top of page
Writer's pictureDavid Goodwin

Sheffield Mice Study | Hair Growth

Updated: Oct 4

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, Universityof 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

journal is cited, in accordance with accepted

academic practice. No use, distribution or

reproduction is permitted which does not

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)

Frontiers in Pharmacology 02 frontiersin.org

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

Frontiers in Pharmacology 03 frontiersin.org

Anjum et al. 10.3389/fphar.2024.1370833

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://

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

Frontiers in Pharmacology 04 frontiersin.org

Anjum et al. 10.3389/fphar.2024.1370833

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.

Frontiers in Pharmacology 05 frontiersin.org

Anjum et al. 10.3389/fphar.2024.1370833

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.

Frontiers in Pharmacology 06 frontiersin.org

Anjum et al. 10.3389/fphar.2024.1370833

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.

Frontiers in Pharmacology 07 frontiersin.org

Anjum et al. 10.3389/fphar.2024.1370833

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

Frontiers in Pharmacology 08 frontiersin.org

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.

Frontiers in Pharmacology 09 frontiersin.org

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.

Frontiers in Pharmacology 10 frontiersin.org

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.

References

Alfredo, R., Anzalone, A., Fortuna, M. C., Caro, G., Garelli, V., Pranteda, G., et al.

(2016). Multi-therapies in androgenetic alopecia: review and clinical experiences.

Dermatol. Ther. 29 (No. 6), 424–432. doi:10.1111/dth.12390

Alfredo, R., Cantisani, C., Melis, L., Iorio, A., Scali, E., and Calvieri, S. (2012).

Minoxidil use in dermatology, side effects and recent patents. Recent Pat. Inflamm.

Allergy Drug Discov. 6 (2), 130–136. doi:10.2174/187221312800166859

Anisa, A., Dikici, S., Waris, T. S., Bashir, M. M., Akhter, S., Chaudhry, A. A., et al.

(2020). Developing affordable and accessible pro-angiogenic wound dressings;

incorporation of 2 deoxy D-ribose (2dDR) into cotton fibres and wax-coated cotton

fibres. J. Tis. Eng. Regen. Med. 14 (No. 7), 973–988. doi:10.1002/term.3072

Badawi, H.M. (2011). Vibrational spectra and assignments of 2-phenylethanol and 2-

phenoxyethanol. Spectrochimica Acta Part A Mol. Biomol. Spectrosc. 82 (1), 63–68.

doi:10.1016/j.saa.2011.06.066

Choi, B. Y. (2018). Hair-growth potential of ginseng and its major metabolites: a

review on its molecular mechanisms. Intern. J. Mol. Sci. 19 (9), 2703. doi:10.3390/

ijms19092703

Frontiers in Pharmacology 11 frontiersin.org

Anjum et al. 10.3389/fphar.2024.1370833

David, E. S., Lindon, C., Kashiwagi, M., and Morgan, B. A. (2010). β-catenin activity in

the dermal papilla regulates morphogenesis and regeneration of hair. Dev. Cell 18 (No.

4), 633–642. doi:10.1016/j.devcel.2010.01.016

Erol, O., Can, G., and Aydıner, A. (2012). Effects of alopecia on body image and

quality of life of Turkish cancer women with or without headscarf. Support. Care Cancer

20 (10), 2349–2356. doi:10.1007/s00520-011-1338-y

Francesca, L., Pallotti, F., Rossi, A., Fortuna, M. C., Caro, G., Lenzi, A., et al. (2017).

Androgenetic alopecia: a review. Endocrine 57, 9–17. doi:10.1007/s12020-017-1280-y

Friedman, E. S., Friedman, P. M., Cohen, D. E., and Washenik, K. (2002). Allergic

contact dermatitis to topical minoxidil solution: etiology and treatment. J. Am. Acad.

