3D Printing Enabled Customization of Functional Microgels - ACS

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Biological and Medical Applications of Materials and Interfaces

3D printing enabled customization of functional microgels Xuan Liu, Jie Tao, Jinlu Liu, Xin Xu, Jiumeng Zhang, Yulan Huang, Yuwen Chen, Jing Zhang, David Yubing Deng, Maling Gou, and Yu-Quan Wei ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b18701 • Publication Date (Web): 12 Mar 2019 Downloaded from http://pubs.acs.org on March 16, 2019

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ACS Applied Materials & Interfaces

3D printing enabled customization of functional microgels Xuan Liu1, Jie Tao1, Jinlu Liu1,2, Xin Xu1, Jiumeng Zhang1, Yulan Huang1, Yuwen Chen1, Jing Zhang3, David Y. B. Deng4, Maling Gou1*, Yuquan Wei1 1State

Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, 610041, China. 2Department of Thoracic Oncology, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, P. R China. 3Department of Neurosurgery, West China Hospital, Sichuan University, Chengdu 610041, China. 4Scientific Research Center, the Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen 518107, Guangdong, China.

ABSTRACT Injectable microgels show great promising applications in cell therapy and drug delivery. Currently, there remains a challenge to rapidly and cost-effectively fabricate customized microgels. Here, we present a digital light processing (DLP) based 3D printing process to fabricate microgels with tailored shapes and sizes. The microgels are constructed by the digital light controlled polymerization of photopolymerizable monomer solution within two seconds. By mixing nanoparticle-encapsulated drugs into the monomer solution, the microgels with sustained drug release can be readily prepared. Also, cells can be printed into microgels with survival and proliferation. In conclusion, this study provides a 3D printing process for customizing functional microgels containing drugs or cells with potential therapeutic applications. Keywords: Microgels; 3D printing; Nanoparticles; Drug Delivery; Biotherapy

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1. INTRODUCTION Hydrogels are composed of polymeric networks with high-water content, showing great potential in biomedical application. Natural and synthetic polymers are the major materials to fabricate hydrogels, including gelatin, hyaluronic acid, chitosan, collagen, alginate, and poly(ethylene glycol) and so on1. Injectable hydrogels attract more and more attention for their minimal invasion2,3. Compared to injectable amorphous hydrogels, there are no free-flowing polymer sols infiltrating into the neighboring region in injectable microgels, which avoids interfering hydrogel formation and hydrogel distribution. In past decades, injectable microgels have already been used in multiple fields such as gene therapy, tissue engineering and cell therapy4–6. Shape is an important factor in drug delivery. For instance, the filtration units of the spleen are asymmetrical slits. The transport of particles is restrained by their size and shape. In this instance, the shape of large-scale particles, like disk-shaped red blood cells, makes them to cross the spleen readily7. Besides, the shape of the particles also affects the endocytosis of macrophages. The activation of endocytosis depends on the local shape of the particles attached to the cells. Compared to rod-shaped particles, spherical particles exhibit significant perinuclear accumulation in macrophages8,9. In addition, particles with optimized shapes were fabricated to modulate foreign body responses10–12. To explore and utilize the effect of shapes in drug delivery, customized microgels are urgently needed. However, the existing techniques for preparing customized microgels are too complex and time consuming. Three-dimensional (3D) printing technology, one of the most popular additive manufacturing technologies, comes to the fore to solve this issue. Among them, digital light processing (DLP) 3D printing technology is a kind of light-assisted printing, which constructs materials through cross-


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linking photopolymers with computer aided design. The technology exhibits capability of high resolution, rapid printing speed and flexibility. Thus, it shows great potential in fabricating customized microgels for drug delivery systems, tissue engineering and prosthetics13–17. In this study, as shown in figure 1, we provided a rapid fabrication method for preparing customized functional microgels based on advanced scalable DLP-3D printing technology. The prepared microgels can be injected via a 1mL syringe in a non-invasive way. Moreover, the microgels can be readily functionalized by incorporating nanoparticles and cells, showing great potential in biomedical applications.

