Research Article

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Use of extracellular matrix hydrogel from

human placenta to restore hair-inductive
potential of dermal papilla cells
Xinyu Zhang1,2, ‡ , Shune Xiao2, ‡ , Bingcheng Liu2 , Yong Miao2 & Zhiqi Hu*,2
1
Department of Plastic, Cosmetic & Maxillofacial Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi ’an, ShanXi,
PR China
2
Department of Plastic Surgery, Nan Fang Hospital, Southern Medical University, Guangzhou, Guangdong, PR China
*Author for correspondence: Tel.: 0086 20 61641862; Fax: 0086 20 87743734; doctorhzqnew@163.com
‡
Authors contributed equally

Aim: To explore the feasibility of human placenta extracellular matrix (HPECM) hydrogel in restoring the
hair-inductive capacity of high-passaged (P8) dermal papilla cells (DPCs) for hair follicle regeneration.
Materials & methods: HPECM hydrogel was prepared following decellularization and enzymatic solubi-
lization treatment. DPCs isolated from human scalp were cultured in 2D and 3D environments. The hair-
inductive ability of DPCs was assessed by quantitative RT-PCR, immunofluorescence staining, immunoblot-
ting and patch assay. Results: DPCs (P8) formed spheres when cultured on the HPECM hydrogel. The ex-
pression levels of Versican, ALP, and β-catenin were restored in the DP spheres. HPECM hydrogel-cultured
DP spheres co-grafted with newborn mouse epidermal cells regenerated new hair follicle. Conclusion:
HPECM hydrogel successfully restores the hair-inductive capacity of high-passaged DPCs.

First draft submitted: 2 September 2018; Accepted for publication: 10 July 2019; Published online:
1 August 2019

Keywords: 3D culture • decellularization • dermal papilla cell • extracellular matrix • hair follicle regeneration • hair
loss • human placenta • hydrogel • thermosensitive • tissue-engineering

Hair has a cosmetic function, affecting an individual’s physical appearance, and consequently hair loss caused by
aging, disease, trauma or medication is one of the most distressing disorders for both men and women [1,2]. Current
therapeutic options for hair loss include either drugs or autologous hair follicle (HF) transplantation [3]. However,
the effectiveness of both treatments is limited because of their inability to regenerate new HFs [4–6]. With recent
progress in regenerative medicine and tissue engineering, rearranging follicle cells in their biomimetic environment
to reconstruct HFs is showing promise [7,8].
HF reconstruction involves complex interactions between epithelial cells and dermal cells [9]. Dermal papilla cells
(DPCs) are believed to play a key role in HF morphogenesis and cycling and have become an essential cell source in
HF reconstruction [10]. However, DPCs tend to lose their hair-inductive capacity during subculture when cells are
cultured in traditional 2D culture conditions [11]. Physiologically, almost all tissue cells reside in extracellular matrix
(ECM). The ECM provides specific physical and chemical cues that regulate cell behaviors such as cell survival,
proliferation, migration and differentiation [12]. Lack of a suitable microenvironment to mimic the 3D structure
and composition of the natural ECM causes DPCs to gradually lose their hair-inductive capacity in 2D culture [13].
Previous studies have shown that a synergistic effect between cells and ECM can be created by constructing a
complex 3D niche including physical–chemical stimulation and soluble signals [14]. Some matrix materials such as
hyaluronic acid [15], collagen [16] and basement membrane matrix [17] have been used to restore the hair-inductive
ability of DPCs by formation of spheroids in 3D culture. However, these methods have limitations, such as the
variable size of spheroids and difficulty of controlling the system, meaning that long-term cultivation of DPCs is
not viable. Additionally, since basement membrane matrix is a heterogeneous tumor cell-derived protein, its safety
in clinical use is unclear.
Human placentas are commonly discarded and can be harvested without harm to the donor. The matrix
components of human placenta extracellular matrix (HPECM) are collagens, laminin, fibronectin, glycoproteins

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Research Article Zhang, Xiao, Shu, Liu, Miao & Hu

and growth factors [18]. HPECM hydrogel can support the culture of cardiomyocytes and stem cells as well as blood
vessel assembly from endothelial cells [19]. In addition, several growth factors found in the placenta are involved
in promoting the wound healing process and hair growth [20,21]. Consequently, the compositional and biological
properties of HPECM hydrogel have the potential to provide a highly favorable environment for 3D culture of
DPCs. In this study, we used an HPECM hydrogel to create a suitable microenvironment for DPCs in vivo and
to explore its potential for restoring the hair-inductive capacity of high-passaged DPCs for HF regeneration. We
expected that HPECM hydrogel could provide not only structural support for DPCs but also mechanical and
chemical cues to induce hair follicle formation.

