecia such as alopecia areata, scarring alopecia or inflammatory scalp disorders; history of hair transplantation; history of scalp tumors; and/or the presence of trichotillomania.

Procedure

Baseline Assessment

As part of our routine practice, patients undergo a structured clinical evaluation to collect demographic and relevant clinical data, followed by dermoscopy to confirm the diagnosis, and global scalp photography following a standardized method and technical settings. Standardized phototrichograms are conducted on all scalps using video-epiluminescence microscopy (FotoFinder Systems, Inc., Columbia, CA, USA) in conjunction with TrichoScan digital image analysis (TrichoScan, Tricolog GmbH, Freiburg, Germany). Trichoscan is a computer‐assisted dermoscopy with dedicated software to diagnose the hair loss and to measure its severity.

Autologous Cellular Micrografts (ACM) Procedure

Under local anesthesia, using anesthesia techniques which were previously described by the author,²² a 2.5 mm punch biopsy was used to extract 3 scalp tissue specimens from the patient’s occiput behind the ear, using Rigeneracons medical device (CE certified class I; Human Brain Wave, Turin, Italy). The collected specimens are placed in Rigeneracons by adding 1.5 mL of sterile physiologic solution to the device. The device then generates a cellular suspension by rotation of Rigeneracons at 80 RPM for 2 minutes. Subsequently, the obtained suspension is diluted with an additional 3 mL sterile physiologic solution. The solution was then injected subdermally into the balding areas of the scalp using a 1mL syringe with a 30-gauge needle; 0.1 mL was injected per point spaced approximately 1 cm apart.

Follow Up and Outcomes

After the intervention, patients were followed up by clinic visits and phone calls for any adverse effects or further concern. The short-term post-treatment assessment including trichometry with TrichoScan digital image analysis was scheduled 1 to 6 months after the intervention, as per the patient’s convenience. Trichometry parameters were divided into positive and negative parameters. Positive parameters correspond to parameters whose increase corresponds to favorable evolution (hair growth) and include hair density (N/cm2), average hair shaft thickness (AHST, μm), percent thick hair (%), cumulative hair thickness (CHT), and number of follicular units (%). Negative parameters correspond to parameters whose decrease corresponds to favorable evolution (hair growth) and include percent thin hair (%), yellow dots (N/cm²), and trichoscopy-derived Sinclair scale (TDSS), which describes the hair midline density calculated from CHT density measured in trichoscopy.²³

Ethical Considerations

The study was approved by the institutional review board of King Abdulaziz University Hospital in Jeddah (Reference No 235–21). Waiver of consent has been obtained because of the retrospective nature of the study. The confidentiality of the patients’ data has been protected and identifiable data of patients have been removed in compliance with the Declaration of Helsinki. The baseline assessments, ACM procedure and follow-up methods described above are part of the routine practice in the dermatology clinic, and were not specifically designed for the present study.

Statistical Methods

Data was managed using Microsoft Excel (version 2017, Microsoft) and statistical analysis was performed with the SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). For all trichometry parameters, absolute and adjusted relative changes were calculated for each participant as follows:

Where,

: pre- to post-intervention change of the parameter value in the given patient,

: baseline population mean of the parameter.

Paired t-test was used to analyze the absolute changes in the total study population and in males and females separately. The correlation between pre- and post-intervention values was analyzed using Pearson’s correlation.

To visually portray the cosmetic effect of the treatment in the study population, we calculated the area coverage index (ACI) as an estimate of the scalp area, in cm², that would be completely covered by 1 cm-long hair, with respect of the hair density and thickness. By assuming a one-μm hair thickness, a 1-cm-long hair would visually cover an area of 1cm × 10⁻⁴ cm = 10⁻⁴ cm². Thus, by considering the hair density (average number of hairs by cm²) and thickness (AHST), the ACI was calculated using the following formula:ACI (cm²hair/cm²scalp) = HD (hair density,N/cm²)*AHST(μm)* 10^-4

Afterwards, a graphical representation was made by generating a 30 × 30 (900-pixel resolution) grid, corresponding to 9cm² of the scalp (covered by 9*ACI), comprising filled and blank 1mm² square pixels that correspond to the covered and uncovered scalp areas, respectively. The number of filled pixels is proportional to ACI and is calculated as N = resolution*ACI. Filled pixels are dispersed over the grid using a random generator. The ACI definition, calculation formula and pixel transformation method are illustrated in Figure 1.

