Dendritic Hydrogel Bioink for 3D Printing of Bacterial Microhabitat

Abstract

A glucose-modified dendritic hydrogel is used as a bioink for bacterial encapsulation. This biocompatible hydrogel is a potentially suitable alternative to conventional alginate hydrogel for bacterial encapsulation, as it readily forms gel in the presence of Na⁺ or K⁺ ions without any additional stimuli such as pH, temperature, sonication, or the presence of divalent metal ions. We created a bacterial microhabitat by adding the gelator to phosphate-buffered saline containing live bacteria at physiological pH and using an additive three-dimensional (3D) printing technique. The bacteria remained viable and metabolically active within the 3D printed bacterial microhabitat, as shown with confocal laser scanning microscopy (CLSM) and scanning electrochemical microscopy (SECM).

Graphical Abstract

INTRODUCTION

Bacterial biofilm contains hundreds of different species of bacteria that stick together in an extracellular polymeric substance (EPS) matrix.^1–3 Within the biofilm, bacteria can participate in cooperative and hostile relationships.⁴ They often transmit infection by forming biofilm, and they interact through short-range chemical signals.⁵ Understanding communication between bacteria in their habitat at short-range distances is an important aspect of microbiology. A simulated three-dimensional (3D) bacterial microhabitat model can be used to create an alternative biofilm without the need for bacteria to produce polysaccharides or to be grown in traditional dental plaque culture. The techniques that are available to make bacterial 3D microhabitats are drop casting, photolithography, spin coating, laser printing, soft lithography, and 3D bioprinting.^6,7 Creation of a simulated 3D bacterial microhabitat offers the potential to study interactions between two or more bacterial species, depending on the distance between them and their population within a biofilm in a simulated environment.⁸

Hydrogels are a unique class of water-swollen polymer, giving hydrogel-based bioinks enormous potential for creating highly hydrated, tissue-like bacterial microhabitats. These bioink materials are ideal for maintaining and extending bacterial viability, can mimic extracellular polymeric substances, and provide porous structures necessary for nutrients to diffuse in and metabolites to diffuse out.^9–15 Recently, there has been a growing need for hydrogel-based bioinks for cell encapsulations.^16–25 Despite the numerous reports on different types of hydrogels since the first publication on hydrogels by Wichterle and Lim in 1960, access to new hydrogel bioinks for 3D printing of cells is limited.²⁶ The most commonly used hydrogel bioinks are alginate, chitosan, silk, collagen, fibrin, polyacrylamide, polyethylene glycol, hyaluronic acid, and polypeptides.^27–36 Each of these hydrogelators has different hydrogelation properties, which define the bioprinting techniques that can be applied.³⁷

Alginate hydrogel, a widely used natural bioink, requires divalent metal ions to form the hydrogel. The alginate hydrogel matrix is not stable over time in solution without the addition of divalent metal ions, thus limiting its use in a variable Ca²⁺-containing solution.³⁸ Chitosan is another naturally occurring hydrogel; however, it is not stable at pH below 6.5.³⁹ Most available synthetic hydrogels, such as those made with acrylate and polyethylene glycol, require additional steps such as exposure to UV radiation, radical initiator treatment, or sonication in order to form hydrogels.^40–42 Dendron-based hydrogels, however, show promising application as an entirely new group of bioinks because their properties can be altered by varying the functional groups in the dendritic structures.⁴³

Dendritic low-molecular-weight gelators show interesting sol-gel transformations in response to external stimuli such as temperature, pressure, light, electric potential, and pH.^44–49 A glucose-modified poly(aryl ether) dendron (Figure 1a, Figure S1) hydrogel was reported for the first time in 2013 by Rajamalli et al.⁵⁰ This hydrogel is highly stable below pH 10, can congeal relatively easily on sonication in deionized water, and does not need any divalent metal ions to form a hydrogel.⁵⁰ We envisioned that this sugar-based dendritic hydrogel could have considerable potential as a bioink.

Figure 1.

