Stimulation of hair regrowth in an
animal model of androgenic
alopecia using 2-deoxy-D-ribose
Muhammad Awais Anjum1, Saima Zulfiqar1,
Aqif Anwar Chaudhary1, Ihtesham Ur Rehman1,2,
Anthony J. Bullock3, Muhammad Yar1* and Sheila MacNeil3*
1Interdisciplinary Research Center in Biomedical Materials, COMSATS University Islamabad, Lahore
Campus, Lahore, Pakistan, 2School of Medicine, University of Central Lancashire, Preston,
United Kingdom, 3Department of Materials Science and Engineering, Kroto Research Institute, University
of Sheffield, Sheffield, United Kingdom
Androgenic alopecia (AGA) affects both men and women worldwide. New blood
vessel formation can restore blood supply and stimulate the hair regrowth cycle.
Recently, our group reported that 2-deoxy-D-ribose (2dDR) is 80%–90% as
effective as VEGF in the stimulation of neovascularization in in vitro models
and in a chick bioassay. In this study, we aimed to assess the effect of 2dDR on hair
growth. We prepared an alginate gel containing 2dDR, polypropylene glycol, and
phenoxyethanol. AGA was developed in C57BL6 mice by intraperitoneally
injecting testosterone (TE). A dihydrotestosterone (DHT)-treated group was
used as a negative control, a minoxidil group was used as a positive control,
and we included groups treated with 2dDR gel and a combination of 2dDR and
minoxidil. Each treatment was applied for 20 days. Both groups treated with 2dDR
gel and minoxidil stimulated the morphogenesis of hair follicles. H&E-stained skin
sections of C57BL/6 mice demonstrated an increase in length, diameter, hair
follicle density, anagen/telogen ratio, diameter of hair follicles, area of the hair
bulb covered in melanin, and an increase in the number of blood vessels.
Masson’s trichrome staining showed an increase in the area of the hair bulb
covered in melanin. The effects of the FDA-approved drug (minoxidil) on hair
growth were similar to those of 2dDR (80%–90%). No significant benefit were
observed by applying a combination of minoxidil with 2dDR. We conclude that
2dDR gel has potential for the treatment of androgenic alopecia and possibly
other alopecia conditions where stimulation of hair regrowth is desirable, such as
after chemotherapy. The mechanism of activity of 2dDR remains to be
established.
KEYWORDS
androgenic alopecia, 2-deoxy-D-ribose, C57BL6 mice, testosterone, minoxidil, hair
regrowth, chemotherapy
1 Introduction
Alopecia can occur due to hormonal imbalance, thyroid problems, certain medications,
and autoimmune diseases. It can be induced by blood thinning medications, contraceptives,
antidepressants, steroidal anti-inflammatory drugs, beta and calcium-channel blockers,
retinoids, and chemotherapeutics (Vicky et al., 2018). Male pattern baldness, also known as
androgenic alopecia (AGA), is one of the most widespread hair loss conditions in the world
(Yohei et al., 2018). In the pathophysiology of AGA, testosterone is converted to
OPEN ACCESS
EDITED BY
Ali Mohammad Sharifi,
University of Malaya, Malaysia
REVIEWED BY
Lannie O’Keefe,
Victoria University, Australia
Shamshad Alam,
University at Buffalo, United States
*CORRESPONDENCE
Muhammad Yar,
Sheila MacNeil,
RECEIVED 15 January 2024
ACCEPTED 24 April 2024
PUBLISHED 03 June 2024
CITATION
Anjum MA, Zulfiqar S, Chaudhary AA,
Rehman IU, Bullock AJ, Yar M and MacNeil S
(2024), Stimulation of hair regrowth in an
animal model of androgenic alopecia
using 2-deoxy-D-ribose.
Front. Pharmacol. 15:1370833.
doi: 10.3389/fphar.2024.1370833
COPYRIGHT
© 2024 Anjum, Zulfiqar, Chaudhary, Rehman,
Bullock, Yar and MacNeil. This is an openaccess
article distributed under the terms of the
Creative Commons Attribution License (CC BY).
The use, distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in this
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comply with these terms.
Frontiers in Pharmacology 01 frontiersin.org
TYPE Original Research
PUBLISHED 03 June 2024
DOI 10.3389/fphar.2024.1370833
dihydrotestosterone (DHT) by 5α-reductase. DHT then binds to
androgen receptors in the dermal papilla cells (DPCs) of sensitive
hair follicles and prolongs the telogen phase, causing hair loss before
the growth of new hair (Izabela et al., 2014). AGA is said to affect
30% of Asian men by age 30 and 50% by age 50 (Yohei et al., 2018). It
also affects 80% of White men and 40% of White women by age 70
(Pietro et al., 2019). Platelet-rich plasma (PRP) injections, topical
drugs, oral medications, and hair transplant operations are currently
being used to treat AGA (Alfredo et al., 2016). Currently, minoxidil
and finasteride are the only FDA-approved drugs used to treat AGA.
Minoxidil facilitates hair growth by facilitating DP survival and
expanding the anagenic phase (Hyun et al., 2004). Finasteride
stimulates the growth of new hair by suppressing the activity of
5α-reductase (Libecco and Bergfeld, 2004). Both medications have
side effects; for example, finasteride has been reported to reduce
sexual drive, while minoxidil can trigger acute anteroseptal
infarction, myocardial infarction, and anorexia (Hiroshi et al.,
2000; Vesoulis et al., 2014).
