Layered Double Hydroxide and Polypeptide Thermogel

Nov 22, 2017 - Layered Double Hydroxide and Polypeptide Thermogel Nanocomposite System for Chondrogenic Differentiation of Stem Cells. Seon Sook Lee ...
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Research Article Cite This: ACS Appl. Mater. Interfaces 2017, 9, 42668−42675

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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 Ewhayeodae-gil, Seodaemun-gu, Seoul, 03760, Korea S Supporting Information *

ABSTRACT: Stem cell therapy for damaged cartilage suffers from 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. Then, tonsil-derived mesenchymal stem cells (TMSCs), arginylglycylaspartic acid-coated LDHs, and kartogenin (KGN) were incorporated into the gel through the thermal-energydriven 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 the mRNA and protein levels in the nanocomposite system, compared to the pure thermogel systems. The inorganic 2D materials increased the rigidity of the 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 hydrogel-based 3D cell culture systems and is eventually expected to be applied as an injectable stem cell therapy. KEYWORDS: sol−gel transition, layered double hydroxide (LDH), stem cell, 3D culture, chondrogenic differentiation

1. INTRODUCTION Cartilage is an avascular tissue, and damaged cartilage with more than a critical size of 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 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 the 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 that 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. © 2017 American Chemical Society

Here, we report a new 2D/3D nanocomposite system incorporating tonsil-derived mesenchymal stem cells (TMSCs), kartogenin (KGN), and arginylglycylaspartic acid (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-energy-driven gelation.8−11 TMSCs separated from the tonsil tissue of an 11-yearold girl were used after obtaining consent.12 Tonsil tissues have been otherwise wasted after a 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 mesenchymal stem cells (MSCs).13−16 Therefore, KGN was used in our study as a chondrogenic differentiation modulator of TMSCs. LDHs are 2D plated materials, and nanosized 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 Received: November 11, 2017 Accepted: November 22, 2017 Published: November 22, 2017 42668

DOI: 10.1021/acsami.7b17173 ACS Appl. Mater. Interfaces 2017, 9, 42668−42675

Research Article

ACS Applied Materials & Interfaces

g, 5.0 mmol) was dissolved in dried toluene (60 mL), and about 50 mL of toluene was distilled out to remove residual water. In order to prepare PEG−PA, NCA-A (8.1 g, 70.4 mmol) was polymerized at 40 °C for 24 h after adding anhydrous chloroform/N,N-dimethylformamide (70 mL; 6/1, v/v) under dry nitrogen conditions. Similarly, NCA-Dz (3.1 g, 12.5 mmol) was polymerized at 40 °C for an additional 48 h 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 PEG−PA−PDz was carried out by using a 0.1 N NaOH solution at 20 °C for 6 h, and then, the 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), gel permeation chromatography (GPC; SP930D, Young Lin), circular dichroism (CD; J-810, JASCO), transmission electron microscopy (TEM; JEM-2100F, JEOL), FTIR spectroscopy (FTS-800 spectrophotometer, Varian), phase diagram (test tube inverting method),8−10,25 and dynamic mechanical analysis (Rheometer RS 1, Thermo Haake)8−10 were performed by the published methods. 2.6. KGN Release. KGN (0.25 mg) 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 preincubated at 37 °C for 10 min. Then, phosphate-buffered saline (PBS; 3.0 mL, 37 °C) at pH 7.4 was added to the gels. KGN-loaded HyStem 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/ min in a thermostatic bath at 37 °C. The whole medium was replaced at sampling intervals. KGN in the medium was analyzed by a highperformance liquid chromatography (HPLC) system (Waters 1525) with a photodiode array detector (Waters 2998) at a wavelength of 274 nm. A Jupiter 5 μm C18 300A column (Phenomenex) and acetonitrile/water (35/65, v/v) were used for 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 min. Solid precipitates were recollected by centrifugation. Then, they were freeze-dried to prepare RGDcoated LDHs after washing with deionized water several times. X-ray photoelectron spectroscopy (XPS; AXIS-NOVA, Kratos) 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 a tonsillectomy at the Ewha Womans University Mokdong Hospital (Seoul, Korea) were kindly donated. The TMSCs from passage 6 were used. Six protocols were compared in this research, where the P0 system is TMSC suspended in PEG− PA−PD solution (0.2 mL, 11.0 wt % in a medium): PI, KGN was added to the P0 system to give a final concentration of 1.0 μM KGN; 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 as in PIII were added to the P0 system; PVI, HyStem was used instead of PEG−PA−PD, and KGN (final concentration 10.0 μM) was added. PIII is the target system and other systems were studied for comparative purposes. The above solutions of suspended stem cells were injected into 24well culture plates, which were incubated at 37 °C 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 (0.6 × 106 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 °C was replaced every third day. 2.9. TMSC Proliferation. After 21 days of incubation, cell proliferation was assayed by a cell counting kit-8 (CCK-8, Dojindo)

