3D Culture of Adipose-Tissue-Derived Stem Cells ... - ACS Publications

Aug 2, 2013 - ... and Reconstructive Surgery, Gachon University Gil Medical Center, ... Zhang , Li Guo , Vijay T. John , Daniel Hayes , and Donghui Zh...
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3D Culture of Adipose-Tissue-Derived Stem Cells Mainly Leads to Chondrogenesis in Poly(ethylene glycol)-Poly(L‑alanine) Diblock Copolymer Thermogel Bora Yeon,†,‡ Min Hee Park,†,‡ Hyo Jung Moon,‡ Seung-Jin Kim,‡ Young Woo Cheon,§ and Byeongmoon Jeong*,‡ ‡

Department of Chemistry and Nano Science, Ewha Global Top 5 Research Program, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul, 120-750, Korea § Department of Plastic and Reconstructive Surgery, Gachon University Gil Medical Center, Incheon, Korea S Supporting Information *

ABSTRACT: Poly(ethylene glycol)-b-poly(L-alanine) (PEG-L-PA)s with L-PA molecular weights of 620, 1100, and 2480 Da and a fixed molecular weight of PEG at 5000 Da were synthesized to compare the thermosensitive behavior, and to investigate their potential as a three-dimensional (3D) culture matrix of adipose-tissue-derived stem cells (ADSCs). The sol-to-gel transition temperature and the concentration ranges where the transition was observed decreased as the L-PA molecular weight increased. ADSCs were cultured in the 3D matrixes of in situ formed PEG-L-PA hydrogels, which were produced by increasing the temperature of cell-suspended PEG-L-PA aqueous solutions. The spherical morphology was maintained in the PEG-L-PA hydrogel, while the cells underwent fibroblastic morphological changes in the Matrigel over 14 days of incubation. ADSCs exhibited high expression of type II collagen in the PEG-L-PA thermogel. In addition, they also moderately expressed the biomarker of myogenic differentiation factor 1 as the same mesodermal lineages, as well as the type III β-tubulin as a cross-differentiation biomarker. Similar to the in vitro study, the ADSCs predominantly exhibited chondrogenic biomarkers in the in vivo study. The study demonstrates that the polypeptide thermogel of PEG-L-PA is promising as a 3D culture matrix of ADSCs and as an injectable tissue engineering biomaterial.

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INTRODUCTION Thermogelling polymer aqueous solutions undergo sol-to-gel transition as the temperature increases. The polymer is typically amphiphilic, and the poly(ethylene glycol) (PEG) has commonly been used as a hydrophilic block due to its safety for human use. Biodegradable polymers such as polyesters, polycarbonates, polyphosphazenes, polyorthoesters, and polypeptides have been used as a hydrophobic block.1−8 The delicate balance between hydrophilicity and hydrophobicity is a key molecular parameter in designing a thermogelling polymer. The amphiphilic polymers are to be self-assembled into nanostructures such as micelles, vesicles, nanofibers, or nanoribbons, depending on the block length of hydrophilic PEG and hydrophobic biodegradable polymers.9−12 Polyalanine, poly(alanine-co-phenyl alanine), and poly(alanine-coleucine) conjugated to PEG have been developed as the first generation of thermogelling polypeptides.13−15 Secondary structures such as α-helix, β-sheet, and random coil in addition to the aforementioned nano assemblies of the polypeptide could be controlled by the composition and molecular weight of the polypeptides, which affect the transition temperature, phase diagram, and modulus of the gel.8,19−21 Thermogelling polymer aqueous solutions have been extensively used in drug delivery, cell culture, postsurgical © 2013 American Chemical Society

treatments for adhesion prevention, and embolization due to their unique characteristics, which include simple sterilization through microfiltration, minimally invasive depot preparation, and the lack of an organic solvent for formulation.1−8,16−21 In particular, the simple fabrication of scaffolds by temperature sensitive sol-to-gel transition makes them attractive for in situ 3D cell culture and injectable tissue engineering applications. Matrigel, a biologically derived thermogelling system, has some limitations in practical applications for tissue engineering due to immunogenic or pathogenic concerns.22,23 Polypeptide-based synthetic thermogelling systems not only have the aforementioned advantages of thermogelling systems but also have additional advantages for cells due to the fact that there is no pH drop during the degradation of the polymers.8,24 In a polyalanine-based thermogel, the 3D culture of chondrocytes demonstrated improved cell proliferation and biomarker expression of articular cartilage, such as collagen type II and sulfated glucosaminoglycan.25 Compared with bone-marrow-derived and umbilical-cordderived mesenchymal stem cells, ADSCs not only have similar Received: June 13, 2013 Revised: August 1, 2013 Published: August 2, 2013 3256