Materials & methods

All experimental procedures in this study were performed in accordance with the guidelines and regulations
established by the medical ethics committee of Southern Medical University (Guangzhou, China).

Preparation of decellularize ECM hydrogel from human placentas

HPECM hydrogel was prepared using previously described methods [19]. Briefly, human placentas were obtained
from donors following normal or cesarean deliveries at Nanfang Hospital of Southern Medical University after
ethical approval. Whole frozen human placentas stored at -80◦ C were allowed to thaw at 4◦ C. The placentas were
then washed in 1 l of 4◦ C distilled water supplemented with 1% penicillin–streptomycin (Gibco, CA, USA) for
30 min at 1200 r.p.m. on an orbital shaker. This process was repeated until excess blood was significantly reduced.
To decellularize placentas, washed tissue was shaken at 200 r.p.m. in 1 l of devitalization buffer supplemented with
2.5 g N-ethylmaleimide (Sigma-Aldrich, MO, USA), 20 g N-lauryl sarcosine (Sigma-Aldrich) and 1% penicillin–
streptomycin overnight at room temperature. Next, 10.2 g of wet tissue was digested in 250 ml (1 mg/ml) pepsin
(Sigma-Aldrich)/ 0.1 M HCl solution for 64–72 h. After approximately 60 h, the pepsin/HCl solution was
brought to pH 8.0 briefly, then to pH 7.0–7.2 using NaOH and HCl. The resulting liquid product of HPECM
digestion appeared viscous and near translucent in appearance and could be induced to form hydrogel at 37◦ C.
The concentrations of FGF and PDGF in the HPECM hydrogel were quantitatively measured with a Quantikine
enzyme-linked immunosorbent assay kit (Sigma–Aldrich) following the manufacturer’s instructions. The HPECM
hydrogel solution was stored at -80◦ C for long-term storage, or at 4◦ C for use within 2 weeks.

Isolation & culture of human DPCs

Human scalp samples were collected from patients who had undergone face-lifting surgery after obtaining informed
consent. Isolation and expansion of DPCs were performed as previously described [22]. Briefly, follicle bulbs were
transected and DP were microdissected from the bulbs, transferred to plastic dishes and cultured in DMEM (Gibco)
supplemented with 1% (v/v) penicillin–streptomycin and 20% (v/v) fetal bovine serum (Gibco) in a humidified
5% CO2 incubator at 37◦ C. Explants were cultured for 7 days, with medium changes every 3 days. Once the
outgrowth reached 80% confluence, human dermal papilla cells (hDPCs) were harvested by incubation with 0.25%
(w/v) trypsin/EDTA (Gibco) and transferred to new culture dishes with a split ratio of 1:2. Thereafter, DPCs were
maintained in DMEM supplemented with 10% fetal bovine serum. DPCs at passage 2 (P2) and passage 8 (P8)
were used in the following experiments.

Decellularization assessment
Hematoxylin–eosin (H&E) staining was used to evaluate the effectiveness of the decellularization process. Native
and decellularized human placentas were fixed in 10% formaldehyde at room temperature for 24 h then paraffin
sections were prepared and stained with H&E.

DPCs cultured on HPECM hydrogel

For 3D cell cultures, hydrogel was formed by adding 50 μl of liquid HPECM hydrogel solution to wells of a 96-well
plate and incubating at 37◦ C for 15–30 min. Following gelation, 1 × 104 DPCs/well, a cell number commonly
used for the Matrigel R
morphogenesis assay [13], were cultured on HPECM hydrogels. The cells were cultured at
◦
37 C and 5% CO2 and imaged under a reverse phase-contrast microscope (IX71 FL, Olympus, Tokyo, Japan).

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 Use of HPECM hydrogel to restore hair-inductive potential of DPCs Research Article

Cell viability assay

DPCs cultured on HPECM hydrogels were stained using a commercial live/dead viability/cytotoxicity kit (Sigma–
Aldrich) according to the manufacturer’s instructions. After 20 min incubation at 37◦ C, the stained samples were
visualized using a fluorescence microscope (Olympus CK40, Olympus) equipped with a digital camera.

