Article pubs.acs.org/Biomac
3D Tissue Engineered Supramolecular Hydrogels for Controlled Chondrogenesis of Human Mesenchymal Stem Cells Hyuntae Jung,† Ji Sun Park,‡ Junseok Yeom,§ Narayanan Selvapalam,∥ Kyeng Min Park,∥ Kyunghoon Oh,∥ Jeong-A Yang,§ Keun Hong Park,*,‡ Sei Kwang Hahn,*,†,§ and Kimoon Kim*,†,∥ †
School of Interdisciplinary Bioscience and Bioengineering, §Department of Materials Science and Engineering, and ∥Center for Self-Assembly and Complexity, Institute for Basic Science, Department of Chemistry, Division of Advanced Materials Science, Pohang University of Science and Technology (POSTECH), San 31, Hyoja-dong, Nam-gu, Pohang, Kyungbuk 790-784, Korea ‡ Department of Biomedical Science, College of Life Science, CHA University, Bundang-gu, Seongnam, 463-840, Korea S Supporting Information *
ABSTRACT: Despite a wide investigation of hydrogels as an artificial extracellular matrix, there are few scaffold systems for the facile spatiotemporal control of mesenchymal stem cells (MSCs). Here, we report 3D tissue engineered supramolecular hydrogels prepared with highly water-soluble monofunctionalized cucurbit[6]uril−hyaluronic acid (CB[6]-HA), diaminohexane conjugated HA (DAH-HA), and drug conjugated CB[6] (drug-CB[6]) for the controlled chondrogenesis of human mesenchymal stem cells (hMSCs). The mechanical property of supramolecular HA hydrogels was modulated by changing the cross-linking density for the spatial control of hMSCs. In addition, the differentiation of hMSCs was temporally controlled by changing the release profiles of transforming growth factor-β3 (TGF-β3) and/or dexamethasone (Dexa) from the hydrolyzable Dexa-CB[6]. The effective chondrogenic differentiation of hMSCs encapsulated in the monoCB[6]/DAH-HA hydrogel with TGF-β3 and Dexa-CB[6] was confirmed by biochemical glycosaminoglycan content analysis, real-time quantitative PCR, histological, and immunohistochemical analyses. Taken together, we could confirm the feasibility of cytocompatible monoCB[6]/DAH-HA hydrogels as a platform scaffold with controlled drug delivery for cartilage regeneration and other various tissue engineering applications.
■
INTRODUCTION Mesenchymal stem cells (MSCs) have been widely investigated for tissue engineering applications. Especially, MSCs are the promising cell source for cartilage regeneration because of their remarkable capability to self-replicate and differentiate to chondrocytes.1,2 One of the major challenges is to maintain the cell population at delivered sites and to deliver adequate growth factors for efficient tissue regeneration.3−5 Several kinds of scaffolds including hydrogels have been developed for the spatiotemporal control of MSCs.6−10 The hydrogels serve as 3dimensional (3D) constructs capable of templating tissue formation with adequate mechanical properties, intercellular interactions, and biomolecular delivery.11,12 Transforming growth factor-β (TGF-β) superfamily members play important roles in the chondrogenesis of MSCs, whose exposure to MSCs in the early time is very important for the effective chondrogenesis.13,14 In contrast, continuous stimulation with dexamethasone, a synthetic glucocorticoid, is known to induce the differentiation of MSCs to chondrocytes.15,16 Physical properties of hydrogels are also important to modulate the 3D cellular environment. For example, various mechanical properties with a different cross-linking density lead to the alteration of the fate of encapsulated cells.17,18 As physically cross-linked cytocompatible hydrogels, supramolec© 2014 American Chemical Society
ular hydrogel systems have been exploited for biomedical applications using macrocyclic host molecules such as cyclodextrin (CD) and cucurbituril (CB). However, CD-based hydrogel systems have intrinsic limitation for in vivo applications because of its low binding affinity to guest molecules.19,20 CB[8] was also used as a cross-linking agent for the preparation of hydrogels by ternary complex formation with two guest molecules,21 which might be too complicated to be used for practical applications to tissue engineering. We previously reported the proof-of-concept of supramolecular hyaluronic acid (HA) hydrogels using the host−guest chemistry between CB[6] and diaminohexane (DAH), which provided a stable 3D microenvironment and tunable postsynthetic modification of the hydrogels by the treatment with “tag”attached CB[6] molecules.22 In this work, we developed 3D tissue engineered supramolecular hydrogels for controlled chondrogenesis of human MSCs (hMSCs) using highly water-soluble monofunctionalized CB[6]-HA, DAH-HA, and drug-CB[6]. The modification degree of monoCB[6]-HA could be easily controlled by Received: July 29, 2013 Revised: February 5, 2014 Published: February 18, 2014 707
dx.doi.org/10.1021/bm401123m | Biomacromolecules 2014, 15, 707−714
Biomacromolecules
Article