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20000 Da were used as molecular weight standards. A styragel HR 4E column (Waters) was used. Phase Diagram. The sol−gel transition of the polymer aqueous solution was investigated by the test tube inverting method.27 The polymer aqueous solution (0.5 mL) was placed in a test tube with an inner diameter of 11 mm. The transition temperature was determined by the flow (sol)-nonflow (gel) criterion by an increment of 1 °C per step. Each data point is the average of three measurements. Dynamic Mechanical Analysis. Changes in the modulus of the PEG-L-PA aqueous solutions that underwent sol-to-gel transition were investigated by dynamic rheometry (Rheometer RS 1; Thermo Haake).28 The aqueous polymer solutions (P50−06, 17.0 wt %; P50−11, 4.6 wt %; and P50−25, 2.0 wt %, respectively) were placed between parallel plates of 25 mm in diameter, with a gap of 0.5 mm. During the dynamic mechanical analysis, the samples were placed inside of a chamber with water-soaked cotton to minimize the water evaporation. The moduli of the polymer aqueous systems were collected under a controlled stress (4.0 dyn/cm2) with a frequency of 1.0 rad/s at 5 °C (sol) and 37 °C (gel). Dynamic Light Scattering. The apparent sizes of the polymer and polymer assemblies in water (0.5 wt %) were studied by a dynamic light scattering instrument (ALV 5000−60 × 0) at 15 °C. A YAG DPSS-200 laser (Langen, Germany) operating at 532 nm was used as a light source. Measurements of the scattered light were taken at an angle of 173° to the incident beam. The results of dynamic light scattering were analyzed by the regularized CONTIN method. The decay rate distributions were transformed to an apparent diffusion coefficient. The apparent hydrodynamic size of a polymer aggregate can be obtained from the diffusion coefficient by the Stokes−Einstein equation. Transmission Electron Microscopy (TEM). The microscopy image of the polymer was obtained by JEM-2100F (JEOL) after evaporating the water of the PEG-L-PA aqueous solution (0.5 wt %) at room temperature on a carbon grid. Sodium phosphotungstate dibasic hydrate was used as a staining agent. Circular Dichroism (CD) Spectroscopy. The ellipticities of the PEG-L-PA aqueous solutions (0.01 wt %) were studied by using a circular dichroism instrument (J-810; JADSCO) as a function of temperature in a range of 5−60 °C. The system was equilibrated at each temperature for 20 min. Scanning Electron Microscopy (SEM). To compare gel morphologies, the polymer solutions (P50−06, 17.0 wt %; P50−11, 4.6 wt %; and P50−25, 2.0 wt %, respectively) were dropped onto a silicon wafer and kept at 37 °C in an oven for 10 min to form a gel. Then the gels were quenched in liquid nitrogen at −196 °C and then freeze-dried. Scanning electron microscopy images were obtained by using a field emission scanning electron microscope (FE-SEM; JSM6700F, JEOL Ltd., Japan). Isolation and Characterization of ADSCs. ADSCs were isolated from the subcutaneous adipose tissue of an 18 year old male donor who had undergone liposuction at the Ewha Womans University Mokdong Hospital (Seoul, Korea) following the ethical guidelines of the University. After collagenase digestion and differential centrifugation, the isolated cells were monolayer-cultured in a low-glucose Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a 5% CO2 atm at 37 °C and then subcultured to passage 5. To confirm the molecular phenotype of the ADSCs, surface markers of the cells were quantified by flow cytometry. A human multipotent mesenchymal stromal cell multicolor flow cytometry kit (R&D Systems, U.S.A.) containing conjugated antibodies to CD105-PerCp (Clone 166707), CD90-APC (CloneThy-1A1), and CD45-PE (Clone ICRF 2D1) was used. For the measurement, 1.0 × 106 cells in an eppendorf tube were washed with phosphate buffered saline (PBS) and resuspended in 100 μL of staining buffer containing bovine serum albumin and sodium azide. A total of 10 μL of each antibody in the kit with a concentration of 25 μg/mL was added and incubated for 45 min at 4 °C in the dark. The cells were washed and resuspended in 200−400 μL of the staining buffer. Then they were transferred into a flow cytometry tube and analyzed using a flow cytometry instrument (FACS calibre, U.S.A.).

differentiation capacity to that of fat, bone, cartilage, and skeletal muscle, but also have easy availability of adipose tissue through a surgical procedure.26 The control of differentiation into a specific tissue has been an important issue and several factors have been identified for this purpose. However, the development of a biocompatible and cytocompatible 3D scaffold is essential for the success of the stem cell therapy because the scaffold can localize the stem cells to a target site and improve the effectiveness of the stem cell therapy. We have developed an injectable PEG-L-PA polypeptide system that undergoes sol-to-gel transition in response to temperature changes, as a minimally invasive drug-delivery system.13,24 In this study, we applied the PEG-L-PA thermogel for the 3D culture of ADSCs. The 3D cell culture matrix was constructed by increasing the temperature of the cell-suspended PEG-L-PA aqueous solution to 37 °C. The modulus of the gels was fixed around 600−1000 Pa to minimize the modulus effect for stem cell differentiation. The gene expression level of type II collagen (Col II), type III β-tubulin (βTub III), myogenic differentiation factor 1 (MyoD 1), lipoprotein lipase (LPL), and osteocalcin (OCN) were compared to investigate the stem cell differentiation for chondrogenesis, neurogenesis, myogenesis, adipogenesis, and osteogenesis, respectively. Matrigel, a commercially available thermogelling system, was also used as a control.

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EXPERIMENTAL SECTION

Materials. α-Methoxy-ω-amino poly(ethylene glycol)s (PEG; Mn = 5000 Da; ID Biochem Inc., Korea) and N-carboxy anhydrides of Lalanine (NCA-L-Ala; M & H Laboratory, Korea) were used as received. A cell counting kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan), a Live/Dead kit (Molecular Probes, Invitrogen, Carsbad, CA, U.S.A.), and Matrigel (BD Biosciences, San Jose, CA, U.S.A.) were used as received. Toluene was dried over sodium before use. Synthesis. The PEG-L-PAs were synthesized by the ring-opening polymerization of the NCA-L-Ala in the presence of α-methoxy-ωamino poly(ethylene glycol)s.14 Table 1 summarizes the polymers investigated.

