3D culture of tonsil-derived mesenchymal stem cells in poly(ethylene glycol)-poly(L-alanine-co-L-phenyl alanine) thermogel.

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3D Culture of Tonsil-Derived Mesenchymal Stem Cells in Poly(ethylene glycol)-Poly(L-alanine-co-L-phenyl alanine) Thermogel Min Hee Park, Yeonsil Yu, Hyo Jung Moon, Du Young Ko, Han Su Kim, Hyukjin Lee, Kyung Ha Ryu, and Byeongmoon Jeong*

Poly(ethylene glycol)-poly(L-alanine-co-L-phenyl alanine) (PEG-PAF) aqueous solutions undergo sol-to-gel transition as the temperature increases. The transition is driven by the micelle aggregation involving the partial dehydration of the PEG block and the partial increase in β-sheet content of the PAF block. Tonsil-tissue-derived mesenchymal stem cells (TMSCs), a new stem cell resource, are encapsulated through the sol-to-gel transition of the TMSCsuspended PEG-PAF aqueous solutions. The encapsulated TMSCs are in vitro 3D cultured by using induction media supplemented with adipogenic, osteogenic, or chondrogenic factors, where the TMSCs preferentially undergo chondrogenesis with high expressions of type II collagen and sulfated glycosaminoglycan. As a feasibility study of the PEG-PAF thermogel for injectable tissue engineering, the TMSCs encapsulated in hydrogels are implanted in the subcutaneous layer of mice by injecting the TMSC suspended PEG-PAF aqueous solution. The in vivo studies also prove that TMSCs undergo chondrogenesis with high expression of the chondrogenic biomarkers. This study suggests that the TMSCs can be an excellent resource of MSCs, and the thermogelling PEG-PAF is a promising injectable tissue engineering scaffold, particularly for chondrogenic differentiation of the stem cells.

M. H. Park, H. J. Moon, D. Y. Ko, Prof. B. Jeong Department of Chemistry and Nano Science Ewha Womans University Ewha Global Top 5 Research Program 52 Ewhayeodae-gil, Seodaemun-gu Seoul 120–750, Korea E-mail: [email protected] Y. Yu, Prof. H. S. Kim, Prof. K. H. Ryu Departments of Molecular Medicine Otorhinolaryngology – Head and Neck Surgery and Pediatrics School of Medicine Ewha Womans University Ewha Global Top 5 Research Program Seoul, Korea Prof. H. Lee College of Pharmacy Graduate School of Pharmaceutical Science Ewha Womans University Ewha Global Top 5 Research Program Seoul, Korea

DOI: 10.1002/adhm.201400140

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1. Introduction

Embryonic stem cells or induced pluripotent stem cells have drawn much attention due to their full capacity to differentiate into all kind of cells, but the problems of ethical consideration and teratoma formation remain unsolved for practical uses. Adult stem cells such as mesenchymal stem cells (MSCs) can be an alternative solution with better control of cellular differentiation and proliferation.[1,2] MSCs shed very promising light as a future therapeutic agent for regenerative medicine due to their self-renewal property, homing property to abnormal sites, immunosuppressive property, and ability to differentiate into various type of cells.[3,4] Although there are several reports for clinical applications of MSCs, many hurdles still remain to be overcome for practical uses of these stem cells.[5] First of all, a sufficient amount of MSCs from various sources is necessary. Up to now, MSCs have been isolated from bone marrow, muscles, dermises, trabecular bones, adipose tissues, periostea, pericytes, blood, synovial membranes, umbilical cords, and amniotic fluid.[6] Typical bone marrow isolation requires an intentional surgical procedure of the donor, which accompanies pain and potential morbidity of the donor. In addition, during the procedure, the collected MSCs are often exposed to viral and bacterial infection. At last, a significant loss of differentiation and proliferation ability of MSCs with donor ages was also reported.[7] Recently, tonsil-tissue-derived mesenchymal stem cells (TMSCs) have been isolated from human palatine tonsils.[8,9] Unlike bone marrow isolation, TMSCs can be easily obtained from otherwise waste tissues of tonsils. Tonsillectomy is the most common pediatric operation and about 40 000 tonsillectomies are practiced per year in Korea.[9] Therefore, above agerelated problems of MSCs can be also avoided. In addition, the nucleated cell numbers from the tissues are 2 × 106 cells cm−3 and 2 × 107–2 × 108 cells cm−3 for bone marrow and tonsil tissues, respectively, indicating about 10–100 times greater for tonsil tissues than bone marrow.[9,10] Proliferation rate of TMSCs is 2–3 times faster than that of bone-marrow-derived

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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were investigated to prove 1) the potential of the PEG-PAF as an injectable tissue engineering scaffold, and 2) usefulness of TMSCs as a new resource of mesenchymal stem cells.