In Vitro Cell Viability in the Hydrogels. The live/dead assay was performed to visualize the distribution of live and dead cells in monoCB[6]/DAH-HA hydrogels. Calcein AM (green for live cells) and ethidium homodimer-1 (red for dead cells) were added to PBS at a concentration of 2 and 4 μg/mL. The cell-entrapped hydrogels were then placed in the solution for 30 min and imaged under a fluorescence microscope. In Vitro Release Tests of Dexamethasone, Dexa-CB[6], and TGF-β3. The release profile of free dexamethasone, Dexa-CB[6], or TGF-β3 in monoCB[6]/DAH-HA hydrogels was monitored in vitro for up to 34 days. Each sample (n = 3) was weighed in 10 mg and placed in a vial containing 1 mL of PBS at 37 °C. At the predetermined time points, the whole volume of PBS was collected and a fresh one was replenished. The released amount of dexamethasone and TGF-β3 was determined by measuring the optical density with a UV spectrophotometer at 242 nm and the ELISA. Subcutaneous Injection of hMSCs in MonoCB[6]/DAH-HA Hydrogel Precursor Solutions. Three kinds of hMSCs were cultured in a 75 cm2 flask containing minimum essential medium αmodification (α-MEM) supplemented with 10% heat-inactivated FBS, penicillin, streptomycin, amphotericin B, ascorbate, and glutamine at 37 °C in a humid 5% CO2. After 7 days, nonadherent cells were discarded and adherent cells were cultured to confluence with medium changes every 3 days. The prepared hMSCs were detached from the culture substrate using 0.05% trypsin-EDTA and suspended in the solution of monoCB[6]-HA (2.0 wt %, 5 × 106 cells in 0.2 mL). DAHHA (2.0 wt %) and Dexa-CB[6] (20 μL, 10 mg/mL) solutions were directly added to the solution of monoCB[6]-HA containing hMSCs. Six-week old Balb/c nude mice were divided into six groups (n = 12 for each group) for the assessment of chondrogenesis after injection of the following samples into the back subcutis of the mice: Group I, the monoCB[6]/DAH-HA hydrogel precursor solution with cells; Group II, the hydrogel precursor solution with cells and free dexamethasone; Group III, the precursor solution with cells and Dexa-CB[6]; Group IV, the precursor solution with cells and TGF-β3; Group V, the precursor solution with cells, TGF-β, and dexamethasone; Group VI, the precursor solution with cells, TGF-β3, and Dexa-CB[6], respectively. Four weeks postinjection, the mice were sacrificed by the overdose injection of anesthetic and an area of skin including the treated site was removed carefully for subsequent biological analysis. The excised monoCB[6]/DAH-HA hydrogel samples were fixed in 4% para-formaldehyde solution. The animal experiments were approved by the Animal Care Committee of CHA University. In Vivo Assessment of Chondrogenic Differentiation of hMSCs. Specific gene expression was measured via RT-qPCR in an ExiCycler (Bioneer, Daejeon, Korea). In brief, 1 μL of each cDNA was amplified in 20 μL PCR assay containing 2.0 mM MgCl2, 20 pM of each primer, and 1× Takara PCR Master Mix (Takara, Otsu, Japan). Samples were then subjected to the following conditions in an ExiCycler: initial denaturation at 94 °C for 10 min, followed by 45 cycles of 94 °C for 40 s, 58 °C for 30 s, and 72 °C for 30 s. To confirm the amplification of specific transcripts, melting curve profiles were generated at the end of each PCR by cooling the sample to 40 °C and then heating it slowly to 95 °C while continuously measuring the fluorescence. The relative quantification was calculated via the 2-delta delta cycle-threshold (ct) method and normalized against the housekeeping gene β-Actin. The following primers were used: COLII, 5′-CCGAGGCAACGATGGTCAG-3′ (sense) and 5′-TTCACCCTTGGCTCCAGGAG-3′ (antisense); SOX9, 5′-ACGCTGGGCAAGCTCTGG-3′ (sense) and 5′-TCCGGGTGGTCCTTCTTGTG-3′ (antisense); COMP, 5′-AGGAGGACTCAGACCAC GATG-3′ (sense) and 5′-CTCCTGGCCGGGGTTAGG-3′ (antisense); Aggrecan, 5′-CCTTGGAGGTCGTGGTGAAAGG-3′ (sense) and 5′-AGGTGAACTTCTCTGGCGACGT-3′ (antisense); β-Actin, 5′-ACAGAGCCTCGCCTTTGCC-3′ (sense) and 5′-ACATGCCGGAGCCGTTGTC-3′ (antisense). Histological and Immunohistochemical Analyses. The excised monoCB[6]/DAH-HA hydrogel samples were embedded in OCT compound and then frozen for histological and immunohistochemical analyses. The specimens were sliced into 10 μm thick
changing the amount of monoallyloxy CB[6] added for the conjugation to HA in aqueous solution. The monoCB[6]/ DAH-HA hydrogels with controlled cross-linking density and feasible mechanical property were used for the effective chondrogenesis of hMSCs in the presence of TGF-β3 and dexamethasone. Dexamethasone conjugated CB[6] (DexaCB[6]) with a hydrolyzable ester linkage was synthesized for long-term sustained release of dexamethasone after its modular modification to monoCB[6]/DAH-HA hydrogels. The chondrogenic differentiation of hMSCs encapsulated in monoCB[6]/DAH-HA hydrogels was assessed for the formation of neocartilage in vivo by the biochemical analysis for glycosaminoglycan (GAG) content, real-time quantitative PCR (RT-qPCR), Western blot, histological, and immunohistochemical analyses.