Table 1. List of Polymers code

PEG-L-PAa

PEG-L-PAb (Mn)

polydispersityb (Mw/Mn)

P50−06 P50−11 P50−25

5000−620 5000−1100 5000−2480

4720 4780 5070

1.3 1.2 1.3

a

Molecular weight determined by 1H NMR spectra in CF3COOD. Molecular weight of the polymers relative to poly(ethylene glycol)s was determined by gel permeation chromatography in N,N-dimethyl formamide. Poly(ethylene glycol)s in a molecular weight range of 400−20000 Da were used as the molecular weight standards.

b

NMR Spectroscopy. 1H NMR spectra of the PEG-L-PA in CF3COOD (500 MHz NMR spectrometer; Varian, U.S.A.) were used to determine the composition and number average molecular weight (Mn) of the polymers. 13C NMR spectra of the PEG-L-PA in D2O (P50−06, 17.0 wt %; P50−11, 4.6 wt %; and P50−25, 2.0 wt %, respectively) were used to investigate the molecular motion of the PEG block in a sol or gel state at 5, 15, and 30 °C. The system was equilibrated at each temperature for 20 min. Gel Permeation Chromatography. A gel permeation chromatography system (Waters 515) with a refractive index detector (Waters 410) was used to obtain the molecular weights and molecular weight distributions of the polymers. N,N-Dimethyl formamide was used as an eluting solvent. PEGs with molecular weight in the range of 400 to 3257

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In Vitro 3D Culture of ADSCs. ADSCs (passage 6, 2.0 × 105 cells) were suspended in polymer aqueous solutions (0.2 mL) of P50− 06 (17.0 wt %), P50−11 (4.6 wt %), P50−25 (2.0 wt %), or Matrigel (as received). They were incubated in a 24-well culture plate at 37 °C for 30 min to form a cell-encapsulating 3D matrix by the sol-to-gel transition of the system. DMEM (1.5 mL) containing 10% FBS and 1% penicillin/streptomycin were added to the cell-encapsulated hydrogel at 37 °C in a humidified atmosphere containing 5% CO2, and the medium was replaced every three days. Cell Proliferation and Viability. The proliferation of ADSCs in hydrogels was assessed by CCK-8 methods (n = 3). CCK-8 solution (1.5 mL; 10% v/v in medium) was added to each well of the plate. After 2 h of incubation, the absorbance value at 450 nm was measured with an ELISA reader (Model 550; Bio-Rad, Hercules, CA, U.S.A.), where the absorbance at 655 nm was used as a baseline. The viability of ADSCs in hydrogels was determined using a Live/Dead kit. Briefly, samples were incubated at room temperature for 30 min in a solution of 4 M ethidium homodimer-1 (EthD-1) and 2.0 M calcein AM in PBS. Labeled cells were then viewed under a Nikon Eclipse E600 fluorescence microscope, and images were captured using Lucia software. Live cells were stained with calcein AM (green), whereas dead cells were stained with EthD-1 (red).29,30 For the quantitative analysis, a total of 200 cells were counted in each sample over three randomly chosen areas, and the live and dead cell counts were recorded. RNA Extraction and Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR). After the 3rd, 7th and 14th days of 3D culture, the total RNA was extracted from the cellencapsulating hydrogels using the TRIZOL reagent (Invitrogen, Burlington, Canada), according to the manufacturer’s protocol. The extracted RNA pellet was dissolved in nuclease-free water, and the RNA concentration was determined using a NanoDrop (ND-1000) spectrophotometer (Thermo Scientific, U.S.A.). After synthesizing the cDNA from isolated RNA, cDNA samples were stored at 20 °C until needed. Real-time RT-PCR was performed using a Rever Tra Ace qPCR RT Kit (Toyobo, Japan). The PCR procedure consisted of an initial denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing and extension at 65 °C for 30 s. The PCR products were visualized by SYBR green. The primers used for amplification are listed in Table 2. The relative expression level of target genes was calculated as 2−ΔΔCt, where target gene expression was normalized as ΔΔCt = (Gene A − GAPDH)t − (Gene A − GAPDH)t0.31 t0 is the data on the day when the experiment started.

In Vivo Study. The ADSC-suspended PEG-L-PA (5000−1100) aqueous solution (0.3 mL, 4.6 wt %) was injected into the subcutaneous layer of BALB/c mice, which were 6 weeks old and had an average body weight of 20 g. 3.0 × 105 cells were used per implant. The biomarker expressions of general macrophages (CD68), pro-inflammatory macrophages (CCR7), and pro-healing macrophages (CD163) around the implant were investigated for inflammatory responses. Hematoxylin and eosin (H&E) staining agents were used to study the features of tissue around the implant. To study the differentiation of the ADSCs in the implant, the gel was stained by alcian blue, Masson’s trichrome, toluidine blue, alizarin red S, and oil red O. Statistical Analysis. Data are presented as the means ± standard deviation. The real-time RT-PCR result is the average of triplicate experiments. Statistical analyses were performed with the Student ttest. The statistical significance, denoted as # (or s) and ## (or ss) were defined as p < 0.05 and p < 0.01, respectively. Animal Procedure. All experimental procedures using animals were conducted in accordance with the NIH guideline for the Care and Use of Laboratory Animals and were approved by the Committee of Ewha Womans University.

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RESULTS AND DISCUSSION PEG-L-PA was synthesized by the ring-opening polymerization of NCA-L-Ala in the presence of α-amino-ω-methoxy-poly(ethylene glycol) (PEG), where the molecular weight of PEG was fixed at 5000 Da and the molecular weight of L-PA was varied by adding different amounts of NCA-L-Ala during the polymerization. The molecular weight of the PEG-L-PA was determined by comparing the methyl peak of Ala (-NHCH(CH3)CO-) at 1.4−1.9 ppm, and ethylene (-CH2CH2O-) peak of PEG at 3.8−4.2 ppm (Supporting Information, Figure S1).14 CH3O‐[CH 2CH 2O]m ‐CH 2CH 2[NH‐COCH(CH3)]n ‐NH 2 A1.4 − 1.9 /A3.8 − 4.2 = 3n/(4m + 2) = n/150

