Polyamidoamine dendrimer-PEG hydrogel and its mechanical property on differentiation of mesenchymal stem cells

1. Introduction

Hydrogels are three-dimensional (3D) crosslinking hydrophilic polymeric networks that can hold large amounts of water or biological fluids. Due to their structural similarity to the natural extracellular matrix (ECM), hydrogels have gained considerable interest in biomedical fields. Since the pioneering research of Wichterle and Lim in 1960 [1], hydrogels have been extensively investigated and applied in diverse applications such as tissue engineering [2,3], 3D bioprinting [4,5], drug delivery [6,7], cancer research [8], and gene delivery [9,10]. In particular, in situ-forming hydrogels have demonstrated numerous advantages over conventional hydrogels because of their distinctive property of easy management in encapsulating biologically active materials and cells by mixing aqueous solutions of crosslinking components. They can be formed by minimal invasive injection of solutions into the target sites for sustained drug delivery or injectable tissue engineering. This provides a simple implantation method to avoid complicated surgical procedures [11,12]. Among the broad spectrum of investigations on in situ-forming hydrogels, many studies have recently shown that mechanical signals transmitted from ECM to cells, together with other genetic and molecular mediators have significant influences on many aspects of cellular behavior such as cell growth, spreading, migration, motility, and differentiation [13–17]. Therefore, the development of highly biocompatible in situ-forming hydrogel systems with tunable mechanical properties could be a powerful tool to manipulate cell functions and differentiations, which could possibly lead to useful clinical applications.

With this research, we present the synthesis and characterization of an in situ-forming hydrogel system containing polyamidoamine (PAMAM) dendrimer and polyethylene glycol (PEG), and the study of hydrogel stiffness on differentiation of MSCs. PAMAM dendrimers are a class of water-soluble, non-immunogenic biocompatible star-burst polymers that possess highly branched structures and ample active surface groups for chemical modification and bioconjugation. Due to their unique properties, PAMAM dendrimers have been widely applied in various biomedical fields including drug delivery [18,19], cancer targeting and treatment [20,21], catalysis [22,23], encapsulated nanoparticles [24,25], and imaging [26,27]. They have also been employed as a component in hydrogels primarily for drug delivery purposes [28–30]. In this study, we functionalize PAMAM dendrimer generation 5 (PAMAM G5) with vinyl sulfone functional groups, which crosslink with thiolated multi-armed PEG to produce in situ-forming hydrogels. PEG is a hydrophilic polyether with a backbone that forms hydrogen-bonds to water molecules [31]. It is a synthetic polymer with inherent biocompatibility that is commonly used in hydrogels for biomedical applications [32–34]. The fibronectin-derived peptide sequence RGD is incorporated in the hydrogel system in order to improve MSC cell adhesion to hydrogel matrix. The viability assay of MSCs cultured in PAMAM-PEG hydrogels after 48 hours indicates that the hydrogel system developed is biocompatible. By mixing different concentrations of PAMAM dendrimer and thiolated multi-armed PEG, hydrogels of varied stiffness (complex modulus from 77 Pa to 5,663 Pa) and gelation times (from 0.65 minutes to 4 hours) are prepared. Differentiation of MSCs atop soft and hard hydrogels in adipogenic and osteogenic induction media for 21 days indicates that the soft hydrogel favors adipogenic differentiation of MSCs, whereas hard gel favors osteogenic differentiation.

