Regenerative Therapy 3 (2016) 108e113

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Regenerative Therapy
journal homepage: http://www.elsevier.com/locate/reth

Original article

Functional peptide KP24 enhances submandibular gland tissue
growth in vitro
Atsushi Ikeda a, b, Hiroaki Taketa a, Gulsan Ara Sathi a, Yoshiaki Hirano c, Seiji Iida b,
Takuya Matsumoto a, *abcDepartment of Biomaterials, Okayama University, 2-5-1 Shikata-Cho, Okayama, 700-8558, Japan
Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University, 2-5-1 Shikata-Cho, Okayama, 700-8558, Japan
Department of Chemical Engineering, Kansai University, 3-3-1 Yamate, Suita 564-8680, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 28 November 2015
Received in revised form
16 February 2016
Accepted 18 February 2016

Introduction: Salivary gland hypofunction, also known as xerostomia, occurs as a result of radiotherapy
for head and neck cancer, autoimmune diseases, or aging. Xerostomia leads to oral health problems andthus affects the quality of life. Biological salivary gland tissue generated in vitro would provide analternative mode of treatment for this disease.
Methods: To develop a novel method for modulating salivary gland tissue growth in vitro, we prepared a
KP24 peptide-immobilized hydrogel sheet, wherein the peptide comprised repeating proline and lysine
sequences, and evaluated the effect of this peptide on salivary gland tissue growth.
Results: We found that the KP24 peptide has the potential to enhance glandular tissue growth in vitro.
This enhancement is associated with neurite outgrowth and increasing neural innervation.
Conclusion: KP24 peptide modiﬁed material would be a promising material for the modulation of salivary gland tissue growth in vitro.
© 2016, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords:
Submandibular gland
Tissue manipulation
Functional peptide
Hydrogel

1. Introduction
Saliva has multiple functions in the mouth, including an antibacterial effect, mucous protection, pH buffering, mastication offood, and re-calciﬁcation of teeth. Previous research has indicatedthat the number of patients with xerostomia increased in recent
years [1]. Salivary hyposecretion is caused by aging, autoimmune
diseases, and salivary gland damage due to malignant tumors andradiotherapy [2,3]. Since xerostomia can cause the induction andprogression of dental caries, periodontitis, oral candidiasis, anddysgeusia and also cause a burning sensation in the mouth, it leads
to a decline in quality of life (QOL). For example, xerostomia patients may have severe problems performing everyday tasks suchas eating or holding a conversation [1]. Palliative care, such as
gargling, parasympathomimetic drugs, and artiﬁcial saliva sabstitutes, are currently used for xerostomia treatment [4,5].

* Corresponding author. Tel.: þ81 86 235 6665; fax: þ81 86 235 6669.
E-mail address: tmatsu@md.okayama-u.ac.jp (T. Matsumoto).
Peer review under responsibility of the Japanese Society for Regenerative
Medicine.

However, these approaches provide relief for very short periods oftime. Therefore, a radical alternative treatment such as the development of tissue regenerative therapy is required.
Recently, the fabrication of salivary gland tissue in vitro has been
paid attention as an alternative approach for tissue regeneration.
Wei et al. demonstrated that submandibular gland (SMG) epithelial
cells can self-organize into a tissue in a synthesized environment[6]. Furthermore, Ogawa et al. have established an in vitro method
for the generation of SMG-like tissue, making use of the condensation of mesenchymal and epithelial cells in type I collagen gel [7].
These studies demonstrated that SMG tissue generation could
occur in vitro; however, the tissue generated in this manner still
exhibited several problems concerning the size and morphology
necessary for the transplantation. Hence, developing a method formodulating tissue growth in vitro is required. Previously, we haveaimed to develop an original substrate that can be used formodulating SMG tissue growth in vitro, and prepared hydrogels
with different mechanical stiffness [8]. Also, we have prepared a
substrate modiﬁed by the Arginine-Glycine-Aspartic-acid (RGD)
peptide, which is a ﬁbronectin motif. In both case, the developedmaterials were valuable to modulate SMG tissue growth in vitro [9].

http://dx.doi.org/10.1016/j.reth.2016.02.006
2352-3204/© 2016, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

