Synthesis, characterization and chondrocyte culture of polyhedral oligomeric silsesquioxane (POSS)-containing hybrid hydrogels

Abstract

Polyhedral oligomeric silsesquioxanes (POSS)-based hybrid hydrogels were successfully prepared via a fast azide-alkyne click reaction between octa-azido-functionalized POSS (OAPOSS) and alkyne-functionalized poly(ethylene glycol) (PEG). A series of hybrid POSS-PEG hydrogels with the highly ordered porous structures were achieved and the pore size could be well controlled by varying the PEG chain length to 3k, 6k and 10k. After immersing in phosphate buffered saline (PBS), POSS-PEG hydrogels exhibited a good water absorption capacity, and their swelling ratio increased with the increase of the PEG chain length. Rheological measurements indicated that all hydrogels exhibited the characteristics of an elastomer. The elastic modulus and ultimate stress (at break) were significantly enhanced with the increase of the PEG chain length which has been revealed by stress–strain mechanical analysis. In vitro cell culture showed that all POSS-PEG hydrogels could well support chondrocytes attachment, spreading and proliferation, especially for the POSS-PEG (10k) hydrogel. Thus, POSS-PEG hydrogels exhibited the potential for cartilage tissue engineering.

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Introduction

Hydrogels are three-dimensional (3D) cross-linked networks that are capable of absorbing and retaining water.¹ During past decades, owing to their unique advantages such as biocompatibility, water storage ability alongside softness which is similar to the natural extracellular matrix (ECM), they have played an increasingly important role in biological and biomedical fields including drug delivery, regenerative medicine and tissue engineering.^2–6

Polyhedral oligomeric silsesquioxanes (POSS), with a unique cubic cage-shaped nanostructure and a multiplicity of functional vertices, have been attracting great attention for the preparation of inorganic–organic hybrid hydrogels since they exhibit enhanced properties such as good biocompatibility, special surface properties and high mechanical stability.^7–12 In general, POSS-containing hybrid hydrogels could be fabricated by chemical and physical cross-linking approaches.^13,14 For example, POSS bearing one reactive group could be introduced into polymer hydrogels using chemical crosslinking. Wang et al. reported photo-crosslinkable poly(propylene fumarate) (PPF)-co-POSS copolymers with POSS molecules chemically linked to the PPF backbone via polycondensation, and then the mechanical and biological performance of crosslinked PPF-co-POSS were also studied.¹⁵ What's more, the typical POSS molecule with multi-functional groups could be utilized as a crosslinker to prepare POSS-containing hybrid hydrogels. We previously prepared thermal-responsive hybrid poly(N-isopropylacrylamide) (PNIPAM) hydrogels with octa-methacrylate POSS (OMAPOSS) as a crosslinking agent via γ-ray irradiation, and further studied their swelling/deswelling behavior.¹⁶ Jiang et al. utilized yne-ended hyperbranched poly(ether amine) (hPEA-yne) and thiol-containing polyhedral oligomeric silsesquioxane (PEG-POSS-SH) to generate a patterned hydrogel, which was facile to be further functionalized.¹⁷ Nischang et al. fabricated hybrid hydrogels based on OMAPOSS and poly(ethylene glycol) (PEG) diester macromonomers by in situ radical-mediated thiol-ene photopolymerization, and their potential applications in biomedical areas were further evaluated.¹⁸ On the other hand, much efforts have also been focused on POSS-containing hybrid hydrogels where POSS moieties were physically linked. Reno et al. prepared the POSS-containing hybrid hydrogels where octa-ammonium POSS (POSS-NH₃⁺) was incorporated into chemical crosslinked gelatin–polyglutamic acid hydrogels. POSS-NH₃⁺ could uniformly dispersed through the electrostatic interactions between the positive charges of POSS-NH₃⁺ and the residual negative charges of gelatin chains.¹⁹ Zheng and co-workers constructed physical interpenetrating polymer networks (IPNs) by incorporation of POSS-containing linear hybrid polymer into cross-linked polymeric network.²⁰

