a novel poly(amido amine)-dendrimer-based hydrogel as a mimic for the extracellular matrix

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A Novel Poly(amido amine)-Dendrimer-Based Hydrogel as a Mimic for the Extracellular Matrix

Yao Wang , Qiang Zhao , He Zhang , Sheng Yang , * and Xinru Jia *

nanostructures for creating bio-related materials. This is due to the easily modifi ed surface groups, which not only provide multiple cross-linkingsites but also provide the possibility of conjugating various bioactive components.

The in situ photo-cross-linking ability is a highly desirable property for hydrogels used in tissue engineering, especially for trauma repair. [ 13 ] Advantages of photo-initiative hydro-gels [ 14 ] include the possibility of controlling both the spatial and temporal aspects of cross-linking, the ability to conduct cross-linking at physiological temperature and pH, and the ability to fi ll irregularly shaped defect sites. In-situ cross-linking the hydrogel can also provide better adhesion and mechanical integrity to the defect sites.

Herein, we report a novel hydrogel constructed from two components: a linear co-polymer of poly(lactic acid)- b -poly(ethylene glycol)- b -poly(lactic acid) with acrylate end-groups (PEG-LA-DA), and a fourth-generation poly(amido amine) den-drimer (G4.0 PAMAM) peripherally modifi ed by polyethylene glycol (PEG) with terminal arginine–glycine–(aspartic acid)–( D -tyrosine)–cysteine (RGDyC) and acryloyl groups. We have combined the superior properties of PAMAM dendrimers, PEG-LA-DA co-polymers, and in-situ photo-cross-linking to create a hydrogel with a highly interconnected porous 3D net-work structure. Our hydrogel scaffold offers several advan-tages over other systems because 1) the multiple cross-linking sites present on the dendrimers can increase the cross-linking density at lower concentrations; 2) the spherical dendrimers may provide discrete ‘molecular islands’ in the network to limit swelling and improve mechanical properties; and 3) the multiple end-groups on the dendrimers facilitate the introduc-tion of functional groups into the system at the nanoscale level. To the best of our knowledge, this is a rare report of a PAMAM-dendrimer-based hydrogel with a highly porous structure, which exhibits reduced swelling, enhanced mechanical stiff-ness, and better cell adhesion, differentiation, and proliferation than hydrogels formed from PEG-LA-DA alone.

As shown in Scheme 1 , the hydrogel network was fabricated via a photo-induced cross-linking reaction using the modifi ed sphere-like PAMAM dendrimers as the ‘molecular islands’ and the linear co-polymer of PEG-LA-DA as the linkage moiety. PEG-LA-DA comprises a PEG chain (molecular weight: 6000 Da) with an average of fi ve repeat units of lactic acid grafted at each side (see the Supporting Information (SI): Figure S1, S2, and S3 and Table S1). Initially, we modifi ed the periphery of the G4.0 PAMAM dendrimers with methoxy-PEG-succinimidyl carbonate ester (mPEG1000-NHS) and maleimide-PEG-succinimidyl carbonate ester (MAL-PEG5000-NHS), in order to make them amenable to derivatization with bioactive components and cross-linking groups and to enhance the biocompatibility and disso-lution properties of the dendrimers. The RGDyC and acryloyl DOI: 10.1002/adma.201400323

Y. Wang, Q. Zhao, Prof. X.-R. Jia Beijing National Laboratory for Molecular Sciences and the Key Laboratory of Polymer Chemistry and Physics of the Ministry of Education College of Chemistry and Molecular EngineeringPeking University Beijing , China Fax: +86-010-62751708 E-mail: [email protected] H. Zhang, Dr. S. Yang Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences College of StomatologyChongqing Medical University Chongqing , China Fax: +86-023-88860085 E-mail: [email protected]

Artifi cial materials that mimic the extracellular matrix (ECM) of mammalian tissues are clinically needed, but their produc-tion remains a challenge. [ 1 ] Natural macromolecules—such as collagen, [ 2 ] alginate, [ 3 ] fi brin, [ 4 ] and chitosan [ 5 ] —have been used in tissue engineering strategies. These polymers, however, have limited clinical applicability; for example, they may have insuffi cient mechanical properties, and in the case of collagen-based gels and fi brin matrices, there is inappropriate shrinkage and rapid degradation. [ 6 ] In addition, it is diffi cult to precisely control and modify their macro-structures and to synthesize them at a large scale. In hopes of meeting clinical demands, the development of polymer-based scaffolds containing adhe-sive peptide ligands as cell recognition sites has attracted much attention. The properties of synthetic polymers can be tailored to match tissue growth in the artifi cial ECM, and cell recog-nition sites within the polymers provide biological activity to mimic the physiological environment. [ 7 ]