A. Ikeda et al. / Regenerative Therapy 3 (2016) 108e113

Recently, a peptide called KP24, composed of repeating proline andlysine sequences, was shown to be a trigger for cell aggregate formation in mesenchymal cell culture [10]. This repeating motif wasimportant for cell aggregation not for cell adhesion caused by lysine
repeating. Therefore, to investigate the effect of KP24 modiﬁed
substrate on SMG tissue growth in vitro, we attempted to culture
SMG tissue on KP24 immobilized alginate hydrogel sheets in thisstudy.
2. Materials and methods
2.1. Preparation of alginate hydrogel sheet
The preparation of alginate hydrogel sheets has been described
elsewhere [8]. Brieﬂy, sodium alginate solution (4 wt.%, Wako Pure
Chemical, Japan) was poured into a porous alumina mold. The moldwas soaked in calcium chloride solution (5wt.%) for 1.5 h to obtain
the alginate hydrogel sheet, which was then washed with ethanol
and Milli-Q ultrapure water (Millipore, MA, USA), and stored in
Dulbecco's modiﬁed Eagle's medium/Nutrient Mixture F-12
(DMEM-F12) (Wako Pure Chemical) supplemented with 1% penicillin/streptomycin(PS) (Nacalai Tesque, Japan), for at least 24 h (gelsize: 10  10  1.5 mm). We had previously found that stiffer
alginate hydrogel (Young's modulus of 184 kPa) attenuates SMG
growth in vitro. Therefore, in the current study, we used this stifferhydrogel as a control, in order to evaluate the effect of the peptideimmobilized substrate on SMG tissue growth.
2.2. Fabrication and analysis of KP24-modiﬁed alginate gel
H-(Lys-Pro)12-OH is a linear peptide synthesized using the
Fmoc peptide synthesis method. Carbodiimide chemistry was used
for the fabrication of KP24-conjugated alginate solution. Brieﬂy, 1ethyl-3-(-3-dimethylaminopropyl)-carbodiimidehydrochloride(3  103 mol/l, EDC, Watanabe Chemical, Japan) was dissolved incooled ultrapure water, and agitated for 10 min. N-hydroxysulfosuccinimide (3  103 mol/l, Sulfo-NHS, Thermo Fisher Scientiﬁc, IL, USA) and KP24 peptide (7.5  104 mol/l) were added tothe solution and agitated overnight at room temperature.
The solution was puriﬁed for 4 days by membrane dialysis(Biotech CE, Spectrum, CA, USA), and lyophilized before storing. To
conﬁrm the immobilization of KP24 peptide, the powder obtained
after lyophilization was dissolved in citrate buffer solution (pH 2.2),
and amino acid analysis was performed.

109USA). Terminal end bud numbers were obtained from ﬁve SMG
tissue samples per group. Also, the SMG tissue size was measured
by using Image J software at 72 h. Each experiment was repeated atleast three times.
2.5. Immunoﬂuorescence staining
The cultured SMG tissues were ﬁxed with 4% paraformaldehyde(PFA) and incubated with ﬂuorescein isothiocyanate (FITC)-conjugated PNA (1:200; SigmaeAldrich, MO, USA). The ﬁxed tissues weretreated with anti-b III-tubulin (1:1000; R&D Systems, MN, USA),
Antibody binding was detected with Alexa Fluor-conjugated secondary antibodies (1:200; Life Technologies, NY, USA), and thesamples were imaged by confocal microscopy (C1; Nikon).
2.6. MC3T3-E1 cell and mesenchymal stem cell culture on KP24modiﬁed gel sheets
Osteoblast-like MC3T3-E1 cells (Riken BRC, Japan) and mousemesenchymal stem cells (MSCs) were used for cell culture study.
The SMG development is greatly regulated by mesenchymal cells.
Since both 3T3E1 cells and mesenchymal stem cells were derived
from mesenchymal tissue, we decided to use these cells in thisstudy. MC3T3-E1 cells that had undergone 5e6 passages were
harvested and used in this study. These cells were cultured at 37  C
in a humidiﬁed 5% CO2 atmosphere for 7 days, in the a-minimal
essential medium (a-MEM) supplemented with 10% fetal bovine
serum (FBS, Life Technologies) and 1% PS. MC3T3-E1 cells wereseeded on KP24 modiﬁed alginate gels or non-modiﬁed alginategels at initial densities of 5000 cells/cm2. Mesenchymal stem cells(MSCs), isolated from the femurs of BALB/c mice (6e10 weeks ofage, Shimizu Laboratories, Japan), were cultured as well. Cell
adhesion and proliferation on the sheet were observed under amicroscope (TE2000, Nikon, Japan).
2.7. PC12 cell culture on KP24-modiﬁed gel sheets
PC12 cells were used as a model for nerve cells (ATCC, VA, USA).
PC12 cells were seeded at a density of 5000 cells/cm2 and cultured
on KP24-modiﬁed and non-modiﬁed alginate gels with High
Glucose-DMEM (Wako Pure Chemical) supplemented with 10%
horse serum (Life Technologies), 5% FBS, 1% PS, and nerve growth
factor (NGF, 50 ng/ml, Millipore) at 37  C in a humidiﬁed 5% CO2
atmosphere for 7 days. Cell number was measured under a2.3. Animal experiments
ICR mice were purchased from Charles River Laboratories (MA,USA). All animal procedures strictly adhered to the Guidelines for
Animal Experiments at Okayama University. All experimental
protocols were approved by Okayama University Animal Care and
Use Committee (OKU-2013033 and OKU-2013341).
2.4. SMG tissue culture on KP24-modiﬁed gel sheets
SMG tissue was extracted at ICR mouse embryonic day (E) 12.5,
and was placed directly on non-modiﬁed alginate hydrogel sheetswith a mechanical stiffness of 184 kPa, on the KP24-immobilized
alginate hydrogel sheets with a mechanical stiffness of 184 kPa,
or on tissue culture dishes. The SMG culture was maintained at
37  C in a humidiﬁed atmosphere with 5% CO2 for 3 days, in DMEMF12/PS medium. Growth and morphological changes of the SMG
tissue in culture were observed by using a microscope (TE2000,
Nikon, Japan). The number of buds in the developing SMG tissue
was counted at 0, 24, 48, or 72 h using Image J software (NIH, MD,