(a) Molecular structure of the glucose-modified poly(aryl ether) dendron hydrogelator. Photograph of bulk hydrogel in (b) 1 mM NaCl and (c) 1 mM KCl and of (d) S. mutans dendron (0.4 w/v%) hydrogel in DMSO-PBS (3:17 v/v). (e) gfp-tagged E. coli bacteria on single hydrogel fiber after 24 h incubation in hydrogel. (f) gfp-tagged E. coli bacteria along with hydrogel fibers. (g) Schematic of 3D structure of the bacteria-encapsulated hydrogel. (h) Viability of bacteria grown in the hydrogel: S. mutans growth in alginate and dendron gel in bulk.

Here we report a new bioink for encapsulation of bacteria. We used a synthetic glucose-modified poly(aryl ether) dendron-based biocompatible hydrogelator as an ink to print a 3D microhabitat with bacteria. One of the important aspects of this dendron molecule is that it can form biocompatible hydrogel with Na⁺ or K⁺ ions in the absence of additional stimuli (Figure 1b, 1c, Table S1). We believe that the Na+ and K+ ions present in phosphate-buffered saline (PBS) facilitate the cross-linking between dendron molecules so that they stack together to form hydrogel fibers. With this unique property of the dendron hydrogelator, hydrogel is easily formed by adding the dendron solution to a dispersion of a wide variety bacteria in 10 mM PBS at physiological pH (7.2). The PBS buffer consists of 137 mM NaCl, 2.7 mM KCl, 1.5 mM NaH₂PO₄, and 10 mM Na₂HPO₄. This hydrogel preparation does not require additional steps, such as UV radiation, ultrasonication, heating, radical initiators, pH adjustment, or the use of divalent metal ions. Thus, this dendron-based hydrogel provides an excellent matrix to study bacterial metabolism in a solution containing variable divalent metal ions such as Ca²⁺. The detailed synthetic procedure and hydrogelation properties with bacteria are described in the Supporting Information.

MATERIALS AND METHODS

Materials.

The poly(aryl ether) dendron derivatives were synthesized according to reported procedures.⁵⁰ All starting materials were obtained from Sigma-Aldrich. PBS (137 mM NaCl, 2.7 mM KCl, 1.5 mM NaH₂PO₄, 10 mM Na₂HPO₄) and artificial saliva (0.70 mM CaCl₂, 0.43 mM MgCl₂, 4 mM NaH₂PO₄, 20 mM HEPES, 30 mM KCl) solutions were freshly prepared in deionized water (18 MΩ) and stored at room temperature. Brain heart infusion (BHI) medium was purchased from Becton Dickinson Biosciences.

Instrumentation.

All electrochemical measurements were performed by using a CHI bipotentiostat and scanning electrochemical microscope (SECM) (Model # 920D, CHI, Austin, TX, USA). A previously developed pH microprobe⁵¹ and a 0.5 mm Pt wire were used as the working and counter electrodes, respectively, along with Ag/AgCl (1 M KCl) reference electrodes. Potentiometric experiments were performed with a separate high-impedance unit (EA Instruments, UK) interfaced with SECM. For rheology measurement, we used Anton Paar Rheometer MCR 302.

Bacterial strains and growth conditions.

The streptococcal species Streptococcus mutans (UA159) was cultured on BHI agar plates at 37 °C in 5% CO₂ environments. Green fluorescent protein (gfp)-tagged Escherichia coli were cultured on LB agar plates at 37 °C in the presence of air. Prior to all biofilm mapping experiments, S. mutans were grown for 12 h in BHI and gfp-tagged E. coli were cultured on LB liquid media with added ampicillin for 12 h.

Bulk hydrogel preparation with bacteria.

Dendron hydrogel was prepared by adding 150 μL of dendron solution (0.4% w/v) in dimethyl sulfoxide (DMSO) to 850 μL of a dispersion of a wide variety of bacteria (E. coli, S. mutans, S. gordonii) (O.D. = 0.1) in 10 mM PBS at pH 7.2 (Table 1).

Table 1.

Hydrogelation of dendron with bacteria.