There has been increasing interest in hydrogels as scaf-folds for cell encapsulation [ 8 ] because the high water content in hydrogels may facilitate the diffusion of nutrients and oxygen to the cells and the removal of waste products and carbon dioxide from the cells. [ 9 ] In addition, gels with 3D networks create an environment that simulates the native state of cells. [ 10 ] A typical example is the hydrogel derived from poly(lactide)- b -poly(ethylene glycol)- b -poly(lactide) co-polymers, which has adjustable degradation properties and favorable biocompat-ibility. [ 11 ] However, the limitations of such polymers include excessive volumetric swelling after cross-linking, inferior mechanical properties, and the absence of bioactivity.

Hydrogels derived from dendrimers with their unique struc-tures offer an alternative to linear polymers. Dendritic architec-tures [ 12 ] —the most pervasive topologies in biological systems at various dimensional scales—are optimal molecular-level

Adv. Mater. 2014, DOI: 10.1002/adma.201400323

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groups were then covalently conjugated to the MAL-PEG5000-NHS through an effective Michael addition reaction to obtain the fi nal modifi ed dendrimers, named acryloyl-G4.0-PEG-RGDyC (AGPR) (see SI: Scheme S1). G4.0-PEG-RGDyC (GPR) (without acryloyl groups) and acryloyl-G4.0-PEG (AGP) (without RGDyC groups) were prepared as controls for comparison.

1 H NMR measurements were performed to determine the grafting ratio of PEG, RGDyC, and acryloyl groups (SI: Figure S4) on the exterior of the PAMAM dendrimers. According to the integration ratio of the tyrosine protons from RGDyC at 7.02 and 6.72 ppm, the acryloyl protons at 5.76 and 5.10 ppm, the PEG protons at 3.60 ppm, and the PAMAM den-drimer protons at 3.05–2.10 ppm, there are about 10 RGDyC groups, 10 double bonds, and 40 mPEG1000 moieties grafted onto the periphery of the G4.0 PAMAM dendrimers. The dis-appearance of the peak at 6.76 ppm indicated that most of the maleimide groups of the MAL-PEG5000-NHS chain ends reacted with RGDyC and allyl mercaptan. Differential scanning calorimetry (DSC) and Fourier-transform (FT)-IR measure-ments were also performed, and their results supported the 1 H NMR data (SI: Figure S5, S6).

The gelation properties were examined by mixing 20% w/v aqueous solution of PEG-LA-DA with different amounts of AGPR (PEG-LA-DA:AGPR = 10/1, 5/1, 2/1, 1/1, or 1/2) in phosphate buffered saline (PBS) followed by UV irradiation at 365 nm for 10 min (SI: Table S2). This resulted in three con-clusions: 1) The PEG-LA-DA co-polymer is essential for gela-tion. Although the sphere-like AGPR contained cross-linking groups in the structure, no gelation occurred as a result of the possible intramolecular and/or intermolecular cross-linking of the acryloyl units on the exterior of the dendrimers (SI: Figure S7). As shown by scanning electron microscopy (SEM) images, the morphology of AGPR (20% w/v solution, after irradiation) was totally different from that of the hydrogel (SI: Figure S8). This result is consistent with that reported for a clay/polymer hydrogel by Aida and co-workers, [ 15 ] who found that the mechanical modulus of hydrogels could be enhanced by using long-chain polymers because they can more easily bridge different clay sheets. In contrast, short-chain polymers lead to cross-linking within the same sheet, which reduces the mechanical modulus. 2)

The acryloyl groups on the exterior of the G4.0 PAMAM dendrimers are also neces-sary for gelation. Gels were not observed as a result of mixing the control samples of G4.0 PAMAM or GPR with the PEG-LA-DA co-polymers at ratios of 1/10 and 1/5, respec-tively. 3) It was established that transparent and robust gels can be obtained with AGPR concentrations in the range of 2%–20% with PEG-LA-DA ( Figure 1 a,b); otherwise, softer gels were observed.