2D material, and they were conjugated with RGD. The RGDcoated LDHs and KGN were coencapsulated 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/ Scheme 1. Schematic Presentation of the Researcha

a

Stem cells (green circles with a blue core), KGN (red dots), and RGD-coated LDHs (yellow hexagons) are incorporated in a hydrogel during the thermal-energy-driven gelation of the polymer aqueous solution. Stem cells adhere to the LDH surface by RGD, and KGN is continuously released in the system and induces chondrogenic differentiation of TMSCs.

poly(lactide/glycolide) thermogel with a gel modulus of 1000− 1200 Pa is an excellent scaffold for chondrogenic differentiation of bone marrow mesenchymal stem cells.15 In particular, PEG− polypeptide thermogels with a modulus of 300−1000 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 RGDcoated LDH and KGN into the PEG−PA−PD thermogel to further enhance cell adhesion and chondrogenic differentiation of the TMSCs.

2. EXPERIMENTAL SECTION 2.1. Materials. Magnesium nitrate hexahydrate, aluminum nitrate nonahydrate, urea, and triphosgene were purchased from SigmaAldrich. α-Amino-ω-methoxy PEG (Pharmicell), peptide GRGDC (Peptron), N-carboxy anhydrides of L-alanine (NCA-A; Onsolution), and 4-benzyl-L-aspartate (Tokyo Chemical Industry) were used as received. 2.2. Synthesis of LDH. 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 °C) for 72 h. The products were collected by centrifugation and then freeze-dried for 24 h. 2.3. Characterization of LDH. The scanning electron microscopy (SEM) images (JSM-6700F, JEOL), X-ray diffraction (XRD) pattern (D/max2000, Rigaku), and ζ-potentials of LDHs and RGD-coated LDHs (Zetasizer NanoZS, Marvern) were investigated. 2.4. Synthesis of PEG−PA−PD. N-Carboxy anhydrides of 4benzyl-L-aspartate (NCA-Dz) were synthesized from 4-benzyl-Laspartate and triphosgene by the published method.24 The 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 (MW = 1 kDa, 5.0 42669

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ACS Applied Materials & Interfaces according to the manufacturer’s protocol. the 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 the mRNAs and protein levels was analyzed by realtime polymerase chain reaction (RT-PCR; CFX96TM, Bio-Rad) 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 1.

Table 1. Primer Sequences and PCR genesa

primer sequences

COL II

F: 5′-CAA ACC CAA AGG ACC CAA GTA-3′ R: 5′-TGT GAG AGG GTG GGA TGA A-3′ F: 5′-ACC TTT GGG CTG CCT TAT ATT-3′ R: 5′-TCC CTC ACT CCA AGA GAA GAT-3′ F: 5′-ACC CAA GGA CTG GAA TCT TTA C-3′ R: 5′-GCC ATT CTT ATA CAG GCC TAC-3′ F: 5′-CTC CTC ACA GTT GCC ATG TA-3′ R: 5′-GTT GAG CAC AGG GTA CTT TAT TG-3′

SOX 9 COL X GAPDH a

COL II, SOX 9, COL X, and GAPDH indicate type II A1 collagen, transcription factor SOX 9, type X collagen, and glyceraldehyde-3phosphate-dehydrogenase, respectively.

Figure 1. (a) SEM images of LDH. (b) XRD pattern of LDH.

2.11. Glycosaminoglycan Assay. DNA and sulfated glycosaminoglycan (GAG) were assayed by the Picogreen assay and dimethyl methylene blue assay, respectively.28 Lambda phage DNA and chondroitin 4-sulfate were used as standards for the 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. The symbols * (p < 0.05) and ** (p < 0.01) express the statistical significance of the differences in mean values by the one-way ANOVA with Tukey tests.

3. RESULTS Micrometer-sized LDHs were prepared by a hydrothermal process at 100 °C for 72 h. SEM images of the LDHs show the uniform size of two-dimensional (2D) hexagonal plates with a lateral length of about 2−3 μm (Figure 1a). The X-ray diffraction (XRD) peaks at 2θ = 12° and 24°, corresponding to the 003 and 006 planes, respectively, indicate that LDHs with a well-defined 2D structure were synthesized (Figure 1b). LDHs can have positive charges due to the presence of trivalent cations that replace some of the divalent metal cations of a brucite-like layer.29,30 Polymerization of N-carboxy anhydrides of L-alanine (NCAA) initiated by α-amino-ω-methoxy PEG led to PEG−PA diblock copolymers.31 Then, the subsequent polymerization of N-carboxy anhydrides of L-4-benzyl-L-aspartate (NCA-Dz) led to PEG−PA−PDz triblock copolymers. After the deprotection of the benzyl ester groups, the final PEG−PA−PD triblock copolymers were prepared (Figure 2). Progress of the reactions was monitored by 1H NMR spectroscopy and GPC. The appearance of PA (1.40−1.80 ppm) and PDz (7.22−7.47 ppm) peaks and the disappearance of the benzyl peaks of the protecting group (7.22−7.47 ppm) in the 1H NMR indicate that the reactions successfully took place (Figure 3). The final composition of the PEG−PA−PD was calculated with the use of the PEG (3.80−4.10 ppm), PA (1.40−1.80 ppm), and PD (3.00−3.39 ppm) peaks. The molecular weight of each block of PEG−PA−PD is 1000−1210−225 Da, indicating that the numbers of the repeating units of ethylene glycol, alanine, and