Minoxidil usually causes early shedding of hairs that are already
in the telogen phase (telogen effluvium) because of shortening of the
telogen phase before refreshing the growth of healthy hair. This
requires long-term application of minoxidil for sustained effect
(Alfredo et al., 2012). The common side effects of topical
minoxidil are dermatological, such as contact dermatitis, pruritus,
and erythema. To date, there is only one case report of heart failure
with use of topical minoxidil (Hiroshi et al., 2000; Friedman et al.,
2002). Many intracellular signaling molecules encourage the
proliferation of dermal papillary cells—including kinases and
growth factors—and hence play an essential part in stimulating
hair growth. The signaling protein vascular endothelial growth
factor (VEGF) is secreted from the vascular epithelium and
stimulates angiogenesis through intracellular pathways. This
extends the vascular network surrounding the hair follicle,
supporting hair regrowth (Mauro et al., 2001; Choi, 2018). Shin
et al. demonstrated that Rg3 (one of the major components in Panax
ginseng) stimulated VEGF mRNA levels while also increasing the
proliferation of human dermal papillary cells. Rg3 activated stem
cells in mouse hair follicles in vivo by upregulating factor-activating
CD34 and was found to promote hair growth better than minoxidil
(Hyun et al., 2014). Theoretically, any agents that can stimulate the
production of VEGF could be useful in the regeneration of hair in
the telogen phase.
2-Deoxy-D-ribose (2dDR) is a D-isomer of a deoxypentose
monosaccharide, in which a hydrogen atom is present with a
hydroxyl group at the C-2 position, in place of the hydroxyl
group. 2dDR is known to enhance tubulogenesis (Ikeda et al.,
2006), prevent hypoxia-induced apoptosis (Yuichi et al., 2004),
and boost VEGF and IL-8 production (Serkan et al., 2021) of ECs
in vitro, which is consistent with the stimulatory effects of 2dDR on
cell proliferation and migration. Our group has found that 2dDR
stimulated angiogenesis, proliferation of endothelial cells, and
accelerated wound healing in rat models (Yar et al., 2017).
These studies (Yar et al., 2017; Serkan et al., 2019; Anisa et al.,
2020; Serkan et al., 2020; Serkan et al., 2021) collectively make a
substantial contribution to the understanding of the biological
(pro-angiogenic) activity of 2dDR. Additionally, we have
demonstrated that 2dDR can be loaded into several biomaterials
for prolonged release over several days to promote the growth of
neonatal blood vessels (Yar et al., 2017; Maryam et al., 2019; Serkan
et al., 2021). Sodium alginate, a biodegradable, non-toxic, and
water-soluble macromolecule, is an ideal polymer for gels (Wang
andWang, 2010). Propylene glycol (PG) is a viscous liquid used in
pharmacological delivery systems for its hydrophilic penetration
and antiseptic properties (Mujica et al., 2016). It improves fluid
retention time in hydrogels and interacts with intercellular lipids
for stratum corneum barrier penetration (Trommer and Neubert,
2006; Victor et al., 2020). Phenoxyethanol is an approved stabilizer
and antimicrobial agent, preventing contamination in hydrogels
(Nolan and Nolan, 1972).
Our aim in the current study was to investigate whether delivery
of 2dDR from hydrogels would stimulate hair regeneration via
angiogenesis. For this study, we prepared hydrogels comprising
sodium alginate and propylene glycol, with phenoxyethanol
added as a stabilizer (blank-SA). It was found that 2dDR-SA
hydrogels, when supplemented with 2dDR, showed sustained hair
growth in AGA mice, demonstrating 2dDR-SA hydrogel as a
potential therapeutic agent for treating AGA.
2 Materials and methods
2.1 Materials
Sodium alginate (Cat. No. 7528-1405) with a molecular weight of
120,000–190,000 g/moL was purchased from Daejung Chemicals,
Korea. Propylene glycol (99.5% pure; Cat. No. 24300-11000PE) was
purchased from Penta Chemicals Limited, Czech Republic. 2-
Phenoxyethanol (94% pure; Cat. No. A10786.30) was purchased
from Alfa Aesar, United States. 2-Deoxy-D-ribose sugar (Cat. No.
00613) was purchased from Chem-Impex International,
United States. Minoxidil (Hair Max® 2%) was purchased from Sante
(Private) Limited, Karachi, Pakistan, and testosterone enanthate (brand
name Testoviron Depot®) was purchased from BAYER Pharmaceuticals,
Germany. Autoclaved deionized water was used in the manufacturing
process in the current research. All chemicals and reagents were of
analytical grade and used without any further purification.
2.2 Preparation of control and
2dDR-loaded hydrogels
Two different hydrogels, blank-SA and 2dDR-SA, were prepared
by simple manual mixing of the constituents in autoclaved sterilized
water at RT by using a spatula. The blank-SA hydrogel was
composed of 1.4 g of sodium alginate (6.4% w/w), 250 mg of
propylene glycol (1.15% w/w), and 82.5 mg of 2-phenoxyethanol
(0.377% w/w) as a stabilizer in 20 mL water. The 2dDR-SA hydrogel
was composed of 1.4 g sodium alginate (6.416% w/w), 250 mg
propylene glycol (1.146% w/w), 82.5 mg of 2-phenoxyethanol
(0.375% w/w), and 86.62 mg of 2-deoxy-D-ribose sugar (0.394%
w/w) in 20 mL water. The prepared hydrogels (blank-SA and 2dDRSA)
were stored in glass vials at RT.
For FTIR and 2dDR release studies, 10 mL each of blank-SA and
2dDR-SA hydrogels were poured into Petri dishes (100 Å~ 15 mm)
and covered with a perforated aluminum foil. These were frozen
at −20°C for 20 h and then placed in Labconco’s FreeZone (4.5 L)
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Anjum et al. 10.3389/fphar.2024.1370833
at −105°C for 24 h. These freeze-dried hydrogels (FD-blank-SA and
FD-2dDR-SA) were stored at RT until used for analyses.