More recently, click chemistry have demonstrated to be a useful technique to construct functional materials with well-defined architectures, including hydrogels.^21–24 Typically, the copper-catalyzed 1, 3-dipolar cycloaddition click chemistry (CuCC) between azides and alkynes has attracted much attention due to the mild reaction conditions, fast reaction speed, high yielding with no by-products and a good tolerance of functional groups.^25–28 For instance, Hawker et al. designed a PEG hydrogel based on diacetylene-functionalized and tetraazide-functionalized PEG derivatives, which has been proved to have a more ideal structure and improved properties in comparison to traditional chemically crosslinked PEG hydrogels.²⁹ Huerta-Angeles et al. respectively synthesized alkynyl and azide functionalized hyaluronic acid (HA), and they were further applied to construct HA based hydrogels which could support chondrocytes proliferation.³⁰ Overviewing the past approaches utilized for the preparation of POSS-containing hydrogels, we found alkyne-azide click chemistry have not been involved.

In the past decades, hydrogels have been proved to be routine biomaterials for tissue engineering since they could be prepared from different raw materials (natural or synthetic materials) in terms of various architectures (particles, films or porous scaffolds), which endow them with alterable structures and biological properties.^31–35 Therefore, various hydrogels have been developed and applied for cartilage tissue engineering.^36–38 Gao et al. designed a biological hydrogel synthesized from modified HA, chondroitin sulfate and gelatin via click chemistry. This hydrogel was further utilized to mimic extracellular matrix (ECM) and exhibited positive effects on the adhesion and proliferation of chondrocytes.³⁹ Bryant et al. designed PEG hydrogels by free-radical photopolymerization of acrylates or thiol-norbornenes, and then confirmed that both polymerization mechanism and network structure could affect the quality of engineered cartilage.⁴⁰ Detamore et al. prepared interpenetrating networks (IPNs) of PEG and agarose with controllable mechanical performance for cartilage tissue engineering.⁴¹ In view of the fact that POSS processes good biocompatibility and high mechanical stability, POSS-containing hydrogels might be alternative materials for cartilage tissue engineering.

In this contribution, we first constructed POSS-containing hybrid hydrogels via the azide-alkyne click reaction between octa-azido-functionalized POSS (OAPOSS) and alkyne-functionalized PEG, which provided a simple and efficient strategy to construct POSS-containing hybrid hydrogels. The thermal stability, swelling and mechanical properties of POSS-PEG hybrid hydrogels were studied, respectively. Moreover, in vitro chondrocytes culture on POSS-PEG hydrogels were performed, and the cell viability, attachment, spreading and proliferation of chondrocytes were evaluated (Scheme 1).

Scheme 1 Preparation and in vitro cell culture of inorganic–organic hybrid POSS-PEG hydrogels based on OAPOSS and alkyne-functionalized PEG.

Experimental

Materials

Poly(ethylene glycol) (PEG) with different molecular weight of 3000 (3k), 6000 (6k) and 10 000 (10k) g mol⁻¹ were purchased from Sigma-Aldrich. Propargyl bromide, sodium azide (NaN₃) and sodium ascorbate were purchased from Aldrich and used as received. Other reagents such as sodium hydride (NaH), calcium hydride (CaH₂), copper sulfate (CuSO₄) and magnesium sulfate (MgSO₄) were purchased from Sinopharm Chemical Reagent Co. and used as received. Solvents such as tetrahydrofuran (THF), N,N-dimethylformamide (DMF), methanol, concentrated hydrochloric acid (HCl) in analytical grade were purchased from Sinopharm Chemical Reagent Co. before use, THF was refluxed above sodium and then distilled.

Dulbecco's modified eagle's medium (DMEM) and phosphate-buffered saline (PBS) were purchased from HyClone (USA). Fetal bovine serum (FBS) was purchased from GIBCO (USA). Collagenase type II, L-proline, non-essential amino acid and L-ascorbyl acid and streptomycin/penicillin were purchased from Beyotime Institute of Biotechnology (China). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Japan). Ethylenediamine tetraacetic acid (EDTA)/trypsin, calcein-AM, propidium iodide (PI), rhodamine-phalloidin and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma (USA).

Preparation of POSS-PEG hydrogels

POSS-PEG hydrogels were prepared using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction with CuSO₄ and sodium ascorbate as the catalyst system. First, 100 mg of alkyne-functionalized PEG was dissolved in 1 mL DMF, and then OAPOSS and CuSO₄·5H₂O (dissolved in 50 μL deionized water) were added into above PEG solution with the molar ratio of alkyne-PEG : OAPOSS : CuSO₄·5H₂O = 4 : 1 : 2. After stirring for few minutes, sodium ascorbate in 50 μL deionized water (at a molar ratio of sodium ascorbate : CuSO₄·5H₂O = 1 : 1) was added to the mixture. Hydrogels were obtained in a few minutes and followed washed in a large volume of PBS for several days to remove THF and the residual copper ions.

