Innovative Method of Alopecia Treatment by Using Adipose-derived Stromal Vascular Fraction (SVF) Cells

Background Androgenetic alopecia (AGA) is characterized by progressive reduction of hair density on the scalp through gradual conversion of terminal hairs into vellus hairs. Stromal vascular fraction (SVF) cells harvested from fat cells are one of the latest breakthroughs in the aesthetic eld. This study aimed to present clinical cases for the treatment of alopecia areata by transplantation of SVF into the scalp. Objective To evaluate the ecacy of the use of the stromal vascular fraction (SVF) in androgenetic alopecia patients. Methods


Introduction
Androgenetic alopecia (AGA) is a genetically determined and androgen in uenced progressive condition, which is characterized by progressive hair loss of the scalp, and its prevalence increases signi cantly with age 1,2 . Although AGA is hereditary, circulating androgen hormones may trigger its onset by miniaturization of hair follicles resulting in the conversion of terminal hair to vellus hair. AGA becomes a medical problem when the hair loss is subjectively considered as excessive, premature, and distressing. AGA is similar to tissue damage, and repair processes are in uenced by growth factors that in turn stimulate homing of cells and chemotaxis 3,4,5 .
At the cellular level, a decrease in hair follicle size is due to loss of stem cells or progenitors at the bulge region of the hair follicle. Conventional approaches for hair refurbishment include medications and hair follicle transplantation surgery 6 . However, these strategies are mostly ineffective due to drawbacks including high cost, numerous side effects, unsatisfactory results, and the requirement for long-lasting use of medicines. In addition, their e cacy is dependent on gender. Contemporary therapies with promising results are required that should be effective in both sexes, and the outcomes should be long-lasting.
Treatment options for men and women with hair loss include medical therapy, hair transplant surgery, low-level laser therapy, hair systems, micropigmentation of the scalp, and topical concealer bers 7,8 . These options are not without shortcomings, and thus researchers are always looking for new and alternative therapies 9 . One emerging area of clinical and scienti c focus lies in exploring the role of adipose tissue (fat), and speci cally autologous adipose transplantation 10 , in the complex, hair growth cycle. Although platelet-rich plasma (PRP) injections are frequently used for AGA patients and overall results suggest that it has a good therapeutic effect 11 , the treatment has to be repeated several times leading to poor compliance of the patient.
The repair of hair follicles in AGA could be improved using a combination of regenerative cells (i.e., stromal vascular fraction (SVF)) as a source of growth factors. Adipose tissue-derived SVF contains non cultured regenerative cells, such as mesenchymal stem cells (MSCs) or adipose derived stem cells(ADSCs), that can "home" to the site of injury 12,13,14 . Abundant research has supported the fact that adipose is biologically active, complex, and an important tissue. In the context of the scalp, adipocyte lineage cells support the stem cell niche and help drive the hair growth cycle 15  Today, autologous fat is transplanted primarily for an esthetic and reconstructive volume effect, and traditionally, rates of graft retention have been widely varied. A number of strategies have been applied to increase this rate of graft take. One such strategy is to enrich the adipose with SVF, a heterogeneous group of generally well-characterized multinucleated cells that can be reliably extracted from adipose by using automated systems. These cells work largely by paracrine mechanisms to support adipocyte viability.
SVF is applied to restore hair growth because it contains several types of regenerative cells such as MSCs that are highly proliferative, have multi-lineage differentiation potential, and are immunomodulatory and immunosuppressive 14 . Furthermore, the cells in SVF also secrete various growth factors and proteins which can perform several functions including activation of hair follicles 18 . SVF can have multiple effects on miniaturized hair follicles by homing to the hair follicles and by their paracrine effects. Stromal vascular fraction-based treatment for AGA can open a new avenue for the development of therapies for hair restoration 19 .
In a group of AGA patients, SVF was injected into the affected area of scalp. After 6 months, the patients were assessed by a physician, and patient global assessment scores were recorded. Photographs of the affected areas were taken. The results show an increase in hair density, decreased hair fall, and improvement in physician and patient global assessment score after SVF treatment.

Subjects
The medical records of patients were reviewed to collect subjects treated with only SVF at the plastic surgery clinic (TOP Plastic Surgery, Seoul, Korea). A thorough medical and hair history was collected, and a physical examination was performed to diagnose male or female pattern alopecia. Patients in this clinical series were healthy men and woman aged 43-64 years with androgenic baldness rated using the Norwood-Hamilton grades and Ludwig scale. Over a period of 6 months, a total of nine subjects (4 men and 5 women) were enrolled. The investigators reviewed the inclusion and exclusion criteria (Table 1) to screen patients accordingly. Importantly, patients who reported the administration of any agent aimed at affecting hair growth within 6 months prior to presentation, regardless of whether they were prescribed by a physician or obtained over the counter, were excluded from participation. All patients had a body mass index (BMI) within normal limits. All patients were considered generally healthy, and no patients had comorbidities such as diabetes or high blood pressure. Written informed consent for the procedure, including photographing and publication in a medical and scienti c journal for educational purposes, was obtained from all subjects, and the protocol was reviewed and approved by the hospital's Internal Medical Advisory Committee. Table 2 summarizes patient demographics, including the amount of adipose harvested, processed, and injected SVF. Table 1 Inclusion and exclusion criteria for enrollment of patients in the study.