Dendron (mg)	DMSO (mL)	Bacteria (O.D. 0.1)	PBS (mL)
4	0.125	-	0.85
4	0.125	E. coli	0.85
4	0.125	S. mutans	0.85
4	0.125	S. gordonii	0.85

Viability test of bacteria inside microhabitats.

For viability testing of the bacteria, we printed the hydrogel microhabitat on a glass substrate and then placed it inside a petri dish. The petri dish was then filled with 8 mL of 30 mM sucrose solution in artificial saliva (pH 6.0), incubated for 2 h and 8 h at 37 °C, and the plate count experiment performed. For this experiment, the hydrogel microstructure was homogenized in 10 mL of BHI media, serially diluted in 10 mL BHI, and successively transferred into seven centrifuge tubes. Then 20 μL of dispersion from each tube was removed, plated in seven different agar plates, incubated at 37 °C for 12 h, and the number of colonies were counted. Using the dilution factor, we calculated colony-forming units (CFUs) per milliliter for both samples.

Substrate preparation for 3D printing.

Glass coverslips were first cleaned in a sonic bath for 10 min in deionized water, acetone, and ethanol and dried with filtered nitrogen gas by using a 0.2 μm syringe filter. The glass substrates were then autoclaved for 2 h at 120 °C and 17 psi by submerging them in deionized water. We then incubated the glass substrates for 2 h at room temperature in 2 wt% aqueous (3-aminopropyl) triethoxysilane (APTES) solution. The substrates were washed with ethanol and completely dried with filtered nitrogen gas. The APTES-modified glass substrates were further incubated for 24 h in alginate anchoring solution containing 0.1 M HEPES, 50 mM sodium chloride, 1 wt% alginate, N-hydroxysuccinimide (molar ratio of 30:1 to alginate), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (molar ratio of 25:1 to alginate). Finally, the glass substrates were washed with deionized water and completely dried by using filtered nitrogen gas.

Fabrication of 3D bacterial microhabitat.

Five layers of Kapton tape were stacked together (total thickness of 300 μm), punched with two 2.4 mm diameter holes connected by a rectangular channel (length 4 mm, width 2 mm), and affixed to the top of the alginate-modified glass substrate. The two cavities on the glass substrate were meant for infusing the bacteria-PBS dispersion from one cavity (infusion chamber) to the other (printing chamber) by using a syringe pump with a flow rate of 10 μL/min. The printing cavity was first filled with the bacteria-PBS dispersion and then the 3D printing process was carried out. For printing of the microhabitat, we loaded 2 wt% of dendron solution in DMSO in the ink reservoir of the inkjet printer and kept it at 0 Pa pressure by using a pressure controller. The droplets were then dispensed at a rate of 1 Hz with a droplet diameter of 25 μm by using a piezo dispenser. The droplet size and frequency were controlled by using a Jetdrive III controller (Table 2). The bacteria become physically entrapped after the dendron droplet comes into contact with the bacteria-PBS dispersion, forming a hydrogel (Figure 2a). To make a cubical microhabitat, we created a macro command to automate the movement of the 3D stepper motor in a predefined movement cycle. The motor resolution was 0.1 μm and the nozzle velocity (3D printing speed) was 25 μm /s. Each microhabitat was made by repeating the movement cycle 10 times, so that the total number of droplets equaled 1200. Using the piezo movement, we printed the cube-shaped bacterial microhabitat with the dendron hydrogel on the surface of the alginate-modified glass and then performed all SECM experiments.

Table 2.

3D inkjet printing parameters.

Voltage pulse (V)	Rise time (μs)	Dwell time (μs)	Fall time (μs)
10–30	3	15	3

Figure 2.

(a) Schematics of 3D printing setup. (b) Proposed mechanism of hydrogel droplet formation.

Confocal imaging of immobilized bacteria.

After 3D printing of the microstructure by using the dendron hydrogel with the gfp-tagged E. coli bacteria, we performed confocal fluorescence imaging of the microhabitat with a Zeiss LSM 780 NLO confocal microscope equipped with an argon laser source with a 40X objective. We used a 488 nm laser source, and its emission was collected with a 500–544 nm filter to visualize the gfp-tagged E. coli bacteria. A 561 nm laser source was used to obtain the reflection from the hydrogel, and it was collected by using a 551–574 nm filter. The bulk hydrogel (Figure S2) was also studied by using same parameter. The volume fraction of bacteria inside the hydrogel microhabitat was calculated from the z-stack confocal image of the microhabitat by using ImageJ software.