Immunofluorescent staining
Immunofluorescent staining was performed according to established methods. Briefly, samples including 2D-P8-
DPCs, 2D-P2-DPCs and HPECM hydrogel-P8–DP spheres were collected, washed and fixed in 4% paraformalde-
hyde (Gibco). The following rabbit monoclonal primary antibodies were used: ALP (1:100; Abcam, Cambridge,
UK); β-catenin and Versican (1:200; Abcam). After incubation with primary antibodies overnight at 4◦ C, sam-
ples were incubated with the corresponding goat antirabbit IgG antibodies (1:200; Abcam) and DAPI (1:500;
Sigma-Aldrich) for 1 h at room temperature. Images were captured using a fluorescence microscope (IX81 FL,
Olympus).

Quantitative real-time PCR

Total RNA was extracted from 2D-P8-DPCs, 2D-P2-DPCs and HPECM hydrogel-P8–DP spheres using RNAiso
Plus reagent (TaKaRa, Dalian, China); cDNA was synthesized from 2 μg of total RNA with a SYBR PrimeScript
RT-PCR Kit (TaKaRa), and quantitative real-time PCR (qRT-PCR) was carried out using a SYBR PrimeScript
RT-PCR Kit on a Stratagene MX3005P qRT-PCR system (Agilent Technologies, CA, USA). All the foregoing steps
were performed according to the manufacturer’s protocol. The fold-change of each target gene was normalized
to GAPDH mRNA. The primers used in this study are listed below:

1. Versican: 5 -TGTCCGATTCATAGTCCTGTCC-3 and 5 -CTCACAGCGATAAGTGCCCTC-3 ;

2. β-catenin: 5 -AGGCCCAGAGCAAGAGAG-3 and 5 -GGAGAGCATAGCCCTCGTAG-3 ;
3. ALP: 5 -ATTGACCACGGGCACCAT-3 and 5 -CTCCACCGCCTCATGCA-3 ;
4. GAPDH: 5 -GCACCGTCAAGGCTGAGAAC-3 and 5 -TGGTGAAGACGCCAGTGGA-3 .

Western blotting
Total protein was extracted using RIPA lysis buffer (Gibco) according to the manufacturer’s instructions. The
extracted protein (30 μg) from each sample was subjected to sodium dodecyl sulfate/polyacrylamide gel elec-
trophoresis and transferred to a polyvinylidene difluoride membrane. Primary antibodies were: ALP (1:10,000),
β-catenin (1:5,000), versican (1:5,000) and GAPDH (1:1,000) monoclonal antibody (all from Santa Cruz Biotech-
nology, Inc., CA, USA). Immune complexes were detected using an enhanced chemiluminescence kit (Santa Cruz
Biotechnology, Inc.) and further quantified by the analyst/PC densitometry software (Bio-Rad Laboratories, CA,
USA).

In vivo HF induction ability of DP microtissues

To determine the ability of DP microtissues to induce new HFs, the patch assay was performed as previously
described [23]. Briefly, cultured cells were divided into four groups as follows: dermal cells (1 × 106 cells) mixed
with epidermal cells (1 × 106 cells) as the positive control group; epidermal cells (1 × 106 cells) alone as the
negative control group; 2D-P8-DPCs (1 × 106 cells) mixed with epidermal cells (1 × 106 cells) as experimental
group 1; HPECM-P8-DPC spheroids (60 spheroids) mixed with epidermal cells (1 × 106 cells) as experimental
group 2. HPECM-P8-DP spheroids were recovered from HPECM hydrogel by incubating at 4◦ C for 1 h then
collecting by centrifugation. Fresh dermal cells and epidermal cells were isolated from newborn C57BL/6 mice
as previously described [24]. The cells of each group were injected subcutaneously into the dorsal side of 7- to
9-week-old nude mice (n = 10 mice per group). Two weeks after injection, all animals were sacrificed and HF
induction was evaluated by gross observation and histological examination. The number of HF of each group
was counted under the microscope and statistical analysis was performed. All experiments were approved by the
Institutional Animal Care and Use Committee and were carried out three times.

Statistical analysis
Data are expressed as mean ± standard deviation. One-way analysis of variance was performed for comparison
between different groups using SPSS17.0 software (IBM, NY, USA). Differences in p-values of less than 0.05 were
regarded as statistically significant.