2. MATERIALS AND METHODS 2.1. Materials Gelatin from porcine skin, paclitaxel (PTX), collagenase I, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT), deuterium oxide were purchased from Sigma-Aldrich (St Louis, MO, USA). Tween-80, dimethyl sulfoxide (DMSO) were obtained from KeLong Chemicals (Chengdu, China). Monomethoxy poly(ethylene glycol)-block-poly(Ò D,L-lactide) (MPEGPDLLA) was purchased from Daigang Biomaterial (Shandong, China). Acetonitrile was purchased from Kermel (Tianjin, China). Cell-Counting Kit-8 was purchased from SUNBAO BIOTECH (Shanghai, China). LIVE/DEAD Cell Imaging Kit was purchased from KeyGEN BioTECH (Jiangsu, China). NIH/3T3 cells (mouse embryo fibroblast) were purchased form ATCC (No. CRL1568). The 1mL syringe with 26G needle was purchased from SHIFENG MEDICAL (Chengdu, China). All chemical reagents were of analytical grade. 2.2. Preparation and characterization of 3D printed microgels Gelatin methacryloyl (GelMA) was synthesized from gelatin and methacrylic anhydride (MA)



as mentioned in the previous study18. 14.65 g sodium bicarbonate and 7.95 g sodium carbonate were dissolved in 1 L distilled water to prepare 0.25M carbonate-bicarbonate buffer (CB buffer). Firstly, 1 g gelatin from porcine skin was dissolved into 10 mL 0.25M CB buffer. The pH was adjusted at 9 using 6M hydrochloric acid or 5M sodium hydroxide. Afterwards, 0.2 mL of MA was dripped dropwise into the solution and mechanically stirred at 50 ℃. After 3 hours, the reaction was stopped by adding 6M hydrochloric acid to change the solution pH of the solution to 7.7. The samples were dialyzed utilizing a dialysis bag (12-14kDa) for 4 days at 40 ℃ to eliminate the MA and salts, followed by a 2-day lyophilization. Subsequently, the materials were stored at -20 ℃ until further use. The synthetic method of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) remained the same as mentioned before19. To prepare the microgels, 10% (w/v) of GelMA and 0.7% (w/v) of LAP were completely dissolved in distilled water. As shown in figure 1a, we sandwiched two narrow silicon membranes between two holders to obtain a gap which controlled the thickness of microgels. Setting up the digital 3D images of microgels in the computer, fluorescein isothiocyanate (FITC) labeled GelMA was subsequently added to the designed gap. After exposure for two seconds, the microgels were obtained. The microgels treated with gradient dehydration were observed by scanning electron microscope (SEM). Besides, lyophilized microgel samples were prepared. Based on the SEM images of lyophilized samples, the pore size and porosity were quantitatively analyzed using ImageJ software. 2.3. Biocompatibility and biodegradation NIH/3T3 cells were cultivated separately in 96-well plates and co-cultivated with microgels. After 48 hours, the cellular viability was assessed by MTT assays. Besides, the morphology and density of cells were evaluated by LIVE/DEAD Cell Imaging Kit and SEM. The enzymatic


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degradation property of microgels was determined in PBS containing collagenase I (1 mg/mL) on an orbital shaker at 37 ℃. The mass loss was calculated by the ratio of mass difference at certain time to the original weight. 2.4. Preparation and characterization of drug loaded microgel nanocomposites PTX/MPEG-PDLLA nanoparticles were prepared as described in previous study20. Brieﬂy, 3 mg PTX and 97 mg MPEG-PDLLA were dissolved in 5 mL dichloromethane under stirring. Afterwards, the obtained solution was vaporized reelingly under reduced pressure for 20 minutes at 60 ℃. When dichloromethane was removed, PTX and MPEG-PDLLA co-polymers formed a thin film. The film was subsequently rehydrated in water to prepare PTX/MPEG-PDLLA nanoparticles. Then, a micromembrane filter with 0.22 μm pore size was employed to purify the solution. The Tyndall effect was observed by a laser pointer. Ultimately, the solution was reserved at 4 ℃ for further use. To evaluate the size and zeta potential of the PTX/MPEG-PDLLA nanoparticles, dynamic light scattering (Nano-ZS, Malvern Instruments, Malvern, UK) was used. All results were taken as mean value of three repeated tests. X-ray diffractometer (XRD) (X’Pert Pro, Philips, Netherlands) was employed to carry out crystallographic assays on paclitaxel powder, blank MPEG-PDLLA nanoparticles and PTX/MPEGPDLLA nanoparticles by using Cu Kα radiation. The cytotoxicity of PTX/MPEG-PDLLA nanoparticles on SKOV3 cells was measured by MTT assays. Firstly, 5×103 cells per well were incubated in a 96-well plate and grown for 24 hours. Then, the cells were treated with various concentrations of PTX or PTX/MPEG-PDLLA nanoparticles for 48 hours. At the same time, controlled group was treated with the same volume of