Figure 2. Synthetic scheme of block copolymer PEG−PA−PD; p, q, and r were determined by 1H NMR and calculated to be 22, 17, and 2, respectively.

aspartic acid of the polymer are 22, 17, and 2, respectively (Table 2). The GPC chromatograms of PEG−PA, PEG−PA− PDz, and PEG−PA−PD confirmed the progress of the reactions, and the molecular weight distribution (Mw/Mn) of the polymers was 1.12, 1.16, and 1.14, respectively (Supporting Information, Figure S1). Upon increasing the concentration of aqueous PEG−PA− PD solution to 1.0 wt % at 20 °C, the peptide band in the circular dichroism (CD) spectra shifted from 218−220 to 230− 240 nm (Figure 4a,b). The red-shift is an indication of the selfassembly of the polypeptide-based amphiphilic block copolymers into micelles.13,32 The critical micelle concentration (cmc) of the PEG−PA−PD is in the range of 0.03−0.08 wt %. TEM images of the PEG−PA−PD triblock copolymers also exhibited spherical micelles (Figure 4b, inset). The carbonyl band at 1620−1630 cm−1 in the FTIR spectrum of the PEG−PA−PD 42670

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concentration from 12.0 to 8.0 wt % (Figure 5a). In particular, the 11.0 wt % aqueous system selected for 3D cell culture

Figure 3. 1H NMR spectra (CF3COOD) of PEG, PEG−PA, PEG− PA−PDz, and PEG−PA−PD. The peaks corresponding to benzyl oxy carbonyl (Z-benzyl and Z-benzyl-CH2 at 7.2−7.5 and 5.0−5.4 ppm, respectively) protecting groups disappeared after the deprotection reaction. The chemical structure of the PEG−PA−PD is inset; p, q, and r are calculated to be 22, 17, and 2, respectively.

Table 2. Molecular Weight and Polydispersity of Polymers polymer

Mna

polymer structurea

polydispersityb

PEG−PA PEG−PA−PDz PEG−PA−PD

1000−1190 1000−1230−425 1000−1210−225

EG22A17 EG22A17Dz2 EG22A17D2

1.12 1.16 1.14

a

Molecular weight of each block, calculated via the 1H NMR (CF3COOD) spectra. The small change in Mn comes from the integration error in the 1H NMR as well as the purification of the polymer in each step. bPolydispersity (Mw/Mn) was determined by GPC against PEG standards.

Figure 5. (a) Phase diagram of aqueous PEG−PA−PD solutions. (b) Storage modulus (G′) and loss modulus (G″) of an aqueous PEG− PA−PD solution (11.0 wt %) as a function of temperature. (c) Storage modulus (G′) of a PEG−PA−PD thermogel (prepared from 11.0 wt % aqueous polymer solution) and a PEG−PA−PD thermogel containing LDH (1.0 wt % of the polymer) at 37 °C. (d) Release profile of KGN from the PEG−PA−PD thermogel and LDH/PEG−PA−PD nanocomposite system. LDH (1.0 wt % of the polymer) and KGN (0.25 mg) were incorporated in the system (11.0 wt %, 0.5 mL). KGN release profile from HyStem gel is also shown for comparison.

Figure 4. (a) CD spectra of PEG−PA−PD as a function of polymer concentration at 20 °C. The legends show the polymer concentration (wt %). (b) Red-shift of the band. The cmc is determined by the two extrapolated lines. TEM image of the PEG−PA−PD assemblies developed from an aqueous polymer solution (0.10 wt %) is inset.

exhibited an appropriate gelation temperature and modulus of 640−680 Pa at 37 °C (Figure 5b). The storage modulus (G′) became greater than the loss modulus (G″) during the thermalenergy-driven gelation.33 The addition of LDHs (1.0 wt % of polymer) increased the gel modulus at 37 °C from 640−680 to 750−820 Pa (Figure 5c). The gel modulus was strong enough to maintain its physical integrity over the 3D cell culture period

(11.0 wt % in D2O) indicates that the polypeptide has a β-sheet structure (Supporting Information, Figure S2). Aqueous PEG−PA−PD solutions undergo a thermal-energydriven gelation when the temperature increases. The gelation temperature increased from 22 to 55 °C by decreasing the 42671