Fig. 1. Different concentrations of peptide introduced into sodium alginate. The
amounts introduced into the gel increased in proportion to the initial concentration of
peptide in the reactive solution.

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A. Ikeda et al. / Regenerative Therapy 3 (2016) 108e113

Fig. 2. a,b) SMG on 184 kPa hydrogel sheets cultured in medium containing various KP24 concentrations (0e100 ml/l, Bar ¼ 100 mm, *:p < 0.05).

microscope (TE2000, Nikon, Japan). We also measured the length of
the neurites growing out from the cell body. The length wasmeasured by using Image J software from obtained images. Fiveneurites from single cells were found to be the average neurite
length in this study.

Firstly, KP24 was solely dissolved in media and added to theisolated SMG tissue cultured on the stiff hydrogels. This promoted
tissue culture growth suggested that KP24 probably has a speciﬁc
inﬂuence on SMG tissue growth (Fig. 2).

3.2. Effect of KP24 immobilized hydrogel on SMG tissue growth
3. Results
3.1. Effect of KP24 on SMG tissue culture
Amino acid analysis results showed that, depending on theconcentration of the peptide added, the amount of immobilized
peptide in the sodium alginate gels increased (Fig. 1).

We then cultured SMG explants on KP24 peptide-immobilizedhydrogel (184 kPa) for 72 h. In a previous study, we had observedthat stiff hydrogel attenuated SMG growth. However, in the current
study, we found that SMG tissues cultured on KP24 immobilizedhydrogel exhibited enhanced growth even when the stiff hydrogel
was used. The number of buds in the control group did not increase

Fig. 3. aec) SMG cultured on hydrogel sheets modiﬁed by introducing various concentrations of KP24 (0e5.0 mol/l, Bar ¼ 100 mm). The SMG growth changed in accordance with
the introduced amount of KP24 (*: p < 0.05).

A. Ikeda et al. / Regenerative Therapy 3 (2016) 108e113

111

Fig. 4. Neural growth in SMG tissue cultured for 72 h on hydrogel sheets with or without KP24 modiﬁcation (red: anti-bIII tubulin, green: PNA, bar ¼ 100 mm).

with increase in the duration of SMG tissue culture. However, in the
SMG tissue samples cultured on the peptide-immobilized hydrogels,the bud numbers increased on an average from 3 to 30 during 72 h ofculture (Fig. 3). Moreover, the epithelial area of SMG tissue was greatly
enhanced when it's cultured on KP24 immobilized surface (Fig. 3).
Immunoﬂuorescence staining of the cultured tissue showedthat the neurons were well distributed among the proliferating
epithelial tissue in the experimental SMG tissue cultures. In
contrast, in the control samples, attenuated neuronal growth
without cell proliferation was observed (Fig. 4).

was used, decrease in cell adhesion was observed, which suggested
that extent of cell adhesion depended on the cell type (Fig. 5). On
the other hand, when we cultured PC12 cells on the non-modiﬁed
or modiﬁed hydrogels, we found that cell adhesion maintained on
the peptide-modiﬁed substrate. Moreover, the neuronal outgrowth
was signiﬁcantly higher in PC12 cells cultured on the peptidemodiﬁed substrate, which suggested that this peptide was able toinﬂuence neuronal cell behavior (Fig. 6).