2.3 Fourier-transform infrared spectroscopy
(FTIR) analysis
The FTIR technique is used to estimate a sample’s interference
pattern of emission or absorption. Chemical characteristics of
manufactured sodium alginate-based 2dDR hydrogel and sodium
alginate Blank-Gel were observed on a spectroscopic analysis (FTIR)
system in the frequency range of 4,000–400 cm−1 with 256 sequential
scans at 8 cm−1 rulings on a photo audio mode, which is a way to
examine materials with no specimen preparation (Thermo Nicolet
6700P, United States spectrometer).
2.4 Analysis of 2dDR release from the
2dDR-SA hydrogel
2dDR release from the 2dDR-SA hydrogel was quantitatively
determined by Dische’s diphenylamine assay (Jennifer and Mura,
2013). A stock solution of Dische’s reagent was prepared by
dissolving 0.75 g diphenylamine in a solution of 50 mL glacial
acetic acid supplemented with 750 μL of concentrated sulfuric
acid in dark conditions. A fresh solution of 2% v/v ethanol in
distilled water was prepared for the assay, and 50 μL of the ethanol
solution was added to 10 mL of the diphenylamine solution.
Room-temperature dried 2dDR-SA hydrogel was cut into 2 Å~
2 cm patches and immersed in PBS in a six-well plate in a sterile
environment. The plate was tightly sealed with Parafilm in order to
avoid evaporation of PBS and incubated at 37°C and 55% humidity.
The release of 2dDR was monitored at different time points (4 h and
1 d, 2 d, 3 d, 4 d, and 7 d). At each time point, the release medium
(PBS) from each well was extracted and replaced with fresh media.
Dische’s reagent (500 μL) was added to 500 μL of release media and
incubated for 10 min at 100°C. Samples were then allowed to cool to
room temperature for 10 min before centrifugation at 10,000 rpm
for 5 min. The absorbance of the resulting supernatant at 590 nm
was measured. The concentration of the released 2dDR was obtained
from a calibration curve of known dilutions of 2dDR. Cumulative
drug release vs. time was calculated.
2.5 Development of the androgenic alopecia
model in male C57BL/6 mice and testing of
blank-SA and 2dDR-SA hydrogels against
androgenic alopecia
Seven-week-old male C57BL/6 mice were purchased from the
Center of Excellence in Molecular Biology, Punjab University
Lahore, Pakistan. The mice were housed in plastic cages at
ambient temperature (25°C ± 2°C) under controlled light/dark
cycles of 12 h/12 h and fed standard mouse chow and water ad
libitum. They were acclimated to the laboratory environment for
1 week. All the principles of laboratory animal care were followed at
the Pre-Clinical Research Facility, IRCBM, CUI, Lahore, and all
animal experiments were carried out following the Guidelines for
the Care and Use of Laboratory Animals by CUI, Lahore Ethical
Committee with Ethical approval certificate number EC/
MY/007/22.
In brief, animals were randomly divided into six groups of three
mice in each NC and T-1, while four in all other four treatment
groups. Treatment groups were referred to as T1–T5, as described in
Table 1. The androgenic alopecia model was developed in C57BL/
6 mice by intraperitoneally injecting testosterone enanthate at
0.1 mL of 5 mg/mL (20 mg/kg) three times per week for 2 weeks,
as previously described in the literature, with slight modifications
(Fu et al., 2021). After 2 weeks, hair on the treated back skin (2 Å~
3 cm) of mice were depilated using a hair removing cream under
general anesthesia using ketamine at 80 mg/kg I/P and xylazine at
10 mg@/kg I/P. Approximately, 0.5 mL of each hydrogel (blank-SA
and 2dDR-SA) was applied topically on the dorsal side of mice (2 Å~
3 cm) once daily for 20 days, and the experiment was terminated on
day 21. Digital photographs were taken on days 0, 7, 14, and 21 using
a DSLR camera. Animals were sacrificed by cervical dislocation
before hair and tissue samples were collected.
The treatment groups in experimental animals are given
in Table 1.
2.6 Hair shaft and hair length analysis
On day 21, at least 70 hairs per mouse were collected from three
mice in each group, as described (Valerie et al., 2001). There are
usually four types of hair present in the scalp of C57BL6 mice, which
are described as guard, awl, auchene, and zigzag. Large guard hairs
are first formed during the embryogenesis of hair. We selected
20 guard hairs from 70 random hair samples. The guard hairs were
distinguished mainly by their length and the diameter of the hair
shaft or thickness of hair. The length of hair was measured by using a
digital vernier caliper, while the hair shaft analysis of guard type
hairs was done by taking pictures of the hair shaft portions using an
inverted microscope (Optika, Italy) at Å~40 magnification.
2.7 Histological evaluation
To evaluate skin morphology and hair regrowth, 2 Å~ 3 cm skin
samples were collected on day 21 (after depilation). Samples were
fixed in 4% paraformaldehyde for 24 h and then embedded in
paraffin blocks. Longitudinal and horizontal sections of 5.0-μmthick
skin were prepared and stained with hematoxylin and eosin
(H&E). Photographs of H&E-stained sections were taken using an
inverted microscope (Optika, Italy). The longitudinal sections were
stained using Masson’s trichrome staining to evaluate the area
covered bymelanin in the hair bulb. The sections were evaluated for
follicular length, follicular diameters, and follicular density. The
horizontal sections were evaluated for anagen follicle (A) and
telogen follicle (T), and the A/T ratio was calculated (Ying et al.,
2017) using an inverted microscope (Optika, Italy), and images
were captured. Four parameters, 1) number of hair follicles; 2) hair
bulb diameter; 3) follicular density (number of hair follicles per
mm2); and 4) number of anagen (A) and telogen (T) follicles per
millimeter area, were measured to evaluate hair growth using
ImageJ software (NIH, MD, United States). To assess the
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angiogenesis potential of 2dDR-SA hydrogels in promoting hair
growth in AGA-induced mice, blood vessels were counted in crosssections
of H&E staining.