■
EXPERIMENTAL SECTION
Materials. Sodium hyaluronate, the sodium salt of hyaluronic acid (HA), with a molecular weight (MW) of 100 kDa, was obtained from Shiseido (Tokyo, Japan) and HA with a MW of 234 kDa was purchased from Lifecore (Chaska, MN). Phosphate buffered saline (PBS) tablet, N-hydroxysulfosuccinimide (sulfo-NHS), cystamine, 1,6diaminohexane (DAH), proteinase K, chondroitin sulfate, fluorescein isothiocyanate (FITC), and rhodamine B isothiocyanate (RBITC) were purchased from Sigma (St. Louis, MO). Spermine (SPM) and 1ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) were obtained from Tokyo Chemical Industry (Tokyo, Japan). The acetomethoxy derivative of calcein (Calcein AM), ethidium homodimer-1 (EthD-1), and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Molecular Probes (Carlsbad, CA). Snake-skin pleated dialysis tube was obtained from Thermo Scientific (Rockford, IL). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin (PS) were obtained from HyClone (Logan, UT). Primary antibodies were obtained as follows: anti-SOX9 (cat. AB5535, Chemicon, Temecula, CA), anticollagen type II (cat. MAB1330, Chemicon, Temecula, CA), anti-COMP (Abcam, Cambridge, U.K.), aggrecan (cat. AB3778, Abcam, Cambridge, U.K.), and anti-GAPDH (Sigma, St. Louis, MO). Trizol, aminoethylcarbazole (AEC), chromogen substrate (Histostain Plus, Broad Spectrum), and CyQuant kit were purchased from Invitrogen Co. (Carlsbad, CA). Radioimmunoprecipitation assay (RIPA) buffer was purchased from Pierce (Rockford, IL) and complete protease inhibitor cocktail was obtained from Roche Applied Science (Indianapolis, IN). OCT compound (TISSUE-TEKs 4583) was purchased from Sakura Finetek (Torrance, CA). We purchased and used three different human MSCs from Lonza (Basel, Switzerland). All reagents were used without further purification. The details for the following synthesis and characterization are described in the Supporting Information. Preparation of MonoCB[6]/DAH-HA Hydrogel. MonoCB[6]HA was synthesized by thiol−ene “click” photoreaction between monoallyloxy CB[6] and thiol-functionalized HA (HS-HA, MW = 100 kDa).22 The photoreactions were performed in a quartz tube by UV irradiation in a RMR-600 photochemical reactor (Rayonet, Branford, CT) equipped with four 254 nm lamps and four 300 nm lamps. A counterpart of DAH-HA to monoCB[6]-HA for the hydrogel formation was synthesized and characterized as described elsewhere.22 Then, a solution of monoCB[6]-HA (200 μL, 2.0 wt %) in water or PBS was added to the equal volume of DAH-HA solution (200 μL, 2.0 wt %), which was vortexed for the formation of monoCB[6]/DAHHA hydrogels. Rheological Analysis of the Hydrogels. Rheological analysis was performed on a TA ARES rheometer with a parallel-plate geometry (20 mm diameter) at 25 °C. The storage and loss moduli of monoCB[6]/DAH-HA hydrogel in a round shape (1.0 cm in diameter and 0.3 mm in thickness) were monitored at a constant strain amplitude (10%) as a function of frequency to assess the mechanical property of the monoCB[6]/DAH-HA hydrogels. 708
dx.doi.org/10.1021/bm401123m | Biomacromolecules 2014, 15, 707−714
Biomacromolecules
Article
Figure 1. Schematics of (a) supramolecular monoCB[6]/DAH-HA hydrogels encapsulating hMSCs and TGF-β3 with modularly modified DexaCB[6] by the strong host−guest interaction between CB[6] and DAH. The chemical structures of (b) monoCB[6]-HA and (c) DAH-HA. sections at −20 °C, and then the nucleus and cytoplasm were stained with H&E, respectively. Additionally, cryosections (10 μm) of implanted monoCB[6]/DAH-HA hydrogels were stained with Alcian Blue and Safranin-O for histological analysis. Immunofluorescence analysis was conducted for COL II, SOX9, and aggrecan by applying antibodies for each of the aforementioned compounds in a humid environment. The following primary antibodies were diluted in PBS: mouse monoclonal COL II antibody, rabbit monoclonal SOX9 antibody, and mouse monoclonal aggrecan antibody. For the sample preparations, the sections were washed three times with PBS for 2 min per wash. Endogenous peroxidases were blocked by incubation in 3% hydrogen peroxide in a humidified chamber at 37 °C for 20 min. The sections were washed three times with PBS for 2 min each. To reduce the background staining, the sections were then