Dermatol. 46 (2), 309–312. doi:10.1067/mjd.2002.119104

Fu, D., Huang, J., Li, K., Chen, Y., He, Y., Sun, Y., et al. (2021). Dihydrotestosteroneinduced

hair regrowth inhibition by activating androgen receptor in C57BL6 mice

simulates androgenetic alopecia. Biomed. Pharma. 137, 111247. doi:10.1016/j.biopha.

2021.111247

Hiroshi, S., Morikawa, S., Fujiwara, C., Terada, H., Uehara, A., and Ohno, R. (2000). A

case of acute myocardial infarction associated with topical use of minoxidil (RiUP®) for

treatment of baldness. Jpn. Heart J. 41 (4), 519–523. doi:10.1536/jhj.41.519

Hyun, H. J., Kwon, O. S., Chung, J. H., Cho, K. H., Eun, H. C., and Kim, K. H. (2004).

Effect of minoxidil on proliferation and apoptosis in dermal papilla cells of human hair

follicle. J. Dermatol. Sci. 34 (No. 2), 91–98. doi:10.1016/j.jdermsci.2004.01.002

Hyun, S. D., Cha, Y. J., Yang, K. E., Jang, S., Son, C.-G., Kim, B. H., et al. (2014).

Ginsenoside Rg3 up-regulates the expression of vascular endothelial growth factor in

human dermal papilla cells and mouse hair follicles. Phytotherap. Res. 28 (No. 7),

1088–1095. doi:10.1002/ptr.5101

Ikeda, R., Che, X. F., Ushiyama, M., Yamaguchi, T., Okumura, H., Nakajima, Y., et al.

(2006). 2-Deoxy-D-ribose inhibits hypoxia-induced apoptosis by suppressing the

phosphorylation of p38 MAPK. Biochem.Biophy. Res. Commu 342 (1), 280–285.

doi:10.1016/j.bbrc.2006.01.142

Izabela, U.-C., Kmieć, M. L., and Broniarczyk-Dyła, G. (2014). Assessment of the

usefulness of dihydrotestosterone in the diagnostics of patients with androgenetic

alopecia. Postepy Dermatol. Alergol. 31 (No. 4), 207–215. doi:10.5114/pdia.2014.40925

Jayde, V., Boughton, M., and Blomfield, P. (2013). The experience of chemotherapyinduced

alopecia for A ustralian women with ovarian cancer. Eur. J. Cancer Care 22 (4),

503–512. doi:10.1111/ecc.12056

Jennifer, P., and Mura, C. (2013). Rapid colourimetric assays to qualitatively

distinguish RNA and DNA in biomolecular samples. JoVE J. Vis. Exp. 72, 50225.

doi:10.3791/50225

Jing, H., Zhou, Z., Yin, R., Yang, D., and Nie, J. (2010). Alginate–chitosan/

hydroxyapatite polyelectrolyte complex porous scaffolds: preparation and

characterization. Intern. J. Biol. Macromol. 46 (No. 2), 199–205. doi:10.1016/j.

ijbiomac.2009.11.004

Libecco, J. F., and Bergfeld, W. F. (2004). Finasteride in the treatment of alopecia.

Expert Opin. Pharmacother. 5 (4), 933–940. doi:10.1517/14656566.5.4.933

María, C. J., Leirós, G. J., and Balañá, M. E. (2018). Androgens and androgen receptor

action in skin and hair follicles. Mol. Cell. Endo. 465, 122–133. doi:10.1016/j.mce.2017.09.009

Maryam, A., Dikici, S., Roman, S., Mehmood, A., Chaudhry, A. A., Rehman, I. U.,

et al. (2019). Addition of 2-deoxy-d-ribose to clinically used alginate dressings

stimulates angiogenesis and accelerates wound healing in diabetic rats. J. Biomater.

Appl. 34 (No. 4), 463–475. doi:10.1177/0885328219859991

Maurizio, C., Radicioni, M., Leuratti, C., Terragni, E., Iorizzo, M., and Palmieri, R.