DMEM medium. Subsequently, 20 μL of MTT solution (5 mg/mL) was supplied to cells and incubated for 4 h at 37 ℃ to assess cell viability. After adding 150 μL of DMSO, formazan crystals were dissolved to show and measure viable cells at 570 nm wavelength on a microplate reader (BioTek, USA). Cell viability was presented as percentage of controlled group. All the results were obtained by five repeated experiments to improve the accuracy. Based on the specific fluorescence property and hydrophobic nature, coumarin-6 was utilized as a model fluorescent drug to evaluate the distribution of drugs in the microgel nanocomposite. The preparation method of coumarin-6/MPEG-PDLLA nanoparticles was similar to PTX/MPEGPDLLA. The distribution of the nanoparticles in microgel nanocomposites was evaluated by fluorescence microscopy. 2.5. Release study in vitro The amount of PTX released from PTX/MPEG-PDLLA nanoparticles and PTX-loaded microgel nanocomposites was measured using the dialysis method. The PTX/MPEG-PDLLA nanoparticles and PTX-loaded microgel nanocomposites were dissolved in phosphate buffer saline (PBS). Both of them were sealed in a dialysis bag (3.5 kDa) and immersed in 10 mL of 0.1M PBS containing 0.5% (w/w) Tween-80 respectively, at 37 °C, 100 rpm. At predesigned time points, 1 mL sample was collected from the incubation medium. Immediately after sampling, an equal volume of fresh PBS was supplemented to keep the volume equal. The amount of released paclitaxel was quantified by high-performance liquid chromatography (1100 HPLC, Agilent Technologies, Santa Clara, CA). Finally, the concentration of paclitaxel released from PTX/MPEG-PDLLA nanoparticles and PTX-loaded microgel nanocomposites was shown as a percentage of the total paclitaxel and plotted as a function of time, respectively.


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To study the nanoparticle release from the microgel nanocomposites, the microgel nanocomposites were immersed in 2 mL PBS at 37 °C with a gentle shaker. Then, the supernatant was collected to observe at day 1, 3 and 7, respectively. The morphology of the PTX/MPEGPDLLA nanoparticles was imaged by a transmission electron microscope (TEM) (H-6009IV; Hitachi Ltd., Tokyo, Japan). In addition, the coumarin-6 loaded hydrogel was dipped in PBS on an orbital shaker at 37 ℃. After 7 days, it was analyzed by confocal microscopy. 2.6. The preparation and characterization of cell micro-scaffolds In the sterile environment, the NIH/3T3 cell suspension was co-printed with GelMA solution to prepare cell micro-scaffolds, which were stained with rhodamine phalloidin and DAPI. At the same time, the survival of cells was estimated by LIVE/DEAD Cell Imaging Kit on the first day and the seventh day. Cell growth was determined with the Cell-Counting Kit-8 by measuring the absorbance at 490nm. 2.7. Statistical analysis Statistical analysis was performed with two-tailed Student’s t-test using GraphPad Prism 5 Software (GraphPad Software, Inc., San Diego, CA).