2. Results and Discussion The PEG-PAF was synthesized by ring opening copolymerization of N-carboxy anhydrides of L-alanine and N-carboxy anhydrides of L-phenyl alanine in the presence of the α-aminoω-methoxy poly (ethylene glycol). The composition and molecular weight of PEG–PAF were calculated by the peaks of 1H NMR spectra at 1.1–2.1 ppm ( CH3 of alanine), 3.5–3.6 ppm ( OCH3 of PEG end group), 3.7–4.1 ppm ( OCH2CH2 of PEG), and 7.1–7.5 ppm ( C6H5 of phenyl alanine) (Figure S1-a, Supporting Information). The molecular weight of each block of PEG-PAF was 1000–650 Daltons with the structure of (ethylene glycol)22.7-[(L-alanine)5.6(L-phenyl alanine)1.7]. The gel permeation chromatogram of PEG-PAF showed a narrow unimodal distribution of the molecular weights with a polydispersity index ( M w / M n ) of 1.3 (Figure S1-b, Supporting Information). The polymer aqueous solutions in a concentration range of 3.0–7.0 wt% underwent sol-to-gel transition as the temperature increased. The transition temperature of the polymer aqueous solutions was determined by the test-tube inverting method by an increment of 1 °C per step and it was reproducible within ±2. The sol–gel transition was reversible in that the PEG-L-PAF hydrogel formed by increasing the temperature of the polymer aqueous solution became a sol by lowering the temperature, and the sol-to-gel transition was observed at the same temperature. The sol-to-gel transition temperature decreased from 28 °C to 12 °C as the polymer concentration increased from 3.0 to 7.0 wt% (Figure 1a). The sol-to-gel transition was also confirmed by the dynamic mechanical analysis. A drastic change in the modulus of the polymer aqueous solution (4.0 wt%) was observed as the temperature changed from 4 °C (sol) to 37 °C (gel) (Figure 1b). In a sol state at 4 °C, the polymer aqueous solution exhibited low modulus (G′) less than 0.02 Pa, whereas the modulus increased to 1200–1400 Pa at 37 °C. We recently reported PEG-PAF thermogels as sustained release systems of insulin and human growth hormone.[30,31] The polymers exhibited gel modulus at 37 °C of 100–150 Pa. Current our PEG-PAF was developed as a 3D cell culture matrix and the gel modulus at 37 °C increased about 10 times by increasing PAF molecular weight from 430 to 650 Daltons and decreasing PEG molecular weight from 2000 to 1000 Daltons. The gel was stiff enough to hold the cells as a 3D hydrogel matrix and the hydrogel showed an excellent durability over the 3D culture period of 3 weeks. The storage modulus (G′) crossed over the loss modulus (G′′) during the sol-to-gel transition at about 20 °C. G′ and G′′ are measures of the elastic component and the viscous component of a complex modulus, respectively. G′ < G′′ in a sol state and G′ > G′′ in a gel state, therefore the crossing of G′ over G′′ was suggested to be an evidence of sol-to-gel transition.[32] The mechanism of sol-to-gel transition of the polymer aqueous solution was investigated by AFM, CD spectroscopy, DLS, FTIR spectroscopy, and 1H NMR spectroscopy. The polymer consisting of hydrophobic PAF and hydrophilic PEG



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MSC.[10] It is also noticeable that the isolated TMSCs undergo not only mesodermal differentiations of osteogenesis, chondrogenesis, and adipogenesis, but also express endodermal genes such as fork head box A2, SIX home box, and chemokine ligand 21 in appropriate induction media.[9] During the traditional 2D culture, they preferentially undergo adipogenic differentiation, with less extent of osteogenic and chondrogenic differentiation.[11] Recently, chimerism from several donors was reported, therefore TMSCs can be mixed without a significant loss of their stem cell properties.[12] Though TMSCs have the above advantages as a resource of MSCs, few reports of TMSC use exist, and more intensive studies should be explored urgently on the unique tissue-derived stem cell. Biochemical growth factors of proteins and small molecules have been investigated to regulate the stem cell differentiation into a specific cell type.[13] For example, TGFβ, dexamethasone, and ascorbic acid glycerol phosphate induce osteogenesis of MSCs.[14] In addition, it is found that physicochemical characteristics of scaffolds can affect stem cell differentiation. Chemical functional groups, stiffness of the hydrogel, shape and size of nano- and micropatterned substrates, diameter of incorporated nano fibers, etc have been reported to affect the stem cell differentiation.[15–18] However, the development of a new bio-inspired scaffold that can direct the stem cell differentiation into a specific type of cells or tissues remains technically challenging. Thermogelling polymer aqueous solutions undergo sol-to-gel transition as the temperature increases. Therefore, cells can be encapsulated in the 3D hydrogel matrix by increasing the temperature of the cell-suspended polymer aqueous solution.[19–28] The thermogelling system avoids the use of surgical procedures, and the 3D scaffold can be prepared by a conventional syringe injection at a target site through the sol-to-gel transition of the system. The in situ gelling system of thermogel can easily take the irregular shape of the cavity or the disease site, and differentiating factors including growth factors can be also simply incorporated in the 3D matrix through sol-to-gel transition. Therefore, the thermogelling polymer aqueous solution is considered to be very promising as a minimally invasive injectable tissue engineering scaffold. Compared with chemically or photochemically crosslinking systems, thermogelling systems can reversibly undergo sol-gel transition and provide a mild procedure for stem cell encapsulation without exposure to radical, UV light, or specific functional groups, which might affect the viability, proliferation, or differentiation of the stem cells.[29] Considering advantages and urgency of TMSCs research and potential of novel polypeptide-based thermogels as a tissue engineering scaffold, here we report the 3D culture and differentiation of TMSCs in a PEG-PAF thermogel. The thermogelling PEG-PAF system is particularly important in that there is no change in pH during the degradation and can be degraded by mammalian enzymes such as elastase, cathepsin B, and cathepsin C, and matrix metalloproteinase.[30,31] In this paper, the characteristics of the sol-to-gel transition were studied using various instruments such as dynamic mechanical analysis, atomic force microscope (AFM), circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), FTIR spectroscopy, and 1H NMR spectroscopy. In addition, the characteristics of the TMSCs under in vitro and in vivo 3D culture conditions

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Gel

30 20

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10

2

3 4 5 6 7 8 Concentration (wt. %)