Figure 1 Area coverage index (ACI) definition, calculation formula and pixel transformation method. The figure describes the method proposed by the author to portray the visual aspect of the scalp as a function of the hair density and average hair thickness, by the (A) unit definition, (B) calculation and (C) pixel transformation of an area coverage index (ACI).

For each parameter and scalp region, the percentage of participants who had favorable outcome (defined as the desired change occurring within the given parameter) was calculated along with the respective mean absolute change and mean adjusted relative change.

A p value of <0.05 was considered to reject the null hypothesis.

Results

Baseline Demographic and Clinical Characteristics

A total 140 patients were included, 113 (80.7%) of them were female, and mean (SD) age was 32.1 (10.1) years. The distribution of males according to Hamilton–Norwood classification showed Class II (48.1%), Class III (18.5%) and Class III Vertex (33.3%). The distribution of females according to Sinclair classification showed Grade 2 (32.7%), Grade 3 (56.6%), and Grade 4 or 5 (10.7%). The median follow-up time was 94 days (range = 39–197 days).

Efficacy of Autologous Cellular Micrografts in the Total Population

Absolute Change

There was increase in all positive parameters including hair density (+4.5 to 7.12 hair/cm²), AHST (+0.96 to 1.88 μm), % thick hair (+1.74 to 3.26%), CHT (+0.48 to 0.56 unit), and number of follicular units (+1.30 to 2.77), depending on the scalp region, and the improvements were more remarkable in frontal region where all these changes were statistically significant (p < 0.05). Additionally, negative parameters decreased, notably in frontal region in % thin hair (−1.81%, p = 0.037) and yellow dots (−1.93 N/cm², p = 0.003). In the majority of parameters, pre- and post-intervention values were moderately to strongly correlated (R = 0.539–0.807) (Table 1).

Table 1 Pre- and Post-Intervention Trichometry Findings by Scalp Region (Intrasubject Analysis)

Adjusted Relative Change

Depending on scalp region, there was relative increase in hair density (up to 4.1%), AHST (up to +3.6%), % thick hair (up to +6.0%), CHT (up to +6.7%), and number of follicular units (up to +3.8%), with relative decrease in % thin hair (down to −7.6%), as adjusted to the population’s baseline. Besides, a substantial relative decrease in yellow dot density was observed, notably in frontal (−36.0%) and temporal (−42.9%) regions. This was associated with decline in TDSS (Figure 2).

Figure 2 Change in key indicators of hair growth, by scalp region, in male and female androgenic alopecia after treatment with autologous cellular micrografts (ACM). Pictures depict: 1) relative pre- to post-intervention change (in percent, left panels) of the 5 positive (green bars) and 2 negative (Orange bars) parameters as adjusted by the respective baseline population mean, that is, bar heights represent the percentage of change in the given parameter by reference to the baseline population mean; 2) mean derived Sinclair grade (right panels) in baseline versus after treatment with ACM. Panels correspond to findings in different scalp regions including frontal (A), temporal (B), and occipital (C) regions.

ACI and the Cosmetic Effect

The mean hair-covered scalp area in the total population increased in all three scalp regions including frontal (from 0.85 to 0.91 cm² hair/cm² scalp), temporal (0.70 to 0.75 cm² hair/cm² scalp) and occipital (0.96 to 1.02 cm² hair/cm² scalp), and all results were statistically significant (p < 0.001). Results were depicted in a randomly generated 900-pixel grid representing 9cm² of the scalp (Figure 3).