2. Materials and methods

Ethylenediamine-cored PAMAM G5 with amino end groups was purchased from Dendritech Inc. (Midland, MI, USA). The eight-armed thiol functionalized PEG (8arm-PEG-SH, 20 kDa) was ordered from NOF America Corporation (White Plains, NY, USA). The peptide CGRGDS with 98.3% purity was synthesized by Peptide 2.0 Inc. (Chantilly, VA, USA). 2-Iminothiolane hydrochloride and divinyl sulfone (DVS) were ordered from Fisher Scientific and were used as received. Rat bone-marrow MSCs were obtained from the Medical University of South Carolina and were originally isolated from Sprague-Dawley rats following a reported procedure [35]. Cells were used for experimentation at passage 2. Live/dead staining kit and primary culture medium containing Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 v/v% FBS, 1 v/v% penicillin, streptomycin, and amphotericin B were ordered from Life Technologies (Carlsbad, CA, USA). Osteogenic induction medium, adipogenic induction medium, and adipogenic maintenance medium were ordered from Lonza (Houston, TX, USA). Nucleus staining agent 4 ^′ ,6-diamidino-2-phenylindole (DAPI) and F-actin labeling agent phalloidin were ordered from Sigma-Aldrich and Molecular Probes (Eugene, OR, USA), respectively.

3. Nuclear magnetic resonance (NMR) analysis of PAMAM dendrimers

¹H-NMR spectra of dendrimer species were recorded using a Varian 400 MHz spectrometer (Santa Clara, CA, USA) in D₂O solvent.

4. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis of PAMAM dendrimers

An aliquot of 1 mg/mL solution of each dendrimer in methanol was mixed in a ratio of 1:1 with a saturated 𝛼-cyano-4-hydroxycinnamic acid matrix in acetonitrile containing 0.1% trifluoroacetic acid. The mixture (1 μL) was spotted on the MALDI plate, air-dried and analyzed by MALDI-TOF mass spectrometry (Bruker Ultraflex MALDI-TOF/TOF). The spectrometer was operated in a linear positive ion mode with a laser frequency of 20 Hz and 80% relative energy. External calibration was done based on the average value of [M + H⁺] of bovine serum albumin (BSA), m/z 33,336. A total of 2,000 shots were used to generate a spectrum.

5. Measurement of gelation time and mechanical properties

The in situ PAMAM dendrimer-PEG hydrogel was formed by mixing vinyl sulfone functionalized PAMAM dendrimers with 8arm-PEG-SH. A representative procedure of gelation time measurement is described in the following steps. Dendrimer 4 (9.0 mg) and 8arm-PEG-SH (17.6 mg) were dissolved in deionized water (300 μL) separately. The resultant dendrimer solution was mixed with PEG solution at 37 °C to form a hydrogel crosslink. The molar stoichiometry of vinyl sulfone functional group in dendrimer 4 to thiol group in PEG was calculated as 1:1. The actual gelation time was determined using capillary force following the method described in our earlier report [36].

Mechanical properties were measured using a DHR-3 rheometer (TA instrument) operated under a 12 mm parallel-plate geometry, a 2 mm plate spacing, and temperature controlled peltier plate. After preliminary amplitude sweep experiments to identify the linear viscoelastic range of the hydrogels, isothermal frequency sweep tests (1% strain amplitude) were performed with a frequency range from 0.1 Hz to 10 Hz. Typically, 200 μL of the hydrogel solution was used with a gap width of 2 mm between the plates. Each test was performed for eight minutes to avoid loss of water during measurement. The experiments were performed in triplicate.

6. Scanning electron microscopy (SEM)

The microstructures of hydrogels were analyzed using a JEOL 5600LV SEM. Samples were freeze-dried and placed on an aluminum stub with double stick tape then coated with 0.1 nm of gold-palladium using a Denton Desk II sputter coater.

7. MSC cell culture

Rat bone-marrow derived MSCs were obtained from the Medical University of South Carolina and used at passage 2. Primary culture medium containing DMEM supplemented with 10 v/v% FBS, 1 v/v% penicillin, streptomycin, and amphotericin B. Cells were cultured in a CO₂ incubator at 37 °C with 5% CO₂:95% air. MSCs with passage number 1 were seeded in T75 flasks. Media was replaced every 2 to 3 days. All polymer, peptide, cell culture reagents, and pipet tips were either sterile filtered, autoclaved, or sterilized via germicidal UV irradiation prior to cell culture.