10

60

37 oC

40

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50

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G"

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-CH2CH2O-

HDO

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0.07 0 10 20 30 40 50 Tem perature (oC) o

0.4

10 C o 20 C 30 oC o 40 C 50 oC

50 oC

0.3 1700 1680 1660 1640 1620 1600 -1 Wavenumber(cm )

5.0

4.7

4.4

4.1

3.8

3.5

ppm

Figure 1. a) Phase diagram of PEG-PAF aqueous solutions. b) Modulus of the PEG-PAF aqueous solution (4.0 wt%) as a function of temperature. c) CD spectra of the PEG-PAF aqueous solution (0.10 wt%) as a function of temperature. d) Distribution of apparent size of PEG–PAF nanoassemblies in water (0.10 wt%) measured by DLS as a function of temperature. e) FTIR spectra of PEG-PAF aqueous solution (4.0 wt% in D2O) as a function of temperature. f) 1H NMR spectra of PEG-PAF aqueous solution (4.0 wt% in D2O) as a function of temperature.

formed micelles in water. The PEG formed a shell and the PAF formed the core of the micelles. Spherical micelle assemblies with a diameter of 20–30 nm were observed by the AFM (Figure S2, Supporting Information). The AFM samples were prepared through the spin-coating of a polymer aqueous solution (0.01 wt%) on the silicone wafer, followed by air-drying of the solution at 20 °C. Even though there might be some 1784


distortion of the micelle during the air-drying process, the spherical micelles were apparent in the images. CD spectra of PEG-PAF aqueous solutions were investigated at 0.01 wt% and 0.10 wt%. CD spectra at low polymer concentration (0.01 wt%) exhibited a minimum at 218 nm, suggesting that the dominant secondary structure of PAF of PEG-PAF is a β-sheeted structure (Figure S3, Supporting Information).[33]





ammonium-chloride-potassium (ACK) lysis, and adhesion to culture plate. The surface markers of TMSCs were analyzed using flow cytometry, where they exhibited negative expressions of hematopoietic stem cell markers of CD14, CD34, and CD45, but positive expressions of mesenchymal stem cell markers for CD73, CD90, and CD105, indicating that the isolated TMSCs are a kind of mesenchymal stem cells (Figure S6, Supporting Information).[37,38] The TMSCs were encapsulated in the gels by increasing temperature of the polymer aqueous solution to the cell culture temperature of 37 °C. The TMSCs were cultured in the in situ formed gel by using specific media inducing adipogenesis (A), osteogenesis (O), chondrogenesis (C). As a control, the TMSCs cultured in the hydrogels by using the basal growth medium (G) without addition of any specific inducing medium was also compared. A detailed composition of each induction media is listed in Table 1. Live/Dead assay was conducted to investigate the cell viability during the 3D culture of the TMSCs. Cell viability and proliferation over 21 d were monitored by staining live cells (green) with calcein AM and dead cells (red) with ethidium homodimer-1 (ethD-1). Over the 21 d of incubation, the TMSCs in the gels showed excellent viability

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On the other hand, the secondary structural information is lost in the CD spectra at high concentration (0.10 wt%). Instead, a red-shifted single band with a minimum at 223–226 nm was observed in the CD spectra, indicating the formation of selfassembled micelles.[34] The ellipticity at 223–226 nm drastically decreased as the temperature increased from 5 °C to 50 °C, indicating that there was a change in self-assemblies as the temperature increased (Figure 1c). DLS study showed the increase in the size from 20–70 nm to 60–150 nm as the temperature increased from 5 °C to 40 °C. As the temperature further increased to 45 °C and 50 °C, the micelle size increased to 800–2300 nm and 1400–3000 nm, respectively, indicating that the extensive aggregation of micelles is a thermally driven process caused by the PEG dehydration, and accompanies changes in secondary structure of polypeptide, as will be discussed (Figure 1d). At high concentrations, the aggregation of micelles is to be more extensive to form a 3D network of gel, suggesting that sol-to-gel transition is driven by the micellar aggregation. FTIR spectra at 1625 cm−1, 1640 cm−1, and 1650 cm−1 are the measure of the β-sheet, random coil, and α-helix structures of a polypeptide, respectively.[35] To avoid the spectral interference in amide I band, D2O was used as a solvent because it has no absorbance around 1600–1800 cm−1. Basically, the sharp peak at 1625 cm−1 of PEG-PAF aqueous solution (4.0 wt%) suggested that the dominant secondary structure of PAF is a β-sheet structure over the temperature range of 5–50 °C (Figure 1e). A partial increase in the absorbance at 1625 cm−1 relative to 1640 cm−1 suggested that β-sheet structure was strengthened as the temperature increased from 5 °C to 50 °C (Figure 1e insert). 1 H NMR spectra of PEG-PAF in D2O showed the collapsing of PEG peak and down field shift of the PEG peak from 3.72 to 3.74 ppm as the temperature increased from 10 °C to 50 °C. Interestingly, the water (HDO) peak at 4.95 ppm upfield shifted to 4.59 ppm as the temperature increased from 10 °C to 50 °C (Figure 1f). Such changes indicated that dehydration of the PEG and consequent changes in the water–PEG interactions.[36] In addition, the alanine peak at 1.1–1.7 ppm and phenyl alanine peak at 7.1–7.6 ppm of PAF of the 1H NMR spectra of PEG-PAF (in D2O) also collapsed as the temperature increased from 10 °C to 50 °C (Figure S4, Supporting Information). Based on above information, it can be concluded that solto-gel transition of the PEG-PAF aqueous solution is driven by the micelle aggregation involving the partial dehydration of the PEG block and the partial increase in β-sheet content of PAF block. Microstructure of the PEG-PAF thermogel was obtained by quenching the gel at 37 °C into the liquid nitrogen (−196 °C), followed by freeze-drying of the gel. The scanning electron microscopy (SEM) image exhibited highly porous structures with micropores (Figure S5, Supporting Information). Considering that the PEG-PAF aqueous solution (4.0 wt%) formed a transparent hydrogel with no shrinkage at 37 °C, the remaining 96% of the gel was filled with water. It is likely that the environment of encapsulated cells in the gel is physiologically relevant to native tissues that pores can act as a channel for exchange of nutrients, oxygen, and cell waste. From palatine tonsil tissues of 11 years old female donor, TMSCs were isolated through collagenase digestion,