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ACS Applied Materials & Interfaces of 21 days, as will be discussed in the following section. PEG− PA−PD is stable under the neutral condition at pH 7.4, and no significant degradation was observed over 21 days (Supporting Information, Figure S3). In this study, KGN was used as a stem cell modulator for chondrogenic differentiation. Sustained release of KGN was observed from the thermogel over 21 days (Figure 5d). Incorporation of LDHs by 1.0 wt % of the polymer decreased the drug release rate, and about 90% of loaded KGN was released over 21 days from the LDHincorporated thermogel. The positively charged surfaces of the LDHs might interact with the negatively charged KGN and PEG−PA−PD, which could affect the release rate of the drug as well as the modulus of the gel. KGN was quickly released over a couple of days from a hydrophilic HyStem which is a hyaluronic acid-based gel. KGN in a concentration range of 0−10.0 μM is tolerable for TMSCs without significant cell death (Supporting Information, Figure S4). Therefore, 10.0 μM KGN was used as an initial concentration in designing the 3D culture system in this study, and thus, KGN released from the system is within the safety range to induce chondrogenic differentiation without detrimental effects on TMSCs, as will be discussed in this study. The weakly positively charged LDHs (ζ = +12.4 mV) were coated with cell-adhesion peptide RGD through ionic interactions between GRGDC and LDH. GRGDC has two positive charges from the terminal amine of glycine (G) and arginine (R) and two negative charges of aspartate (D) and the terminal carboxylic acid of cystine (C) at pH 7.0. After washing with an excess amount of water, the RGD-coated LDHs were prepared. XPS spectra of LDHs and RGD-coated LDHs were compared (Figure 6a). A specific peak with a binding energy of 400 eV was observed for the RGD-coated LDHs. The peak was assigned to an electron in the 1s orbital of nitrogen of RGD.34 The RGD-coated LDHs were used as a 2D nanomaterial in constructing the 2D/3D nanocomposite systems for 3D cell culture. The ζ-potential of the RGD-coated LDHs slightly increased to +13.2 mV (Figure 6b). To investigate the effect of the RGD-coated LDHs and KGN content on chondrogenic differentiation of TMSCs, six cell culture systemsPI, PII, PIII, PIV, PV, and PVIwere constructed, as described in the Experimental Section. PI and PII contain a small amount of KGN (1.0 μM), whereas PIII, PIV, PV, and PVI contain a large amount of KGN (10.0 μM). PII and PIII are composite systems of RGD-coated LDH and thermogel. PI, PIV, and PV are thermogel systems. PVI is a commercially available HyStem hydrogel system. The same number of TMSCs (0.6 × 106 cells/well) was used for all systems. The characteristics of cell proliferation and biomarker expressions related to the chondrogenic differentiation of TMSCs were compared for the above systems. Live and dead assay is a standard assay method in which live cells (green) are contrasted with dead cells (red).35 The cell proliferation images prove the cytocompatibility of the current systems (Supporting Information, Figure S5). In particular, cell aggregates were observed in RGD-coated LDHs incorporating composite systems of PII and PIII, which contrast with the scattered cells in the pure hydrogel systems of PI, PIV, PV, and PVI. A LDH plate with multiple RGD molecules might attract cells and trigger the cell aggregation. After 21 days of incubation, the number of cells was determined by the CCK-8 method. The relative cell density of the PII and PIII systems was 30−40% higher than that of the PI system, suggesting that the incorporated RGD-coated LDHs increased the number of

Figure 6. XPS (N 1s) spectra (a) and ζ-potential (b) of LDH and RGD-coated LDH. RGD (1.0 wt % of LDH) was coated on the surfaces of the LDH. (c) Relative cell density in the 3D cell culture systems compared to that of the PI system after 21 days of culture.

remaining cells in the system (Figure 6c). No statistically significant difference in the cell densities among the hydrogel systems of PI, PIV, PV, and PVI was observed. Chondrogenic biomarkers of COL II and Sox 9 were compared at the mRNA level after 21 days of incubation of TMSCs in each system. COL II is the main extracellular molecule in articular cartilage and is used as a typical biomarker of chondrogenic differentiation of stem cells.36−39 Binding of chondrocytes to COL II ligands upregulates SOX 9 and induces the chondrogenic differentiation of stem cells.40 Compared to the PI system, COL II mRNA expression was 14- and 40-fold higher in the PII and PIII systems, respectively (Figure 7a). In addition, the PIV system, with a high concentration (10.0 μM) of KGN, showed 20-fold higher expression of COL II than the PI with a low concentration (1.0 μM) of KGN. PIV and PV showed that soluble RGD did not increased the COL II expression, suggesting that the hydrophilic RGD was release out of the gel and did not affect stem cell differentiation. HyStem, a hyaluronic acid-based gel, is hydrophilic, and KGN was quickly released over a couple of days (Figure 5d). The COL II expression in the PVI system (HyStem) was similar to that of the PI system. Compared to the PI system, SOX 9 mRNA expression was 4and 7-fold higher in the PII and PIII systems, respectively (Figure 7b). PIV and PV systems showed 3−4-fold higher SOX 9 expression than the PI system. The PVI system was similar to the PI system in its SOX 9 mRNA expression. In all cases, the PIII system was highest in chondrogenic biomarker expression 42672