8. Cell viability of MSCs in hydrogels

Hydrogels were prepared under sterile conditions. 8arm-PEG-SH (12.4 mg) was dissolved in 200 μL primary culture media. 100 μL of 2 × 10⁶ cell/mL MSCs was mixed with 100 μL of 4.0 w/v% dendrimers in primary culture media. The mixture of cells and dendrimer was then mixed with 8arm-PEG-SH solution to obtain 5 × 10⁵ cell/mL as the final cell concentration in the gel. The cell-hydrogel mixture (100 μL) was then injected into 96-well plate with triplet for each sample and incubated at 37 °C in a CO₂ incubator with 5% CO₂: 95% air for 48 hours. Live/dead staining kit was used to label the cells encapsulated in hydrogel based on the manufacturer’s manual. After 48 hours of cell culture, 150 μL of a mixture of 2 μM calcein AM and 4 μM EthD-1 working solution were added to each cell-hydrogel culture well and incubated for 45 minutes at room temperature. Hydrogels in each well were then removed and placed on microscope slides and covered with coverslips. A TCS SP5 AOBS laser scanning confocal microscope was used to collect fluorescent images of cells. ImageJ software was used to quantitatively determine the viability of cells based on the green fluorescent intensity of live cells.

9. Cell differentiation and immunofluorescent staining

MSCs were seeded atop hydrogel thin films in 48-well plates at a density of 3 × 10³ cells/cm² for osteogenic differentiation and 2 × 10⁴ cells/cm² for adipogenic differentiation. For the osteogenic plate, cells were cultured in the primary culture medium after being seeded on the hydrogel. After 24 hours of culture, the media was replaced by osteogenic induction medium. The MSCs were fed every 3 days for 3 weeks. For the adipogenic plate, cells were cultured in the primary culture media until the cultures reached confluence. At 100% confluence, three cycles of induction/maintenance were conducted for optimal adipogenic differentiation. Each cycle consisted of feeding the rat MSCs with supplemented adipogenic induction medium and culturing for three days followed by 1–3 days of culture in supplemented adipogenic maintenance medium. After three complete cycles of induction/maintenance, the rat MSCs were cultured for seven more days in supplemented adipogenic maintenance medium, with the medium replaced every 2–3 days. At the end of the induction, cells were fixed in 4% paraformaldehyde solution for 30 minutes. After washing (3 times at 10 minutes each) with PBS buffer containing 0.1% Triton X-100 (PBST), the fixed cells were then incubated with rhodamine phalloidin (Molecular Probes, Eugene, OR) for two hours at room temperature, followed by washing with PBST 3 times at 10 minutes each. Cells stained with phalloidin were further counterstained with DAPI for 2 hours at room temperature, followed by washing with PBST buffer 3 times at 10 minutes each. Fluorescent images of the stained osteogenic samples were obtained using a TCS SP5 AOBS laser scanning confocal microscope, and the images of adipogenic samples were recorded with an Olympus IX73 inverted microscope.

10. Results and discussion

Figure 1 displays the synthesis of PAMAM dendrimers with multiple vinyl sulfone groups which in situ crosslink with thiol groups on commercial 8arm-PEG-SH to form a hydrogel. Among the varied generations and terminal functional groups in the PAMAM dendrimer family, the ethylenediamine-cored PAMAM G5, bearing 110 amine groups as analyzed by potentiometric titration, has been selected for hydrogel synthesis due to its optimal size, molecular weight, and ample available terminal amino groups for chemical reactions. The numerical subscript outside the parenthesis of each functional group represents the number of that functionality intended to be attached per dendrimer. PAMAM dendrimer G5 is first surface modified by partial acetylation with acetic anhydride, yielding G5-Ac(75) (2) with 75 terminal amine groups acetylated and 35 remaining amino groups available for further chemical modification. Thiolated dendrimer G5-Ac(75)-SH(35) (3) is achieved by reaction of G5-Ac(75) with Traut’s regent in aqueous conditions. Further reaction of G5-Ac(75)-SH(35) with DVS produces vinyl sulfone functionalized dendrimer G5-Ac(75)-DVS(35) (4). For enhancement of cell attachment and proliferation, RGD peptide is attached to dendrimer by conjugation of different equivalents of CGRGDS peptide to PAMAM dendrimer in aqueous conditions to accomplish bifunctional dendrimers 5-7. All dendrimer species are purified by membrane filtration through a 10,000 molecular weight cut-off (MWCO) membrane and extensively washed with PBS buffer and water, followed by lyophilization.

