Layered Double Hydroxide and Polypeptide Thermogel

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Layered Double Hydroxide and Polypeptide Thermogel Nanocomposite System for Chondrogenic Differentiation of Stem Cells Seon Sook Lee, Goeun Choi, Hyun Jung Lee, Yelin Kim, Jin-Ho Choy, and Byeongmoon Jeong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17173 • Publication Date (Web): 22 Nov 2017 Downloaded from http://pubs.acs.org on November 24, 2017

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ACS Applied Materials & Interfaces

Layered Double Hydroxide and Polypeptide Thermogel Nanocomposite System for Chondrogenic Differentiation of Stem Cells

Seon Sook Lee, Go Eun Choi, Hyun Jung Lee, Yelin Kim, Jin-Ho Choy, and Byeongmoon Jeong*

Department of Chemistry and Nanoscience, Ewha Womans University, 52 Ewhayeodaegil, Seodaemun-gu, Seoul, 03760, Korea

* E-mail: [email protected]

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ABSTRACT: Stem cell therapy for damaged cartilages suffers from the low rates of retention, survival, and differentiation into chondrocytes at the target site. To solve these problems, here we propose a two dimensional/three dimensional (2D/3D) nanocomposite system. As a new two dimensional (2D) material, hexagonal layered double hydroxides (LDHs) with a uniform lateral length of 2–3 µm were prepared by a hydrothermal process. And then, tonsil-derived mesenchymal stem cells (TMSCs), RGD-coated LDHs, and kartogenin (KGN) were incorporated into the gel through the thermal energy-driven gelation of the system. The cells exhibited a tendency to aggregate in the nanocomposite system. In particular, chondrogenic biomarkers of type II collagen and transcription factor SOX 9 significantly increased at both mRNA and protein levels in the nanocomposite system, compared to the pure thermogel systems. The inorganic 2D materials increased the rigidity of matrix, slowed down the release of a soluble factor (KGN), and improved cell-material interactions in the gel. The current 2D/3D nanocomposite system of bioactive LDH/thermogel can be a new platform material overcoming drawbacks of a hydrogel-based 3D cell culture systems and is eventually expected to be applied for an injectable stem cell therapy.

Keyword: sol-gel transition, layered double hydroxide (LDH), stem cell, 3D culture, chondrogenic differentiation

1. INTRODUCTION

Cartilage is an avascular tissue, and a damaged cartilage with more than a critical size of 2


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0.6 cm cannot be spontaneously recovered.1 Currently, microfracture operation and stem cell therapy have been applied as clinical treatments for the regeneration of the damaged cartilage.2-4 However, stem cell therapy suffers from the low rates of retention and survival of the cells at the target site. In particular, it is reported that paracrine effects of the secretions from stem cells, rather than the chondrocytes differentiated from stem cells, are therapeutically working for cartilage repair by the clinically approved stem cell therapy.3,4 Therefore, an effective system to drive chondrogenic differentiation of stem cells is very important for better cartilage repair. Cells are in a three dimensional (3D) microenvironment in biological systems. The cells cultured in a 3D system tend to exhibit biomarkers and phenotypes different from the cells cultured in a two dimensional (2D) system.5-7 Considering the fact that polystyrene plates (2D material) have been traditionally used for cell culture, a 2D/3D composite system which incorporates 2D materials in a hydrogel (3D culture system) can be a new platform for stem cell culture, carrying knowledge of previous 2D culture systems to a 3D culture system. In this regard, a well-defined 2D material and a cytocompatible 3D hydrogel are key components of the system. Here, we report a new 2D/3D nanocomposite system incorporating TMSCs, kartogenin (KGN), and RGD-coated layered double hydroxides (LDHs) in a poly(ethylene glycol)-poly(L-alanine)-poly(L-aspartate) (PEG-PA-PD) triblock copolymer thermogel for effective chondrogenic differentiation of the incorporated TMSCs. Thermogel is an aqueous polymer solution that undergoes thermal energydriven gelation.8-11 TMSCs separated from the tonsil tissue of an 11 years girl were used after obtaining consent.12 Tonsil tissues have been otherwise wasted after a 3