Figure 3 Short-term hair growth in male and female androgenic alopecia after treatment with autologous cellular micrografts (ACM). Pictures depict pre- to post-intervention change in area coverage index (ACI) in the frontal (A), temporal (B), and occipital (C) regions. Each grid represents 9cm2 (3×3cm) of scalp area; where gray cells represent the scalp area covered by hair, white cells represent the scalp areas not covered by hair, and dark gray cells represent highly dense areas indicating ACI > 1cm2.

Effect Size in Favorable Responders

Two-thirds of patients (63.6–66.4%) had a favorable change in CHT, with a mean absolute change 1.2–1.4 unit depending on the scalp region. Further, more than 50% had favorable change in the other parameters, except yellow dots that decreased in up to 43.6% of the participants depending on the scalp region (Table 2).

Table 2 Change in Hair Growth Indicators Among Favorable Responders

Efficacy by Gender

In Females

The mean CHT increased by 0.52 to 0.57 units, associated with a significant increase in both AHST (+1.41 to 2.17μm) and % thick hair (+2.64% to 4.16%) in all 3 scalp regions. Increase in hair density was not statistically significant in the frontal region. Further, all negative parameters decreased, but statistical significance was not consistent throughout scalp regions (Table 3). The pre and post treatment scalp photographs of a sample female patient are depicted in Figure 4-Patient 1.

Table 3 Hair Growth Indicators by Gender and Scalp Region in Male and Female Androgenic Alopecia After Treatment with Autologous Cellular Micrografts (ACM)

Figure 4 Pre and post treatment scalp photographs of a sample female and male patients with androgenetic alopecia, before and after treatment with autologous cellular micrografts (ACM). Photographs show cosmetic improvement after autologous cellular micrografts in a female (1) and a male (2) patient. In the female patient (1), there was increase in CHT from 10.3, 7.5 and 9.9 to 10.5, 8.3 and 12 mm/cm2 in frontal, temporal and occipital areas, respectively. In the male patient (2), there was increase in CHT from 12.7 to 13.2 mm/cm2 in the frontal and 8.8 to 12.3 mm/cm2 in the occipital area, while CHT decreased from 8.5 to 7.5 mm/cm2 in the temporal area.

In Males

The frontal region showed a statistically significant increase in mean CHT (+0.66 units, p = 0.045) associated with a remarkable increase in mean hair density (+11.2 hair/cm², p = 0.031) in reference to baseline (Table 3). The pre and post ACM scalp photographs of a sample male patient are depicted in Figure 4-Patient 2.

Discussion

Summary of Findings

The present study provides the largest series of AGA patients treated with ACM to date. Pre- to post-intervention assessment showed favorable changes in all positive and negative trichometry parameters, including growth of an average 5–7 new hairs per cm² of scalp and an increase in the average hair thickness by 1.6–1.9μm, which is reflected by an increase in the percentage of thick hairs and proportional decrease in the percentage of thin hairs. Additionally, there was considerable reduction of yellow dots by 1–2 per cm² of scalp combined with increase in the percentage of follicular units by up to 3%. All these changes resulted in the enhancement of the CHT by an average 0.5–0.6 units, representing 5–6 mm² increment in hair coverage index per cm² of scalp, which induced a significant cosmetic effect as demonstrated by the visual model developed by the author in this study. Such favorable outcomes were observed in up to two-thirds of patients, depending on the parameter, and the magnitude of changes among positive responders was more remarkable, notably in the frontal region. These findings are very encouraging and indicate multiple effects of ACM in improving the hair of AGA patients in the short term.