Characterization

Nuclear magnetic resonance (NMR). All ¹H-NMR spectra were performed on a BRUKER AV400 spectrometer (400 MHz) in CDCl₃ with tetramethylsilane (TMS) as an internal reference.

Fourier transform infrared (FT-IR). All FT-IR spectra were recorded in the range of 4000 to 400 cm⁻¹ using a Perkin-Elmer Spectrum One FT-IR spectrophotometer equipped with an ATR sampling unit (25 °C).

Thermogravimetric analysis (TGA). The thermal properties of the POSS-PEG hydrogels were measured using a Perkin-Elmer Pyris-1 thermogravimetric analyzer in a nitrogen atmosphere from 100 to 700 °C at a heating rate of 10 °C min⁻¹.

Scanning electron microscopy (SEM). Morphology of POSS-PEG hydrogels was characterized by scanning electron microscope (SEM, JSM-6360LV, Jeol). All hydrogel samples were dehydrated by freeze-drying for 48 h after immersing in PBS to reach swelling equilibrium. Afterwards, hydrogel surfaces were sputter-coated with a thin layer of gold and imaged at an accelerating voltage of 10 kV.

Swelling measurements

To study swelling properties of hydrogels, freeze-dried POSS-PEG hydrogel samples were immersed in PBS at room temperature for 36 h. After each predetermined time interval, the hydrogel was taken out, and the weight was measured after removing excess surface deionized water with filter paper. The swelling ratio (SR) was defined as follows:

SR = (W_t − W₀)/W₀ × 100% (1)

where, W_t and W₀ are the weight of the swollen hydrogel and the initial weight of the freeze-dried hydrogel, respectively.

Mechanical characterizations of POSS-PEG hydrogels

Rheological measurements were performed using a rotational rheometer (RS600, Thermo Hakke, USA). A special mold was used to fabricate hydrogel disks with dimensions of 20 mm × 1.0 mm (diameter × thickness). Storage and loss moduli were measured using a constant strain mode with dynamic frequency range of 0.01 to 10 Hz (rad per s) at a proper strain (1%). The stress–strain mechanical analysis was performed using a mechanical testing system (GT-TCS2000, GOTECH, China) equipped with a computerized control and measurement system. POSS-PEG hydrogel samples for the measurement were prepared with dimensions of 10 mm × 5.0 mm (diameter × thickness), and then all samples were compressed with a crosshead speed of 1 mm min⁻¹ until the hydrogels fractured. Finally, the elastic modulus was calculated according to the initial 10% portion of the stress–strain curve which was nearly linear.

Chondrocyte culture

Chondrocytes were isolated from the cartilage tissue of bovine. In brief, the cartilage tissue was cut into small pieces and then washed with PBS containing 0.25% EDTA/trypsin at 37 °C for 90 min, followed by incubation in DMEM containing 0.2% collagenase type II at 37 °C for 4 h. The isolated chondrocytes were cultured in DMEM supplemented with 10% fetal calf serum, 1% streptomycin/penicillin, 1% L-proline, 1% non-essential amino acid, and 1% L-ascorbyl acid under standard conditions at 37 °C, 5% CO₂ in a humidified incubator, and the culture medium was exchanged every 2 days. Chondrocytes were sub-cultured at 90–95% confluency and used for experiments with passage 2.

Hydrogel films (1 mm thickness) were prepared by a special mold, and then cut into small disks with dimensions of 15.6 mm diameter which could completely cover the well bottom of 24-well plates after reaching swelling equilibrium. POSS-PEG hydrogel films were sterilized with 75% ethanol for 4 h and followed by UV-illumination from both sides for 30 min. The sterile hydrogel films were washed with PBS for three times and then incubated in a small volume of growth medium for 12 h before cell seeding. Afterwards, cell suspensions were added directly into each well. For cell growth and live/dead assay, chondrocytes were seeded at a density of 2 × 10⁴ cells per well. For cytoskeleton staining, the seeding density was 5 × 10⁴ cells per well.