tonsillectomy. Small molecular compounds such as KGN and an oxopiperazine derivative of 5{i,2} are reported to be as effective as TGF-β in inducing chondrogenic differentiation of MSCs.13-16 Therefore, KGN was used in our study as a chondrogenic differentiation modulator of TMSCs. LDHs are 2D plated materials, and nano-sized LDHs have been studied as a biocompatible drug delivery carrier into the cells.17-19 However, the LDH nanoparticles are easily internalized into the cell through endocytosis. In this study, we developed well-defined micrometer-sized LDHs as a new 2D material, and they were conjugated with RGD. The RGD-coated LDHs and KGN were co-encapsulated with TMSCs in the gel during thermal energy-driven gelation. The in-situ formed 2D/3D nanocomposite system provides a new cell culture niche, where the encapsulated RGD-coated LDHs are expected to provide 2D surfaces to which stem cells are anchored through binding to the RGD while KGN is continuously provided for chondrogenic differentiation of the stem cells (Scheme 1). Recent studies also proved that PEG/poly(lactide/glycolide) thermogel with a gel modulus of 1,000−1,200 Pa is an excellent scaffold for chondrogenic differentiation of BMSCs.15 In particular, PEG-polypeptide thermogels with a modulus of 300−1,000 Pa already proved their effectiveness for chondrogenic differentiation of incorporated MSCs.20-22 The current system is a continuation of the above research by incorporating RGD-coated LDH and KGN into the PEG-PA-PD thermogel to further enhance cell adhesion and chondrogenic differentiation of the TMSCs.

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2. EXPERIMENTAL SECTION

2.1. Materials. Magnesium nitrate hexahydrate, aluminum nitrate nonahydrate, urea, and triphosgene were purchased from Sigma-Aldrich (USA). α-Amino-ω-methoxy PEG (Pharmicell, Korea), GRGDC (Peptron, Korea), N-carboxy anhydrides of Lalanine (NCA-A, Onsolution, Korea), and 4-benzyl-L-aspartate (Tokyo Chemical Industry, Japan) were used as received. 2.2. Synthesis of LDH. An Mg2Al(OH)6(CO3)0.5 dihydrate was synthesized by modifying the urea hydrolysis method.23 An aqueous solution of magnesium nitrate hexahydrate (0.03 M), aluminum nitrate nonahydrate (0.06 M), and urea (0.30 M) was mixed in a molar ratio of 2:1:10, and then treated under hydrothermal conditions (100 o

C) for 72 hours. The products were collected by centrifugation and then freeze-dried

for 24 hours. 2.3. Characterization of LDH. The scanning electron microscopy (SEM) images (JSM-6700F, JEOL, Japan), X-ray diffraction (XRD) pattern (D/max2000, Rigaku, Japan), and zeta potentials of LDHs and RGD-coated LDHs (Zetasizer NanoZS, Marvern, UK) were investigated. 2.4. Synthesis of PEG-PA-PD. N-carboxy anhydrides of 4-benzyl-L-aspartate (NCA-Dz) were synthesized from 4-benzyl-L-aspartate and triphosgene by the published method.24 Final yield was 71 %. PEG-PA-PD was synthesized by sequential polymerization of NCA-A and NCA-Dz, followed by deprotection of the benzyl ester groups. α-Amino-ω-methoxy PEG (M.W. = 1 kDaltons, 5.0 g, 5.0 mmol) was dissolved in dried toluene (60 mL), and about 50 mL of toluene was distilled out to remove 5



residual water. In order to prepare PEG-PA, NCA-A (8.1 g, 70.4 mmol) was polymerized at 40 oC for 24 hours after adding anhydrous chloroform/N,N-dimethyl formamide (70 mL; 6/1 v/v) under dry nitrogen conditions. Similarly, NCA-Dz (3.1 g, 12.5 mmol) was polymerized at 40 oC for additional 48 hours to prepare PEG-PA-PDz triblock copolymers. The polymer was precipitated into diethyl ether, and the residual solvent was removed under vacuum. Deprotection of benzyl ester groups of the PEGPA-PDz was carried out by using a 0.1 N NaOH solution at 20 oC for 6 hours, and then, pH was adjusted to 7.0 by using a 1.0 N HCl solution. The PEG-PA-PD triblock copolymers were purified by dialysis and freeze-drying. The yield was about 72 %. 2.5. Polymer Characterization. 1H-NMR(CF3COOD) spectroscopy (500 MHz NMR spectrometer, Varian, USA), gel permeation chromatography (GPC, SP930D, Younglin, Korea), circular dichroism (CD) spectroscopy (J-810, JASCO, Japan), transmission electron microscopy (TEM, JEM-2100F, JEOL, Japan), FTIR spectroscopy (FTIR spectrophotometer FTS-800, Varian, USA), phase diagram (testtube inverting method),8-10,25 and dynamic mechanical analysis (Rheometer RS 1, Thermo Haake, Germany)8-10 were performed by the published method. 2.6. KGN Release. KGN (0.25mg) was dissolved in an aqueous PEG-PA-PD solution (11.0 wt.%, 0.5 mL) without or with LDH (0.55 mg). The vial containing each formulation was pre-incubated at 37 oC for 10 minutes. Then, phosphate buffered saline (PBS, 3.0 mL, 37 oC) at pH=7.4 was added to the gels. KGN-loaded HyStemTM gel was also prepared by the manufacture’s protocol, and the release profile of KGN was investigated. The vials were shaken at a stroke of 40 strokes/minute in a thermostatic bath at 37 oC. The whole medium was replaced at sampling intervals. KGN in the 6