SEM was used to visualize the networks of the hydrogel. The gel from PEG-LA-DA/AGPR (at a ratio of 5/1) possessed a highly porous structure. As shown, the morphology

consisted of pores in the range of 20–50 µm containing sev-eral smaller holes inside (≈5 µm) (Figure 1c–e; SI: Figure S9). Hydrogels with differently sized pores are useful in tissue engi-neering because pores with a minimum size of 100 µm are ben-efi cial for tissue growth and vascularization in bone-grafting, while pores in the nanometer range can enhance cell adhesion and proliferation at the implant site and can potentially absorb proteins and growth factors. [ 16 ]

The swelling properties of the hydrogel were examined by immersing the freshly cross-linked hydrogel in PBS at 37 °C for 48 h. AGPR was found to play a key role in controlling the swelling ratio. Adding 2% AGPR to the system effectively decreased the swelling ratio to 300% ( Figure 2 ), as compared to a swelling ratio of 1000% for the hydrogel consisting of only linear PEG-LA-DA. The swelling ratio was further reduced to 180% when AGPR was increased to 20%. The results suggest that the reduction in swelling observed in the PEG-LA-DA/AGPR hydrogel is a consequence of the “dendritic effect”. The sphere-like PAMAM molecules function as discrete multivalent junction points that limit the expansion of the networks, leading to less swelling. Restricted expansion of the hydrogel is highly desirable for clinical applications to ensure that the gel does not exceed the trauma boundaries and detach from the wound site.

The degradation time of the PEG-LA-DA/AGPR hydrogels at a PEG-LA-DA:AGPR ratio of 10/1, 5/1, 2/1, and 1/1 was measured to be 30, 36, 39, and 45 days, respectively, indicating a controllable manner of degradation. In comparison, the gel composed only of PEG-LA-DA co-polymer degraded in 27 days (SI: Figure S10).

The storage modulus G ′ and loss modulus G ′′ were measured to assess the mechanical stiffness of the samples ( Figure 3 a,b;


Scheme 1. Schematic description of the hydrogel from AGPR and PEG-LA-DA. PLA refers to poly(lactic acid) units.

Figure 1. a,b) Photographs of the hydrogel at room temperature. c–e) SEM images of the hydrogel with scale bars of 500 (c), 100 (d), and 20 (e) µm. The hydrogel was prepared from PEG-LA-DA/AGPR (at a 5/1 ratio) and fully swelled at 37 °C for 48 h.

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SI: Table S3) as a function of the angular frequency at a fi xed strain, γ = 10%. The G ′ values of the samples were larger than that of G ′′ over the range of frequencies tested (from 0.1 to 100 rad s −1 ), and all samples showed a single plateau region in the dynamic moduli. Notably, the mechanical stiffness of the hydrogels was obviously enhanced by adding AGPR to the system; particular enhancement was attained when the AGPR content reached 4%. This is due to 1) the increase in cross-linking density, resulting from multiple cross-linking sites on the periphery of the PAMAM dendrimers; and 2) the amide structures of PAMAM, which may allow for better gel stiff-ness than gels containing ester bonds. [ 17 ] However, intra- and/or intermolecular cross-linking reactions may occur among the AGPR themselves (SI: Figure S7) at higher concentration ratios (PEG-LA-DA:AGPR = 2/1, 1/1, 1/2), thus leading to a looser network, and consequently lower elastic moduli.

Strain amplitude sweeps demonstrated a broad elastic response range (Figure 3 c). The G ′ and G ′′ values were constant until the strain increased to 1000% (yielding point), indicating that the hydrogel could withstand a wide range of deforma-tion. This is a desirable property for bone repair or tissue engi-neering applications because the gel would need to withstand environmental changes resulting from mechanical loading. We assume that the PEG-LA-DA co-polymers act as soft ductile components to dissipate the stress, while the dendrimers are the hard units that enhance the modulus of the network. [ 18 ]

In order to better understand the properties of the hydrogel, we also evaluated the modulus of samples prepared by mixing 4% AGPR or 4% AGP with 20% PEG-LA-DA (SI: Figure S11). A similar storage modulus was observed for the two gels,

indicating that the incorporation of bio active components, such as RGDyC, did not nega-tively infl uence mechanical strength. This result is consistent with the report of Lev-ental and co-workers, who found that the mechanical stiffness of hydrogels was main-tained despite the introduction of various peptides. [ 19 ]

To evaluate the ability of the hydrogel to mimic ECM cues that support stem-cell pro-liferation and differentiation, we encapsulated mouse bone marrow mesenchymal stem cells (mMSCs) within the hydrogel. Hydrogels at a 5/1 ratio for both PEG-LA-DA/AGPR and PEG-LA-DA/AGP were evaluated, and PEG-LA-DA alone was used as the control. For the hyrdogel of PEG-LA-DA/AGPR, evidence of cell toxicity was not observed within the 7 days

that the cells were incubated in the hydrogel (SI: Figure S12); it showed more cell viability than the control sample. In contrast, hydrogels without RGDyC (PEG-LA-DA/AGP) had reduced cell viability, suggesting that introducing RGDyC into the hydrogel increased cell attachment and proliferation.