The value of m is 112 for the 5000 Da PEG. The number average molecular weights of each block of the PEG-L-PAs determined by 1H NMR spectra (CF3COOD) were 5000−620, 5000−1100, and 5000−2480, which were coded as P50−06, P50−11, and P50−25, respectively. The relative molecular weight of PEG-L-PA to PEG standards and the molecular weight distribution could also be determined by gel permeation chromatography. N,N-Dimethyl formamide was used as an eluting solvent. The molecular weight and molecular weight distribution were in the ranges of 4720−5070 Da and 1.2−1.3, respectively. The fact that the molecular weight (Mn) from gel permeation chromatography is less than the theoretical value indicates that the PEG-L-PA has a rather shrunken conformation in comparison with the corresponding molecular weight of the poly(ethylene glycol) in N,N-dimethyl formamide solvent. Table 1 summarizes the polymers investigated. The PEG-L-PA aqueous solutions underwent sol-to-gel transition as the temperature increased. The sol-to-gel transition temperatures decreased from 47 to 20 °C, from 34 to 14 °C, and from 18 to 6 °C as the concentrations were increased from 8.0 to 17.0 wt %, from 2.0 to 6.0 wt %, and from 1.0 to 2.0 wt % for P50−06, P50−11, and P50−25, respectively (Figure 1a). The concentration ranges for sol−gel transition were 8.0− 17.0 wt %, 2.0−6.0 wt %, and 1.0−2.0 wt % for P50−06, P50− 11, and P50−25, respectively. At concentrations below the sol− gel transition being observed, the polymer aqueous solution

Table 2. Primer Sequences and PCR Conditions for RealTime RT-PCRa gene Col II βTub III MyoD 1 LPL OCN GAPDH

primer sequences F: 5′-AGGAGGCTGGCAGCTGTGTGC-3′ R: 5′-CACTGGCAGTGGCGAGGTCAG-3′ F: 5′-GCCTCTTCTCACAAGTACGTG-3′ R: 5′-CCCCACTCTGACCAAAGATGAA-3′ F: 5′-GCAGGTGTAACCGTAACC-3′ R: 5′ACGTACAAATTCCCTGTAGC-3′ F: 5′-GAGATTTCTCTGTATGGCACC′ R: 5′-CTGCAAATGAGACACTTTCTC-3′ F: 5′-TCAACCCCGACTGCGACGAG-3′ R: 5′-TTGGAGCAGCTGGGATGATGG-3′ F: 5′-ATGGGGAAGGTGAAGGTCG-3′ R: 5′TAAAAGCAGCCCTGGTGACC-3′

annealing temperature (°C) 64 58 49 51 63 57

a Col II, βTub III, MyoD 1, LPL, OCN, and GAPDH indicate type II collagen, type III β-tubulin, myogenic differentiation factor 1, lipoprotein lipase, osteocalcin, and glyceraldehyde-3-phosphate-dehydrogenase, respectively. F and R indicate indicate forward and reverse primers, respectively.

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wt % for P50−06, P50−11, and P50−25, respectively) was less than 0.1 Pa (data not shown). The nanoassemblies of the polymer aqueous solution were compared using dynamic light scattering and transmission electron microscopy. The dynamic light scattering study shows micelles with peak average diameters of 21, 18, and 16 nm for P50−06, P50−11, and P50−25, respectively. Interestingly, the micelle size decreased as the molecular weight of the hydrophobic block (L-PA) increased. Changes in the aggregation number can be considered in this trend. By assuming a hard sphere model, the aggregation number per micelle (N agg ) can be estimated using the following equations.35,36 R = (3M mν2/4πNA )1/3 Nagg = M m /M 0

Mm and NA are the molecular weight of a micelle and Avogadro’s number, respectively. ν2 is the partial specific volume of the polymer, which is assumed to be 0.92 cm3/g.37 M0 is the molecular weight of a polymer. Based on this calculation, the aggregation numbers are 566, 327, and 187 for P50−06, P50−11, and P50−25, respectively. The aggregation number per micelle is comparable with that of PEG-poly(Llactic acid) (5000−3000 Da), which is 593.38 The aggregation number decreased as the hydrophobic block of L-PA increased. The micelle formation of the PEG-L-PAs was also confirmed by TEM images of the polymer developed from the polymer aqueous solutions (0.5 wt %). The peak average micelle sizes determined by dynamic light scattering were 21, 18, and 16 nm for P50−06, P50−11, and P50−25, respectively (Figure 2a).

Figure 1. (a) Phase diagrams of PEG-L-PA aqueous solutions determined by the test tube inverting method. The data are the average of three measurements. (b) The moduli of gels at 37 °C prepared from PEG-L-PA aqueous solutions of P50−06 (17.0 wt %), P50−11 (4.6 wt %), P50−25 (2.0 wt %). As-received Matrigel (M) at 37 °C as an aqueous solution is also compared. The modulus in sol states was less than 0.1 Pa at 5 °C (not shown).

increased its viscosity as the temperature increased. However, the system is too weak to stop the flow when the vial containing the polymer aqueous solution is inverted. At higher concentrations than the sol−gel transition being observed, the aqueous polymer system was a gel over the investigated temperature range of 0−60 °C. As expected, the concentrations showing sol−gel transition decreased as the hydrophobic block of L-PA increased. The sol-to-gel transition of P50−25 was observed in a narrow concentration range of 1.0−2.0 wt %. Additionally, in contrast to the aqueous solutions of PEG-L-PA with 5000−620 Da (P50−06), which underwent sol-to-gel transition (thermogelling behavior) in the range of 8.0−17.0 wt %, PEG-poly(L-lactic acid) (5000−720 Da) aqueous solutions that underwent gel-to-sol transition (gel melting behavior) as the temperature increased within a high concentration range of 30.0−50.0 wt %.32 The intrinsic properties of the polypeptide with rigidity and specific secondary structures might contribute the unique phase behavior of the polypeptide aqueous solution. Dynamic mechanical analysis was carried out on the gel (37 °C) and sol (5 °C) states. A gel modulus of about 600−1000 Pa was observed for 17.0 wt %, 4.6 wt %, and 2.0 wt % aqueous solutions of P50−06, P50−11, and P50−25, respectively (Figure 1b). Considering the practical application as an injectable system, the gel time is important. In case the polymer aqueous solutions are in contact with a 37 °C environment, they undergo sol-to-gel transition within 10 s; however, even the gel is soft (G < 1000 Pa) enough to be injected through a 20 gauge needle. Concentrations of 17.0 wt %, 4.6 wt %, and 2.0 wt % were selected for P50−06, P50−11, and P50−25, respectively, for considering 3D culture of ADSCs. Because the gel modulus affects the differentiation of the stem cells, we controlled the polymer concentration to have a similar gel modulus.33,34 As a control, Matrigel as received as an aqueous solution showed the gel modulus of 60−80 Pa at 37 °C. In the sol state, the modulus of the polymer aqueous solutions (17.0 wt %, 4.6 wt %, and 2.0