Inclusion
Male and female patients with androgenic alopecia Subject who has previously failed or has been deemed nonresponsive to a previous experimental hair loss treatment, prior surgery in the treatment area and subject who has a sensitive, irritated, or abraded scalp area Clinically signi cant medical or psychiatric illness currently or within 30 days of study screening as determined by the investigator Any disease or condition (medical or surgical) that in the opinion of the investigator, might compromise hematologic, cardiovascular, pulmonary, renal, gastrointestinal, hepatic, or central nervous system function; or any condition that would place the subject at increased risk Korea). Once the HuriCell disposable kit is placed within the device, the system performs an auto-check to ensure the integrity of the closed system. Tissue is then introduced into the processing canister where it is weighed and then washed with the saline solution to remove the residual waste solution and extravasated blood. The HuriCell device calculates the amount of HRR reagent to be used (based on tissue weight) and then, at the appropriate stage, prompts the operator to add the required volume of HRR. The tissue is continuously agitated during enzymatic digestion of the connective tissue. Once digestion is complete, the SVF fraction is pumped into a centrifuge chamber where it is washed and concentrated. The nal cell product can then be aspirated from the chamber in a volume of 13 mL.
The stromal cell fraction was ltered through a 70 µm cell strainer (BD Biosciences, Inc., San Jose, CA, USA). The number and relative viability of SVF recovered from tissue processing samples in each study were determined using a Semiautomated Cell Counter (ADAM MC Cell Counter; NanoEnTek, Seoul, Korea). (2) to reach the hair follicle area, the injection into the scalp area was performed with the following attributes: 3cc syringe; gauge, 30; and depth, 4 mm ; (3) 0.15 ml per injection was delivered perpendicularly, separated by 2 cm in a square shape all over the scalp marked previously; a total of 4 ml were injected in 48 spots; (4) after the injection was administered, the needle was kept in the scalp for 2 s. After the transplantation, the patient was prescribed nonsteroid anti-in ammatory and cephalexin antibiotics for 3 days. Patients were advised not to shower until 24 h after the procedure, not to sunbathe until after 1 week, and not to engage in sports until after 1 week; however, return to work can be on the same day. Follow-up for hair evaluation was based on the hair cycles and was performed 1, 3 and 6 months after injection, using Aroma Smart Wizard system (ASW200, Aram Huvis, Seoul, Korea).

Measurements and statistical analysis
Hair density (hair count per cm 2 ) was calculated by counting the total number of hairs in the target area. Hair thickness was calculated as the average diameter of hairs, and scalp status, keratin of scalp, scalp sensitivity, scalp sebum, hair pore status, and cuticle status measured automatically on the Smart Wizard System. Mean differences were then tested by paired t-tests. It should be noted that the sample size decreases over time, so less weight should be given to observed differences at time points. Wilcoxon signed rank tests were applied to detect the difference in the rates between different groups. P values of less than 0.05 were considered signi cant.

Results
In the current study, patients enrolled were divided into two groups: Male group (n = 4) and female group (n = 5). Given that current clinical practice guidelines on the treatment of AGA, we treated nasteride 1 mg, dutasteride 0.5 mg for men and 3% minoxidil foam for women.