(2016). Effects of a novel finasteride 0.25% topical solution on scalp and serum

dihydrotestosterone in healthy men with androgenetic alopecia. Intern. j.clin.

pharma. Ther. 54 (No. 1), 19–27. doi:10.5414/CP202467

Mauro, M., Barboni, B., Turriani, M., Galeati, G., Zannoni, A., Castellani, G., et al.

(2001). Follicle activation involves vascular endothelial growth factor production and

increased blood vessel extension. Bio. Repro. 65 (No. 4), 1014–1019. doi:10.1095/

biolreprod65.4.1014

Mujica, A. S., Choe, C., Meinke, M. C.,Müller, R. H.,Maksimov, G. V., W-Alberti,W.,

et al. (2016). Confocal Raman microscopy and multivariate statistical analysis for

determination of different penetration abilities of caffeine and propylene glycol applied

simultaneously in a mixture on porcine skin ex vivo. Euro. J. Pharma. Biopharma. 104,

51–58. doi:10.1016/j.ejpb.2016.04.018

Munirah, M. S., Ahmad, A., and Anuar, F. H. (2016). Characterization of poly

(L-lactide/Propylene glycol) based polyurethane films using ATR-FTIR spectroscopy.

AIP Conf. Proc. 1784 (1), 030020. doi:10.1063/1.4966758

Nolan, R. A., and Nolan,W. G. (1972). Phenoxyethanol as a fungal enzyme extractant

and preservative. Mycologia 64 (6), 1344–1349. doi:10.1080/00275514.1972.12019388

Pietro, G., Scioli, M. G., Bielli, A., Angelis, B. D., Sio, C. D., Fazio, D. D., et al. (2019).

Platelet-rich plasma and micrografts enriched with autologous human follicle

mesenchymal stem cells improve hair re-growth in androgenetic alopecia.

biomolecular pathway analysis and clinical evaluation. Biomedicines 7 (2), 27.

doi:10.3390/biomedicines7020027

Purba, T. S., Ng’andu, K., Brunken, L., Smart, E.,Mitchell, E., Hassan, N., et al. (2019).

CDK4/6 inhibition mitigates stem cell damage in a novel model for taxane-induced

alopecia. EMBO Mol. Med. 11, e11031. doi:10.15252/emmm.201911031

Rúben, P., Tojeira, A., Vaz, D. C.,Mendes, A., and Bártolo, P. (2011). Preparation and

characterization of films based on alginate and aloe vera. Intern. J. Polym. analy.

Character. 16 (7), 449–464. doi:10.1080/1023666x.2011.599923

Serkan, D., Bullock, A. J., Yar, M., Claeyssens, F., and MacNeil, S. (2020). 2-

deoxy-d-ribose (2dDR) upregulates vascular endothelial growth factor (VEGF)

and stimulates angiogenesis. Microvas. Res. 131, 104035. doi:10.1016/j.mvr.2020.

104035

Serkan, D., Mangır, N., Claeyssens, F., Yar, M., and MacNeil, S. (2019). Exploration of

2-deoxy-D-ribose and 17β-Estradiol as alternatives to exogenous VEGF to promote

angiogenesis in tissue-engineered constructs. Reg. Med. 14 (No. 3), 179–197. doi:10.

2217/rme-2018-0068

Serkan, D., Yar, M., Bullock, A. J., Shepherd, J., Roman, S., and MacNeil, S. (2021).

Developing wound dressings using 2-deoxy-d-ribose to induce angiogenesis as a

backdoor route for stimulating the production of vascular endothelial growth factor.

Inter. J. Mol. Sci. 22 (21), 11437. doi:10.3390/ijms222111437

Supino, R. (1995). “MTT assays,” in In vitro toxicity testing protocols (Springer),

137–149.

Sven, M. R., Foitzik, K., Paus, R., Handjiski, B., D Veen, C. V., Eichmüller, S., et al.