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Research Article Zhang, Xiao, Shu, Liu, Miao & Hu

Figure 1. Preparation and characterization of the

human placenta extracellular matrix hydrogel. (A) Native
human placental tissue. (B) Hematoxylin and eosin
staining of microscopic sections before decellularization.
(C) Human placental tissue after decellularization
treatment. (D) Hematoxylin and eosin staining of
microscopic sections after decellularization treatment.
(E) Sol–gel transition behavior of the human placenta
extracellular matrix hydrogel. Scale bars = 100 μm.

Results
Characterization of HPECM hydrogel
Human placentas were washed several times to remove blood components (Figure 1A). Numerous cell nuclei were
visible in the tissue before decellularization (Figure 1B). After decellularization treatment (Figure 1C), few nuclei
were visible (Figure 1D). The absence of nuclei demonstrated the complete decellularization of the tissue compared
with native human placenta. Pepsin-solubilized HPECM remained as a viscous liquid at room temperature, but
gelation occurred in <30 min when incubated at 37◦ C (Figure 1E). These thermosensitive HPECM hydrogels
displayed sol–gel properties similar to those of commercial purified collagen gels. The mean concentrations of
PDGF and FGF in supernatants of HPECM hydrogels were 0.39 ± 0.06 and 1.27 ± 0.08 ng/ml, respectively.

DP spheroid formation & cell viability

As expected, DPCs proliferated in an adherent fashion with spindle-like morphology in conventional 2D culture
(Figure 2A), while DPCs cultured on HPECM hydrogels showed spherical morphology during the culture period
(Figure 2B). On day 0 (12 h after seeding), day 1 and day 3, cell viability was determined using live/dead staining.
The majority of cells exhibited green fluorescence showing the high viability of DPCs (Figure 2C).

Hair induction-related gene & protein expression

Compared with 2D-P2-DPCs which acted as positive control, qRT-PCR showed that mRNA expression of genes
associated with hair-inducing ability – ALP, β-catenin and Versican – was lost in 2D-P8-DPCs, whereas expression
of these characteristic markers was restored when cells formed spheres on HPECM hydrogels (Figure 3A). The
protein expression of ALP, β-catenin and Versican was verified by immunofluorescence staining (Figure 3B), and
results were similar those of qRT-PCR, confirming that expression of these three proteins was higher in HPECM
hydrogel-P8-DP spheres compared with 2D-P8-DPCs. Further quantitation by immunoblotting showed that the
HPECM hydrogel-P8-DP spheres expressed all these signature markers, whereas in 2D-P8-DPCs they were barely
expressed (Figure 3C).

HF induction ability of DP microtissues

Two weeks after implantation, large numbers of newly formed HFs were observed within the hypodermis of nude
mice in the positive control group (Figure 4A). However, nothing could be seen in the negative control group

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 Use of HPECM hydrogel to restore hair-inductive potential of DPCs Research Article

12 h 24 h 72 h

Figure 2. Morphological observation and cell viability of dermal papilla cells cultured on conventional 2D culture
plates and on human placenta extracellular matrix hydrogels. (A) Dermal papilla cells proliferate in an adherent
fashion with spindle-like morphology on conventional 2D plates. (B) Dermal papilla cells showed spherical
morphology on human placenta extracellular matrix hydrogels. (C) Live/dead staining showed that the cells were
alive when cultured on human placenta extracellular matrix hydrogel. Scale bars = 200 μm.

(Figure 4B) or the 2D-P8-DPCs group (Figure 4C). A few de novo HFs and pigmented hair shafts were visible in
the HPECM hydrogel-P8-DP sphere group (Figure 4D); H&E staining images further revealed the structure of
HFs induced by HPECM hydrogel-P8-DP spheres and epidermal cells (Figure 4E). Quantification of the number
of HF per graft site of the different groups is shown in Figure 4F.