Figure 2. (a) Size of the micelle determined by dynamic light scattering of polymer aqueous solutions (0.5 wt %). (b) TEM images of the PEG-L-PAs developed from their aqueous solutions (0.5 wt %). The scale bar is 100 nm.

Similar spherical micelles were observed in TEM images (Figure 2b). The exact size can be slightly different from that obtained by the dynamic light scattering due to the potential shrinkage of the micelle during the evaporation of the water. However, the decrease in micelle size is clear in the TEM images as well as in dynamic light scattering, as the molecular weight of the hydrophobic block increases. 3259

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water molecules, which are dissociated as the temperature increases.44 When the PEG is conjugated to a hydrophobic block, the dehydration temperature decreases.40−43 The decrease in solubility of PEG and/or the change in secondary structure of the L-PA contribute to the sol-to-gel transition of current PEG-L-PA block copolymer aqueous solutions (Figure 4).

To investigate the sensitivity of the secondary structure of the polypeptide in response to the temperature change, circular dichroism spectra of the PEG-L-PA aqueous solutions were obtained as a function of temperature. At high concentrations where the amphiphilic polymers assemble into micelles, the secondary structural information of the polypeptides cannot be obtained by CD spectra.14,39 Therefore, the CD spectra were obtained at 0.01 wt % as a function of temperature. The characteristic α-helical structures with a positive band at 190− 195 nm and a negative band with two minima at 205−210 and 215−225 nm were observed for all P50−06, P50−11, and P50−25 aqueous polymer solutions (Figure 3). From the CD

Figure 4. 13C NMR spectra of PEG-L-PA aqueous solutions as a function of temperature. The concentrations of the polymer in D2O are 17.0 wt %, 4.6 wt %, and 2.0 wt % for P50−06, P50−11, and P50− 25, respectively.

Figure 3. Ellipticities of the PEG-L-PA aqueous solutions (0.01 wt %) as a function of temperature.

Based on the dynamic light scattering, TEM, CD, and 13C NMR spectroscopy, the temperature-sensitive transitions of the PEG-L-PA are presented as a scheme in Figure 5. PEGs at low temperatures and dehydrated PEGs at high temperature are presented as thin blue curves, thick green (P50−06) or thick yellow-green (P50−25). L-PAs are presented as black curves. The amphiphilic polymers of PEG-L-PA form micelles in water, where the hydrophilic PEGs form a shell, and the hydrophobic L-PAs form a core of the micelles. P50−06 produces a larger micelle size than P50−25. The thermal stability of the L-PA block of PEG-L-PA depends on the molecular weight of L-PA. The α-helical secondary structure of P50−06 is stabilized in a temperature range of 5−60 °C. A water-soluble polypeptide with a highly stabilized α-helix was also reported by elongating the water solubilizing charged amino acid on an α-helical polypeptide backbone.45 The PEG-L-PA block copolymer is another way to position a water-solubilizing PEG moiety on an α-helical L-PA backbone. The α-helical secondary structure of P50−25 was sensitive to the temperature change, and the L-PA underwent a transition from α-helix to random-coil structure as the temperature increased. The thermosensitivity of the current

spectra, the thermal stability of the L-PA of P50−06 contrasts with the high temperature-sensitivity of the L-PA of P50−25. The small L-PA is stabilized in the micelle core surrounded by the large PEG of P50−06, whereas the relatively large L-PA of P50−25 exhibited a secondary structure that was vulnerable to the temperature change. Compared with P50−06 and P50−11, the high temperature-sensitivity of the secondary structure of P50−25 and the high hydrophobicity of the polymer might facilitate the sol-to-gel transition of the P50−25 aqueous solutions at lower temperatures and concentrations. The temperature sensitivity of PEG of the PEG-L-PA was compared using 13C NMR spectra of PEG-L-PA in D2O. As the temperature increased, the polymer aqueous solution underwent sol-to-gel transition. The PEG peak was collapsed and downfield-shifted in a gel state, similar to previous thermogelling polymers such as PEG/poly(lactic acid-co-glycolic acid), PEG/polycaprolactone, and PEG/phosphazene, suggesting that the molecular motion of PEG decreases, and that the PEG dehydrates as the temperature increases.40−43 Each repeating unit of PEG has been reported to be hydrogen-bonded to two 3260

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Figure 6. SEM images of gels at 37 °C prepared from PEG-L-PA aqueous solutions of P50−06 (17.0 wt %), P50−11 (4.6 wt %), and P50−25 (2.0 wt %). The SEM image of Matrigel at 37 °C was also compared. The gel formed in situ at 37 °C was quenched in liquid nitrogen (−196 °C), followed by freeze-drying. The scale bar is 10 μm.