Fabrication of pH microsensor for SECM chemical probe.

A dual-barrel theta pipette was pulled with a pipette puller. Two 1.5 cm long, 25 μm diameter Pt wires were inserted into each barrel of the theta pipette before sealing. Conductive silver epoxy was used to connect the Pt and Cu wires. The electrode surface was then polished with sandpaper and fine polished with a polishing pad, with 1.0, 0.3, and 0.05 μm alumina powder being used successively. The electrode was tested by cyclic voltammetry and further characterized by running probe approach curves on an insulating substrate in the presence of 1 mM ferrocenemethanol solution (0.1 M KCl as supporting electrolyte). Polyaniline was deposited in one of the Pt electrodes to make a pH-sensing electrode. Polyaniline deposition was performed on one of the two Pt surfaces by cycling the potential from −0.2 to 1.0 V (vs. Ag/AgCl reference) in 0.1 M aniline in 1 M aqueous HCl at 100 mV/s for 50 cycles. pH calibration was performed with a high-impedance potentiometer at room temperature (23 °C) by addition of 1 M lactic acid to artificial saliva to bring the pH from 7.2 to 4 and showed a Nernstian slope of 59 mV/pH (Figure S3).

pH mapping using SECM.

After printing the S. mutans microhabitat on a glass substrate by using the 3D printing method, we transferred it to a petri dish and filled it with 1 mM ferrocenemethanol solution. The petri dish was then placed on an SECM stage (Figure 3) to measure the pH change due to production of lactic acid from the bacteria microhabitat by using the SECM pH probe (Figure S3). The height of the microhabitat was obtained by using a negative feedback approach mode in SECM, where one of the Pt electrodes in the dual tip was used as a working electrode and the Ag/AgCl and Pt wires were used as reference and counter electrodes, respectively. To measure the height of the microhabitat, we recorded several negative feedback approach curves on glass and on the microhabitat by applying +0.4 V with respect to the Ag/AgCl reference electrode in the presence of 1 mM ferrocene methanol solution in artificial saliva. The average height of the microhabitat was measured by converting the current to height from the negative approach curve. The dual pH probe was later positioned at 20 μm above the bacterial microhabitat and connected to the SECM by using a high-impedance unit (EA Instruments). The solution in the petri dish was then changed to artificial saliva (pH 6.0) and the temperature of the solution raised to 37 °C by using a temperature controller. A 1 M sucrose solution was added to attain a final concentration of 30 mM. The potential (mV) was measured against the Ag/AgCl reference electrode and converted to pH by using the pH calibration from the post-SECM experiment.

Figure 3.

(a) Schematic diagram of SECM experimental setup to measure metabolic profile of S. mutans. (b) Zoomed-in view of the substrate.

RESULTS AND DISCUSSION

For a hydrogel to act as a bioink, one of the necessary criteria is that the hydrogel must be biocompatible. A bacteria-encapsulated (S. mutans) dendron hydrogel (Young’s modulus=9.92×10³ Pa) sample was prepared by adding the dendron solution (0.4% w/v) in DMSO (15% w/v) to a dispersion of bacteria in PBS (84.6% w/v) (Figure 1d). The hydrogel was homogenized by vortexing bacteria dispersion with the dendron (NMR characterization-Figure S4 and S5) immediately after addition. Aliquots of the bacteria-hydrogel sample were then incubated in BHI media at 37 °C for either 2 h or 8 h. Later, a plate count experiment was performed by using a bacteria-encapsulated gel matrix (Figure 1e–1g) and compared with that of the most commonly used naturally occurring alginate hydrogel bioink. The bar chart in Figure 1h shows the biocompatibility of this hydrogel. The bacteria number was almost 30 times the original number in the dendron hydrogel within 6 h (CFU/mL of bacteria were 5.4×10⁷ at 2h and 1.6×10⁹ at 8 h). One might speculate that such a rapid increase in bacterial number inside the hydrogel could cause it to significantly swell. To address this concern, we performed a detailed fractional volume analysis by using a confocal laser scanning microscopy technique. We used the gfp-tagged S. mutans bacteria to quantify the volume occupied by the bacteria and reflection mode to visualize and quantify the volume of dendron hydrogel. We found that most of the space inside the hydrogel matrix was filled with water (the volume fraction was about 99%) and only 1% was filled with bacteria (TableS2). This observation suggests that the nutrients can freely diffuse through the porous hydrogel matrix and assist the bacteria in multiplying and occupying the voided space by replacing the water. The biocompatibility data were analyzed using Welch’s unpaired t-test (experiments were performed n=3 times). We have obtained p-value=0.04, which suggests that the biocompatibility of dendron hydrogel is significantly higher than that of alginate hydrogel within 95% confidence.