(2001). A comprehensive guide for the accurate classification of murine hair follicles in

distinct hair cycle stages. J. Investig. Dermatol. 117 (No. 1), 3–15. doi:10.1046/j.0022-

202x.2001.01377.x

Thaned, P. (2009). Alginate–magnesium aluminum silicate films: importance of

alginate block structures. Intern. J. pharma. 365 (No. 1-2), 100–108. doi:10.1016/j.

ijpharm.2008.08.025

Trommer, H., and Neubert, R. (2006). Overcoming the stratum corneum: the

modulation of skin penetration. A review. Skin. Pharma. Pphysio. 19 (2), 106–121.

doi:10.1159/000091978

Valerie, A. R., Nigel, A., Hibberts, M., Thornton, J., Merrick, A. E., Hamada, K., et al.

(2001). Do androgens influence hair growth by altering the paracrine factors secreted by

dermal papilla cells? Eur. J. Dermatol. 11 (No. 4), 315–320.

Van-Long, T., Bak, M. J., Lee, C., Jun, M., and Jeong, W.-S. (2017). Hair regenerative

mechanisms of red ginseng oil and its major components in the testosterone-induced

delay of anagen entry in C57BL/6 mice. Molecules 22 (9), 1505. doi:10.3390/

molecules22091505

Vesoulis, Z. A., Stephanie, J., Zeller, A. B., and Cole, F. S. (2014).Minoxidil-associated

anorexia in an infant with refractory hypertension. Pharmacother. J. Hum. Pharm. Drug

Ther. 34 (No. 12), e341–e344. doi:10.1002/phar.1495

Vicky, Y., Juhász, M., Chiang, A., and Mesinkovska, N. A. (2018). Alopecia and

associated toxic agents: a systematic review. Skin. Appendage Disord. 4 (No. 4), 245–260.

doi:10.1159/000485749

Victor, C., Alonso, C., Pont, M., Zanuy, M., Córdoba, M., Espinosa, S., et al. (2020).

Effect of propylene glycol on the skin penetration of drugs. Archives Dermatological Res.

312 (No. 5), 337–352. doi:10.1007/s00403-019-02017-5

Wang, W., and Wang, A. (2010). Synthesis and swelling properties of pH-sensitive

semi-IPN superabsorbent hydrogels based on sodium alginate-g-poly (sodium acrylate)

and polyvinylpyrrolidone. Carbohyd. Polym. 80 (4), 1028–1036. doi:10.1016/j.carbpol.

2010.01.020

Yar, M., Shahzadi, L.,Mehmood, A., Raheem, M. I., Román, S., Chaudhry, A. A., et al.

(2017). Deoxy-sugar releasing biodegradable hydrogels promote angiogenesis and

stimulate wound healing. Mat. tod. Commun. 13, 295–305. doi:10.1016/j.mtcomm.

2017.10.015

Ying, F., Ma, L., Li, X., Ding, W., and Chen, X. (2017). Hair growth promoting effect of

white wax and policosanol from white wax on themousemodel of testosterone-induced

hair loss. Biomed. Pharma. 89, 438–446. doi:10.1016/j.biopha.2017.02.036

Yohei, T., Aso, T., Ono, J., Hosoi, R., and Kaneko, T. (2018). Androgenetic alopecia

treatment in Asian men. J. Clin. Aesthet. Dermatol. 11 (No. 7), 32–35.

Yuichi, N., Gotanda, T., Uchimiya, H., Furukawa, T., Haraguchi, M., Ikeda, R., et al.

(2004). Inhibition of metastasis of tumor cells overexpressing thymidine phosphorylase

by 2-deoxy-L-ribose. Canc. Res. 64 (No. 5), 1794–1801. doi:10.1158/0008-5472.can-

03-2597

Frontiers in Pharmacology 12 frontiersin.org

Anjum et al. 10.3389/fphar.2024.1370833

31 views0 comments

Recent Posts

See All

Comments


bottom of page