3. RESULTS 3.1 Preparation, and characterization of 3D printed microgels The setup schematic diagram of DLP-3D printer for microgels customization is shown in figure 1a. In this setup, 405 nm visible light LED is selected to initiate the polymerization of the monomer solution. Compared to conventional UV, the 405 nm visible light has less harm to cells or drugs. Selective photopolymerization is realized by modulating the optical pattern with a digital



micromirror device (DMD) chip. In detail, the DMD chip consists of about two million micromirrors (1824*1140), which get command of computer-aided design models. In the process of DLP-3D printing, designed images are projected onto the monomer solution, leading to the controlled polymerization within two seconds. Thus, DLP-3D printing technology provides a method for fabricating customized microgels by readily adjusting the images. Figure 1b illustrated the designed 3D sketch of various shapes of microgels, included square, round, triangle and star. The SEM results proved that the morphology of 3D printed microgel was consistent with the design. According to quantitative analysis by ImageJ software, the average pore size and porosity of lyophilized microgels were 3.52 ± 0.54 μm and 62.63 ± 1.31% respectively, which was showed in figure 1c. To verify whether the microgels could be injected completely through a 1mL syringe, the FITC-labeled microgels were collected to observe their morphology before and after injection. As shown in figure 1d, the 3D-printed customizable microgels regained their original shape after the mechanical extrusion through a 1mL syringe. 3.2. Biocompatibility and degradation In terms of degradation, microgels were immersed in PBS solution (containing 1 mg/mL collagenase I). Figure 2a indicated that microgels had a property of slow degradation (68.8% after 510 mins). Figure 2b demonstrated that the microgel extract had little toxicity by MTT assay. Then the NIH/3T3 cells were cultivated in 24-well plates alone and with microgels respectively. After 48 hours’ incubation, cells were stained by LIVE/DEAD Cell Imaging Kit. For cell morphology and viability in figure 2c, there was no significant difference between two groups. In addition, the cytoskeleton of NIH/3T3 cells spread out and clung tightly to the microgel in figure 2d. The satisfactory adhesion of NIH/3T3 cells to the microgel revealed that it had an excellent


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biocompatibility. In conclusion, the results proved that microgels had good biocompatibility and biodegradability. 3.3. Preparation and characterization of the PTX/MPEG-PDLLA nanoparticles and microgel nanocomposites As shown in figure 3a, the PTX/MPEG-PDLLA nanoparticles were prepared via a selfassembly manner. The Tyndall effect was observed by a laser pointer in figure 3b, which indicated the solution we prepared was colloidal. Figure 3c demonstrated the mean hydrodynamic particle size (22 nm) with PDI of 0.197 and zeta potential (-3.8 mV) of the PTX/MPEG-PDLLA nanoparticles. For the XRD spectra, shown in figure 3d, the typical XRD peaks of PTX vanished in the XRD spectrum of nanoparticles. It was implied that the PTX are molecularly dispersed in MPEG-PDLLA nanoparticles. In our study, cytotoxicity of free PTX and PTX/MPEG-PDLLA nanoparticles on SKOV3 cells was estimated in vitro. The cells were incubated with various concentrations of nanoparticles and drugs for 48 hours, then determined by MTT assays. In figure 3e, cells were significantly inhibited in a dose-dependent manner by free PTX and PTX/MPEG-PDLLA nanoparticles. The half maximal inhibitory concentration (IC50) of the PTX/MPEG-PDLLA nanoparticles (41.9 ng/mL) was slightly lower than that of free PTX (51.4 ng/mL). Therefore, we suggest nanoparticles may strengthen cellular uptake and rapid efflux of PTX. To observe the distribution of the nanoparticles in the microgel directly, coumarin-6 was utilized as a model drug. In figure 3f, the nanoparticle-associated green fluorescence dots were evenly distributed in the 3D-pinted microgels with square, circular, triangle or star shapes. It revealed that PTX/MPEG-PDLLA nanoparticles could also distribute uniformly in the microgel