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biological system. COL X is a biomarker related to the hypertrophic differentiation of chondrocytes.26,27 PIII and PV systems exhibited a higher, with no statistical significance, expression of type X collagen compared with other systems (Figure 7c). The biomarker expressions at the protein level indicated a parallel trend with the mRNA level. An immunofluorescence study also exhibited the enhanced chondrogenic differentiation in the nanocomposite systems. Immunofluorescence images of the proteins secreted by the cells, COL II and SOX 9, were compared (Figure 8a,b). Both COL II and SOX 9 were stained

Figure 7. mRNA expression of COL II (a), SOX 9 (b), and COL X (c) after 21 days of 3D culture in each system. The mRNA expression levels were determined by RT-PCR, and normalized by GAPDH and zeroth day data.

at the mRNA level than the other systems, suggesting that both KGN and RGD-coated LDHs contributed to the chondrogenic differentiation of the TMSCs. RGD is a well-known cell adhesion peptide; however, an excess amount of the peptide interferes with chondrogenic differentiation, and thus, an optimal concentration of RGD is important in the chondrogenic differentiation of stem cells.41 In the current research, 1.0% of RGD-coated LDHs was used in the PII and PIII system, which significantly improved both cell density and chondrogenic differentiation without detrimental side effects. In particular, PIII, with a high content of KGN and exhibiting cell aggregation, was pronouncedly higher in chondrogenic biomarker expression at the mRNA level, suggesting that the supply of KGN and cell morphology during the 3D cell culture are important factors for the chondrogenic differentiation of TMSCs. We previously reported that the chondrogenic differentiation is preferred to adipogenic or osteogenic differentiation of MSCs in the PEG−polypeptide thermogels.21,22,42 Here, we proved that chondrogenic biomarkers were even more highly expressed in the nanocomposite system, suggesting the significance of the 2D material of RGD-coated LDHs in the cell culture system. Chondrocytes might further differentiate into hypertrophic cells around which the cartilage tissue becomes calcified in a

Figure 8. Immunofluorescence images of COL II (a) and SOX 9 (b). (c) GAG content. The images were taken for each system 21 days after 3D culture of TMSCs.

red in the fluorescence images. The COL II and SOX 9 exhibited particularly higher intensity in the PIII nanocomposite system than in the pure thermogel systems of PI, PIV, PV, and PVI. The immunofluorescence study also confirmed the significance of RGD-coated LDHs as well as KGN in chondrogenic differentiation of TMSCs. In particular, the cell aggregates found in the composite systems of PII and PIII suggest that the cell aggregation might positively contribute to the chondrogenic differentiation of the TMSCs. Aggregation of the cells increases cell−cell contacts and communications and affects cell geometry and oxygen content in cell body, thus enhancing the cell signaling appropriate for chondrogenic differentiation of the stem cells.39,43,44 The 42673

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ACS Applied Materials & Interfaces Notes

spontaneous aggregation of the stem cells in the composite systems is noteworthy of comments in this regard. As another biomarker of chondrogenic differentiation of TMSCs, GAG content was assayed. GAG content of PIII was 2.6-fold higher than that of PI (Figure 8c). GAG content of PIV and PV was about 2-fold higher than that of PI. These findings also indicate that a high concentration of KGN and incorporation of RGD-coated LDHs increased the GAG content. The higher expressions of the chondrogenic biomarkers in the nanocomposite systems than in the hydrogel systems suggest that RGD-mediated cell adhesion and aggregation in the 2D/3D composite cell culture systems play a role and that KGN is a synergistic modulator for chondrogenic differentiation of the incorporated stem cells.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (2017R1A2B2007356, 2017R1A5A1015365, and 2014M3A9B6034223).