Cell viability, attachment and proliferation of chondrocytes on POSS-PEG hydrogel films were separately measured. Chondrocytes seeded on 24-well plates at the same seeding density were performed in parallel as the control. The cell viability of chondrocytes on different POSS-PEG hydrogel films was assessed by live/dead assay on day 1 and day 3. After removals of culture medium, samples were directly rinsed with PBS containing 2 mM calcein-AM and 2 mM PI in dark at 37 °C for 30 min. The images were then acquired using a Ti-5 fluorescence microscope (Nikon, Japan). In addition, CCK-8 assay was performed to further evaluated cell viability of chondrocytes. After each predetermined time interval, each sample was incubated with DMEM containing 10% (v/v) CCK-8 in dark at 37 °C for 3 h. The optical density (OD) values of each supernatant at 450 nm were measured by a microplate reader (PowerWave XS2, BioTek, USA) in a 96-well plate. Cytoskeleton staining of chondrocytes on POSS-PEG hydrogel films was performed to visualize the actin cytoskeleton (morphology) of chondrocytes. After culturing for 4, 12 and 24 h, hydrogel films were removed from cell culture plates and rinsed with PBS for three times. Samples were successively fixed with 4% paraformaldehyde in PBS for 20 min and soaked in 0.25% Triton X-100 for 10 min. Afterwards, samples were washed with PBS again and then rinsed with PBS containing 0.06 mmol L⁻¹ rhodamine-phalloidin fluorescent dye and 2 μg mL⁻¹ DAPI in dark at 37 °C for 30 min. Then the samples were washed with PBS to remove unbounded phalloidin conjugate and DAPI. Finally, the cytoskeleton and nucleus of chondrocytes were viewed by a Ti-5 fluorescence microscope. Cell areas at 4, 12 and 24 h were further measured to determine the cell spreading of chondrocytes. In brief, 20 non-overlapping cells were chosen and their areas were calculated and averaged by ImageJ software (National Institutes of Health, USA). Cell numbers of chondrocytes after 1 and 3 day incubation were also measured by CCK-8 assay. After each predetermined time interval, cell-seeded POSS-PEG hydrogel films were washed with PBS for three times to remove the unattached cells. Samples were incubated with DMEM containing 10% (v/v) CCK-8 and the optical density (OD) values of each supernatant were further determined. Cell attachment of chondrocytes at 4 h was further evaluated by normalizing the attached chondrocytes on POSS-PEG hydrogel films to those on well bottom.

Statistical analysis

All experiments were in triplicate (n = 3) and each value represents the mean ± standard error. Statistical significance was evaluated by ANOVA using GraphPadPrism 6.0 with a level of significance p < 0.05.

Results and discussion

Synthesis of OAPOSS and alkyne-functionalized PEG

The synthesis routes for OAPOSS and alkyne-functionalized PEG were separately illustrated in Scheme 2. OCPOSS was prepared via hydrolytic condensation of 3-chloropropyltrimethoxysilane as described by Marsmann et al.⁴² Afterwards, the terminal chlorine group was then converted to azide moiety by substitution reaction between OCPOSS and excessive NaN₃ in DMF. The ¹H-NMR spectra of OCPOSS and OAPOSS were shown in Fig. S1.† It could be clearly seen that after azidation reaction the resonance of all methylene proton signals had a relatively high-field chemical shift, revealing that the reaction was completely carried out. Similarly in Fig. S2,† a strong absorbance peak appeared in the FT-IR spectrum of OAPOSS (∼2100 cm⁻¹), which was assignable to the characteristic of terminal azide group of OAPOSS, indicating that the complete substitution of chlorine with azide groups. All these results confirmed that OAPOSS was successfully synthesized.

Scheme 2 Synthesis of OAPOSS and alkyne-functionalized PEG.