nanocomposites. 3.4. Release study in vitro The dialysis method was used to calculate the amount of PTX released from PTX/MPEGPDLLA nanoparticles and microgel nanocomposites in vitro. As shown in figure 4a, approximately 56% PTX was released from nanoparticles after 168 hours, while 17% PTX was released from microgel nanocomposites at the same time. The significant difference indicated that microgels encapsulating PTX/MPEG-PDLLA nanoparticles inside had a stable and effective slow-release effect. From figure 4b, TEM results showed that nanoparticles existed in the supernatant during the release period. It was implied that the microgel nanocomposites could release nanoparticles. In addition, the coumarin-6 loaded hydrogel was immersed in PBS solution (containing 0.5% Tween80). After 7 days, a small piece of the hydrogel was cut and scanned by confocal microscope in figure 4c. The fluorescent intensity of coumarin-6 weakened gradually from the core to the margin in the same cross section, while it weakened gradually from the interior to the surface in different levels. 3.5. The preparation and characterization of cell micro-scaffolds The schematic of cell micro-scaffolds preparation is shown in figure 5a. In figure 5b, NIH/3T3 cells were evenly distributed in the microgel without obvious aggregation. After seven-day culture, results of the LIVE/DEAD Cell Imaging Kit study showed that almost all cells survived. The cells on the surface of the microgel proliferated rapidly and differentiated obviously. Meanwhile, the cells inside the microgel were restricted to the 3D networks and the proliferation speed was slower than that on the surface of the microgels. Furthermore, cell proliferation was quantitatively determined by Cell Count Kit-8. The number of cells increased to 1.8 times within 14 days. In


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summary, a cell micro-scaffold was successfully developed which was capable to sustain the survival and proliferation of cells.

4. DISCUSSION In this work, a rapid fabrication technique was proposed to construct customized functional microgels via a DLP-3D printing technology. The 3D printed customized microgels could be injected with mini-invasion and remained their original shape after they were extruded out of a 1mL syringe. On top of that, to meet requests of different applications, the designed microgels could be functionalized with nanoparticles and cells, showing great potential in biomedical applications. Currently, many researchers are focusing on developing the fabrication of particles with various shapes via molds, microfluidics and lithography21–23. However, these methods require specialized equipment with custom geometry, namely molds, microfluidics or masks, usually cannot produce a variety of shapes in one batch. At the same time, the mold method lacks the scalability for high throughput production. The output can only be expanded by increasing the size of molds. Fast 3D printing technology with high flexibility and good controllability comes to solve above issues. It offers a great promising approach for the mass fabrication of customized microgels. With improved flexibility and versatility, 3D printing has become an excellent manufacturing platform for preparing complex tissue structures and biological scaffolds. Inkjet and extrusive bioprinters offer great advantages due to its simplicity and low cost24,25. However, they have many limitations in bioprinting, like cell damage, cell aggregation, slow fabrication speed, weak binding force between the interface, and low printing resolution24,26. In the meantime, there is annoyance caused by nozzle clogging, which reduce printing speed27,28. Recently, advanced DLP-3D printing



technology which possesses high precision, low cost and fast speed has attracted widespread attention29,30. DLP-3D printed hydrogel scaffolds can be stably integrated with contents, like cells, nanoparticles and bio-factors, to realize functions. In this study, we utilized DLP-3D printing to design and fabricate uniform microgels with two-seconds exposure. The microgels have good biocompatibility and degradability which could be functionalized by enabling nanoparticles and cells. It provides a fast and efficient method for the preparation of customized functional microgels. The obtained microgels are versatile which can encapsulate drugs as well as proteins, cells and other biomolecules to prepare functional microgels31. Many studies have verified its feasibility. An injectable multifunctional microgel co-delivering cells and bio-factors together was developed. The system is able to carry therapeutic agents or cells, which is beneficial to developing future treatment for vascular diseases6. Greg a. Foster et al. designed an injectable poly (ethylene glycol) microgel taking advantage of microfluidics-based polymerization, which controlled the release of proteins by cross-linking with protease degradation peptides32. It promoted the development of microgels in the field of controlling protein delivery. Thus, it indicates that the microgels fabricated by DLP-3D printing technology is able to match with various drugs or cells, showing great potential applications in the future.