Table 1. List of media compositions. Basal medium Growth medium (G)

DMEM-HG FBS

10%

PS

1%

AA

1%

Adipogenesis induction medium (A)

MEM

50 µL adipogenic supplement/5 mL basal medium

FBS

10%

PS

1%

AA Osteogenesis induction medium (O)

Chondogenesis induction medium (C)

Induction medium

1% MEM

250 µL osteogenic supplement/ 5 mL basal medium

FBS

10%

PS

1%

AA

1%

DMEM/F-12

25 µL chondrogenic supplement/2.5 mL basal medium

FBS

1%

PS

1%

AA

1%

DMEM-HG, Dulbeccos modified eagles medium, high glucose; MEM, minimum essential medium; DMEM/F-12, Dulbecco’s modified eagle medium: nutrient mixture F-12 FBS, fetal bovine serum; PS, penicillin/streptomycin; AA, antibioticantimycotic. Supplements in the induction media are as follows. Adipogenic supplement contains hydrocortisone, isobutyl methyl xanthine, indomethacin, and other factors. Osteogenic supplement contains dexamethasone, ascorbate phosphate, β-glycerol phosphate, and other factors. Chondrogenic supplement contains dexamethasone, ascorbate phosphate, proline, pyruvate, TGF- β3, and other factors. However, the exact concentrations of each component are not disclosed by the vendor (R&D systems, USA).



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4

Relative cell density

in all incubating conditions of A, O, C, and G (Figure S7, Supporting Information). The cell density increased about two times over 21 d of incubation in the A, O, C, and G media except the C medium at 21st day (Figure 2a). The decrease in cell density in the C medium at 21st day might be related to the differentiation of the stem cells instead of proliferation, as will be discussed in biomarker expression in the C medium.[39] Differences in the cell morphology of encapsulated cells were observed depending on the induction media after staining with calcein AM (Figure 2b). After 21 d of cell culture, spherical cells with protruded fiber-like morphologies were observed. In particular, such fibrous branches formed as early as 3 d of incubation in C medium, and the number of branches from the cell increased with incubation time (Figure 2c). In the adipogenesis induction medium, the cells maintained their spherical shape up to 7 d, which is longer than in the osteogenesis or chondrogenesis induction medium. Previously, it was reported that MSCs tend to differentiate into adipocytes while maintaining their round morphology.[5] Changes in cell morphology can induce changes in cytoskeletal organization and affect cell differentiation.[40,41] Therefore, variations in the cell proliferation rate and cell morphologies might indicate the differentiation of the stem cells to different lineages. Regulatory molecules such as growth factors and cytokines are important for the control of stem cell differentiation. They can be added to the culture media as an excipient or can be secreted by the cells. TMSCs were cultured in induction media supplemented with adipogenic (A), osteogenic (O), or chondrogenic (C) factors provided (R&D systems, USA). TMSCs were also cultured in the basal growth medium (G) in the absence of induction supplements. The gene expressions from TMSCs were investigated for adipogenesis, osteogenesis, and chondrogenesis. The sequences of primers for real-time reverse transcription polymerase chain reaction (RT-PCR) are shown in Table 2. As for the TMSCs cultured in the adipogenesis induction medium, specific genes of adipogenic differentiation including PPARγ, LPL, and aP 2 were investigated. Expression levels of those specific genes were not apparent except for aP 2 in 21st day of culture (Figure 3a). Specific genes of osteogenic differentiation including Runx 2, ALP, and OCN were investigated for the TMSCs cultured in the osteogenesis induction medium. Expression levels of osteogenic genes were not significant except for third days of culture (Figure 3b). The gene expression of adipogenesis and osteogenesis was about two times higher in each induction medium than basal growth media in the absence of induction supplement (G) (Figure S8-a and S8-b, Supporting Information). To investigate the chondrogenic differentiation, gene expression of AGG, Col II, and Col X was studied. The small expression levels for AGG (early marker) and Col X (late maker) were observed, whereas a very high expression level for Col II was observed (Figure 3c). The Col II expression level in the chondrogenesis induction medium was four times higher than the basal growth medium (G) (Figure S8-c, Supporting Information). Tissue-specific ECM production in the 3D hydrogel matrix by differentiated cells was analyzed for evaluating the lipid globules, calcium deposits, and sulphated glycosaminoglycans (sGAGs), which are biomarkers of adipogenesis, osteogenesis, and

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14 12

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A

O

C

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*

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21d

8 6 4 2 0 0d

3d

7d

Figure 2. a) Relative cell density in the hydrogels normalized by the cell density at the zeroth day. The cell density was assayed by the image analysis of the cells developed by the Live/Dead assay kit. (mean ± SE, n = 3; ANOVA: p < 0.05; * vs G). b) Morphologies of cells cultured in the different media of G, A, O, and C which is defined in the Table 1. The scale bar is 10 µm. c) Number of branches per cell. (mean ± SE, n = 5; ANOVA: p < 0.05; * vs. G). 0d (1 hour), 3d (3 days), 7d (7 days), 14d (14 days), and 21d (21 days) indicate time elapsed after 3D culture of the TMSCs started.