Pathophysiological Basis of ACM Mechanisms of Action

Mature HFSCs, located in the hair follicle bulge, are multipotent cells that play a key role in the regeneration of hair follicles and other scalp skin structures. They have the ability to self-regenerate between the telogen and anagen phases of the hair cycle; or migrate down the hair matrix and become progenitor cells, forming the internal hair follicles and hair stem. The activation of bulge-located stem cells is controlled by the surrounding microenvironment “niche” including their daughter cells, as well as by dermal papilla cells (DPCs) signaling pathways, notably the Wnt/β-catenin pathway which is crucial for entry into the anagen phase. DPCs further contribute in regulating hair growth by secreting hair growth-stimulating factors such as insulin-like growth factor 1 (IGF-1), basic fibroblast growth factor, and vascular endothelial growth factor, which respectively have an autocrine effect on the dermal papilla itself and paracrine effect on hair follicle epithelial cells.^19,24–30

The most supported pathophysiological mechanism of AGA involves a suppressive effect of systemic or locally produced (by balding DPCs) dihydrotesterone on follicular keratinocyte growth, which leads to catagen. This effect is mediated by the upregulation of transforming growth factor beta (TGF-β) and Wnt antagonist DKK-1 production by DPCs. These alterations result in a number of pathohistological anomalies including impaired HFSC differentiation, oxidative stress and perifollicular fibrosis and inflammation, which altogether lead to the chronic hair miniaturization resulting in smaller dermal papillae and a reduction in CD200⁺ progenitor cells.^31–37 On the other hand, although progenitor cells are damaged in AGA, the HFSCs remain preserved, which explains the reversibility of the condition. These observations constitute the foundation for ACM, which consist of the transplantation of mature multipotent stem cells in the balding areas, thereby enabling hair follicle regeneration.¹⁷ Besides unaffected areas of the scalp, ACM may use multipotent stem cells originating from adipose tissue.³⁸ Another plausible mechanism of action of ACM is the reactivation of already existing stem cells and progenitor cells of miniaturized follicles, by reinstating the hair growth signaling via the injection of growth factors.¹⁸

Reports on Efficacy of ACM in AGA

The few available reports on ACM efficacy are encouraging. In a small placebo-controlled trial (N = 11), the use of ACM showed ~29% relative increase in hair density in treated scalp areas, approximately 6 months after treatment.¹⁹ Another study including 17 patients showed patient-perceived increase in hair thickness (12/17 patients) and reduction in hair fall (8/17), while the remaining cases observed no change for either parameter.²⁰ More recently, Ruiz et al studied the efficacy of ACM at 4, 6 and 12 months, among 100 AGA and showed significant increase in hair density (+33.3/cm²) and percentage of thick hair (+5.6%) at 2 months.²¹

Efficacy of ACM by Gender

Findings from the present study suggest a gender-specific effect of ACM in AGA. The most remarkable effect in males was the increase in hair density, while in females it was the promotion of hair thickness and reduction of the number of yellow dots. Interestingly, the two patterns resulted in a comparable improvement of CHT across genders. Additionally, although the occipital area was not treated in any of the study patients, women experienced significant improvement in all hair growth indicators in the occipital region with a single ACM session, which could have an implication in improving the donor area in females requiring a hair transplantation.

Pixel-Based Representation of Cosmetic Effect

The author proposes the use of a pixel-based graphical method to portray the cosmetic effects of treatments in hair loss in a standardized fashion. This method provides an intuitive visualization of the change in scalp area coverage after treatment. It can be used in all types of hair loss and treatments (transplantation, topical treatments, etc.) and can have interesting clinical and research applications.

Limitations

The external validity of the present study may be limited by the retrospective and noncontrolled design. Further, the present design does not demonstrate the final clinical improvement due to the short-term endpoint of the study outcomes. A longer follow-up study will be needed in the future.

Conclusion

Two-thirds of patients with AGA would respond favorably to a single treatment session with ACM in the first 6 months following treatment. The pre- to post-ACM trichometry analysis showed significant improvement in hair regrowth, hair thickening, promotion of follicular units and reduction of yellow dots; all combined, these effects result in an increase in the hair area coverage index, representing a noticeable cosmetic change. There is a probable gender-specific effect of ACM in AGA that should be further studied.