incubated in 10% normal goat serum for 30 min. The sections were incubated in the primary antibody at 4 °C for 90 min, which were washed 3 times for 5 min with PBS. After washing, the biotinylated secondary antibody was applied and incubated. For the color reaction, slides were treated with aminoethylcarbazole (AEC) chromogen substrate (Histostain Plus, Broad Spectrum) for 10−15 min, which yielded a red-brown color, indicating a positive reaction. The reaction was stopped by washing the slides in distilled water. Then, the sections were counterstained with Mayer’s hematoxylin for 20 s, washed with distilled water, and mounted with coverslips. Biochemical Analysis for GAG Production. Excised monoCB[6]/DAH-HA hydrogel samples were digested with proteinase K (0.25 mL of 1 mg/mL protease K in 50 mM Tris with 1 mM EDTA, 1 mM iodoacetamide, and 10 μg/mL pepstatin-A) at 56 °C overnight. The sulphated GAG content was measured by dimethylmethylene blue dye binding assay using chondroitin sulfate as a standard. The cellularity was measured based on the DNA content with a CyQuant kit using lambda DNA as a standard. Cellularity (DNA) and GAG contents of pellets were quantified and expressed as the GAG/DNA ratio. Western Blot Analysis. For Western blotting, the cells were lysed in RIPA buffer supplemented with complete protease inhibitor cocktail. Approximately 30−50 μg of protein was loaded on 8−10% SDS polyacrylamide gel (SDS-PAGE) and transferred to an Immobilon-P membrane (Millipore Co., Bedford, MA). The membranes were blocked in 5% skim milk in TBS-Tween 20 (0.02%) and incubated with primary antibodies of anti-SOX9, anticollagen type II, anti-COMP, aggrecan, and anti-GAPDH. The blots via chemiluminescence were developed using Amersham ECL reagents.
Statistical Analysis. The data are expressed as means ± standard deviation from several separate experiments. Statistical analysis was carried out via the t-test using the software of SigmaPlot10.0. A value for *P < 0.05 was considered statistically significant.
■
RESULTS AND DISCUSSION Synthesis and Characterization of MonoCB[6]/DAHHA Hydrogel. Hydrogels are generally prepared by chemical cross-linking with highly reactive cross-linkers, and physical cross-linking with noncovalent interactions such as hydrophobic and ionic interactions. However, the chemical crosslinking causes a significant cytotoxicity in some cases, and physical cross-linking is difficult to control and unstable for long-term applications in the body. In order to prepare supramolecular HA hydrogels, monoCB[6]-HA and DAH-HA were synthesized as described in Supporting Information. Monoallyloxy CB[6] was prepared by the reaction between monohydroxy CB[6] and allylbromide. The successful synthesis of monoallyloxy CB[6] was confirmed by MALDI-TOF mass and 1H NMR analyses. MonoCB[6]-HA was then synthesized by thiol−ene “click” reaction between HS-HA and monoallyloxy CB[6] (Figure S1a). Because monoallyloxy CB[6] is highly soluble in aqueous solution, the HA backbone can be easily modified with a wide range of monoallyloxy CB[6] for various tissue engineering applications. The synthesized monoCB[6]-HA was characterized by the integration ratio of specific peaks of CB[6] and HA in the 1H NMR spectrum (Figure S1b). The complex formation of monoCB[6]-HA with spermine was also confirmed by 1H NMR analysis.22 As schematically shown in Figure 1, monoCB[6]/DAH-HA hydrogels were prepared by simple mixing of DAH-HA and monoCB[6]-HA solutions with a different degree of CB[6] in the presence of hMSCs. The host−guest interaction between CB[6] and DAH molecules in the precursor solution is the driving force for the hydrogel formation acting as a cytocompatible specific cross-linker. The number of CB[6]/DAH pairs represents the cross-linking density of the hydrogels. We previously reported multifunctional CB[6]/DAH-HA hydrogels,22 but the number of 709
dx.doi.org/10.1021/bm401123m | Biomacromolecules 2014, 15, 707−714
Biomacromolecules
Article
Figure 2. (a) Degree of CB[6] modification on HA backbone by 1H NMR analysis with increasing ratio of CB[6] to HS-HA added for the conjugation reaction (n = 3). (b) Storage modulus of monoCB[6]/DAH-HA hydrogels prepared with 2.0 wt % solutions of 50 mol % DAH-HA and 10 mol % monoCB[6]-HA or 3 mol % multiCB[6]-HA.