Discussion
Although HF regeneration through tissue engineering has shown promise for the treatment of hair loss, many
unresolved questions remain and technical limitations need to be overcome before widespread clinical application
can be expected [24,25]. The HF is composed of epidermal (epithelial) and dermal (mesenchymal) compartments
and the interaction between these two parts is essential for HF regeneration [26]. The epidermal components include
HF stem cells, hair germinal matrix cells and the inner and outer root sheath, while the dermal components mainly
contain DP (DPC) and connective tissue sheath [27]. In essence, DPCs are a cluster of specialized mesenchymal
cells surrounded by epithelial cells at the base of HFs, which persist as the DP [11]. When HF formation first
occurs during embryogenesis, the epithelial part requires the guidance of DPCs to organize into the complex hair
structure [10,28]. In other words, the DPC is capable of induction of new HFs, the regulation of morphogenesis
and the control of the hair growth cycle. Because of their inherent inductive capacity, DPCs are considered to be
the optimal seeding cells source for HF reconstruction [29]. However, while cultured DPCs at early passages retain
their intrinsic property of inducing HF formation, those at high passages inevitably lose their inductive capacity,
leading to failure of HF formation [10,30].
Studies suggested that the hair-inducing ability of DPCs is closely related to their aggregative growth pattern.
There are several 3D culture systems which aim to maintain the DPCs’ inductive capacity through DP spheroid
formation [31]. The existing 3D culture systems include low binding plates or plates with synthetic membranes [32,33],
hanging drop methodology [34], the layer-by-layer (LbL) nanoencapsulation technique [35] and artificial scaffolds
such as collagen, gelatin-sponge or Matrigel R
[17]. Due to the low cell adhesion, ultra-low attachment substrates can
facilitate self-assembly of DPCs into 3D spheroids. However, the size of DP spheroids formed by low-binding plates
is often too large, which may easily lead to cell necrosis. The hanging drop is a simple method for forming spheroids
through surface tension of the droplet and the microgravity environment in each drop, but external forces such

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Research Article Zhang, Xiao, Shu, Liu, Miao & Hu

2D – P2 HPECM – P8 2D – P8
2D – P2
HPECM – P8
2D – P8
****
Versican
1.5 NS ****
**** ****
Relative expression

NS **** NS ****

1.0
β-catenin

0.5

0.0 ALP
Versican β-catenin ALP

****
0.6 2D – P2

Ratios of proteins to GAPDH

NS
Versican **** HPECM – P8
2D – P8
0.4 **** ****
β-catenin NS **** NS ****

ALP
0.2

GAPDH
0.0
Versican β-catenin ALP
2D – P2

HPECM – P8

2D – P8

Figure 3. Signature gene and protein expression of dermal papilla cells cultured in 2D conditions and in human
placenta extracellular matrix hydrogel. (A) Gene expression of dermal papilla cells (DPCs) related to hair-inductive
capacity – ALP, β-catenin and Versican – was well maintained in the human placenta extracellular matrix (HPECM)
hydrogel-P8 group. (B) Similar to the qRT-PCR result, HPECM hydrogel-P8 DP spheres exhibited greater protein
expression compared with 2D-P8-DPCs. (C) The result of immunoblotting was in accordance with the results of
qRT-PCR and immunofluorescence. Expression levels of each marker were normalized to the internal control of
GAPDH.
****p < 0.05 compared with the 2D-P8-DPCs group. n = 3.
2D – P2: Passage 2 DPCs cultured on tissue culture plate; 2D – P8: Passage 8 DPCs cultured on tissue culture plate;
HPECM hydrogel-P8: Passage 8 DP spheres formed on HPECM hydrogel.

as collision or shaking will make these liquid drops more vulnerable to fall or spread out. LbL nanoencapsulation
is performed by employing gelatin and alginate for ultrathin nanocoated matrices on a single DPC, then DPCs
tend to form DP spheroids through electrostatic interactions between them, but the gelatin and alginate annex
additives to DPCs, which may affect the DPC’s functionality through some unknown cytokines. Matrigel R
, the
most commonly used scaffold, consists of proteins and ECM molecules extracted from basement membrane of a
mouse sarcoma, and thus cannot accurately represent the native ECM microenvironment in somatic tissues, while
the risk of tumor formation should also be taken into consideration [36]. More importantly, besides their respective
problems and limits, none of the above-mentioned methods truly represent the DPC niche in vivo [37,38].
A suitable microenvironment incorporating various physiological and chemical functions plays an indispensable
role in functional cell activity. It has been recognized for decades that the ECM is critical for cell proliferation,

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 Use of HPECM hydrogel to restore hair-inductive potential of DPCs Research Article

Mouse epidermal cells Mouse epidermal cells Mouse epidermal cells Mouse epidermal cells
+ dermal cells + 2D-P8 DPCs + HPECM-P8 DP microtissues