chondrogenesis, respectively. Oil red O stains intracellular lipid globules as a red color, alizarin red S stains calcium deposits as



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Genes

Primer sequences

PPARγ

F: 5′-GCTGTTATGGGTGAAACTCTG-3′ R: 5′-CTCGGACGTAGAGGTGGAAT-3′

LPL

F: 5′-AACAATCTGGGCTATGAGATC-3′ R: 5′-TGAATCTTTACTTGGTAATGGA-3′

aP 2

F: 5′-TGGTTGATTTTCCATCCCAT-3′ R: 5′-CAGTTTAAGGACCGGGTCAT-3′

Runx 2

F: 5′-GGGCACAAGTTCTATCTGGAA-3′ R: 5′-CGGTGTCACTGCGTGAA-3′

ALP

F: 5′-GACATCGCCTACCAGCTCAT-3′ R: 5′-TCACGTTGTTCCTGTTCAGC-3′

OCN

F: 5′-AGGAGGCTGGCAGCTGTGTG-3′ R: 5′-CACTGGCAGTGGCGAGGTCA-3′

Col X

F: 5′-CAAGGCACCATCTCCAGGAA-3′ R: 5′-AAAGGGTATTTGTGGCAGCA-3′

GAPDH

F: 5′-ATGGGGAAGGTGAAGGTCG-3′ R: 5′-TAAAAGCAGCCCTGGTGACC-3′

PPARγ, peroxisome proliferator-activated receptor-gamma; LPL, lipoprotein lipase; aP 2, adipocyte protein 2; Runx 2, Runt-related transcription factor 2; ALP, alkaline phosphatease; OCN, osteocalcin; AGG, aggrecan; Col II, collagen type II; Col X; collagen type X; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase.

Relative mRNA expression

F: 5′-CCCGCTACGACGCCATCTG-3′



an orange color, and alcian blue stains sGAGs as a blue color. The histological analysis confirmed that there was no significant expression for lipid globules and calcium deposits, however, a significant expression of sGAGs was seen (Figure 4a). Immunofluorescence staining of PPARγ, OCN, and Col II developed in the TMSCs encapsulated in the 3D hydrogel matrix was carried out to further evaluate adipogenesis, osteogenesis, and chondrogenesis, respectively. 4′,6′-diamidino2-phenylindole (DAPI) and Alexa Fluor 488 phalloidin stain the nucleus and the filamentous actin of the cell as blue and green colors, respectively.[43] The PPARγ, OCN, and Col II, if any, should appear as a red color by the immunofluorescence staining. Both PPARγ and OCN were not apparently observed, whereas the Col II was highly stained as a red color (Figure 4b). Both histology and immunofluorescence staining indicated that TMSCs underwent chondrogenic differentiation in the chondrogenesis induction medium, whereas there were no significant increases in adipogenesis and osteogenesis in each induction medium. Stem cell differentiation into a specific cell type can be affected by biophysical parameters of the scaffold as well as the soluble factors including growth factors or small molecules.[13] Recently, a significant progress on the materials parameters

LPL

aP 2

0.02 0.00

0.08

3d

b)

7d

14d

21d

Runx 2

ALP

OCN

7d

14d

21d

0.06 0.04 0.02 0.00 0d

[42]

PPARγ

0.04

0d

R: 5′-CGGGCGGTAGTGGAAGACG-3′ Col II

a)

0.06

F: 5′-TGAGAGCCCTCACACTCCTC-3′ R: 5′-ACCTTTGCTGGACTCTGCAC-3′

AGG

0.08

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Table 2. Real-time RT-PCR primers for differentiation-specific gene expression analysis.



0.16 0.26 0.14 0.24

3d

c)

AGG

Col II

Col X

7d

14d

21d

0.12 0.22 0.10 0.20 0.08 0.06 0.04 0.02 0.00 0d

3d

Figure 3. Gene expression of 3D cultured TMSCs in the a) adipogenic, b) osteogenic, and c) chondrogenic induction media. Gene expression analyzed by real-time RT-PCR. The data were normalized by the GAPDH. n = 3. 0d (1 hour), 3d (3 days), 7d (7 days), 14d (14 days), and 21d (21 days) indicate time elapsed after 3D culture of the TMSCs started.



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Figure 4. a) Histology of cell-embedded 3D matrix stained by oil red O (A), alizarin red S (O), and alcian blue (C) which stain lipid globule, calcium deposit, and sulfated glycosaminoglycan in the ECM, respectively. b) Immunofluorescence of PPARγ (A), OCN (O), and Col II (C), which are typical biomarkers for adipogenic, osteogenic, chondrogenic differentiation, respectively. DAPI and phalloidin stain nucleus and actin of a cell, respectively. Each anti-antibody (Antibody) of PPARγ, OCN, and Col II stains the corresponding protein by binding to the antibody of each protein. The scale bar is 20 µm. The histology and immunofluorescence were assayed 21 days after 3D culture.