2.8 Statistical analysis
All experiments were conducted in triplicate or more, and their
results were calculated as mean ± S.D. Statistical analysis (unpaired
Student’s t-test) was performed via GraphPad QuickCalcs (https://
www.graphpad.com/quickcalcs/ttest1.cfm). All results with a
p-value ≤ 0.05 were considered statistically significant.
3 Results
This study focused on the development of a 2dDR-releasing
sodium alginate-based tube hydrogel to promote the growth of hair
in AGA-induced mouse model. Hydrogels were synthesized by the
simple mixing of sodium alginate and propylene glycol with 2-
phenoxyethanol in water with and without 2dDR. These were
freeze-dried for FTIR and release studies of 2dDR, while for in
vivo studies, wet hydrogels were used. The results of the FTIR and
release studies of 2dDR are described in the following sections.
3.1 FTIR of blank-SA and 2dDR-SA hydrogels
FTIR analysis was used to determine the essential structural and
chemical properties of the blank-SA and 2dDR-SA hydrogels and
validate the formation of a hydrogel matrix. This method also
records structural changes caused by any effects of the drug on the
carrier hydrogel. The results of the FTIR spectra of the hydrogels shown
in Figure 3A were drawn using Origin software (OriginLab,
United States) and cross-checked against literature. The spectra
obtained for 2dDR, blank-SA, and 2dDR-SA are presented in
Figure 4. Sodium alginate exhibited absorption band characteristics
at 3,410 cm−1, which can be due to hydroxyl group (–OH) (Thaned,
2009; Jing et al., 2010); 1,635 cm−1 due to the asymmetric stretching
vibration of COO groups; 1,419 cm−1 due to the symmetric stretching
vibration of COO groups; 1,050 cm−1 due to the elongation of C–O
groups; and 1,294 cm−1 and 1,024 cm−1 due to C–O and C–O–C
stretching vibrations of alcoholic hydrogen bonding, respectively
(Rúben et al., 2011). Bands at 2,859 cm−1 and 2,970 cm−1
corresponded to C–H stretching, and the bands at 3,410 cm−1
corresponded to the hydroxyl group of propylene glycol (Munirah
et al., 2016). Characteristic intense bands associated with O–C, C–C,
and C–O stretching modes were observed at 1,078 and 1,042 cm−1. The
very intense Raman spectra at 996 and 1,006 cm−1 were the
characteristic ring band of 2-phenoxyethanol. The observed intense
bands at 750 and 688 cm−1 were assigned to the C–H ring wagging
modes of 2-phenoxyethanol (Badawi, 2011). In comparison to the peaks
of scaffolds without 2dDR, FTIR spectra show that 5% 2dDR-loaded
hydrogel has no peak shift. Thus, 2dDR has no effect on the sodiumalginate
structure (Figure 1A).
3.2 2dDR release analysis from 2dDRSA
hydrogel
Developing hydrogels with controlled release of drugs is
desirable for wound healing applications. The 2dDR drug
release was measured using Dische’s diphenylamine assay.
This assay is based on 2dDR being changed by the acidic
environment into an aldehyde molecule, which then interacts
with diphenylamine to form a blue complex that can be easily
measured using UV/Vis absorption spectroscopy. For this
purpose, the 2dDR release from room temperature-dried
2dDR-SA was performed in PBS at 37°C for different time
intervals (4, 24, 48, 72, 96, and 168 h), and the results are
shown in Figure 1B. After 4 h, the 2dDR-SA hydrogel released
70.3% (1.33 mg/2 mL) of the total drug. The release was 79.4%
(2.045 mg/2 mL) by day 1; by day 2, 84% release (2.60 mg/2 mL);
by day 3, 87% release (2.88 mg/2 mL); and by day 4, 89% release
[3.50 mg/2 (Figure1B)]. The 2dDR-SA displayed the same release
of 89% (3.50 mg/2 mL) on day 7 as it did on the 4th day, and this
was the release of essentially all of the 2dDR from the hydrogel.
This shows that 2dDR is slowly released at 37°C for up to 96 h,
thus reducing the need for repeated treatments on a daily basis.
3.3 Promoting regrowth of hair in the
testosterone-induced androgenic alopecia
mouse model
Hair growth was measured according to the method developed
by Fu et al. (2021), by determining the skin color of mice. As melanin
accumulated in the mouse skin, the color gradually changed from
pink to pinkish white, white, grayish white, gray, and finally dark
gray. This is because melanogenic activity of hair follicles (HFs) is
TABLE 1 Different treatment groups of C57BL/6 mice subjected for hair regrowth evaluation.
Sr. no. Groups Androgenic alopecia (inj. of testosterone) Treatment No. of mice
1. NC (normal control) No No 3
2. T-1 (model group) Yes No 3
3. T-2 (blank-SA) Yes Blank-SA 4
4. T-3 (2dDR-SA) Yes 2dDR-SA 4
5. T-4 (positive group) Yes Minoxidil 2% spray 4
6. T-5 (mixed group) Yes 2dDR + Minoxidil 4
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tightly correlated with the hair cycle. After 7 days of full anagen
phase, the skin retained a dark gray color before turning white once
more due to melanocyte death, which denoted the beginning of the
catagen phase. This suggests that skin color is a useful indicator of
hair regrowth following depilation. The telogenic dorsal skins of
C57BL/6 mice were treated topically with blank-SA and 2dDR-SA
hydrogel, minoxidil 2% spray, and a combination of minoxidil 2%
spray and 2dDR-hydrogel. The hydrogel was applied 5 min after
minoxidil spray in the TE-induced mice in order to assess the hair
regenerative activities in the AGA-induced androgenic alopecia
(AGA) mouse model. It is well known that the dorsal hair of
C57BL/6 mice has a time-synchronized hair growth cycle
(Figure 2A). The skin color, which is bright pink during the
telogen phase and turns gray or black during the anagen phase,
is used as an indication that hairs are growing and is recorded in a
skin color index (Figure 2C) (Van-Long et al., 2017). Until day 14,
the treatment group with TE only (T-1) had extensive patches of
skin lacking pigmentation, and the skin was pinkish white, scoring a
2 on the skin color score. By day 21, 30%–40% of shafts were formed
in skin areas of T-1, while T-2 containing only the blank-SA
hydrogel showed slightly higher skin color scores than T-1, with
a skin color score of 2 on day 14 and 4 on day 21 when applied
topically, however, the hair growing ability of T-3 (2dDR-SA
hydrogel) in the AGA mouse model was significantly higher than
that of T-1 and T-2 by day 14. The T3 group expressed a skin color
score of 5 and 60%–70% of the dorsum of AGA mice was covered
with hairs by day 21, and the dorsum of mice in T-3 was 90%
covered with hairs, and the skin color score was 6. By comparing the
T-3 group with the NC and T-4 positive control groups, no
difference in the skin color score and the appearance of hair
shafts at these time points were observed (Figure 2B).