ADSCs expressed CD 105 and CD90, but did not express CD45, suggesting that our ADSCs meet the requirements for MSCs (Supporting Information, Figure S2). ADSCs were encapsulated in a 3D matrix of the hydrogels by increasing the temperature of the cell-suspended PEG-L-PA aqueous solutions to 37 °C. The temperature-sensitive sol-togel transition of the systems produced 3D matrixes for ADSCs in situ. The ADSC were cultured by replacing the DMEM containing 10% fetal bovine serum and 1% penicillin/ streptomycin on the cell-encapsulated hydrogels in 5% CO2 atm at 37 °C every three days. The analysis by the CCK-8 method showed that extent of proliferation of the ADSCs was comparable or better in the P50−11 and P50−25 than Matrigel over 14 days, whereas the P50−06 was completely washed away after 14 days (Figure 7a and Supporting Information, Figure S3). Other gels kept their physical integrity over the experimental period of 14 days. The morphologies of the cells significantly differed. The cells in PEG-L-PA kept their spherical morphology and developed small sprouts in 7−14 days, whereas the cells in Matrigel showed spherical morphology on the zeroth day and then developed fibroblastic morphology over 3−14 days (Figure 7b). The low modulus of Matrigel at 37 °C (60−80 Pa) and bioactive functional groups of the Matrigel might induce such differences. Gene expressions during the 3D culture of the ADSCs were compared for P50−06, P50−11, and P50−25. Matrigel was used as a control for comparison. ADSCs are known to be usually differentiated into mesodermal cells, including chondrocytes, myocytes, adipocytes, osteocytes, and fibroblasts, which belong to the same lineage.49 Therefore, the expression level for Col II, MyoD 1, LPL, and OCN were compared to investigate chondrogenesis, myogenesis, adipogenesis, and osteogenesis, respectively. Even though neurons belong to a different lineage, the gene expression for βTub III was also studied to investigate the neurogenesis, because a gel modulus of 100−1000 Pa is known to be optimal for neurogenesis.33 The sequences of the primers used in the real-time RT-PCR are listed in Table 2. All expressions were normalized by the GAPDH level. The most significant gene expression was observed to be the Col II, while minor expressions of βTub III and MyoD 1 were also observed (Figure 8a−c). The gene expressions for LPL and OCN were undetectable. These findings indicate that ADSCs underwent mostly chondrogenesis with some degree of neurogenesis and myogenesis under the current 3D culture conditions. The neurogenesis can be classified as cross-differentiation of ADSCs, because the

Figure 5. Schematic presentation of temperature-sensitive conformational changes of PEG-L-PA in water. PEGs (thin blue curves) dehydrate and micelles aggregate as the temperature increases. The dehydrated PEG is presented as thick green (P50−06) or yellow-green (P50−25). L-PAs (black curves) undergo α-helix-to-random-coil transition for P50−25, whereas the α-helixes are preserved for P50− 06 as the temperature increases.

PEG-L-PA with α-helical structure is contrasted with that of PA-PEG-PA, for which its β-sheet structure is strengthened as the temperature increases.46 The PEG dehydration might be a major mechanism of sol-to-gel transition for P50−06, whereas the dehydrated PEG of P50−25 might interact with hydrophobic L-PA of P50−25, and the α-helix might be destabilized by increasing the temperature from 5 to 60 °C. Such differences in temperature-sensitivity resulted in difference in phase behavior among the PEG-L-PA aqueous solutions. The physical integrity was maintained for less than 10 days for the P50−06 thermogel, whereas the P50−11 and P50−25 maintained it for more than 2 weeks, which will be discussed in the following section. The relatively weak interactions among the aggregated polymer micelles of P50−06 might be responsible for such behavior. The SEM images of the P50−06, P50−11, P50−25, and Matrigel were obtained by quenching the gels at 37 °C in the liquid nitrogen (−196 °C), followed by freeze-drying the gel. The highly porous structures of the polymer gels were observed for all systems (Figure 6). The pores can act as mass transport channels through which nutrients are supplied and metabolites are discarded during the 3D cell culture. The pore sizes of 3− 50 μm were observed for the in situ formed gels of P50−06, P50−11, and P50−25. The pore size and size distribution are a complex function of polymer composition, rigidity of the polymer, and evaporation temperature of water as well as initial polymer concentration. The P50−06 exhibited smaller pores than P50−11 and P50−25. The large pore size of the Matrigel system is noticeable. To be defined as mesenchymal stem cells (MSCs), the cells should be plastic-adherent and have the ability of osteogenic, chondrogenic, and adipogenic differentiation.26,47 In particular, the cells should express CD73, CD90, and CD105, but should not express CD117 (c-kit), CD11b, CD14, CD19, CD34, CD45, CD79α, and HLA-DR.48 In the current study, the plastic-adherence property of ADSCs was confirmed during the isolation procedure. The flow-cytometry confirmed that the 3261

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Figure 7. (a) Proliferations of the ADSCs in the hydrogels analyzed by the CCK-8 method. 100% is the cell number on the day when experiments started. The cell number is the average of the triplicate experiments; # and ## indicate p < 0.05 and p < 0.01 (Student t test), respectively, relative to Matrigel; s, ss, and i indicate p < 0.05, p < 0.01, and p > 0.05 (Student t test), respectively, between the comparing groups. (b) Viability of cells in the hydrogel analyzed by the Live/Dead kit. Live (green) and dead (red) images of cells are shown. The scale bar is 50 μm. M indicates Matrigel. 0 day indicates 1 h after the injection.