affecting stem cell differentiation has been reported.[15–18] MSCs undergo osteogenesis, adipogenesis, and chondrogenesis in the phosphate, t-butyl, and carboxylic acid functional-groups- modified PEG hydrogel.[15] MSCs undergo preferential osteogenesis in the TiO2 nanotubes with 15 nm in diameter.[16] In addition, neurogenesis, myogenesis, and osteogenesis were observed as the hydrogel stiffness increased from 0.1–1 KPa to 25–40 KPa.[17] However, such a trend observed for polyacrylamide-based hydrogel was not observed in poly(dimethylsiloxane) system.[18] It is important to note that the adhesion between intracellular cytoskeleton and extracellular substrate is an essential factor that mediates the change in cell morphology and triggers the mechanotransduction for stem cell differentiation.[44] Several thermogelling systems have been investigated for stem cell encapsulation and injectable tissue engineering applications. The MSCs undergo osteogenic biomarker expression of osteocalcin and Col I in the RGD-conjugated polyphosphazene thermogel as well as polycaprolactone thermogel.[21,22] When

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VEGF was incorporated in the polycaprolactone thermogel, myogenic biomarker expression was dominantly observed.[23] MSC and BMP-2 were encapsulated in a pH-/temperaturesensitive thermogel of sulfamethazine oligomer end-capped polycaprolactone, where the alkaline phosphatise activity, von Kossa staining, and alizarin red S staining suggested that MSC underwent osteogenic differentiation.[24] MSCs encapsulated in the chitosan/glycerol phosphate thermogel expressed chondrogenic-to-nucleus pulposus biomarkers.[25] The differences in MSC differentiation into a specific cell type in above thermogels are open for discussion. Other than the incorporated growth factors, stem cell differentiation can be affected by the functional groups and stiffness of the gel, density of gel network, cell morphology, and cell–cell contact in a matrix.[15,17,45,46] In addition, the gel modulus can be changed during the cell culture period due to the difference in proteins expressions in the gel matrix, which, in turn, can affect stem cell differentiation. The differences in functional groups such as phosphate and RGD groups in the RGD-conjugated polyphosphazene, ester, and sulfamethazine groups in the sulfamethazine oligomer end-capped polycaprolactone, glucosamine, and phosphate groups in the chitosan/glycerol phosphate, and hydroxyl and ester groups in the polycaprolactone can also affect the stem cell differentiation. Amino groups on the self-assembled monolayer of the ω-functionalized alkane thiol/gold system were reported to induce osteogensis.[47] The amino end group of the PEG-PAF might be responsible for small induction of osteogenesis in the PEG–PAF thermogel system. However, current PEG-PAF thermogelling system is unique in that it is proved to be excellent for chondrogenic differentiation with high expressions of Col II and sGAGs. In particular, we have observed unique branching of the cells in a chondrogenic medium, such changes in cell morphology might be involved in preferential chondrogenic differentiation of TMSCs. In current stage, the exact reason for preferential chondrogenic differentiation of TMSCs in the PEG-PAF thermogel is not clear. However, all above factors including stiffness of the gel, density of gel network, cell morphology, and functional groups of phenyl and amide/amine in the current PEG-PAF might be suggested as potential reasons. Previously, we reported the polypeptide thermogel provided an excellent microenvironment for the proliferation of isolated chondrocytes.[19,48] Therefore, the PEG–PAF can be an excellent platform not only for TMSCs differentiation into chondrocytes but also for proliferation for the chondrocytes therein. Based on the in vitro results, the TMSCs/PEG-PAF thermogel was investigated as an in vivo injectable tissue engineering system. A hydrogel implant containing TMSCs was formed in the subcutaneous layer of mice by injecting TMSCsuspended polymer aqueous solution through the temperature-sensitive sol-to-gel transition. In the in vivo implant, gene expression of AGG, Col II, and Col X was significantly assayed (Figure 5a). In particular, Col II gene was dominantly expressed over the all incubation period. Alcian blue staining of the implanted gel over 21 d in mice exhibited that sGAGs (blue) production around the cells (red) significantly increased, compared with the gel incubated over 3 d (Figure 5b). The in vivo data also suggested that implanted TMSCs in the PEG-PAF hydrogel underwent chondrogenesis.




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For target tissue regeneration, the practical protocol to regulate the stem cell differentiation into a specific cell type should be developed. And, various resources of stem cells and efficient methods to supply a sufficient number of stem cells should be developed. Bio-nanotechnologies and understanding on biochemical cues for material–cell interactions have greatly improved our strategies in designing of a new biomaterial for stem- cell-based therapy. Both in vitro and in vivo studies suggest that TMSCs can be an excellent stem cell resource and the TMSC/PEG-PAF system provides an excellent platform for chondrogenic differentiation of TMSCs. The gel stiffness, chemical functional groups of the polymer, and incorporation of biochemical factors can be further optimized for practical applications as a cartilage tissue repairing system.

4. Experimental Section

Figure 5. Chondrogenic differentiation of TMSCs cultured in in vivo nude mouse model. The PEG–PAF (4.0 wt%) aqueous solution (0.3 mL) containing 0.3 × 106 cells/mouse was injected in the subcutaneous layer of nude mice. a) Gene expression of TMSCs per recovered gels analyzed by real time RT-PCR. n = 3. The data were normalized by the GAPDH data. b) Histological staining of the cell-encapsulated gels by the alcian blue staining method at 3d (3 days) and 21d (21 days) after the injection. The scale bar is 20 µm.