Additionally, mice in the T-5 group that were treated by a
combination of both 2dDR-SA hydrogel and minoxidil showed
white grayish skin by day 14, and hair shafts appeared at day 21.
AGA mice in T-5 showed slightly delayed hair growth and change in
the skin color score compared to NC, T-3, and T-4, but these
parameters are still more promising than those in T-1 and T-
2 (Figure 2D).
3.4 Hair shaft and hair length analysis
Guard, awl, auchene, and zigzag hair are four different types of
hair found in mouse coats. During embryogenesis, large guard hair
develops first, followed by secondary intermediates such as awl,
auchene, and zigzag. Awl hair is long and straight; auchene hair has
one oblique bend; and zigzag hair (shortest and most frequent) is
composed of up to four segments (David et al., 2010). In this
section, we only selected guard hair for measurement of the
length and diameter of the hair shaft. Twenty guard hairs were
plucked from the dorsum of mice, where treatments had been
applied. A total of 60 guard hairs from three mice of each group
were subjected to analysis. The length of guard hairs was
measured by using a digital vernier caliper. The average length
measured in NC was 6.04 mm, while the average length from the
T-1 TE only group (2.410 mm) was lowest of all the treatment
groups, and the T-3 B-SA group showed a slight increase in hair
length to 2.63 mm. The average length of guard hair in T-3, T-4,
and T-5 as 6.20, 6.19, and 6.02 mm, respectively (Figures 3A, B).
On comparing with the normal control, no significant difference
was found between T3, T4, and T5, while there was a significant
difference between T1 and T2 with NC.
The second-most important factor to assess hair morphogenesis
is the thickness of the hairs. We took 20 guard hairs from each
mouse from the area where we had applied the treatments on the
dorsum. The hair samples were observed at Å~4 magnification to
measure the thickness of hair shafts. The diameter of hair shafts of
20 guard hairs from all three mice from all six groups was
examined. Our analysis showed the highest average hair shaft
diameter in the NC group (204 μm) followed by the T-4
(201.2 μm), T-3 (198.60 μm), and T-5 (187.5 μm) groups. The
lowest average hair shaft diameter (138.8 μm) was observed in T-
1, without any topical treatment, and the diameter of hair shaft
in the T-2 group (B-SA) was 152.4 μm. Statistical analysis
showed there was no significant difference between T4 and
T3, while there was a significant difference between NC and
T1, T2, and T5 with the normal control. This clearly shows that
2dDR-SA has affected the morphogenesis of hair by increasing
FIGURE 1
(A) FTIR spectra of blank-SA and 2dDR-SA hydrogels; (B) release and colorimetric detection of 2dDR at different time intervals (4 h and 1, 2, 3, 4, and
7 days) of drug release analysis.
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the length and thickness of mouse hair. The hair length and
thickness in group T-3 (2dDR-SA) were very well-matched to
those of the NC and T-4 positive control groups, which indicated
that 2dDR restored the normal morphogenesis of hair by
supporting the increase in hair length and thickness
(Figures 3C, D).
FIGURE 2
(A) Schematic illustration of the in vivo experiment. (B) Comparison of dorsal hair regeneration of C57BL/6 mice without any treatment (NC),
testosterone (T-1), blank-SA (T-2), 2dDR-SA (T-3), minoxidil (T-4), synergistic 2dDR, and minoxidil (T-5) (n = 04) at different time intervals (days 0, 7, 14,
and 21 of the experiment). (C) Mouse skin color score index. (D) Graphical representation of skin color scored by different treatment groups at various
time intervals (days 0, 4, 8, 12, 16, and 20 of the experiment). Results are presented as mean ± SD, n = 4. ***p ≤ 0.001, **p < 0.01, and ns p > 0.05.
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FIGURE 3
Gross analysis of hair: (A) digital photographs of hair length, (B) hair length analysis of skin sections from different treatment groups. Results are
presented as n = 60 + SD; ****p ≤ 0.0001 denotes NC versus T-1 and T-2; ns p > 0.05. (C) Photographs of hair shafts from different treatment groups. (D)
Hair shaft analysis of skin sections from different treatment groups. Results are presented as n = 60 + SD; ****p ≤ 0.0001 denotes NC versus T-1; ***p ≤
0.001 NC versus T-2; **p ≤ 0.01 denotes NC versus T-5; and ns p > 0.05 denoted NC versus T-3 and T-4.
FIGURE 4
Microscopic analysis of skin sections retrieved on day 21 of experiment after H&E staining: (A) length of hair follicles in skin sections from different
treatment groups (arrows representing the length of the hair follicles). (B) Comparison of the length of hair follicles in skin sections from different
treatment groups. Results are presented as n = 4 + SD; *p ≤ 0.1 denotes NC versus T-1 and T-2; ns p > 0.05. (C) Hair follicle density in skin sections from
different treatment groups (arrows representing the density of the hair follicles). (D) Comparison of hair follicle density in skin sections from different
treatment groups. Results are presented as n = 4 + SD; ****p ≤ 0.0001 denotes NC versus T-1; ***p ≤ 0.001 NC versus T-2 and T-4; **p ≤ 0.01 denotes NC
versus T-3 and T-5.