neurons are nonmesodermal in origin.50 Usually, to induce the neurogenesis in the ADSCs, specific neuroinductive conditions are needed which can be achieved by adding differentiation factors such as azacytidine, valproic acid, insulin, hydroxyanisole, hydrocortisone, epidermal growth factor, or fibroblast growth factor.51 In particular, P50−11 showed significantly higher expression of Col II compared with other polymers and Matrigel (p < 0.01 by the Student t test as denoted ## and ss in Figure 8a). Both P50−11 and P50−25 showed significantly higher gene expressions of βTub III and MyoD 1 compared with Matrigel (Figure 8b,c). The recent findings suggest that not only the stiffness of the gel but also the cell shape, size of cell aggregates, cell−cell contact, and cell density in a matrix affect differentiation of stem cells.52−56 Our hydrogels resulted in different cell morphologies (Figure 7b), which might be a reason for the different differentiation shown in Figure 8. In

addition, cell densities varied among different groups because of the different proliferation rates as shown in Figure 7a. Consequently, the differences in differentiation among the groups are not unreasonable, although the exact reason is not clear. The preferred differentiation of stem cells was reported in cases where the cells are in contact, indicating that a high cell density is usually beneficial for cell differentiation.57 Due to the difference in protein expressions, the gel modulus also can be changed during the cell culture period even though the initial moduli of the gels were similar, which, in turn, can affect cell differentiation. Therefore, the highest expression of Col II in the P50−11 gel might be related to the differences in cell morphologies, high proliferation rate, great cell density, and change in gel modulus among the current hydrogels. Physicochemical cues are reported to induce ADSC differentiation into a specific tissue. Control of the optimal 3262

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in a RGD dose-dependent manner in case RGD-chimeric protein is mixed in the alginate culture system,62 while ADSCs that were cultured in a hyaluronic acid treated poly(lactic acidco-glycolic acid) scaffold showed enhanced expressions of chondrogenic biomarkers including sGAG and Col II in comparison with the use of the untreated polymer scaffold.63 Chondrogenic differentiation was also improved in case TGFβ1 was conjugated to the culture matrix of the hydrogel.64 The ADSCs were cultured in agarose, alginate, and gelatin scaffolds with a modulus of 4−10 KPa, and chondrogenic differentiation was observed in the chondrogenic medium containing TGFβ1.65 ADSCs can be induced into chondrogenesis by pellet culture, which increases cell−cell contacts thereby mimicking the condensation during cartilage development.66 Currently our PEG-L-PA is important in that the chondrogenic differentiation was mainly induced without any other external growth factor, allowing the potential for in vivo application of the system for cartilage repair. To confirm the cell differentiation and tissue compatibility of the ADSC-encapsulated system under in vivo conditions, an ADSC-suspended polymer (P50−11) aqueous solution was injected into the subcutaneous layer of mice, and histological and immunohistochemical analyses were carried out. P50−11 was chosen due to its dimensional stability as a 3D matrix and excellent chondrogenic biomarker expression in the in vitro study. Each system of polymer aqueous solution (0.3 mL) contained 3.0 × 105 cells/mouse, and triplicates for each sampling interval of 0 (1 h after the injection), 3, 7, and 14 days were obtained. The gels remaining on 0, 3, 7, and 14 days after injection are shown in Figure 9a. The gel in the rats decreased

Figure 8. Gene expressions of ADSCs in 3D-cultured samples analyzed by real-time RT-PCR. The biomarkers for chondrogenesis (Col II: a), neurogenesis (β III Tub: b), and myogenesis (Myo D 1: c) are shown. The data were normalized by the GAPDH and 0th day data. The data are presented as the mean ± standard deviation of three independent experiments. # and ## indicate p < 0.05 and p < 0.01 (Student t test), respectively, relative to Matrigel. s, ss, and i indicate p < 0.05, p < 0.01, and p > 0.05 (Student t test), respectively, between the comparing groups. The biomarkers for adipogenesis (LPL) and osteogenesis (OCN) were undetectable. M indicates Matrigel. 0 day indicates 1 h after the injection.

Figure 9. (a) In situ formed gel in vivo mouse model as a function of time. The P50−11 (4.6 wt %) aqueous solution (0.5 mL) containing 3 × 105 cells/mouse was injected in the subcutaneous layer of mice and photos were taken at 0 (0D), 3 (3D), 7 (7D), and 14 (14D) days after the injection. (b) Immunostaining of the tissue around the remaining gel at 14 days after the injection. The biomarkers of general macrophages (CD68), pro-inflammatory macrophages (CCR7), and pro-healing macrophages (CD163) are expressed in brown around the implant. H&E staining is also shown. The scale bar is 100 μm.

calcium concentration of 8 mM in the culture medium induce osteogenic differentiation.58 The addition of VEGF enhanced myogenic differentiation and vascularization.59 The extent of self-renewal, angiogenesis, and adipogenesis in the ADSCs could be controlled by varying the stiffness of the gel.52 The PEG-cyclodextrin inclusion complex has been reported to control the physicobiological properties of the scaffold. By varying the density of the inclusion complex and chemical moieties of the cyclodextrin, the ADSC morphology and gene expression could be controlled.60 The chondrogenic differentiation of ADSCs has been extensively investigated because of the availability of ADSCs as well as the problems of current microfracture-based treatments for cartilage repair. Also, chondrocyte transplantation therapy requires an invasive protocol that damages the joint while obtaining the cells.61 Meanwhile, it has been shown that chondrogenesis is improved

in size by about 30% of its original one over two weeks. Fourteen days after transplantation of the ADSC-encapsulated hydrogel in the subcutaneous layer of mice, immunohistochemical staining for biomarkers of general macrophages (CD68), pro-inflammatory macrophages (CCR7), and prohealing macrophages (CD163) around the implant was performed to investigate the inflammatory response (Figure 9b). In addition, H&E staining was also performed to investigate the features of the cytoplasm, nucleus, and extracellular matrix around the implant (Figure 9b). In Figure 3263

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Figure 10. Histological staining around the implant at 14 days after the injection by alcian blue (A and a), Masson’s trichrome (B and b), toluidine blue (C and c), alizarin red S (D and d), and oil red O (E and e) staining methods. The regions marked by arrows are enlarged (a−e). The gel regions (G) are indicated by the dotted curve. The scale bars in (A−E) and (a−e) are 200 and 40 μm, respectively.

genesis are not significantly developed in the ADSCs encapsulated in the gel. To conclude, the histological investigation suggests that chondrogenesis is the major fate of the ADSCs encapsulated in the gel. The cartilage is not repaired when the injury size is greater than a critical value due to its nonvascular physiology, and thus, the treatments of the cartilage disease have been an important issue for tissue engineering and cell therapy.72 The potential of stem cell therapy using the injectable tissue engineering method has been demonstrated in this study.