150
follicles per graft
Number of hair

**
100

50

0
Positive
control

Negative
control

Experimental
group 1

Experimental
group 2

Figure 4. In vivo hair follicle regeneration assay. The subcutaneously implanted grafts were observed 2 weeks later.
(A) Positive control: neonatal mouse epidermal cells and dermal cells were co-transplanted. Many hairs were observed
in the recipient site of nude mice. (B) Negative control: neonatal mouse epidermal cells were transplanted alone, and
no hair induction was observed. (C) Experimental group 1: passage 8 DPCs grown on tissue culture plates were
co-transplanted with neonatal mouse epidermal cells, and no hairs were evident; (D) experimental group 2: passage 8
DP spheres cultured on HPECM hydrogel surface were co-transplanted with neonatal mouse epidermal cells.
Formation of several hairs could be seen at the recipient site. (E) H&E staining images showing HFs induced from
HPECM hydrogel-P8 DP spheres (scale bars = 200 μm). (F) Number of hair follicles formed per graft.
**p < 0.05 compared with the experimental group 1.
DPC: Dermal papilla cell; H&E: Hematoxylin and eosin; HF: Hair follicle; HPECM: Human placenta extracellular matrix.

morphogenesis and differentiation [12,39]. The ECM interacts with cells through its structural features and other
cues such as its bound growth factors and intracellular signaling, each of which result in cell fate decisions [40].
Thus, for the purpose of HF reconstruction, it is crucial to maintain the hair-inducing property of DPCs by
providing them with an instructive microenvironment comparable to that in vivo. Recent efforts have focused on
decellularization techniques to obtain DECM for use as a biologic scaffold which is similar to the original ECM
environment [41,42]. DECM scaffolds can be harvested from almost every tissue and organ. Several studies have
evaluated the effect of decellularized human placenta in therapeutic applications [43].
According to Choi et al., decellularized placental ECM sheets can be designed as a dermal substitute to support
the regeneration of full-thickness wounds with newly-formed HFs and microvessels [20]. Francis et al. reported that
human placenta-derived hydrogel reduced scarring in a rat model of cardiac ischemia and enhanced cardiomyocyte
and stem cell cultures [19]. Other studies have used human placenta extract as a cell culture supplement to promote
the hair-inductive capacity of DPCs by increasing β-catenin and Wnt3a expression levels [44,45]. Thus, we believe
HPECM hydrogel harvested from human placenta through decellularization techniques is suitable for HF tissue
engineering.
The decellularization methods implemented in this study effectively removed the cellular components, while
retaining bioactive molecules within the HPECM hydrogel after decellularization. Among these preserved bioactive
factors, FGF and PDGF are known to play crucial roles in promoting the hair-inducing ability of DPCs [46,47].
Pepsin-digested HPECM solution can self-assemble into hydrogel when brought to physiological conditions.
Formation of the HPECM hydrogel is a temperature- and/or pH-controlled collagen-based self-assembly process
without additional crosslinking agent that is initiated by the spontaneous reformation of the ECM protein monomer.

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Research Article Zhang, Xiao, Shu, Liu, Miao & Hu