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3.5 Histological evaluation
After 21 days of treatment, H&E- and Masson’s trichromestained
skin slices were microscopically analyzed, and the following
factors responsible for hair growth were examined.
3.5.1 Length of hair follicles
The length of the hair follicle was measured, microscopically,
from horizontal sections stained with H&E skin sections. To
evaluate hair follicle length, the distance between the follicular
bulb residing in the dermal layer of skin and/epidermis (the
upper most layer of skin) was measured. Calculations were made
by counting the hair follicles per histological slide view under an
inverted microscope from each of three samples in each group by
keeping the scale bar at 300 μm at Å~40 magnification. After day 21,
the penetration of the maximum number of hair follicles into the
epidermis was observed in NC, T-3, T-4, and T-5. The length (μm)
of 20 hair follicles was measured by ImageJ. The average length
measured in NC, T-1, T-2, T-3, T-4, and T-5 was 221 ± 0.19 μm,
134.92 ± 0.22 μm, 154.72 ± 0.32 μm, 248 ± 0.19 μm, 248 ± 0.19 μm,
and 204 ± 0.29 μm, respectively. Statistically, we have found no
significant difference among NC, T3, T4, and T5, while there was a
significant difference among NC, T1, and T2.
The histomicrographs of these two groups indicated that the
hair follicles still resting in the dermis were miniaturized by the effect
of AGA, while the hair follicles from T-3, T-4, and T-5 are greater in
length and penetrated into the epidermis, indicating the
antagonizing effects of AGA in mice (Figures 4A, B).
3.5.2 Hair follicle density
To measure the hair follicle density, cross-sectional views of the
skin tissue of mice stained by H&E were examined under an inverted
microscope. It was observed that treatment groups T-3, T4, and T-5
had higher hair follicular densities than NC, T-1, and T-2 groups.
The follicular density in the T-3, T4, and T-5 groups was recorded as
58.1, 57.6, and 56.9 hair follicles per view, while the follicular density
in NC, T-1, and T-2 was 48.9, 24.34, and 38.2, respectively. The hair
follicle density in T-3 (2dDR-SA hydrogel-treated group) was
comparable with that of the T-4 positive control and NC group,
which showed a strong hair regenerating capacity of 2dDR-SA
against AGA (Figures 4C, D). There was a statistically significant
difference between NC, T3, T4, and T5, with a higher hair follicle
density in all these three treatment groups than in NC. The hair
follicle density was significantly reduced between NC
and T1 and T2.
3.5.3 Diameter of the hair bulb
The diameter of the hair bulbs in all groups was measured from
the cross-sections of H&E-stained skin sections of mice. For the
diameter of the hair bulbs, the same trend was observed as that of
hair follicle density. Diameters of the hair bulbs in the NC, T-1, T-2,
T-3, T-4, and T-5 groups were calculated as 17.07 ± 0.17 μm, 13.85 ±
0.34 μm, 14.16 ± 0.17 μm, 17.52 ± 0.11 μm, 16.68 ± 0.20 μm, and
16.41 ± 0.41 μm, respectively. Among all these treatment groups, T-
3 (treated with the 2dDR-SA hydrogel) exhibited the maximum
diameter of bulbs in regenerated hair, as compared to treatment with
minoxidil. Statistical analysis showed no significant difference
between NC and T3, but there was a slight difference between
NC and T4 and T5, and there was a clear significant difference
between NC and T1 and T2 (Figures 5A, B).
3.5.4 Anagen and telogen ratio (A/T ratio)
The A/T ratio indicates how long the anagen phase is persisting.
The A/T ratio of all groups was calculated by counting the number of
telogen hair follicles (T) located in the epidermis and the number of
anagen hair follicles (A) located in the dermis and subcutis. On
microscopic analysis, it was observed that hair follicles in the mouse
skin of NC, T-1, and T-2 groups were in the epidermis and shifted
earlier to the telogen phase, while T-3, T-4, and T-5 groups had
follicles in the dermis and anagen phase. A/T ratios of NC, T-1, T-2,
T-3, T-4, and T-5 groups were 1.2, 0.63, 0.87, 1.87, 1.69, and 1.83,
respectively. This showed that hair was affected by AGA in the case
of NC, T-1, and T-2, but hair regeneration was seen in T-3, T-4, and
T-5 groups.
Statistical analysis showed an upward significant difference
between NC, T3, T4, and T5 supporting hair follicles in the
anagen phase and a downward significant difference between
NC, T1, and T2. Based upon these findings, it was concluded
that the 2dDR-SA hydrogel kept hair in the anagen phase
(Figures 5C, D). The anagen and telogen hair follicles are
identified on the basis of the presence of their outer root sheath
and inner root sheath. The hair follicles in anagen show a welldefined
outer and inner root sheath, while in telogen, the inner root
sheath has a desiccated or wrinkled outer root sheath. Pictures of
anagen and telogen phases are shown in Figure 5E.