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Fig. 1.

Synthetic scheme of vinyl sulfone and RGD functionalized PAMAM dendrimer conjugates.

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Fig. 2.

¹H NMR spectra of G5-Ac(75)-DVS(35) (4) and G5-Ac(75)-DVS(29)-RGD(6) (7).

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Fig. 3.

MALDI-TOF measurement of average molecular weights of dendrimers. (a) G5-NH₂; (b) G5-Ac(75); (c) G5-Ac(75)-SH(35); (d) G5-Ac(75)-DVS(35); (e) G5-Ac(75)-DVS(34)-RGD(1); (f) G5-Ac(75)-DVS(32)-RGD(3); (g) G5-Ac(75)-DVS(29)-RGD(6).

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Table 1

Summary of molecular weights and numbers of functionalities on dendrimer

Species	Structure	Mw a	# of functionality per dendrimer
Dendrimer 1	G5-NH2	26,400	NH2 (110)
Dendrimer 2	G5-Ac(75)	29,600	Ac(74 a , 72 b )
Dendrimer 3	G5-Ac(75)-SH(35)	33,300	SH(37 a , 40 b )
Dendrimer 4	G5-Ac(75)-DVS (35)	37,270	DVS(33 a , 29 b )
Dendrimer 5	G5-Ac(75)-DVS(34)-RGD(1)	37,900	DVS(20 b ); RGD (1.2a)
Dendrimer 6	G5-Ac(75)-DVS(32)-RGD(3)	39,100	DVS(26 b ); RGD (3.4a)
Dendrimer 7	G5-Ac(75)-DVS(29)-RGD(6)	41,200	DVS(18 b ); RGD (7.2a)

^a measured by MALDI-TOF; ^b measured by ¹H-NMR integration.

The chemical composition and success of sequential conjugation of dendrimer species are characterized by ¹H NMR spectroscopy. Figure 2 displays the representative ¹H NMR spectra of G5-Ac(75)-DVS(35) (4) and G5-Ac(75)-DVS(29)-RGD(6) (7). Proton spectra of other dendrimer species can be found in our earlier report [36]. The alkenyl protons in vinyl sulfone (a, b and c) are distinctively observed at 6.3 and 6.8 ppm in both spectra, indicating the success of functionalization of dendrimer with DVS. The RGD peaks, even though small, are clearly seen at 4.1, 3.8 and 3.7 ppm (Fig. 2B), which are assigned to protons on 𝛼-carbons of the peptide backbone, and protons next to oxygen and nitrogen. The number of vinyl sulfone groups per dendrimer can be calculated from proton integration of vinylic protons (a, b and c) relative to the acetyl methyl group at 1.8 ppm, which can be quantitatively determined by the proton integration following a reported procedure [37]. MALDI-TOF is an additional powerful strategy to characterize PAMAM dendrimers by determining the average molecular weights (Fig. 3). The consistent increase of average molecular weights demonstrates the success of sequential conjugation of functional groups on PAMAM dendrimers. In addition, the number of functional groups per dendrimer can also be quantitatively determined based on the increase of average molecular weights. Table 1 summarizes the numbers of each functionality on PAMAM dendrimers characterized by ¹H NMR and MALDI-TOF. It is clearly seen that the numbers of functional groups on dendrimers determined by ¹H NMR integration are consistent with those determined by MALDI-TOF, which demonstrates the reliability of both analytical methods for dendrimer characterization.