multiCB[6] conjugated to HA was limited because of its low solubility in aqueous solution. The hMSCs encapsulated in the monoCB[6]/DAH-HA hydrogels can be efficiently differentiated to chondrocytes by the codelivery of TGF-β3 and Dexa-CB[6] modularly modified to the residual DAH in the hydrogels. The degree of CB[6] modification in monoCB[6]-HA almost linearly increased with the ratio of added monoallyloxy CB[6] to HS-HA (Figure 2a). Among them, monoCB[6]/ DAH-HA hydrogels with a modification degree of 10 mol % was chosen for the following application to the chondrogenesis. The storage modulus of monoCB[6]/DAH-HA hydrogels measured by the frequency sweep rheological analysis was about four times higher than the previous multiCB[6]/DAHHA hydrogels22 in the same concentration mainly due to the difference in cross-linking density (Figure 2b). The monoCB[6]/DAH-HA hydrogels had a highly porous microstructure according to scanning electron microscopy (SEM) (Figure S2). More than 95% of the hMSCs in CB[6]/DAH-HA hydrogels survived and proliferated even after incubation for 10 days (Figure S3). MonoCB[6]/DAH-HA hydrogels can be easily functionalized with drug-CB[6] by noncovalent and modular modification. Within the monoCB[6]/DAH-HA hydrogels prepared with an equal volume of monoCB[6]-HA (CB[6] content of 10 ± 2 mol %) and DAH-HA (DAH content of 50 ± 2 mol %) solutions, there are remaining DAH moieties more than 40 mol % in the hydrogel, which can further interact with drug-CB[6]. The fluorescence of monoCB[6]/DAH-HA hydrogels modularly modified with FITC-CB[6] was maintained for more than 10 days (Figure S4). To induce chondrogenesis of MSCs encapsulated in monoCB[6]/DAH-HA hydrogels, we chose and conjugated dexamethasone to CB[6]. Dexamethasone is a synthetic corticoid and a potent modulator for the chondrogenic differentiation of hMSCs. Because Dexa works within the cells, Dexa-CB[6] was designed to have an ester-linkage between Dexa and CB[6] for its slow release by hydrolysis from the Dexa-CB[6]/monoCB[6]/DAH-HA hydrogels. DexaCB[6] was synthesized by the activation of carboxyl group of Dexa-suc23 and the subsequent reaction to the amineCB[6]24
(Figure S5). The successful synthesis of Dexa-CB[6] was also confirmed by 1H NMR analysis. Then, Dexa-CB[6] was modularly modified to monoCB[6]/DAH-HA hydrogels by strong host−guest interaction between CB[6] of Dexa-CB[6] and DAH in the hydrogels. This on-demand modular modification is also the big advantage of monoCB[6]/DAHHA hydrogels for tissue engineering applications. In Vitro Release Tests of TGF-β3 and Dexamethasone. According to in vitro release tests, the release profile of DexaCB[6] was significantly different from those of free dexamethasone and TGF-β3 (Figure 3). The release of dexametha-
Figure 3. In vitro release profiles of dexamethasone (black, inset), Dexa-CB[6] (red), and TGF-β3 (blue) from monoCB[6]/DAH-HA hydrogels (n = 3).
sone from Dexa-CB[6] showed an initial burst of about 50% within a week and then continuous release for more than 3 weeks. In contrast, almost all the free dexamethasone was rapidly released out from monoCB[6]/DAH-HA hydrogels within 2 h. TGF-β3 was released from the hydrogels for a week, which was slower than free dexamethasone because of its higher molecular weight and positive charge, but much faster than Dexa-CB[6] due to its physical loading. The results indicate that Dexa-CB[6] stably bound to the monoCB[6]/DAH-HA hydrogels can consistently stimulate hMSCs for the chondrogenesis of encapsulated hMSCS. When treated with DexaCB[6], the number of proliferated hMSCs was much higher than that with free dexamethasone (Figure 4). The continuous 710
dx.doi.org/10.1021/bm401123m | Biomacromolecules 2014, 15, 707−714
Biomacromolecules
Article
stimulation of hMSCs with dexamethasone might be advantageous for cell proliferation as well as differentiation.
Figure 5. GAG/DNA contents in monoCB[6]/DAH-HA hydrogels with free dexamethasone or Dexa-CB[6] in the absence and presence of TGF-β3 (n = 3, **P < 0.01 vs free dexamethasone).