3.5.5 Area of the hair bulb covered in melanin and
evaluation of blood vessels
Area of the hair bulb covered by melanin was measured using
Masson’s trichrome stained cross-sectional slices of C57BL/6 mouse
skin from all groups using ImageJ software. The highest percentage
of area of the hair bulb covered by melanin in hairs was found in NC,
T-1, T-2, T-3, T-4, and T-5 and measured as 1,490 ± 1.29 μm2, 920 ±
0.32 μm2, 1,025 ± 0.19 μm2, 1,507 ± 0.917 μm2, 1,482 ± 1.18 μm2, and
1,489 ± 1.5 μm2, respectively. There was no statistically significant
difference between NC and T3 (2dDR-SA hydrogel-treated group),
but there was a significant difference between T1, T2, and NC. It was
observed that there was less melanin in hair bulbs of T-1 and T-2
groups, while maximum melanin was seen in T-3, where there was
hair regeneration due to the accumulation of melanin around
anagenic hair bulbs (Figures 6A, B).
The effect of 2dDR on neovascularization was demonstrated by
microscopic analysis of the mouse skin, and the number of blood
vessels was counted using ImageJ software. The highest number of
blood vessels in the dermis of the skin was found in NC (4 ± 0.191)
and T-3 (4.06 ± 0.19), in the same manner as follicular density. The
blood vessel count in T-1, T-2, T-4, and T-5 was 1.05 ± 0.216, 2 ±
0.32, 3.5 ± 0.21, and 3.5 ± 0.26, respectively. In summary,
angiogenesis was reduced in the DHT model, but significantly
increased in 2dDR and minoxidil models (Figures 6C, D).
4 Discussion
In our first study of the effects of 2dDR on wound healing in
animals (Serkan et al., 2021), we observed apparent stimulation of
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Anjum et al. 10.3389/fphar.2024.1370833
the hair follicles adjacent to the wound. We did not investigate this
further at that time, but now, we critically observed the effect of
2dDR on hair regrowth. To do this, we used a well-established
mouse model of androgen-induced hair loss. This study shows a
positive effect of 2dDR on hair regrowth in this animal model using
the alginate hydrogel as a carrier to deliver 2dDR in a
sustained manner.
Blank-SA and 2dDR-SA hydrogels were prepared by simple
manual mixing of alginate, propylene glycol, phenol ethanol, and
2dDR in water. These were characterized by FTIR, which confirmed
no evidence of chemical changes in gels on the addition of 2dDR. In
vitro drug release showed sustained release of 2dDR for 7 days.
Male pattern baldness, or androgenic alopecia, is themost common
type of hair loss worldwide (María et al., 2018). It is caused by abnormal
androgen expression and metabolic effects, with key components being
dihydrotestosterone (DHT) and 5α-reductase. The DHT-AR complex
triggers TGF-2, inhibiting cell proliferation, leading to early hair follicle
entry into the catagen phase (Francesca et al., 2017).
The treatment of AGA remains challenging, with only two FDAapproved
drugs being used, minoxidil and finasteride (Maurizio
et al., 2016). Finasteride and minoxidil stimulate hair growth, but
both have some negative effects.
Angiogenesis is known to help hair regrowth, and our
previous studies showed the ability of 2dDR to promote
angiogenesis (Serkan et al., 2019). In our current research, we
tested the effect of 2dDR on AGA in a mouse model. For the
topical delivery of 2dDR, the alginate gel was used as a carrier.
This alginate-based 2dDR-SA hydrogel showed sustained
release of 2dDR.
To develop the AGA model in C57BL/6 mice, we injected
testosterone intraperitoneally three times a week. We found that
the 2dDR-SA hydrogel-treated mice showed a similar effect of hair
regrowth as the positive control group (minoxidil) by 21 days. We
quantified the hair regrowth activity by investigating gross
morphogenesis parameters such as a change in skin color, hair
length, and hair shaft diameter. The 2dDR-SA hydrogel supported
hair regrowth.
Anagen, catagen, and telogen are the three stages of hair follicle
growth. The anagen phase is characterized by an increase in follicle
length, robust hair growth, and thickening of the hair shaft. In the
telogen and catagen phases, the length of the follicle decreases, the
hair shaft becomes thinner and wrinkled, and hair loss develops
(Sven et al., 2001). The more hair follicles in the anagen phase, the
better the hair growth. We noted that 2dDR-SA hydrogel-treated
FIGURE 5
Microscopic analysis of skin sections retrieved on day 21 of experiment after H&E staining: (A) diameter of hair follicles in skin sections from different
treatment groups (arrows representing the diameter of the hair follicles); (B) Comparison of the diameter of hair follicles in skin sections from different
treatment groups. Results are presented as n = 4 + SD; ****p ≤ 0.0001 denotes NC versus T-1; ***p ≤ 0.001 NC versus T-2; **p ≤ 0.01 denotes NC versus
T-5, *p ≤ 0.1 denotes NC versus T-5, and ns p > 0.05. (C) Hair follicle structures of terminal anagen and terminal telogen phases from different
treatment groups (black and orange arrows representing anagen and telogen phases, respectively). (D) Comparison of hair follicle structures of terminal
anagen and terminal telogen from different treatment groups. Results are presented as n = 4 + SD; ****p ≤ 0.0001 denotes NC versus T-1, T-2, T-3, T-4,
and T-5. (E) The difference between the anagen and telogen hair bulb: (E1) Hair bulb in the anagen stage: black, orange, and red arrows indicating the
outer root sheath, inner root sheath, and medulla, respectively, (E2) hair bulb in the telogen stage: black and red arrows indicating the desiccated outer
root sheath and medulla, respectively, with no inner root sheath.
Anjum et al. 10.3389/fphar.2024.1370833
mice showed a high anagen ratio compared to the negative control
group and blank-SA hydrogel group.