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(1) Jackson, D. W.; Lalor, P. A.; Aberman, H. M.; Simon, T. M. Spontaneous Repair of Full-Thickness Defects of Articular Cartilage in a Goat Model. J. Bone Jt. Surg., Am. Vol. 2001, 83, 53−64. (2) Zhu, Y.; Yuan, M.; Meng, H. Y.; Wang, A. Y.; Guo, Q. Y.; Wang, Y.; Peng, J. Basic Science and Clinical Application of Platelet-Rich Plasma for Cartilage Defects and Osteoarthritis: A Review. Osteoarthritis and Cartilage 2013, 21, 1627−1637. (3) Park, Y. B.; Ha, C. W.; Lee, C. H.; Yoon, Y. C.; Park, Y. G. Regeneration in Osteoarthritic Patients by a Composite of Allogeneic Umbilical Cord Blood-Derived Mesenchymal Stem Cells and Hyaluronate Hydrogel: Results from a Clinical Trial for Safety and Proof of concept with 7 Years of Extended Follow-Up. Stem Cells Transl. Med. 2017, 6, 613−612. (4) Jeong, S. Y.; Kim, D. H.; Ha, J.; Jin, H. J.; Kwon, S. J.; Chang, J. W.; Choi, S. J.; Oh, W.; Yang, Y. S.; Kim, G.; Kim, J. S.; Yoon, J. R.; Cho, D. H.; Jeon, H. B. Thrombospondin-2 Secreted by Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Promotes Chondrogenic Differentiation. Stem Cells 2013, 31, 2136−2148. (5) Baker, B. M.; Chen, C. S. Deconstructing the Third Dimension − How 3D Culture Microenvironments Alter Cellular Cues. J. Cell Sci. 2012, 125, 3015−3024. (6) Choi, B. G.; Park, M. H.; Cho, S. H.; Joo, M. K.; Oh, H. J.; Kim, E. H.; Park, K.; Han, D. K.; Jeong, B. In Situ Thermal Gelling Polypeptide for Chondrocytes 3D Culture. Biomaterials 2010, 31, 9266−9272. (7) Cao, L.; Cao, B.; Lu, C.; Wang, G.; Yu, L.; Ding, J. An Injectable Hydrogel Formed by In Situ Crosslinking of Glycol Chitosan and Multi-benzaldehyde Functionalized PEG Analogues for Cartilage Tissue Engineering. J. Mater. Chem. B 2015, 3, 1268−1280. (8) Park, M. H.; Joo, M. K.; Choi, B. G.; Jeong, B. Biodegradable Thermogels. Acc. Chem. Res. 2012, 45, 424−433. (9) Yu, L.; Ding, J. Injectable Hydrogels as Unique Biomedical Materials. Chem. Soc. Rev. 2008, 37, 1473−1481. (10) Dou, Q. Q.; Liow, S. S.; Ye, E.; Lakshminarayanan, R.; Loh, X. J. Biodegradable Thermogelling Polymers: Working Towards Clinical Applications. Adv. Healthcare Mater. 2014, 3, 977−988. (11) Zhang, Z.; Lai, Y.; Yu, L.; Ding, J. Effects of Immobilizing Sites of RGD Peptides in Amphiphilic Block Copolymers on Efficacy of Cell Adhesion. Biomaterials 2010, 31, 7873−7882. (12) Ryu, K. H.; Cho, K. A.; Park, H. S.; Kim, J. Y.; Woo, S. Y.; Jo, I.; Choi, Y. H.; Park, Y. M.; Jung, S. C.; Chung, S. M.; Choi, B. O.; Kim, H. S. Tonsil-Derived Mesenchymal Stromal Cells: Evaluation of Biologic, Immunologic and Genetic Factors for Successful Banking. Cytotherapy 2012, 14, 1193−1202. (13) Johnson, K.; Zhu, S.; Tremblay, M. S.; Payette, J. N.; Wang, J.; Bouchez, L. C.; Meeusen, S.; Althage, A.; Cho, C. Y.; Wu, X.; Schultz, P. G. A Stem Cell-Based Approach to Cartilage Repair. Science 2012, 336, 717−721. (14) Kang, M. L.; Ko, J. Y.; Kim, E. J.; Im, G. I. Intra-Articular Delivery of Kartogenin-Conjugated Chitosan Nano/Microparticles for Cartilage Regeneration. Biomaterials 2014, 35, 9984−9994. (15) Li, X.; Ding, J.; Zhang, Z.; Yang, M.; Yu, J.; Wang, J.; Chang, F.; Chen, X. Katrogenin-Incorporated Thermogel Supports Stem Cells for Significant Cartilage Regeneration. ACS Appl. Mater. Interfaces 2016, 8, 5148−5159. (16) Cho, T. J.; Kim, J.; Kwon, S. K.; Oh, K.; Lee, J. A.; Lee, D. S.; Cho, J.; Park, S. B. A Potent Small-molecule Inducer of Chondrogenic

4. CONCLUSIONS A 2D/3D nanocomposite system incorporating TMSCs, KGN, and RGD-coated LDHs in a thermogel was designed as an injectable scaffold for the chondrogenic differentiation of the incorporated TMSCs. As a new 2D nanomaterial, hexagonal LDHs with a lateral length of 2−3 μm were newly synthesized by a hydrothermal process. To prepare an LDH-interactive and cytocompatible thermogel, PEG−PA−PD was synthesized. SEM images, XRD pattern, and ζ-potential measurements indicated that the LDHs are well-defined 2D materials with weakly positively charged surfaces. A nanocomposite system consisting of TMSCs, KGN, RGD-coated LDHs, and polypeptide hydrogel was prepared in situ by thermal-energydriven gelation of the system. KGN, a condrogenic modulator, was continuously released from the gel over 21 days. RGDcoated LDHs improved cell aggregation in the nanocomposite systems, and expression of chondrogenic biomarkers was significantly enhanced at both the mRNA and the protein levels in the nanocomposite system of PIII, compared to the pure hydrogel systems. Considering that surfaces of the LDHs can be modified by other bioactive molecules through ionic interactions, a variety of bioactive 2D materials can be designed. Thermogels also provide a facile tool to simultaneously entrap the stem cells, 2D materials, and growth factors. Therefore, the current 2D/3D nanocomposite system of bioactive LDH/ thermogel can be a new platform as a one-shot injectable system for stem-cell-based tissue engineering and therapy.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b17173. Gel permeation chromatograms of PEG−PA, PEG− PADz, and PEG−PA−PD; FTIR spectra of an aqueous PEG−PA−PD solution; GPC profiles of PEG−PA−PD; cytotoxicity of KGN for TMSCs; live and dead assay images of the cells (Figures S1−S5) (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +82 2 3277 3411. Fax: +82 2 3277 2384. ORCID