PEG and its derivatives, as a class of promising biomaterials, have been widely applied in medical and biological fields such as drug delivery and tissue engineering due to their biocompatible, non-immunogenic and hydrophilic properties.^43–45 Among these PEG-based biomaterials, the hydrogels were routinely used, and mostly constructed using PEG chains with terminated functional groups. For example, Reichert et al. utilized azide- and acetylene-terminated PEG chains to generate well-defined hydrogels via azide-alkyne click reaction with CuSO₄ and sodium ascorbate in water as a catalytic system.⁴⁶ In current work, alkyne-functionalized PEG was synthesized via substitution reaction between PEG and propargyl bromide. Typically, ¹H-NMR spectrum of alkyne-functionalized PEG (6k) was shown in Fig. S3.† The ratio of the integral intensity of alkyne protons to that of ethylene protons of PEG was consistent with the theoretical value, suggesting that the substitution reaction was performed completely. Moreover, Fig. 1 showed FT-IR spectra of the PEG (6k) and alkyne-functionalized PEG (6k). Notably, the characteristic hydroxyl absorbance peak at ∼3450 cm⁻¹ disappeared, and the new characteristic alkyne absorbance peak appeared at ∼3240 cm⁻¹, which further confirmed that alkyne-functionalized PEG (6k) was successfully synthesized.

Fig. 1 FT-IR spectra of PEG (6k), alkyne-functionalized PEG (6k), OAPOSS and POSS-PEG (6k) hydrogel.

Preparation of POSS-PEG hydrogels

POSS-PEG hydrogels were prepared via the Huisgen 1, 3-dipolar cycloaddition between the azide groups of OAPOSS and the terminal alkynyl groups of alkyne-functionalized PEG.⁴³ The reaction was performed by CuSO₄ and sodium ascorbate as the catalyst system under mild condition. Obviously, it could be seen that characteristic absorbance peaks of the alkynyl and azide groups separately at ∼3240 cm⁻¹ and ∼2100 cm⁻¹ disappeared in the FT-IR spectrum of the POSS-PEG (6k) hydrogel (Fig. 1). Therefore, the reaction between the alkyne groups of PEG and the azide groups of OAPOSS was well performed, and POSS-PEG (6k) hybrid hydrogels were successfully prepared.

Fig. 2A clearly showed that all POSS-PEG hydrogels were strong enough to maintain their physical shape, while a slight increase of the swelling volume could be found with the increase of PEG chain length. Since the pore size and interconnect structure are critical parameters on regulating tissue regeneration, the microstructures of POSS-PEG hydrogels prepared with different PEG chain length were characterized by scanning electron microscopy (SEM). As shown in Fig. 2B, all POSS-PEG hydrogels had a porous structure. The average pore size of POSS-PEG (3k), POSS-PEG (6k) and POSS-PEG (10k) was 7.43 ± 1.9, 13.2 ± 2.2 and 17.4 ± 3.6 μm, respectively. Moreover, an increased pore size and network density could be clearly seen with the increase of PEG chain length, due to the lower crosslinking density. Therefore, they could provide sufficient surface area for cell binding, cell migration, cell growth, cell-to-cell contact and nutrient exchange.⁴⁷

Fig. 2 (A) The optical images of rhodamine dyed (I) POSS-PEG (3k), (II) POSS-PEG (6k) and (III) POSS-PEG (10k) hydrogels. All samples were prepared by a special mold with dimensions of 15 mm × 1.0 mm (diameter × thickness), and then immersed in PBS to reach swelling equilibrium. (B) SEM images of POSS-PEG hydrogels with different magnifications, (a) ×1k, scale bar: 50 μm; (b) ×2k, scale bar: 20 μm.

Properties of POSS-PEG hydrogels

The thermal property of POSS-PEG hydrogels with different PEG chain length was determined by thermogravimetric analysis (TGA) in nitrogen, and their TGA curves were shown in Fig. 3. All POSS-PEG hydrogels exhibited nearly the same thermal decomposition behavior with a similar on-set thermal degradation temperature (∼360 °C), and the most weight loss occurring at 420–430 °C. Compared to the thermal stability of the precursor PEG, which was revealed in our previous report,⁴⁸ the on-set thermal degradation temperature did not increase whenever for POSS-PEG (3k), POSS-PEG (6k) or POSS-PEG (10k). This result was also in line with our previous result, where thermal stability of POSS-containing PEO hybrids in nitrogen has not been obviously enhanced by the introduction of POSS molecules.⁴⁸ Moreover, when the temperature was raised to 700 °C, the organic substances were completely decomposed and the residual was ascribed to the ceramics formed during the thermal decomposition and oxidation.^20,49 The residual yield of POSS-PEG (3k), POSS-PEG (6k) and POSS-PEG (10k) hydrogels was 5.68, 2.53 and 1.67 wt% (at 700 °C), respectively, which was agreeable with the theoretical POSS content calculated according to the feed ratios.