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First, based on a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT) published in 2013, which point out the minimal phenotypic criteria to characterize the uncultured SVF population from adipose tissue, these freshly isolated cells were characterized ( Table 3). The immunophenotyping of the transplanted cells showed a clearly heterogeneous population expressing not only the mesenchymal stem cell markers but also the panhematopoietic/monocyte/macrophage/endothelial/pericyte markers along with particularly high levels of CD34 19 .   The images were taken from the same affected area of the scalp at each visit in both groups, and the number of hair/cm 2 was counted in each patient. In addition, in one subject, half of the affected area of the scalp was treated with SVF, while the other half was not treated. Mean density of hair on the pre-injection visit in the nontreated site was 44.44 ± 5.09 versus 43.33 ± 3.11 in the treated site (Fig. 1). On the nal visit (after 3 months of last session), it was 68.88 ± 2.97 in the nontreated site versus 90 ± 2.35 in the treated site. Overall, the percentage of density increased in the treated site by 48.11% as compared to the nontreated site density of 35.48%. Hair density was signi cantly increased on the treated side at 3 months after pre-injection (P = 0.01 and P = 0.009 respectively, n = 9).
Hair thickness was calculated with one average value obtained for analysis per patient. The increase in thickness was signi cant at 6 months post injection (P = 0.02) (Fig. 2). The average hair thickness on the preinjection visit was 0.032 ± 0.053 mm in the designated nontreated site compared to 0.029 ± 0.003 mm in the designated treated site. On the 6 months post-injection visit, hair thickness was 0.053 ± 0.003 mm in the nontreated site compared to 0.058 ± 0.003 mm in the treated site.
The treated areas did not show any overall signi cant changes in scalp status, scalp sensitivity, scalp sebum, hair pore status, cuticle status, or any of the other parameters measured with skin analysis throughout the 6 months follow up except keratin of scalp (Fig. 3). Although the majority of the patients do not achieve improved scores, a signi cant improvement in the score of keratin of scalp was seen in the treated side as compared to the nontreated side (p = 0.032). Representative global and macrophotographs of a patient at 6 months are shown in Fig. 4.

Discussion
In this study, we investigated the potential effect of the adipose-derived stromal vascular fraction (SVF) on androgenetic alopecia (AGA). Adipose tissue being a biologically active complex is important for tissue engineering and regenerative medicine applications 14 . In the current study, we used SVF which is a mixture of several types of cells including ASCs. Tolerability and safety of using SVF was determined, and no adverse side effects were reported in any patient.
In AGA patients, the basic concept of using SVF is to replenish stem cell repository in the bulge region of hair follicles by homing and to stimulate growth cycle of stem cells by paracrine effects 17 . SVF is known to be one of the most accessible sources of MSCs and has recently emerged as a new therapeutic option for degenerative conditions. 18 With a primary role in the homeostasis of organs and tissues, MSCs maintain the stem cell niche, help tissue recovery after injuries, and ensure healthy aging 13 . In addition to replacing damaged cells in affected tissues, SVF has bene cial effects through its paracrine action via various cytokines and growth factors 18, 20 . A recent study showed the synergic effect of PRP and micrografts enriched with autologous human follicle MSCs on AGA 10,11 . In comparison with PRP or micrografts, ADSC-based therapies have more published evidence of their effect on hair regrowth through clinical trials 21 .
The therapeutic role of SVF was assessed using parameters such as hair density, hair thickness, photographs, and status of scalp. In this case series, the potential effect of a single dose of SVF on AGA was tested. There was a signi cant increase of hair density in the treated site compared to the nontreated site. The average number of hair thickness of the treated side was signi cantly increased at 6 months post injection (P < 0.05).
Furthermore, nonfunctioning hair follicles lled with hyper-keartotic plugs 22 , up to today assumed incapable of forming new hair, showed more signi cant improvement in the score of keratin of scalp in the treated side as compared to the nontreated side (p = 0.032). No side effects were noted after treatment.
Currently, only a few FDA-approved agents for AGA treatment are available 22 . Finasteride and minoxidil, either as monotherapy or in combination, are recommended as the gold standard treatment for AGA 23 According to recent data, promotion of hair growth via ADSCs can be enhanced by combining it with minoxidil, which stimulates the motility of ADSCs and increases the secretion of growth factors and paracrine signaling 29 . This result might suggest that ASCs migrated close to the injection site and enabled hair growth.
Alternatively, ASCs might be capable of migration by making use of the local circulation. The differentiation as well as production and secretion of growth factors that activate neighboring cells are also mentioned as The proposed strategy can provide not only a treatment for AGA patients but also be helpful in the development and success of tissue engineering and regenerative medicine applications. In addition, the results of this study will open a new avenue in dermatology for the treatment of patients with AGA. Taking everything into consideration, we believe that the hallmarks of tissue damage are also present in AGA. Addressing the combination of both cellular as well as intercellular aspects of wound repair as an alternative treatment of AGA seems to deserve further attention.

Conclusions
This initial data experience demonstrates that scalp stem cell-enriched grafting may represent a promising alternative approach for treating baldness in men and women. A single treatment of SVF injected in the scalp of patients with AGA signi cantly increased hair density within 6 months. Further research is required to determine the optimal treatment regimen. Hair density before and after the treatment of the right side of the scalp with SVF. Patients that were analyzed pre-injection, and 1, 3, and 6 months post-injection after SVF treatment. * n = 9; p < 0.05.

Figure 4
Representative photographs documenting the increase in hair counts after SVF cell treatment. Baseline (preinjection) versus 6 months (post-injection) global photographs of treated vertex are shown.