Our histological investigations demonstrated that 2dDR-SA
hydrogel increased hair development by elongating the anagen
phase, which was shortened in AGA. To thoroughly evaluate the
hair regeneration capacity of the 2dDR-SA hydrogel, six parameters
were selected: length of hair follicles, density of hair follicles, A/T
ratio, diameter of hair follicles, area of the hair bulb covered in
melanin, and the number of blood vessels. In the 2dDR-SA group, a
significant increase in hair follicle length and dense hair follicles
were observed, similar to the positive control group (minoxidil)-
treated group. The dermal papilla cells (DPCs) in the hair bulbs are
responsible for follicle growth. The growth phase of the hair follicle
is determined by the rate of dermal papilla cell proliferation, which
helps maintain the diameter of the hair bulb and promotes hair
growth in the anagen phase. The hair bulb is smallest in size when
the follicles are in the telogen phase, where the DPCs are dormant. In
the present study, the mice treated with the 2dDR-SA hydrogel had
hair bulb diameters like those in the normal controls and greater hair
follicle density compared to the negative control and blank-SA
hydrogel-treated groups.
The stimulation of angiogenesis around the hair bulb can control its
size. The better the blood supply to the hair bulb, the larger its diameter
and the more hair growth (Supino, 1995). We counted the blood vessels
in the horizontal sections of H&E-stained tissues using an inverted
microscope. 2dDR-SA hydrogel and minoxidil-treated groups showed
an increase in the number of blood vessels compared to the group
treated with blank-SA hydrogel. Blood vessels were slightly greater in
number in the mice treated with 2dDR-SA.
The synthesis of stem cell factor in DPCs has been known to be
inhibited by testosterone, which prevents bulbar melanocyte
pigmentation and results in pallor in hair of AGA patients. In the
present study, the mouse skin samples were stained with Masson’s
trichrome and examined micromorphologically and photographed
using an inverted microscope. 2dDR-SA hydrogel treatment increased
melanin synthesis inthe hair bulbsof theC57BL/6mousemodel. Overall,
2dDR-SA hydrogel stimulated hair growth in the AGA mouse model,
and its effect was similar to that of minoxidil, an FDA-approved drug.
FIGURE 6
Microscopic analysis in skin sections retrieved on day 21 of experiment after H&E staining: (A) morphology of the area of the hair bulb covered in
melanin in skin sections from different treatment groups (arrows representing the area covered by the hair bulb in melanin). (B) Comparison of the
morphology of the area of the hair bulb covered in melanin in skin sections from different treatment groups. Results are presented as n = 4 + SD; ****p ≤
0.0001 denotes NC versus T-1 and T-2; **p ≤ 0.01 denotes NC versus T-5, *p ≤ 0.1 denotes NC versus T-4, and ns p > 0.05 denotes NC versus T3. (C)
The number of blood vessels from different treatment groups (arrows representing no. of blood vessels per view). (D) Comparison of the number of blood
vessels from different treatment groups. Results are presented as n = 4 + SD; ***p ≤ 0.0001 denotes NC versus T-1 and T-2; **p ≤ 0.01 denotes NC versus
T-4, *p ≤ 0.1 denotes NC versus T-5, and ns p > 0.05 denotes NC versus T3.
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Anjum et al. 10.3389/fphar.2024.1370833
Finally, as the positive effects of 2dDR were possibly associated
with a stimulation of angiogenesis, it would work on other causes of
hair loss such as chemotherapy-induced alopecia (María et al.,
2018). This is responsible for low self-esteem, anxiety, depression,
poor body image, and disturbed thinking (Erol et al., 2012; Jayde
et al., 2013). A bald head and loss of eyebrows and eye lashes can
result from cancer treatment, and this can put patients under great
social distress. This is a badly under-researched area, and hence new
approaches are needed. One example of new thinking in the area is a
recent pivotal study which shows how paradoxically inhibiting
proliferation of hair bulb cells prior to treatment with
chemotherapeutic agents protects these cells from the most
damaging effect of chemotherapeutic agents (Purba et al., 2019).
5 Conclusion
The study showed the effectiveness of the 2dDR-SA hydrogel in
stimulating hair regrowth in an animal model of male androgeninduced
hair loss. Parameters such as the length of hair, hair shaft
diameter, length of hair follicles, density of hair follicles, A/T ratio,
diameter of hair follicles, area of hair bulbs covered in melanin, and
blood vessel count all confirmed effective hair regrowth after the
application of the 2dDR-SA hydrogel. This was found possibly to be
as effective as the FDA-approved drug minoxidil.
In conclusion, this manuscript demonstrates that 2dDR
stimulates hair growth in an animal model of androgenic
alopecia. As such, it is the first study to do so. Our tentative
hypothesis is that 2dDR upregulates VEGF in this animal model,
leading in turn to the stimulation of angiogenesis and stimulation of
new hair growth. However, to study the mechanism of action of
2dDR, further work will be required to investigate the levels of VEGF
in this model, following the addition of 2dDR, and to what extent the
hair follicle stimulation can be blocked by the addition of VEGF
inhibitors.
Data availability statement
The original contributions presented in the study are included in
the article/Supplementary Material; further inquiries can be directed
to the corresponding authors.
Ethics statement
The animal study was approved by COMSATS University
Islamabad, Lahore Ethical Committee, Islamabad, Lahore,
Pakistan. The study was conducted in accordance with the local
legislation and institutional requirements.
Author contributions
MA: writing–review and editing, investigation, methodology,
visualization, and writing–original draft. SZ: investigation,
methodology, visualization, writing–original draft, and
writing–review and editing. AC: project administration,
writing–original draft, and writing–review and editing. IR: project
administration, writing–original draft, and writing–review and
editing. AB: writing–original draft and writing–review and
editing. MY: conceptualization, funding acquisition, investigation,
methodology, project administration, supervision, writing–original
draft, and writing–review and editing. SM: conceptualization,
investigation, supervision, writing–original draft, and
writing–review and editing.
Funding
The authors declare that no financial support was received for
the research, authorship, and/or publication of this article.
Acknowledgments
The authors would like to thank Mubashra Zehra for helpful
discussions. A divisional patent has been filed on this innovation by
COMSATS University Islamabad and the University of Sheffield.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors, and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
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