Byeongmoon Jeong: 0000-0001-9582-1343 42674

DOI: 10.1021/acsami.7b17173 ACS Appl. Mater. Interfaces 2017, 9, 42668−42675

Research Article

ACS Applied Materials & Interfaces

(33) Afrasiabi, R.; Kraatz, H. B. Sonication-Induced Coiled Fibrous Architectures of Boc-L-Phe-L-Lys(Z)-OMe. Chem. - Eur. J. 2013, 19, 1769−1777. (34) Lee, J. S.; Kim, K.; Lee, K.; Park, J. P.; Yang, K.; Cho, S. W.; Lee, H. Adv. Funct. Mater. 2015, 25, 4754−4760. (35) Hayes, A. W. Principles and Methods of Toxicology, 3rd ed.; Raven Press: New York, 1994; pp 1231−1258. (36) Pieper, J. S.; van der Kraan, P. M.; Hafmans, T.; Kamp, J.; Buma, P.; van Susante, J. L. C.; van den Berg, W. B.; Veerkamp, J. H.; van Kuppevelt, T. H. Crosslinked Type II Collagen Matrices: Preparation, Characterization, and Potential for Cartilage Engineering. Biomaterials 2002, 23, 3183−3192. (37) Kye, E. J.; Kim, S. J.; Park, M. H.; Moon, H. J.; Ryu, K. H.; Jeong, B. Differentiation of Tonsil-Tissue-Derived Mesenchymal Stem Cells controlled by Surface-Functionalized Microspheres in PEGPolypeptide Thermogels. Biomacromolecules 2014, 15, 2180−2187. (38) Chen, W.; Chen, S.; Morsi, Y.; El-Hamshary, H.; El-Newhy, M.; Fan, C.; Mo, X. Superabsorbent 3D Scaffold Based on Electrospun Nanofibers for Cartilage Tissue Engineering. ACS Appl. Mater. Interfaces 2016, 8, 24415−24425. (39) Cao, B.; Li, Z.; Peng, R.; Ding, J. Effects of Cell-cell Contact and Oxygen Tension on Chondrogenic Differentiation of Stem Cells. Biomaterials 2015, 64, 21−32. (40) Bosnakovski, D.; Mizuno, M.; Kim, G.; Takagi, S.; Okumura, M.; Fujinaga, T. Chondrogenic Differentiation of Bovine Bone Marrow Mesenchymal Stem Cells (MSCs) in Different Hydrogels: Influence of Collagen Type II Extracellular Matrix on MSC Chondrogenesis. Biotechnol. Bioeng. 2006, 93, 1152−1163. (41) Kloxin, A. M.; Kasko, A. M.; Salinas, C. N.; Anseth, K. S. Photodegradable Hydrogels for Dynamic Tuning of Physical and Chemical Properties. Science 2009, 324, 59−63. (42) Park, M. H.; Yu, Y.; Moon, H. J.; Ko, D. Y.; Kim, H. S.; Lee, H. J.; Ryu, K. H.; Jeong, B. 3D Culture of Tonsil Derived Mesenchymal Stem Cells in Poly(Ethylene Glycol)-Poly(L-Alanine-co-L-Phenyl Alanine) Thermogel. Adv. Healthcare Mater. 2014, 3, 1782−1791. (43) Raghothaman, D.; Leong, M. F.; Lim, T. C.; Toh, J. K.; Wan, A. C.; Yang, Z.; Lee, E. H. Engineering Cell Matrix Interactions in Assembled Polyelectrolyte Fiber Hydrogels for Mesenchymal Stem Cell Chondrogenesis. Biomaterials 2014, 35, 2607−2616. (44) Estes, B. T.; Diekman, B. O.; Gimble, J. M.; Guilak, F. Isolation of Adipose Derived Stem Cells and Their Induction to a Chondrogenic Phenotype. Nat. Protoc. 2010, 5, 1294−1311.