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Table 2

Gelation time and stiffness of PAMAM-PEG hydrogels at varied concentrations

Entry #	G5-Ac(75)-DVS(35) (mg)	H2O (μL)	8arm-PEG-SH (mg)	H2O (μL)	Dendrimer (w/v%)	Gelation time (min)	Modulus (Pa)
1	7.5	300	14.5	300	1.2	263	77 ± 18
2	9.0	300	17.6	300	1.4	7.3	674 ± 65
3	14.2	300	27.6	300	2.2	2.1	2,169 ± 53
4	24.1	300	46.9	300	3.6	0.87	3,929 ± 271
5	36.2	300	70.4	300	5.1	0.65	5,663 ± 701

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Fig. 4.

Mechanical property of hydrogels with various dendrimer concentrations. (A) Complex modulus of hydrogel samples measured from 0.1–10 Hz. (B) Average complex modulus of hydrogel samples (n = 33, ^∗ p < 0.0001).

Hydrogels are formed when aqueous solutions of DVS functionalized PAMAM dendrimer (4) are mixed with aqueous solutions of 8-arm-PEG-SH. Table 2 and Fig. 4 summarizes the gelation time and mechanical property of hydrogels at varied concentrations. The amounts of dendrimers and 8arm-PEG-SH are calculated based on equivalent number of DVS groups on dendrimer and thiol groups on PEG. Under experimental conditions, the gelation time decreases from 263 min to 0.65 min and the complex modulus increased from 77 Pa to 5,663 Pa, as the dendrimer concentrations increase from 1.2% to 5.1 w/v%. Dendrimer concentration lower than 1.2% results in no gel formation after three days at 37 °C when mixed with 8arm-PEG-SH, while dendrimer concentration higher than 5.1% causes solubility problems. Roughly, the dendrimer and PEG concentrations experimented cover the whole range of concentrations applicable for hydrogel formation, with the highest complex modulus being nearly 74 times that of the lowest. It is easily seen that hydrogels formed with higher concentration of dendrimers and 8arm-PEG-SH exhibit less gelation time and higher stiffness. However, no mathematical relationship is observed among dendrimer concentration, gelation time and stiffness.

The representative microstructures of hydrogels are characterized by SEM. Figure 5 illustrates the microstructure of hydrogels with dendrimer concentrations of 1.2% (77 Pa) and 5.1% (5,663 Pa), respectively. It can be observed that the hard gel shows denser and less porous structures than the soft gel.

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Fig. 5.

Scanning electron microscopy (SEM) of PAMAM dendrimer-PEG hydrogels. (A) Soft gel (complex modulus 77 Pa). (B) Hard gel (complex modulus 5,663 Pa). The scale bar is 100 μm.

The biocompatibility of the synthesized hydrogels is tested by viability of MSCs in hydrogels with dendrimer concentration of 4 w/v% crosslinked with 8arm-PEG-SH (Fig. 6A). The columns 1 through 4 in Fig. 6A represent hydrogels from dendrimers conjugated with 0, 1, 3, and 6 RGD peptides, respectively. Live and dead cells are stained green and red with calcein AM and EthD-1, respectively, using a live/dead staining kit after 48 hours of culture. MSCs are found viable in all hydrogel samples with very minimum number of dead cells observed. The viable cells are quantitatively determined by the intensity of green fluorescence using NIH free ImageJ software. The percentage of live cells in Fig. 6B is calculated as the optical density of live cells (green) divided by the total optical density of both live and dead cells (green and red) in the three dimensional hydrogel culture media. Figure 6B indicates that MSCs maintain roughly the same viability in all hydrogel samples. It is worth noticing that MSCs in the hydrogel containing 6 RGD peptides per dendrimer show significant cell spreading as displayed by cell morphology (Fig. 6A column 4), indicating RGD peptide promotes cell attachment and proliferation in hydrogel matrix.

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Fig. 6.

Cell viability of MSCs in PAMAM dendrimer-PEG hydrogels. (A) The hydrogels 1-4 are prepared with PAMAM dendrimers 4-7 which are functionalized with 0, 1, 3, 6 RGD per dendrimer. All hydrogels are formed by mixing 4 w/v% dendrimer solutions with equal volume PEG with equivalent amount of thiol groups. Cell concentration in each sample is 5 × 10⁵ cells/mL. Live and dead cells are stained green and red, with calcein AM and EthD-1, respectively. All images share the same scale bar: 100 μm. The inset is the enlarged picture of a representative single cell for morphology visibility. (B) Cell viability was quantitatively determined by ImageJ based on green fluorescent intensity in images. The percentage of live cells is calculated as the optical density of live cells (green) divided by the total optical density of both live and dead cells (green and red) in the three dimensional hydrogel culture media.