hydrolysis of ester linkage for a few weeks. The long-term treatment of hMSCs with Dexa-CB[6] modularly bound to monoCB[6]/DAH-HA hydrogels might enable hMSCs to differentiate effectively for the chondrogenesis. The temporal codelivery of TGF-β3 and Dexa-CB[6] seemed to be the reason for the synergistic effect on mature chondrocyte-related gene expressions in hMSCs. Immunohistochemical Analysis for the Chondrogenesis. To analyze the proliferation of hMSCs during chondrogenesis, H&E staining was carried out for the cells in the monoCB[6]/DAH-HA hydrogels (Figure 7). In case of monoCB[6]/DAH-HA hydrogels with both TGF-β3 and DexaCB[6], the encapsulated hMSCs were evenly and entirely distributed within the construct, reflecting relatively active proliferation by Dexa-CB[6] and TGF-β3. In contrast, for the case of the control without TGF-β3 and Dexa-CB[6], cells were mainly observed near the edge of the scaffolds (Figure 7), which can be explained by the accelerated metabolism through the edge of hydrogels. The results revealed that the incubation with TGF-β3 and Dexa-CB[6] enhanced not only differentiation but also proliferation of the hMSCs in monoCB[6]/ DAH-HA hydrogels. For the detection of proteoglycans and polysaccharides specific to chondrocytes in the ECM, hMSCs encapsulated in the monoCB[6]/DAH-HA hydrogels were examined by histological analysis with Alcian blue (blue color) and Safranin-O (orange color) staining (Figure 7). In accordance with the results in Figures 5 and 6, blue and orange colors, representing proteoglycans and polysaccharides, were clearly observed in monoCB[6]/DAH-HA hydrogels encapsulating hMSCs. The intensity of staining was more significant in the presence of TGF-β3 and Dexa-CB[6] than other cases without Dexa, Dexa-CB[6], and TGF-β3 (Figure 7). Furthermore, the cartilage tissue specific proteins were detected by immunohistochemical analysis for COL II, SOX9, and aggrecan with the neo-cartilage formation of hMSCs in the monoCB[6]/DAH-HA hydrogels (Figure 8). The hMSCs encapsulated in the monoCB[6]/DAH-HA hydrogels with both TGF-β3 and Dexa-CB[6] were the most positively stained for COL II, SOX9, and aggrecan, followed by those with TGFβ3 and Dexa, Dexa-CB[6], TGF-β3, and the control. In addition, all of the chondrogenic markers were well expressed on Western blot bands in the presence of both TGF-β3 and Dexa-CB[6] (Figure 8b). As reported elsewhere,13,14 the early time delivery of TGF-β3 and the continuous stimulation of MSCs with dexamethasone seemed important for the effective
Figure 4. (a) Fluorescence microscopic images showing hMSCs in monoCB[6]/DAH-HA hydrogels without and with dexamethasone or Dexa-CB[6] after staining with calcein AM for live cells (scale bar = 50 μm). (b) Quantified data for cell numbers in monoCB[6]/DAH-HA hydrogels without and with dexamethasone or Dexa-CB[6] in 3, 7, and 10 days (n = 3, *P < 0.05 vs free dexamethasone).
In Vivo Chondrogenic Differentiation of hMSC in MonoCB[6]/DAH-HA Hydrogels. On the basis of in vitro test results, we investigated in vivo chondrogenic differentiation of hMSCs encapsulated in the hydrogels. The precursor solutions of DAH-HA (2.0 wt %, 0.2 mL PBS) and monoCB[6]-HA mixed with hMSCs (2.0 wt %, 5 × 106 cells in 0.2 mL PBS) were injected into the back subcutis of nude mice with free dexamethasone or Dexa-CB[6]. MonoCB[6]/ DAH-HA hydrogels were formed in situ and maintained even after 4 weeks postinjection (Figure S6). The chondrogenic differentiation of hMSCs in the monoCB[6]/DAH-HA hydrogel was analyzed by the biochemical analysis for GAG contents, RT-qPCR, Western blot, histological, and immunohistochemical analyses. The expression level of GAG was significantly higher for the case of incubation with Dexa-CB[6] than free dexamethasone in the presence of TGF-β3 (Figure 5). This pattern was similar with other chondrogenic markers such as COL II, COMP, SOX9, and aggrecan in the RT-qPCR (Figure 6). Especially, hMSCs treated with TGF-β3 and Dexa-CB[6] in monoCB[6]/DAH-HA hydrogels showed about seven times higher chondrogenic induction than the control in the chondrocyte-specific genes of COL II. These results indicated that physically loaded TGF-β3 and strongly bound Dexa-CB[6] in the hydrogel synergistically activated the hMSCs for the effective chondrogenesis. Considering the in vitro release profile in Figure 3, dexamethasone might be slowly released from Dexa-CB[6] bound to the hydrogels in mice by the 711
dx.doi.org/10.1021/bm401123m | Biomacromolecules 2014, 15, 707−714
Biomacromolecules
Article
Figure 6. RT-qPCR analysis for the chondrogenic gene expression levels of COL II, COMP, SOX9, and aggrecan in 4 weeks. The amount of mRNA was normalized using β-actin as a housekeeping gene. The data represent the mean ± SD (n = 3, *P < 0.05 and **P < 0.01 vs free dexamethasone).