We further wanted to use this bioink in additive 3D printing by exploiting the very fast (<1 s) hydrogel formation kinetics when the dendron-based gelator compound comes into contact with bacteria containing the PBS solution. Figure 2a depicts the schematics of the 3D inkjet printing setup. We envisioned using this newly developed bioink in a 3D printing technique to create a bacterial microhabitat, where bacteria would be physically trapped inside the hydrogel matrix while the nutrients and metabolites produced by the bacteria could diffuse in and out of the microhabitat without any significant hindrance. This microhabitat-based simulated biofilm model will open new opportunities to study bacterial metabolism in a controlled microenvironment, especially the effects of microenvironments on bacterial metabolic behavior between different species and vice versa.^52–59

To achieve this microhabitat, we used a custom-designed 3D inkjet printer (Figure 2a). In this process, when the dendron-DMSO solution comes into contact with the bacterial dispersion in PBS, it forms hydrogel droplets within a fraction of a second.

Figure 2b shows the proposed hydrogelation mechanism, which is based on geometry and volume considerations of the printed microhabitat and the total number of droplets. The dendron-DMSO droplet meets the bacteria-PBS dispersion and then releases some DMSO, which achieves critical gelation concentration and forms the hydrogel droplet by mechanically encapsulating the bacteria within the gel matrix. Bacterial viability was further studied after printing the bacterial microhabitats. Figure 4 is a bar chart showing bacterial growth vs. time inside the printed microhabitats. Bacteria can grow to almost 5.5 times their original number in a 3D printed hydrogel microhabitat within 6 h (CFU/mL of bacteria were 0.96×10⁴, 5.1×10⁴, and 5.5×10⁴ at 2 h, 4 h, and 8 h, respectively). Since it is not possible to print the alginate microhabitat of same density as that of dendron hydrogel, we avoid comparing the bacterial viability test with dendron and alginate hydrogel microhabitat. Instead we compare the bacterial viability inside alginate and dendron hydrogel in larger volume. The hydrogel microhabitats (volume is about 3.0×10⁶ mm³) was printed inside a cavity filled with the phosphate-buffered saline solution (volume is about 2×10¹⁰ mm³). Since the printed glucose-modified hydrogel microhabitat is being within micrometer-scale dimensions, the conventional swelling ratio analysis was not applicable. To measure the swelling of the printed hydrogel (submerged into the buffer solution), we have performed gel height measurement using scanning electrochemical microscopy, and we have not observed any noticeable changes in gel heights over time. This swelling behavior is also indicative of the volume fraction analysis (Table S2). Since the ratio of the volume fraction of bacteria to the void space is only about 1:99 in the printed hydrogel, bacteria are free to grow within the void space of the microhabitat in presence of nutrients, without affecting the volume of the microhabitat.

Figure 4.

Viability of bacteria in printed microhabitat: S. mutans bacteria were grown inside the dendron hydrogel microhabitat in the presence of MOPS-BHI (1:1 v/v) and a 30 mM sucrose solution at 37 °C for 12 h and viability tests were performed.