9b, G indicates the remaining gel region. The dotted curve indicates the boundary between the gel and the surrounding tissue. Macrophages can have M1 (pro-inflammatory) and M2 (pro-healing) phenotypes. M1 is induced by interferon-γ, lipopolysaccharide, and tumor necrosis factor-α, whereas M2 is stimulated by interleukin-4, -10, and -13 cytokines.67 M1 and M2 are signs of inflammation and constructive tissue remodeling or tissue repair, respectively.68 Currently, we use CCR7 as an M1 surface marker and CD163 as an M2 surface marker.69,70 CD68 is also used as a general macrophage surface marker, which might come from the monocyte-macrophage lineage, including activated monocytes, resting-tissue macrophages, and activated macrophages. In the current study, quantitative analysis was not performed, based on the number of cells expressing both CCR7 (M1) and CD163 (M2) markers; however, the increase in number of M2 macrophages relative to M1 macrophages is evident as shown in the Figure 9b as the thicker brown color of CD163 (pro-healing) than CCR7 (pro-inflammatory). This observation suggests respectively mild tissue inflammation and tissue compatibility of the implanted system. Therefore, a favorable remodeling response of tissue around the implant site seems to occur.71 The H&E stained image shows the infiltration of the cells around the implant 14 days after the injection of the ADSC-encapsulated formulation. To investigate the in vivo biomarker expressions by the differentiation of the ADSCs encapsulated in the gel, the tissue around the remaining gel in the subcutaneous layer of mice was stained by alcian blue, Masson’s trichrome, toluidine blue, alizarin red S, and oil red O staining methods. The remaining gel regions (G) two weeks after transplantation of the gel encapsulating ADSCs are indicated by the dotted curve (A−E), and the regions marked by arrows are enlarged (a−e) in Figure 10. Alcian blue staining clearly indicated the formation of sulphated glucoaminoglycan (sGAG; indicated in blue), which is a biomarker for chondrogenesis (Figure 10A and a). Cytoplasm and nuclei are stained in reddish pink. Masson’s trichrome staining shows that muscle and keratin, which are biomarkers for myogenesis, are not developed well (Figure 10B and b). Toluidine blue shows that there is no significant population of Nissl substance, a biomarker for neurogenesis, which should have appeared in dark blue (Figure 10C and c). Alizarin red S (Figure 10D and d) and oil red O (Figure 10E and e) also did not develop orange to red staining of calcium deposits and lipids, suggesting that osteogenesis and adipo-

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CONCLUSIONS PEG-L-PA diblock copolymers that undergo sol-to-gel transition as the temperature increases were synthesized to compare the delicate differences in thermosensitive behavior, and their potential application in the 3D culture of ADSCs. The molecular weight of L-PA was varied to 620, 1100, and 2480 Da with a fixed molecular weight of PEG at 5000 Da. The UV−vis spectroscopy, dynamic light scattering, and transmission electron microscopy suggested that PEG-L-PAs form micelles 10−50 nm in diameter in water with a hydrophobic L-PA core and hydrophilic PEG shell. Circular dichroism spectroscopy suggested that L-PA of PEG-L-PA forms α-helical secondary structure, and the ellipticity of PEG-L-PA varied depending on the molecular weight of L-PA. In addition, the α-helical secondary structure of L-PA of P50−06 was preserved over the temperature range of 5−60 °C, whereas that of P50−25 turned into random coils as the temperature increased. 13C NMR spectroscopy showed collapsing and a downfield shift of the PEG peak as the temperature increased, suggesting that the dehydration of PEG and decreased molecular motion of the PEG at high temperatures. The increased hydrophobicity and the change in secondary structure of the P50−25 make the solto-gel transition occur at lower concentrations and temperatures than with the PEG-L-PAs with a smaller molecular weight of L-PA. 3D culture systems of ADSCs were compared in the PEG-LPA hydrogels with a similar gel modulus of 600−1000 Pa, which was produced by increasing the temperature of cellsuspended aqueous solutions to 37 °C. Even though the PEGL-PA thermogel was comparable or better than Matrigel for proliferation of the ADSCs, the spherical morphology was maintained in the PEG-L-PA hydrogel, while the cells underwent fibroblastic morphological changes in the Matrigel. In vitro study showed that ADSC showed high expression for the type II collagen, as well as moderate expression of type III 3264

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β-tubulin and myogenic differentiation factor 1 in the PEG-LPA. In vivo study also indicated that chondrogenic differentiation of the ADSCs mainly occurred in the gel implanted in the subcutaneous layer of mice. The preferred chondrogenic differentiation of the ADSCs might be related to the cell morphologies, proliferation rate, cell density, and gel modulus during the 3D culture of the cells in the in situ formed PEG-LPA hydrogel. This study suggests that ADSC/PEG-L-PA is a promising system for the injectable tissue engineering application of the ADSCs.

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR spectra (CF3COOD) of poly(ethylene glycol)-poly(Lalanine) block copolymers, characterization of molecular phenotype of the ADSCs, and stability of hydrogels under in vitro cell culture conditions. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions †

These authors equally contributed to the paper (B.Y.; M.H.P.).

Notes

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 (MSIP; 2009-0080447 and 2012M3A9C6049784).

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