3. Conclusions The PEG-PAF aqueous solution underwent sol-to-gel transition as the temperature increased. Mechanism of the sol-to-gel transition was studied by dynamic mechanical analysis, AFM, CD spectroscopy, DLS, FTIR spectroscopy, and 1H NMR spectroscopy. The AFM images and DLS study indicated that the amphiphilic PEG-PAFs formed spherical micelles in water. CD and DLS studies performed as a function of temperature suggested that the micelles underwent significant aggregation as the temperature increased. FTIR and 1H NMR spectroscopy studies carried out as a function of temperature also suggested that β-sheet content of the PAF block partially increased and the PEG block dehydrated as the temperature increased. TMSCs were encapsulated in situ in PEG-PAF gels by increasing the cell-suspended aqueous solution of the PEGPAF. TMSCs were 3D cultured in induction media supplemented with adipogenic (A), osteogenic (O), or chondrogenic (C) factors. The mRNA expressions of the encapsulated cells were investigated for adipogenic (PPARγ, LPL, and aP 2), osteogenic (Runx 2, ALP, and OCN), or chondrogenic (AGG, Col X, and Col II) biomarkers. In addition, immunofluorescence for PPARγ, OCN, and Col II expressions was investigated. Both mRNA and protein expressions suggested that the TMSCs preferentially underwent chondrogenesis with particularly high expressions of Col II and sulfated glycosaminoglycan. In addition, when TMSCs encapsulated in hydrogels were implanted in the subcutaneous layer of mice, similar results were observed.


Materials: α-amino-ω-methoxy PEG (M. W. = 1000 Daltons, IDB Chem), N-carboxy anhydrides of L-alanine, and N-carboxy anhydrides of L-phenyl alanine (KPX life, Korea) were used as received. Chloroform was dried over anhydrous magnesium sulfate (Daejung, Korea) before use. Anhydrous N,N-dimethyl formide was used as received from SigmaAldrich. Toluene was distilled over sodium before use. The cell counting kit-8 (CCK-8, Dojindo Laboratories, Japan), the Live/Dead assay kit (Molecular Probes, Invitrogen, USA) was used as received. Synthesis of PEG-PAF: PEG-PAF was synthesized by the ring opening polymerization of N-carboxy anhydrides of alanine and N-carboxy anhydrides of phenyl alanine in the presence of α-amino-ω-methoxy PEG. 1H NMR Spectroscopy: 1H NMR spectra in CF COOD (500 MHz 3 NMR spectrometer, Varian) were used to determine the composition and number average molecular weight of the polymer. In addition, 1H NMR spectra in D2O was obtained at 10, 20, 30, 40, and 50 °C to study the conformational change of the polymer in water as a function of temperature. The polymer aqueous solution was equilibrated for 20 min at each temperature. Gel Permeation Chromatography (GPC): The 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 a molecular weight range of 400–20 000 Daltons were used as the molecular weight standards. A Styragel HR 4E column (Waters) was used. Phase Diagram: The sol–gel transition temperature of the polymer aqueous solution was determined by the test-tube inverting method. The aqueous polymer solution (0.5 mL) was put in a test-tube with an inner diameter of 11 mm. The transition temperature was determined by the flow (sol)-no flow (gel) criterion when the test tube was inverted by an increment of 1 °C per step. Each data point is an average of three measurements. Dynamic Mechanical Analysis: The storage modulus of the polymer aqueous solution (4.0 wt%) was investigated by dynamic rheometry (Rheometer RS 1; Thermo Haake) as a function of temperature. The temperature program was set at 4 °C for 17 min, heating to 37 °C at a heating rate of 1 °C min−1, followed by maintaining at 37 °C for 30 min. The aqueous polymer solution was placed between parallel plates with 25 mm in diameter and a gap of 0.5 mm. The data were collected under controlled stress (4.0 dyn cm−2) and frequency of 1.0 rad s−1. Atomic Force Microscopy: The polymers solutions (0.01 wt%) were spin-coated on the silicone wafer at 5000 rpm at 20 °C for 60 s, and then dried in air. The images of the polymers on the silicone wafer were obtained in a tapping mode by AFM (Veeco Dimension 3100, Digital instruments Ltd, USA). The tapping mode AFM probe with a force constant of 20 N m−1 was scanned over the sample.