H&E staining indicated that the cells were evenly distributed in the hydrogel pores ( Figure 4 a). The 3D culture within the hydrogel enabled the cells to maintain their natural morphology while communicating with the surrounding microenvironment and responding to each other within an in vitro model system. DAPI–actin staining (DAPI = 4′,6′-diamino-2-phenylindole) showed that the cells in the hydrogel with coupled RGDyC exhibited stronger stress fi bers than those in the control sample (PEG-LA-DA alone); this may be due to enhanced cell adhesion and differentiation supported by the framework (Figure 4 a). [ 20 ]

It has been reported that the RGDyC presence can trigger α5β1 integrin receptor activation in mMSCs, which further promotes osteoblast proliferation and leads to an increased bone regeneration capacity. [ 21 ] Real-time polymerase chain reaction (PCR) analysis showed that the mRNA level of alka-line phosphatase (ALP), an early osteogenic marker of mMSC differentiation, was signifi cantly increased in cells cultured in the PEG-LA-DA/AGPR hydrogel as compared to the hydrogel without RGDyC (Figure 4 b). Expression of the bone differen-tiation markers osterix (OSX), parathyroid hormone 1 receptor (PTH1R), and osteocalcin (OC) was also signifi cantly up-regu-lated. The results suggest that the novel PAMAM-dendrimer-based hydrogel provides chemical and physical cues supporting mMSC osteogenic differentiation.


Figure 2. a) Equilibrium swelling ratio for PEG-LA-DA alone and PEG-LA-DA/AGPR at ratios of 10/1, 5/1, 2/1, and 1/1, measured on the indicated days. b) The average swelling ratio for PEG-LA-DA alone and PEG-LA-DA/AGPR at ratios of 10/1, 5/1, 2/1, and 1/1 ( n , number of tests > 3). The hydrogels were immersed in PBS buffer at 37 °C for 48 h, and then dried for degradation testing; the whole process was sustained for over 50 days.

Figure 3. Plots of the angular frequency ( ω ) versus a) storage modulus ( G ′) and b) loss modulus ( G ′′) of the hydrogels at different PEG-LA-DA:AGPR ratios (1/0 (PEG-LA-DA alone), 10/1, 5/1, 2/1, 1/1, 1/2). c) Oscillatory rheological measurements for PEG-LA-DA/AGPR at a 5/1 ratio.

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In conclusion, we have constructed a novel hydrogel system that mimics the ECM of native tissues. The introduction of AGPR in the gel promotes a highly porous network and effec-tively improves hydrogel properties, including an increase in mechanical stiffness and a reduction in the swelling ratio. The PAMAM-dendrimer-based hydrogel supported mMSC prolif-eration and differentiation in the absence of cytotoxic effects. This dendrimer-based hydrogel may serve as a model for devel-oping advanced materials with novel properties for applications in tissue engineering.

Experimental Section The synthetic procedures and characterization of AGPR and PEG-LA-DA, as well as the preparation and properties of the hydrogels, are described in detail in the SI. The 1 H NMR and FT-IR spectra, the GPC trails, SEM images, degradation profi les, and the cell viability assay are also provided in the SI.

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

Acknowledgements This work was fi nancially supported by the National Natural Science Foundation of China (21174005 and 21274004) through X.R.J. and the Chongqing Science and Technology Commission Natural Science Foundation of China (cstc2012jjA0178) through S.Y.

Received: January 21, 2014 Revised: February 26, 2014

Published online:

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Figure 4. a) H&E staining and DAPI–actin staining of MSCs in the hydrogel 3 days after encapsulation. Control: PEG-LA-DA (Linear); Test: PEG-LA-DA/AGPR = 5/1. b) Real-time PCR analysis for mMSC osteogenic differentiation. Alkaline phosphatase (ALP), osterix (OSX), parathyroid hormone 1 receptor (PTH1R), and osteocalcin (OC) were evaluated by real-time PCR to assess differentiation in the hydrogels at 4 and 7 days. 18S was used as a housekeeping gene. Control: PEG-LA-DA:AGP = 5/1; Test: PEG-LA-DA:AGPR = 5/1.

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a novel poly(amido amine)-dendrimer-based hydrogel as a mimic for the extracellular matrix

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