Fig. 3 TGA curves of POSS-PEG hydrogels.

Swelling ratio is a measurement that related to wettability of materials, and high wettability is favourable for their application in tissue engineering. Fig. 4 presented the swelling properties of the POSS-PEG hydrogels. Obviously, all samples had relatively high wettability which improved the transport of nutrient, and metabolites, thereby avoiding the protein denaturation.⁵⁰ In addition, POSS-PEG hydrogels might have an enhanced substance permeability compared to the conventional organic PEG hydrogels, since the swelling ratios of POSS-PEG hydrogels were relatively higher than those of the conventional organic PEG hydrogels with the same PEG density.⁵¹ Moreover, he swelling ratio of POSS-PEG hydrogels increased with the increase of PEG chain length, which was in line with the increased volume, as presented in Fig. 2A. This could be attributed to the fact that POSS-PEG hydrogels with a shorter PEG chain length had a smaller porous structure, so they could not contain more free water. On the other hand, the content of POSS moieties was also higher in POSS-PEG hydrogels with a shorter PEG chain length. Thus, the water absorption capacity of these hydrogels was further weakened by the highly hydrophobic POSS moieties.

Fig. 4 Swelling behavior of POSS-PEG hydrogels.

The mechanical property of hydrogels is one of the key parameters for their practical applications. The viscoelastic mechanical behavior of POSS-PEG hydrogels could be determined by rheological measurements. The storage modulus (G′) and loss modulus (G′′) of POSS-PEG hydrogels as a function of frequency range from 10⁻² to 10¹ were shown in Fig. 5A. All samples exhibited a solid-like behavior (G′ > G′′) and G′ was nearly independent of the frequency, since the hydrogels were well crosslinked. In addition, all POSS-PEG hydrogels showed that the G′ was an order of magnitude greater than G′′ over the frequency range, indicating that all POSS-PEG hydrogels had the characteristics of an elastomer.³⁹ The stress–strain mechanical analysis of POSS-PEG hydrogels was evaluated by uniaxial compression (Fig. 5B). An increase of both elastic modulus (E) and ultimate stress (at break) could be clearly seen in POSS-PEG hydrogels with the increase of PEG chain length, which was agreeable with G′ as shown in Fig. 5A. According to the previous literatures, the mechanical property of organic–inorganic hybrid hydrogels was in general correlated to the crosslinking density, the content of inorganic component and the inter-chain entanglements of organic component.^6,52,53 POSS-PEG hydrogels with a longer PEG chain length had a lower crosslinking density and a lower content of inorganic POSS units which could not contribute to the higher mechanical property of POSS-PEG hydrogels. However, the longer PEG chain might provide more entanglements in the hydrogels, which could result in the higher mechanical property.

Fig. 5 (A) Storage (G′) and loss modulus (G′′) of POSS-PEG hydrogels. (B) Stress–strain curves and (C) elastic modulus (E) of POSS-PEG hydrogels from uniaxial compression. Results were mean ± SD, n = 3 replicates per experiment.

In vitro culture of chondrocytes on POSS-PEG hydrogels

To evaluate the potential of hybrid POSS-PEG hydrogels as scaffold materials for cartilage tissue engineering, the cell viability, attachment, spreading and proliferation of chondrocytes on these hydrogel films were determined. The viability of chondrocytes on different hydrogel films was determined by live/dead staining and CCK-8 assay. As shown in Fig. 6, a high percentage of viable cells could be clearly seen for chondrocytes on all three hydrogel films and the cell culture plate (control), and the duration of culture almost had no effect on the viability of chondrocytes, indicating that all POSS-PEG hydrogels had a very good biocompatibility. On day 1, chondrocytes were sparsely distributed on hydrogel films or the bottom of the cell culture plate, while chondrocytes tended to cover the most of the surface and cell clusters could form in some places on day 3. The cell viability of chondrocytes on POSS-PEG hydrogels were further evaluated by CCK-8 assay (Fig. 7). Obviously, chondrocytes maintained high cell viability on day 1 and 3 for each sample, which was in line with the result from the live–dead assay.

Fig. 6 Live/dead assay of chondrocytes adhesion on (a) POSS-PEG (3k), (b) POSS-PEG (6k), (c) POSS-PEG (10k) hydrogel films and (d) the cell culture plate for 1 and 3 days (green: live; red: dead); scale bar = 100 μm.