5. CONCLUSION This work demonstrated a digital light processing based 3D printing process to fast construct tailored injectable microgels within seconds. The prepared microgels are biocompatible and can be readily used to deliver drugs and cells, which shows potential therapeutic applications.


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Figure 1 (a) Schematic diagram of DLP-3D printer for microgels customization. After designing the digital 3D images in the computer, the microgels with precise 3D structure are constructed. (b) The digital design and SEM images of 3D printing microgels treated with gradient dehydration. Scale bars, 500 μm. (c) Representative SEM images of lyophilized microgel samples. Scale bar, 10 μm. (d) The morphology of FITC-labeled microgels (before and after the injection). Scale bar, 200 μm.



Figure 2 (a) The degradation profile upon incubation with collagenase I. (b) The MTT result of cells cultured with different concentrations of microgels for 48 hours. (c) The biocompatibility of microgels. NIH/3T3 cells were cultivated separately in 24-well plates and co-cultivated with microgels. After 48 hours, the LIVE/DEAD Cell Imaging Kit was used to evaluate the survival of cells. Scale bar, 100 μm. (d) SEM images of NIH/3T3 cell cultured on the microgel material. Scale bars, 10 μm.

Figure 3 (a) An illustration of the fabrication of PTX/MPEG-PDLLA nanoparticles. (b) Tyndall


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effect. (c) Size and zeta potential of the PTX/MPEG-PDLLA nanoparticles. (d) The XRD of paclitaxel powder, blank MPEG-PDLLA nanoparticles and PTX/MPEG-PDLLA nanoparticles. (e) Cytotoxicity of PTX/MPEG-PDLLA nanoparticles. SKOV3 cells were treated with free PTX and PTX/MPEG-PDLLA nanoparticles in different concentrations. Cell viability was detected after 48 hours by MTT assay. (f) Schematic of coumarin-loaded microgel nanocomposites preparation. Fluorescence microscope images of microgel nanocomposites of different sizes and shapes. Scale bar, 500 μm.

Figure 4 The principle of drug release from microgel nanocomposites. (a)The release profile of the PTX/MPEG-PDLLA nanoparticles and microgel nanocomposites. (b)The TEM images of nanoparticles released from microgel nanocomposites at day 1, 3 and 7. Scale bar, 100 nm. (c)After seven-day drug release in PBS, the hydrogel was cut to be detected by confocal microscopy. The picture on the right shows the merge result. While, the picture below shows the single-layer results. Scale bar, 500 μm.



Figure 5 The survival and proliferation of the cell micro-scaffolds. (a) Schematic of cell microscaffolds preparation. (b) The confocal microscope images of NIH/3T3 cells inside the microscaffolds dyed with DAPI and rhodamine phalloidin. Scale bar, 100 μm. (c) The survival and proliferation of the cells interior and on the surface of the microgels on the first day and seventh day by LIVE/DEAD Cell Imaging Kit (merged images). Scale bar, 100 μm. (d) Cell growth results were shown by the Cell-Counting Kit-8.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]

Author Contributions X.L., J.T. and J.L. contributed equally to this work. All authors have given approval to the final


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version of the manuscript. M.G. and Y.W. supervised the project. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The work is supported by the Key Research and Development Projects of People's Liberation Army (BWS17J036), 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University (ZYYC08007), the fundamental research funds for the central universities (2018SCUH0010). We also appreciate Hui Wang from the Analytical & Testing Center of Sichuan University for her help with SEM characterization.



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Soman, P.; Chung, P. H.; Zhang, A. P.; Chen, S. Digital Microfabrication of User-Defined 3D Microstructures in Cell-Laden Hydrogels. Biotechnol. Bioeng. 2013, 110 (11), 3038–3047.

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Foster, G. A.; Headen, D. M.; González-García, C.; Salmerón-Sánchez, M.; Shirwan, H.; García, A. J. Protease-Degradable Microgels for Protein Delivery for Vascularization. Biomaterials 2017, 113 (1), 170–175.



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3D Printing Enabled Customization of Functional Microgels - ACS

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