Figure 7. Histological analysis of neocartilage in vivo formed by the differentiation of hMSCs in monoCB[6]/DAH-HA hydrogels without and with free dexamethasone or Dexa-CB[6] in the absence and presence of TGF-β3 after staining with H&E, Alcian blue, and Safranin-O in 4 weeks.
supramolecular HA hydrogels seem to be applied as a platform scaffold to a variety of cell therapies and tissue engineering.
chondrogenesis. Taken together, it was convincing that the spatiotemporal control of cells encapsulated in the supramolecular monoCB[6]/DAH-HA hydrogels was successfully carried out for the controlled chondrogenesis. The proliferation and differentiation of hMSCs for tissue engineering applications can be spatially controlled by modulating the cross-linking density of monoCB[6]/DAH-HA hydrogels with the optimized number of CB[6]/DAH pairs. In addition, the encapsulated cells can be temporally controlled by the various release profiles of TGF-β3 and Dexa using the hydrolyzable Dexa-CB[6]. The
■
CONCLUSION 3D tissue engineered supramolecular monoCB[6]/DAH-HA hydrogels were successfully developed for the spatiotemporal control of hMSCs. The controlled chondrogenesis of hMSCs was made possible by modulating the mechanical strength of the hydrogels, and the release profiles of TGF-β3 and dexamethasone using the hydrolyzable Dexa-CB[6]. The 712
dx.doi.org/10.1021/bm401123m | Biomacromolecules 2014, 15, 707−714
Biomacromolecules
Article
Figure 8. (a) Immunohistochemical analysis of neocartilage in vivo formed by differentiation of hMSCs in monoCB[6]/DAH-HA hydrogels without and with free dexamethasone or Dexa-CB[6] in the absence and presence of TGF-β3 (scale bar = 100 μm). (b) Representative Western blots of COL II, COMP, SOX9, and aggrecan gene expressions. GAPDH was used as an internal control.
■
ACKNOWLEDGMENTS We gratefully acknowledge the Acceleration Research, Brain Korea 21, World Class University, and the Bio & Medical Technology Development programs (Project Nos. R31-2008000-10059-0, 2012M3A9C6049791) of the National Research Foundation funded by the Korean government (MEST) for this work.
effective chondrogenic differentiation of hMSCs encapsulated in the monoCB[6]/DAH-HA hydrogels with TGF-β3 and Dexa-CB[6] was corroborated by the biochemical GAG content analysis, RT-qPCR, histological, and immuno-histochemical analyses. From the results, we could confirm the feasibility of cytocompatible monoCB[6]/DAH-HA hydrogels for the spatiotemporally controlled chondrogenesis of hMSCs.
■
■
ASSOCIATED CONTENT
S Supporting Information *
A detailed description of synthesis of HA derivatives and CB derivatives, SEM image of monoCB[6]/DAH-HA hydrogel, viability of hMSCs in monoCB[6]/DAH-HA hydrogel, in vivo formation of monoCB[6]/DAH-HA hydrogel, and GAG/DNA contents are provided. This material is available free of charge via the Internet at http://pubs.acs.org.
■
REFERENCES
(1) Johnson, K.; Zhu, S.; Tremblay, M. S.; Payette, J. N.; Wang, J.; Bouchez, L. C.; Meeusen, S.; Althage, A.; Cho, C. Y.; Wu, X.; et al. A stem cell-based approach to cartilage repair. Science 2012, 336, 717− 721. (2) Alves da Silva, M. L.; Martins, A.; Costa-Pinto, A. R.; Costa, P.; Faria, S.; Gomes, M.; Reis, R. L.; Neves, N. M. Cartilage tissue engineering using electrospun PCL nanofiber meshes and MSCs. Biomacromolecules 2010, 11, 3228−3236. (3) Mooney, D. J.; Vandenburgh, H. Cell delivery mechanisms for tissue repair. Cell Stem Cell 2008, 2, 205−213. (4) Lu, H. F.; Narayanan, K.; Lim, S. X.; Gao, S.; Leong, M. F.; Wan, A. C. A. A 3D microfibrous scaffold for long-term human pluripotent stem cell self-renewal under chemically defined conditions. Biomaterials 2012, 8, 2419−2430. (5) Kraehenbuehl, T. P.; Langer, R.; Ferreira, L. S. Threedimensional biomaterials for the study of human pluripotent stem cells. Nat. Methods 2011, 9, 731−736. (6) Chung, C.; Burdick, J. A. Engineering cartilage tissue. Adv. Drug Delivery Rev. 2008, 60, 243−262.