Differentiation of Human Bone Marrow-derived Mesenchymal Stem Cells. Chem. Sci. 2012, 3, 3071−3075. (17) Li, A.; Qin, L.; Wang, W.; Zhu, R.; Yu, Y.; Liu, H.; Wang, S. The Use of Layered Double Hydroxides as DNA Vaccine Delivery Vector for Enhancement of Anti-Melanoma Immune Response. Biomaterials 2011, 32, 469−477. (18) Choi, G.; Kwon, O. J.; Oh, Y.; Yun, C. O.; Choy, J. H. Inorganic Nanovehicle Targets Tumor in an Orthotopic Breast Cancer Model. Sci. Rep. 2015, 4, 4430. (19) Senapati, S.; Thakur, R.; Verma, S. P.; Duggal, S.; Mishra, D. P.; Das, P.; Shripathi, T.; Kumar, M.; Rana, D.; Maiti, P. Layered Double Hydroxides as Effective Carrier for Anticancer Drugs and Taloring of Release Rate through Interlayer Anions. J. Controlled Release 2016, 224, 186−198. (20) Park, M. H.; Moon, H. J.; Park, J. H.; Shinde, U. P.; Ko, D. Y.; Jeong, B. PEG-Poly(l-alanine) Thermogel As a 3D Scaffold of BoneMarrow-Derived Mesenchymal Stem Cells. Macromol. Biosci. 2015, 15, 464−472. (21) Yeon, B.; Park, M. H.; Moon, H. J.; Kim, S. J.; Cheon, Y. W.; Jeong, B. 3D Culture of Adipose-Tissue-Derived Stem Cells Mainly Leads to Chondrogenesis in Poly(Ethylene Glycol)-Poly(L-Alanine) Diblock Copolymer Thermogel. Biomacromolecules 2013, 14, 3256− 3266. (22) Park, J.; Kim, I. Y.; Patel, M.; Moon, H. J.; Hwang, S. J.; Jeong, B. 2D and 3D Hybrid Systems for Enhancement of Chondrogenic Differentiation of Tonsil-Derived Mesenchymal Stem Cells. Adv. Funct. Mater. 2015, 25, 2573−2582. (23) Yang, M.; Liu, J.; Chang, Z.; Williams, G. R.; O’Hare, D.; Zheng, X.; Sun, X.; Duan, X. Mg/Al-CO3 Layered Double Hydroxide Nanorings. J. Mater. Chem. 2011, 21, 14741−14746. (24) Eckman, A. M.; Tsakalozou, E.; Kang, N. Y.; Ponta, A.; Bae, Y. Drug Release Patterns and Cytotoxicity of PEG-Poly(Aspartate) Block Copolymer Micelles in Cancer Cells. Pharm. Res. 2012, 29, 1755− 1767. (25) Chen, Y.; Luan, J.; Shen, W.; Lei, K.; Yu, L.; Ding, J. Injectable and Thermosensitive Hydrogel Containing Liraglutide as a LongActing Antidiabetic System. ACS Appl. Mater. Interfaces 2016, 8, 30703−30713. (26) Shen, G. The Role of Type X Collagen in Facilitating and Regulating Endochondral Ossification of Articular Cartilage. Orthod. Craniofac. Res. 2005, 8, 11−17. (27) Herlofsen, S. R.; Kuchler, A. M.; Melvik, J. E.; Brinchmann, J. E. Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells in Self-Gelling Alginate Discs Reveals Novel Chondrogenic Signature Gene Clusters. Tissue Eng., Part A 2011, 17, 1003−1013. (28) Levett, P. A.; Melchels, F. P. W.; Schrobback, K.; Hutmacher, D. W.; Malda, J.; Klein, T. J. A Biomimetic Extracellular Matrix for Cartilage Tissue Engineering Centered on Photocurable Gelatin, Hyaluronic Acid and Chondroitin Sulfate. Acta Biomater. 2014, 10, 214−223. (29) Huang, S.; Zhu, G. N.; Zhang, C.; Tjiu, W. W.; Xia, Y. Y.; Liu, T. Immobilization of Co-Al Layered Double Hydroxides on Graphene Oxide Nanosheets: Growth Mechanism and Supercapacitor Studies. ACS Appl. Mater. Interfaces 2012, 4, 2242−2249. (30) Wen, T.; Wu, X.; Tan, X.; Wang, X.; Xu, A. One-Pot Synthesis of Water-Swellable Mg-Al Layered Double Hydroxides and Graphene Oxide Nanocomposites for Efficient Removal of As(V) from Aqueous Solutions. ACS Appl. Mater. Interfaces 2013, 5, 3304−3311. (31) Choi, Y. Y.; Jang, J. H.; Park, M. H.; Choi, B. G.; Chi, B.; Jeong, B. Block Length Affects Secondary Structure, Nanoassembly and Thermosensitivity of Poly(Ethylene Glycol)-Poly(L-Alanine) Block Copolymer. J. Mater. Chem. 2010, 20, 3416−3421. (32) Kim, S. J.; Park, M. H.; Moon, H. J.; Park, J. H.; Ko, D. Y.; Jeong, B. Polypeptide Thermogels as a 3D Culture Scaffold for Hepatogenic Differentiation of Human Tonsil-Derived Mesenchymal Stem Cells. ACS Appl. Mater. Interfaces 2014, 6, 17034−17043. 42675

DOI: 10.1021/acsami.7b17173 ACS Appl. Mater. Interfaces 2017, 9, 42668−42675