In an in vitro study, we demonstrated that the HPECM hydrogel can promote sphere formation of DPCs and
maintain their viability. In order to test the effect of HPECM hydrogels on the expression of hair-inducing-related
genes and proteins in DPCs, ALP [48], β-catenin [49] and Versican [50] were chosen as molecular markers, as they
are closely related to the hair-inducing ability of DPCs [32]. The qRT-PCR, immunostaining, and immunoblotting
results all verified that spheres formed on HPECM hydrogels did maintain expression of these marker genes and
proteins, which is in accordance with previous studies showing that sphere formation can restore the expression of
high-passage DPC markers [30]. In our in vivo study, we showed that mature follicles could be induced by DPCs
cultured on HPECM hydrogels and freshly isolated neonatal epidermal cells. Taken together, our results supported
the hypothesis that the microenvironment provided by HPECM hydrogel can restore the hair-inducing properties
of high-passage DPCs. The sphere formation and the presence of growth factors might account for this effect.
In previous studies, various forms of DECM derived from different tissues and organs have been used as biological
scaffolds for the engineering of functional tissues and organs including cartilage [51], bone [52], bladder [53], heart [54]
and liver [55]. In our study, we chose human placenta as the source. Compared with other source materials, this
HPECM hydrogel enjoys considerable advantages including: human placenta is an abundantly available donated
human organ, which is easy to obtain. The gelation process of HPECM hydrogel is simple, solubilized HPECM
remains liquid at room temperature and shows no gelation activity until the temperature of the material reaches
37◦ C, and no special tools or equipment are required. The HPECM hydrogel is suitable for long-term culture.
The HPECM hydrogel is rich in stemness factors, which can promote the maintenance of the inductive ability of
DPCs.
Another advantage of the HPECM hydrogel is its clinical applicability. Solubilized HPECM remains liquid at
room temperature and shows no gelation activity until the temperature reaches 37◦ C. Consequently, the gelation
property of the HPECM hydrogel greatly facilitates its practicality, especially for injectable delivery via minimally
invasive methods. Further investigations are needed to explore the potential of HPECM hydrogel via catheter
injection to provide a suitable microenvironment for HF regeneration, which may serve as the foundation for the
convenience of clinical application.
However, there is one limitation of our study, in that we did not assess the efficiency of HPECM hydrogel
in maintaining DPCs’ HF inductive capability compared with other 3D culture methods. Together, our research
team has reported on several methods, such as Matrigel R
culture [17], the hanging drop method [34] and the LbL
nanoencapsulation technique [35], mentioned above. We are dedicated to an in-depth comparative investigation
between different current methods to assess which method is more suitable for HF regeneration in future studies.

Conclusion
In summary, we used HPECM hydrogel as a bioactive scaffold which successfully restored the hair-inducing
properties of DPCs both in vivo and in vitro. Experimental results indicated that HPECM hydrogel is likely
to provide a favorable biomimetic microenvironment to regulate the morphology and function of DPCs by
reactivating key genes whose expression was lost under 2D conditions. We will conduct further research into the
exact mechanism of interactions between the environment and gene expression.

Future perspective
Recently, use of DECM scaffolds in tissue engineering and regenerative medicine has gained much interest. It
is important that such a scaffold should provide both mechanical support and abundant nutrients for the cells.
Human placenta contains plenty of ECM components and endogenous growth factors which are well preserved
after decellularization treatment. Thus, HPECM hydrogel derived from human placenta is an original, complex,
human, xeno-free, tumor-material-free ECM hydrogel, which mimics more characteristics of the extracellular
microenvironment in vivo than other 3D-culture systems. We demonstrated that HPECM hydrogel successfully
restored hair inductive capability of high-passaged DPCs, and thus could become a new option for HF tissue
engineering. In addition, considering its gelation property and lower immunogenicity, more research should be
done to explore the feasibility of using HPECM hydrogel for injectable delivery via minimally invasive methods,
which may be clinically helpful in future.

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 Use of HPECM hydrogel to restore hair-inductive potential of DPCs Research Article

Summary points
• Decellularized extracellular matrix hydrogels from human placenta (human placenta extracellular matrix
[HPECM]) has the following advantages: easy to obtain; the gelation process is simple, allowing for long-term
cultivation, and it is rich in stemness factors.
• HPECM hydrogel establishes an instructive 3D environment, including mechanical stimulation and bioactive
molecules, for dermal papilla cells (DPCs) cultured on it.
• We explored the potential of HPECM hydrogel in restoring the hair-inductive capacity of high-passaged DPCs for
hair follicle regeneration both in vivo and in vitro.
• DPCs formed spheres when cultured on the HPECM hydrogel. The gene and protein expressions of Versican, ALP
and β-catenin, which are closely related to the hair-inducing capacity of DPCs, were restored in the HPECM
hydrogel-cultured DPCs compared with the 2D-cultured DPCs.
• High-passaged DPCs (P8) cultured on HPECM hydrogels successfully regenerated new hair follicle in nude mice,
whereas the DPCs cultured in 2D conditions did not.
• HPECM is likely to provide a favorable biomimetic microenvironment to regulate the morphology and function of
DPCs by reactivating key genes whose expression was lost under 2D conditions.

Financial & competing interests disclosure

This study was supported by the Natural Science Foundation of China (grant numbers: 81471900 and 81701929). The authors
have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial
conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

Human placentas were obtained from donors following normal or cesarean deliveries at Nanfang Hospital of Southern Medical
University after ethical approval. The authors state that they have obtained appropriate institutional review board approval or have
followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for
investigations involving human subjects, informed consent has been obtained from the participants involved.

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