In addition, we created 3D printed bacterial microhabitats of different sizes and were able to position them at varying distances on an alginate-coated glass substrate by using a 3D inkjet printing technique. Figure 5a–5e shows optical images of the printed bacterial microhabitats. Varying the size of the microhabitat allowed us to study the early and late stages of bacterial biofilm formation, and immobilizing the bacteria at different distances allowed us to elucidate distance-dependent bacterial interactions within a biofilm.⁵⁷

Figure 5.

(a), (b), (c), (d), and (e) Optical images of the printed bacterial microhabitats. (f) Z-stack confocal fluorescence images of the 3D bacterial microhabitat of gfp-tagged E. coli bacteria.

The bacterial distribution inside the printed microhabitat was examined by using confocal laser scanning microscopy, as shown in Figure 5f. Bacteria remain trapped inside the hydrogel matrix (Figure S1, Figure 1e–1f) compared with bacteria in PBS, where they are free to move, leading to a random distribution of bacteria and providing a hazy image. Figure 1e is a confocal fluorescence microscopic image that shows bacterial growth on gel fiber after gfp-tagged bacteria were incubated for 24 h in hydrogel. We have performed the volume fraction analysis of the bacteria microhabitat just after printing and the same after 12 hours of incubation of the bacterial microhabitat at 37 °C in presence of nutrients (Initial bacterial optical density was 0.1). The bacterial volume fraction was increased from 0.7% to 0.8% after the incubation whereas the hydrogel volume fraction remained constant (0.3%), which suggests that there were no significant changes to the structure of the bacterial microhabitat. Hence, no biodecomposition occurred during the experiment.

The immobilized bacteria within the 3D printed microhabitat of the dendron-based bioink must remain metabolically active. To investigate the metabolic activity of the immobilized bacteria, we used SECM as an analytical tool. Figure 3a and 3b shows details of the SECM experimental setup. The oral pathogenic microbe S. mutans was chosen as a model microorganism. This bacterial species forms a biofilm on the tooth surface, where it consumes sugars to produce lactic acid, decreases the pH, and dissolves the tooth to create dental cavities. We have extensively studied this bacterial species in our laboratory.^60–62 The bacteria showed metabolic activity within the 3D printed bacterial microhabitat that was similar to what we observed in our earlier study.⁶⁰ Figure 6a and 6b shows the metabolic activity of the bacteria obtained from the SECM experiment; the nutrients (sucrose) can diffuse through the hydrogel and the metabolites (lactic acid) can diffuse outside the gel.

Figure 6.

(a) Schematic diagram of SECM experimental setup. (b) pH measured at 20 μm above the S. mutans microhabitat in artificial saliva (pH 6.0) in the presence of 30 mM sucrose at 37 °C.

After recording the pH change above the S. mutans microhabitat over time in artificial saliva (pH 6.0), we observed that the local pH dropped to 5.3 in the presence of 30 mM sucrose at 37 °C. The result is shown in Figure 6(b), suggest that sucrose diffuse through porous dendron-hydrogel microhabitat and consumed by S mutans bacteria to produce lactic acid as a metabolic by-product and subsequently change the local pH in the close vicinity of hydrogel microhabitat from 6.0 to 5.3. A pH microsensor was used in the SECM to study bacterial metabolism above the S. mutans microhabitat, as in our earlier developed method.⁵¹ Detailed pH sensor fabrication and characterization (Figure S3), as well as the SECM experimental procedure, is described in the Materials and Methods section.

CONCLUSIONS

We have successfully demonstrated that a sugar-based dendritic hydrogel can potentially give rise to a new class of 3D printable bioink that provides a good alternative to Ca²⁺-based alginate hydrogel, especially where experimental conditions demand variable or no Ca²⁺ ions in the solution. This novel Na⁺ or K⁺ ion-based dendritic hydrogel bioink was used with 3D printing technology to create a simulated bacterial microhabitat in the presence of PBS buffer at physiological pH. The bioink shows excellent biocompatibility and the bacteria remain metabolically active within the 3D printed microhabitat. This bacterial microhabitat model with dendritic hydrogel bioink allows the study of bacterial metabolism in a simulated biofilm setup and can further be extended to the study of bacterial chemical interaction among multiple bacterial species.