AUTHOR INFORMATION
Corresponding Authors
*Tel.: 82 2 3468 3392. Fax: 82 2 3468 3373. E-mail: pkh0410@ cha.ac.kr. *Tel.: 82 54 279 2159. Fax: 82 54 279 2399. E-mail: skhanb@ postech.ac.kr. *Tel.: 82 54 279 8128. Fax: 82 54 279 8129. E-mail: kkim@ postech.ac.kr. Notes
The authors declare no competing financial interest. 713
dx.doi.org/10.1021/bm401123m | Biomacromolecules 2014, 15, 707−714
Biomacromolecules
Article
(7) Alge, D. L.; Azagarsamy, M. A.; Donohue, D. F.; Anseth, K. S. Synthetically tractable click hydrogels for three-dimensional cell culture formed using tetrazine-norbornene chemistry. Biomacromolecules 2013, 14, 949−953. (8) Kloxin, A. M.; Kasko, A. M.; Salinas, C. N.; Anseth, K. S. Photodegradable hydrogels for dynamic tuning of physical and chemical properties. Science 2009, 324, 59−63. (9) Seliktar, D. Designing cell-compatible hydrogels for biomedical applications. Science 2012, 336, 1124−1128. (10) Burdick, J. A.; Prestwich, G. D. Hyaluronic acid hydrogels for biomedical applications. Adv. Mater. 2011, 23, 41−56. (11) Dawson, E.; Mapili, G.; Erickson, K.; Taqvi, S.; Roy, K. Biomaterials for stem cell differentiation. Adv. Drug Delivery Rev. 2008, 60, 215−228. (12) Drury, J. L.; Mooney, D. J. Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials 2003, 24, 4337−4351. (13) Buxton, A. N.; Bahney, C. S.; Yoo, J. U.; Johnstone, B. Temporal exposure to chondrogenic factors modulates human mesenchymal stem cell chondrogenesis in hydrogels. Tissue Eng., Part A 2010, 17, 371−380. (14) Bian, L.; Zhai, D. Y.; Tous, E.; Rai, R.; Mauck, R. L.; Burdick, J. A. Enhanced MSC chondrogenesis following delivery of TGF-β3 from alginate microspheres within hyaluronic acid hydrogels in vitro and in vivo. Biomaterials 2011, 32, 6425−6434. (15) Hwang, N. S.; Varghese, S.; Lee, H. J.; Zhang, Z.; Ye, Z.; Bae, J.; Cheng, L.; Elisseeff, J. In vivo commitment and functional tissue regeneration using human embryonic stem cell-derived mesenchymal cells. Proc. Natl. Acad. Sci. U.S.A. 2008, 52, 20641−20646. (16) Oshina, H.; Sotome, S.; Yoshii, T.; Torigoe, I.; Sugata, Y.; Maehara, H.; Marukawa, E.; Omura, K.; Shinomiya, K. Effects of continuous dexamethasone treatment on differentiation capabilities of bone marrow-derived mesenchymal cells. Bone 2007, 41, 575−583. (17) Cha, C.; Jeong, J. H.; Shim, J.; Kong, H. Tuning the dependency between stiffness and permeability of a cell encapsulating hydrogel with hydrophilic pedant chains. Acta Biomater. 2011, 7, 3719−3728. (18) Nemir, S.; West, J. L. Synthetic materials in the study of cell response to substrate rigidity. Ann. Biomed. Eng. 2009, 38, 2−20. (19) Kretschmann, O.; Choi, S. W.; Miyauchi, M.; Tomatsu, I.; Harada, A.; Ritter, H. Switchable hydrogels obtained by supramolecular cross-linking of adamantyl-containing LCST copolymers with cyclodextrin dimers. Angew. Chem., Int. Ed. 2006, 45, 4361−4365. (20) Wu, D. Q.; Wang, T.; Lu, B.; Xu, X. D.; Cheng, S. X.; Jiang, X. J.; Zhang, X. Z.; Zhuo, R. X. Fabrication of supramolecular hydrogels for drug delivery and stem cell encapsulation. Langmuir 2008, 24, 10306−10312. (21) Appel, E. A.; Loh, X. J.; Jones, S. T.; Biedermann, F.; Dreiss, C. A.; Scherman, O. A. Ultrahigh-water-content supramolecular hydrogels exhibiting multistimuli responsiveness. J. Am. Chem. Soc. 2012, 134, 11767−11773. (22) Park, K. M.; Yang, J. A.; Jung, H.; Yeom, J.; Park, J. S.; Park, K. H.; Hoffman, A. S.; Hahn, S. K.; Kim, K. In situ supramolecular assembly and modular modification of hyaluronic acid hydrogels for 3D cellular engineering. ACS Nano 2012, 4, 2960−2968. (23) Everts, M.; Kok, R. J.; Asgeirsdottir, S. A.; Melgert, B. N.; Moolenaar, T. J. M.; Koning, G. A.; Luyn, M. J. A.; Meijer, D. K. F.; Molema, G. Selective intracellular delivery of dexamethasone into activated endothelial cells using an E-selectin-directed immunoconjugate. J. Immunol. 2001, 168, 883−889. (24) Choi, J.; Kim, J.; Kim, K.; Yang, S. T.; Kim, J. I.; Jon, S. A rationally designed macrocyclic cavitand that kills bacteria with high efficacy and good selectivity. Chem. Commun. 2007, 1151−1153.
714
dx.doi.org/10.1021/bm401123m | Biomacromolecules 2014, 15, 707−714