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CD Spectroscopy: Ellipticities of the PEG-PAF aqueous solutions (0.01 and 0.10 wt%) were obtained by a circular dichroism instrument (J-810; JASCO) as a function of temperature in a range of 5–50 °C with an increment of 5 °C each step. Dynamic Light Scattering: The apparent size of a polymer or polymer aggregates in water (0.10 wt%) was studied by a dynamic light scattering instrument (ALV 5000–60×0) as a function of temperature. A YAG DPSS200 laser (Langen, Germany) operating at 532 nm was used as a light source. The results of dynamic light scattering were analyzed by the regularized CONTIN method. FTIR Spectroscopy: IR spectra (FTIR spectrophotometer FTS-800; Varian) of the PEG-PAF aqueous solutions (4.0 wt% in D2O) were investigated as a function of temperature in a range of 5–50 °C with an increment of 5 °C each step. Scanning Electron Microscopy: The SEM image of a PEG-PAF gel at 37 °C was obtained after the aqueous polymer solution (4.0 wt%) was dropped on the silicon wafer, and incubated at 37 °C in the oven for 20 min. And then, the gel at 37 °C was quenched into liquid nitrogen at −196 °C, and then freeze-dried. Scanning electron microscopy images were obtained by the field emission scanning electron microscopy (FESEM) instrument (JSM-6700F, JEOL Ltd., Japan). Isolation and Characterization of TMSCs: TMSCs were isolated from palatine tonsils of 11 years old female donor (IRB approval code: ETC 11–53–02) at the Ewha Womans University Mokdong Hospital (Seoul, Korea) following the ethical guidelines of the University. Briefly, after obtaining the informed consent form from the donor, the tonsils were collected from the patient by tonsillectomy. The tonsil tissues were minced and digested in RPMI-1640 (Invitrogen, USA) containing 210 U mL−1 collagenase type I (Invitrogen) and 10 g mL−1 DNase (SigmaAldrich, USA) for 30 min at 37 °C. Digested tissues were subjected to filtration through a wire mesh, and the cells were then washed twice in RPMI-1640/20% normal human serum (NHS; PAA Laboratories GmbH, Austria) and once more in RPMI-1640/10% NHS. From these cells, mononuclear cells were obtained by Ficoll-Paque (GE Healthcare, UK) density gradient centrifugation. Cells were plated at a density of 108 cells in a T-150 culture flask in Dulbecco's modified Eagle’s medium-high glucose (DMEM-HG; Invitrogen) containing 10% fetal bovine serum (FBS; Invitrogen), 100 µg mL−1 streptomycin and 100 U mL−1 penicillin. After 48 h, nonadherent cells were removed and adherent mononuclear cells were replenished with new culture medium. To confirm molecular phenotype of the TMSCs, surface markers of the cells were quantified by flow cytometry (FACS Calibur system, Becton Dickinson, USA). The antibodies used for flow cytometry were hematopoietic cell markers (CD14, CD34 and CD45) and stem cell markers (CD73, CD90 and CD105).[37,38] All antibodies were purchased from Biolegend (San Diego, USA) and used at a concentration of 2 µg mL−1 for staining. 3D Cell Culture: TMSCs were cultured in DMEM-HG containing 10% fetal bovine serum and 1% penicillin/streptomycin under a 5% CO2 atmosphere at 37 °C and were subcultured to passage 5. The cells were maintained during 7 d in basal media before 3D cell culture began. Harvested cells (passage 6, 0.2 × 106 cells) mixed with PEG-PAF aqueous solution (4.0 wt%; 0.2 mL) were incubated in 24 well culture plates at 37 °C for 1 h, during which TMSCs were encapsulated by the sol-to-gel transition. For differentiation studies, TMSCs were 3D cultured in the PEG-PAF gel under a 5% CO2 atmosphere at 37 °C by using adipogenic, osteogenic, or chondrogenic induction media (R&D Systems, USA) described in Table 1, and the media were replaced every 3 d. The morphology in PEG–PAF hydrogels was determined by analyzing the images taken by the Olympus IX71 fluorescence microscope, where live cells (green) were stained with calcein AM and dead cells (red) were stained with ethD-1. Relative cell density was determined using Olympus DP2-BSW by counting the number of live cells in the images and comparing the data at the sampling day with the zeroth day. At the sampling time of 0 (1 h), 3, 7, 14, and 21 d of cell culture, the total RNA content was extracted from the cell encapsulated in gels using the TRIZOL reagent (Invitrogen, USA), according to the manufacturer’s protocol. The extracted RNA pellet was dissolved in nuclease-free water, and the RNA quality and concentration were determined using


the Experion system (Bio-Rad, USA). After synthesizing the cDNA from the isolated RNA, real-time RT-PCR was performed by the CFX96 system using the IQ SYBR Green Supermix. The sequence of primers of peroxisome proliferator-activated receptor-gamma (PPARγ), lipoprotein lipase (LPL), adipocyte protein 2 (aP 2), runt-related transcription factor 2 (Runx 2), alkaline phosphatase (ALP), osteocalcin (OCN), aggrecan (AGG), collagen type II (Col II), collagen type X (Col X), and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) are listed in Table 2. All data were normalized by the GAPDH level. For histology and immunofluorescence staining of the cell-encapsulated 3D matrix, the matrices at each time point were embedded in optimum cutting temperature (OCT) compound (Sakura Finetek USA, USA). The cryosections with 10 µm thickness were stained with oil red O, alizarin red S, and alcian blue for histological evaluation. Immunofluorescense was conducted using PPARγ, OCN, and Col II (Abcam, UK) primary antibody. The samples were rinsed with phosphate buffered saline after which they were incubated in Goat Anti-Rabbit IgG H&L (DyLight 550) and Goat Anti-Mouse IgG H&L (DyLight 549) (Abcam, UK). Secondary antibody was applied, followed by phosphate buffered saline washes and subsequent incubation with Alexa Fluor 488 phalloidin and 4′,6'-diamidino-2-phenylindole (DAPI, Molecular Probes). Labeled cells were then viewed under an Olympus IX71 fluorescence microscope and images were captured using Olympus DP2-BSW. In Vivo Feasibility Study: TMSCs (passage 6, 0.3 × 106 cells with chondrogenic supplements) were mixed with 4.0 wt% of polymer (0.3 mL) in sol state and then subcutaneously injected at the back of female mice (BALB/c) with average body weight of 20 g. After 3, 7, 14, and 21 d of incubation, the mice were sacrificed and the implanted hydrogels were removed. Real-time RT-PCR was conducted for hydrogels according to the manufacturer's instructions for gene expressions of AGG, Col II, and Col X. The hydrogels were also fixed in 10% neutral buffered formalin for histological evaluation. Then, they were embedded in paraffin, followed by sectioning with a blade. The sections were deparaffinized and stained with alcian blue. Animal Procedure: All experimental procedures using animals were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Committee of Ewha Womans University. Statistical Analysis: Quantitative data were expressed as mean ± standard error of the mean (SE). The significance of differences in the mean values was evaluated using the one-way ANOVA with Tukey tests. Differences were considered significant when the p value was less than 0.05 (marked as *).

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (MSIP) (2012M3A9C6049835). Received: March 11, 2014 Revised: June 1, 2014 Published online: June 24, 2014

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