To study the effects of hydrogel stiffness on MSC adipogenic and osteogenic differentiation, the MSCs are cultured on surfaces of both hard and soft gels in 48-well plates for three weeks. The osteogenic differentiation of MSCs in hard gel (5,663 Pa) is measured by confocal microscopy after three weeks in order to observe the details of cell morphology in differentiation. MSCs are found difficult to attach to gel surfaces in nature, which leads to partial cell death. In order to enhance MSC cell attachment to hydrogel surface, the PAMAM-Ac(75)-DVS(29)-RGD(6) is selected to make the hydrogel samples. The cell attachment is improved and the morphology is recorded using a TCS SP5 laser scanning confocal microscope (Fig. 7). Fluorescently-labeled phalloidin is used to stain the F-actin of MSCs in green and DAPI is used to stain the nucleus in blue. It can be seen that a higher degree of osteogenic differentiation is observed in hard gels than in soft gels as demonstrated by the more prominent polygonal, elongated, and detailed filopodia structures at the end of the elongated cells. Despite the fact that some degree of osteogenic differentiation in soft gel is also observed, which is due to the function of differentiation inducing agents in the osteogenic induction media such as ascorbate and beta-glycerophosphate, it can still be concluded that the stiffness of hydrogels facilitates osteogenic differentiation of MSCs.

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Fig. 7.

Morphology of MSC cells with osteogenic media on surface of soft (77 Pa) and hard (5,663 Pa) gels after 21 days. Actin was stained with phalloidin-488 (green) and nuclei with DAPI (blue). All scale bars are 100 μm.

Figure 8 displays the morphology of MSCs in hydrogels of different stiffness after 21 days of culture in adipogenic induction media, with an Olympus IX73 inverted microscope. Like the cells in osteogenic media, some degree of adipogenic differentiation is observed in both soft and hard gels as demonstrated by the spherical cell morphology which is due to differentiation inducing agents in adipogenic induction media such as indomethacin and 3-isobuyl-methyl-xanthine. However, it is clearly seen that MSC cells cultured in soft gel have shown higher viability, proliferation, and more adipogenic differentiation, which leads to the conclusion that soft hydrogel favors adipogenic differentiation of MSC cells over hard gels.

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Fig. 8.

Morphology of MSC cells with adipogenic media on surface of soft (77 Pa) and hard (5,663 Pa) hydrogels after 21 days’ culture. All scale bars are 50 μm.

11. Conclusions

We have successfully developed PAMAM dendrimer-PEG hydrogel system through crosslinking vinyl sulfone functionalized PAMAM G5 with 8arm-PEG-SH. The PAMAM dendrimers are extensively characterized by ¹H NMR and MALDI-TOF. The microstructures of soft and hard gels are characterized by SEM and their mechanical property with various stiffness is measured by a rheometer. Viability assay of MSC cells after 48 hours of culture in hydrogel demonstrated the biocompatibility of PAMAM dendrimer-PEG hydrogel system. The MSC differentiation experiments in hydrogels of different stiffness show that soft hydrogel favors adipogenic differentiation while hard gel favors osteogenic differentiation, which demonstrates that the substrate’s mechanical property has great effects on stem cell behavior and lineage commitment. In addition, the induction media used also influences the differentiation of MSC cells towards its lineage. This in situ-forming hydrogel system with well-defined chemical structures, functionalized RGD peptide, and tunable mechanical property has sufficient biocompatibility and supports cell adhesion, proliferation, and differentiation of MSC cells. The system developed may serve as suitable scaffolds for cartilage tissue engineering and repairs of soft and hard tissue defects.

Footnotes

Conflict of interest

None to report.

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