3.3. Isolated cell activity on peptide immobilized substrate

In vitro generation of biological tissues has been paid attention
in these days [7,11e17]. A speciﬁc morphological change called
branching morphogenesis is observed in the SMG tissue [18,19].
Since such branching morphogenesis can also be observed in otherglandular-like tissues, including the lung, kidney, pancreas, andmammary gland tissues [20,21], methods developed for SMG tissue

4. Discussion

In order to understand the mechanism underlying these observations, MC3T3-E1 cells and isolated primary MSCs were
cultured on the hydrogels with or without peptide modiﬁcation.
The results indicated that even when peptide-modiﬁed hydrogel
Controla)

KP24(0.5 mol/l)b)Control

KP24(2.5 mol/l)

KP24(1.0
mol/l)c)

KP24(2.5mol/l)

1day
d)

2

3day

5day

7day

2

1.5

Con

1.5

Con

1

KP24

1

KP24

0.5

0.5

0

0
day0

day7

day0

day7

Fig. 5. a) MC3T3-E1 cells (osteoblast-like cell) cultured for 7 days on hydrogel modiﬁed by KP24 (0e2.5 mol/l, Bar ¼ 100 mm). b) MSC (Mesenchymal Stem Cells) cultured for 7 dayson hydrogel modiﬁed by KP24 (2.5 mol/l, Bar ¼ 100 mm). c) Ratio of MC3T3-E1 cell adhesion on hydrogel with or without KP24 modiﬁcation. d) Ratio of MSC adhesion on hydrogelwith or without KP24 modiﬁcation.

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A. Ikeda et al. / Regenerative Therapy 3 (2016) 108e113

Fig. 6. a) Neural cell culture for 7 days on hydrogel sheets with or without KP24 modiﬁcation. b) Cell adhesion number on the KP24-modiﬁed sheet showed better than do cells on
the sheet without KP24 modiﬁcation (*p: <0.05). c) Cells on the KP24-modiﬁed sheet showed better neurite growth than do cells on the sheet without KP24 modiﬁcation(Bar ¼ 100 mm, *p: <0.05).

growth modulation may be used for the growth modulation ofother tissues as well.
In this study, we investigated the ability of a functional peptide
to modulate SMG tissue growth in vitro. SMG tissue culture results
showed that tissue growth is enhanced when the peptide solution
is added to the medium. In addition, the peptide-immobilized
substrate enhanced the SMG growth signiﬁcantly in a dosedependent manner. KP24 is a linear peptide with a repeating
lysine (basic amino acid) and proline (neutral amino acid) motif.
This peptide is composed of H-(Lys-Pro)n-OH sequence and promotes cell aggregate formation when it is added to the medium[10]. KP24 (n ¼ 12) exhibits more cell agglutination capacity than
KP20 (n ¼ 10) or KP 28 (n ¼ 14). In order to understand the obtained
phenomena further, different types of cells were cultured on the
peptide-modiﬁed, and unmodiﬁed substrate. MC3T3-E1 osteoblast-like cells and isolated primary MSCs did not attach well, evento the peptide-modiﬁed substrate. These results indicated that theenhanced SMG growth is not related to the interaction between
KP24 and general mesenchymal cells. In contrast, PC12 neuron-likecells adhered to the substrate and showed better neurite outgrowthon the peptide-modiﬁed substrate when compared to the unmodiﬁed substrate.
The importance of neuronal growth in SMG tissue development
has been previously reported. For example, removal of the parasympathetic ganglions in mouse explant organ culture decreased
the number of epithelial buds and slowed down SMG morphogenesis [22,23]. In one of our recent reports, we demonstrated that
attenuation of neurturin (NRTN), a neurotrophic factor, inhibits exvivo SMG growth even when cultured on the RGD-modiﬁed alginate hydrogel. We have also shown that neurite outgrowth iscrucial for increasing SMG growth as well [9]. Therefore, we propose herein that the KP24 peptide-immobilized substrate plays arole in SMG tissue growth in vitro by promoting neuronal survivaland growth, which leads to the growth and expansion of the SMG
tissue in culture.
Since peptide synthesis method is well-established, this
peptide-based approach would be a promising method for themodulation of SMG tissue growth in vitro.

5. Conclusion
KP24 peptide enhanced SMG tissue growth by increasing
neuronal growth and enhancing neural innervation. The peptidemodiﬁed substrate would be a promising tool for the modulationof SMG tissue growth in vitro.
Conﬂict of interest
All authors declare no conﬂict of interest associated with thismanuscript.
Acknowledgment
The work was supported by Grant-in-Aid 24106508, 25220912
and 26106718 from JSPS.
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