Fig. 7 Cell viability of chondrocytes adhesion on POSS-PEG hydrogels on day 1 and 3. Results were mean ± SD, n = 3 replicates per experiment.

Fig. 8 showed the fluorescent images of actin cytoskeleton of chondrocytes on POSS-PEG hydrogel films. Notably, more and more chondrocytes expressed spread-out phenotypes with the duration of culture, and then almost all chondrocytes expressed spread-out phenotypes at 24 h, indicating that an enhanced cellular response could be achieved. Furthermore, cell functions including cell attachment, spreading, and proliferation of chondrocytes on POSS-PEG hydrogel films were evaluated. Normalized cell attachment of chondrocytes at 4 h was similar for all three samples and the control, and no statistically significant was found (Fig. 9A). To further determine the cell spreading of chondrocytes on POSS-PEG hydrogel films, the cell spreading area at 4, 12 and 24 h were evaluated, respectively (Fig. 9B). Obviously, a significantly larger cell spreading area could be found for all samples with the duration of culture, which was in line with the enhanced spread-out phenotypes expression as shown in Fig. 8. Additionally, the spreading area of chondrocytes on the POSS-PEG (10k) hydrogel film at 24 h was slightly larger than those on the cell culture plate. The promoted cell spreading area might be attributed to the augmented wettability of the POSS-PEG (10k) hydrogel film.⁵⁴ Cell numbers on day 1 and 3 were evaluated by CCK-8 assay as shown in Fig. 9C. On day 1, a similar cell number of chondrocytes was found for each experimental group, and there was no statistically significant. On day 3, chondrocyte proliferation of all the samples also had a similar behavior except for POSS-PEG (10k) hydrogel film, which had a slightly larger cell number over control and POSS-PEG (3k) hydrogel film. PEG hydrogels alone are considered to be bio-inert materials which would inhibit nonspecific cell adhesion and spread.^55,56 In current work, all POSS-PEG hydrogel films were non-cytotoxic to chondrocytes, and could well support attachment, spreading and proliferation of chondrocytes. Chondrocytes responded more positive to the POSS-PEG (10k) hydrogel, as revealed by the enhanced cell functions including cell area and cell number. Therefore, the introduction of POSS in PEG hydrogels could be beneficial for the culture of chondrocytes.

Fig. 8 Fluorescent images stained with rhodamine-phalloidin (red) and DAPI (blue) of chondrocyte adhesion on (a) POSS-PEG (3k), (b) POSS-PEG (6k), and (c) POSS-PEG (10k) hydrogel films for 4 h, 12 h and 24 h, respectively. Scale bar = 100 μm.

Fig. 9 Cell attachment, spreading and proliferation of chondrocytes on POSS-PEG hydrogel films. (A) Normalized cell attachment at 4 h. (B) The average spreading area of chondrocytes at 4, 12 and 24 h. (C) Cell numbers of chondrocytes on day 1 and 3. Results were mean ± SD, n = 3 replicates per experiment, and statistical significance was tested with ANOVA.

Conclusions

We have successfully prepared POSS-PEG hybrid hydrogels with different PEG chain length via azide-alkyne click reaction between OAPOSS and alkyne-functionalized PEG. All POSS-PEG hydrogels had a highly ordered porous structure, and they exhibited the characteristics of an elastomer as revealed by their mechanical properties. The swelling ratio of POSS-PEG hydrogels increased with the increase of PEG chain length due to the larger porous structure and the lower content of highly hydrophobic POSS moieties. Moreover, in vitro culture of chondrocytes showed cells could well attach, spread and proliferate on biocompatible POSS-PEG hydrogel films. Additionally, chondrocytes responded more positive to the POSS-PEG (10k) hydrogel, as revealed by the enhanced cell functions including cell area and cell number. Thus, POSS-PEG hydrogels exhibited the great potential for cartilage tissue engineering.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (No. 51173044 and 21574039), Research Innovation Program of SMEC (No. 14ZZ065), Shanghai Pujiang Program under 14PJ1402600, and the Open Funding Project of the State Key Laboratory of Bioreactor Engineering. W. Z. also acknowledges the support from the Fundamental Research Funds for the Central Universities.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental details and characterization data. See DOI: 10.1039/c5ra27989e

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