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medium was analyzed by high performance liquid chromatography system (HPLC, Waters 1525, Korea) with photodiode array detector (Waters 2998, Korea) at a wavelength of 274 nm. A Jupiter 5µ C18 300A column (Phenomenex, USA) and acetonitrile/water (35/65, v/v) were used in HPLC. 2.7. RGD-coated LDH. LDHs (0.1 g) was added to an aqueous GRGDC (0.001 g) solution (4 mL). Then, the solution was thoroughly mixed over 10 minutes. Solid precipitates were recollected by centrifugation. Then, they were freeze-dried to prepare RGD-coated LDHs after washing with deionized water several times. The X-ray photoelectron spectroscopy (XPS) machine (AXIS-NOVA, Kratos, UK) was used for surface analysis of the LDHs and RGD-coated LDHs. 2.8. Cell Culture. TMSCs recovered from the tonsil tissues of a young girl (11 years old) after tonsillectomy in the Ewha Womans University Mokdong Hospital (Seoul, Korea) were kindly donated. The TMSCs with passage 6 were used. Six protocols of PI, PII, PIII, PIV, PV, and PVI were compared in this research. PIII is a target system and other systems were studied for comparative purposes. The P0 system is a TMSC-suspended PEG-PA-PD solution (0.2 mL, 11.0 wt.% in a medium). PI: KGN was added to the P0 system to make a final concentration of KGN 1.0 µM . PII: KGN (final concentration: 1.0 µM) and RGD-coated LDHs (1.0 wt.% relative to PEG-PA-PD) were added to the P0 system. PIII: KGN (final concentration: 10.0 µM) and RGD-coated LDHs (1.0 wt.% relative to PEG-PA-PD) were added to the P0 system. PIV: KGN (final concentration: 10.0 µM) was added to the P0 system. PV: KGN (final concentration: 10.0 µM) and the same amount of soluble RGD with 7



PIII were added to the P0 system. PVI: HyStemTM was used instead of PEG-PA-PD, and KGN (final concentration: 10.0 µM) was added. The stem cell-suspended above solutions were injected into 24-well culture plates and they were incubated at 37 oC under 5 % CO2. Then, the 3D cell culture systems of PI–PVI were prepared. PI, PIV, PV, and PVI are actually 3D culture systems of hydrogel without RGD-coated LDH. PII and PIII are nanocomposite systems consisting of RGD-coated LDHs and thermogel. The same number of TMSCs (0.6x106 cells/well) were used for each system. On top of the 3D culture system, the culture medium (1.5 mL) of high glucose DMEM incorporating fetal bovine serum (10%), penicillin/streptomycin (1%), and antibiotic/antimitotic (1%) at 37 oC was replaced every third day. 2.9. TMSC Proliferation. After 21 days of incubation, cell proliferation was assayed by the cell counting kit-8 (CCK-8, Dojindo, Japan) according to the manufacturer’s protocol. Cell density of each system was compared with that of the PI system. 2.10. mRNA Expression. After 21 days of incubation, expression of biomarkers at mRNAs and protein levels was analyzed by real-time PCR (CFX96TM, Bio-Rad, USA) and immunofluorescence for the chondrogenic biomarkers of type II collagen (COL II) and transcription factor SOX 9, and type X collagen (COL X).14,20-22 COL X is a measure of hypertrophic differentiation and endochondral ossification.26,27 The primer lists used in this study are shown in Table 2. 2.11. Glycosaminoglycan Assay. DNA and sulfated glycosaminoglycan (GAG) 8


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were assayed by Picogreen assay and dimethyl methylene blue assay, respectively.28 Lambda phage DNA and chondroitin 4-sulfate were used as standards for Picogreen assay and GAG content assay, respectively following manufacture’s protocols. Triplicate experiments were carried out for each system. 2.12. Statistical Analysis. Mean ± standard deviation from the triplicate experiments are shown for data. * (p

Layered Double Hydroxide and Polypeptide Thermogel

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