Granular Biphasic Colloidal Hydrogels for 3D Bioprinting.

Granular hydrogels composed of hydrogel microparticles are promising candidates for 3D bioprinting due to their ability to protect encapsulated cells. However, to achieve high print fidelity, hydrogel microparticles need to jam to exhibit shear-thinning characteristics, which is crucial for 3D printing. Unfortunately, this overpacking can significantly impact cell viability, thereby negating the primary advantage of using hydrogel microparticles to shield cells from shear forces. To overcome this challenge, a novel solution: a biphasic, granular colloidal bioink designed to optimize cell viability and printing fidelity is introduced. The biphasic ink consists of cell-laden polyethylene glycol (PEG) hydrogel microparticles embedded in a continuous gelatin methacryloyl (GelMA)-nanosilicate colloidal network. Here, it is demonstrated that this biphasic bioink offers outstanding rheological properties, print fidelity, and structural stability. Furthermore, its utility for engineering complex tissues with multiple cell types and heterogeneous microenvironments is demonstrated, by incorporating ß-islet cells into the PEG microparticles and endothelial cells in the GelMA-nanosilicate colloidal network. Using this approach, it is possible to induce cell patterning, enhance vascularization, and direct cellular function. The proposed biphasic bioink holds significant potential for numerous emerging biomedical applications, including tissue engineering and disease modeling.

Impact of PEG sensitization on the efficacy of PEG hydrogel-mediated tissue engineering.

While poly(ethylene glycol) (PEG) hydrogels are generally regarded as biologically inert blank slates, concerns over PEG immunogenicity are growing, and the implications for tissue engineering are unknown. Here, we investigate these implications by immunizing mice against PEG to stimulate anti-PEG antibody production and evaluating bone defect regeneration after treatment with bone morphogenetic protein-2-loaded PEG hydrogels. Quantitative analysis reveals that PEG sensitization increases bone formation compared to naive controls, whereas histological analysis shows that PEG sensitization induces an abnormally porous bone morphology at the defect site, particularly in males. Furthermore, immune cell recruitment is higher in PEG-sensitized mice administered the PEG-based treatment than their naive counterparts. Interestingly, naive controls that were administered a PEG-based treatment also develop anti-PEG antibodies. Sex differences in bone formation and immune cell recruitment are also apparent. Overall, these findings indicate that anti-PEG immune responses can impact tissue engineering efficacy and highlight the need for further investigation.

Clickable Granular Hydrogel Scaffolds for Delivery of Neural Progenitor Cells to Sites of Spinal Cord Injury.

Spinal cord injury (SCI) is a serious condition with limited treatment options. Neural progenitor cell (NPC) transplantation is a promising treatment option, and the identification of novel biomaterial scaffolds that support NPC engraftment and therapeutic activity is a top research priority. The objective of this study is to evaluate in situ assembled poly (ethylene glycol) (PEG)-based granular hydrogels for NPC delivery in a murine model of SCI. Microgel precursors are synthesized by using thiol-norbornene click chemistry to react four-armed PEG-amide-norbornene with enzymatically degradable and cell adhesive peptides. Unreacted norbornene groups are utilized for in situ assembly into scaffolds using a PEG-di-tetrazine linker. The granular hydrogel scaffolds exhibit good biocompatibility and do not adversely affect the inflammatory response after SCI. Moreover, when used to deliver NPCs, the granular hydrogel scaffolds supported NPC engraftment, do not adversely affect the immune response to the NPC grafts, and successfully support graft differentiation toward neuronal or astrocytic lineages as well as axonal extension into the host tissue. Collectively, these data establish PEG-based granular hydrogel scaffolds as a suitable biomaterial platform for NPC delivery and justify further testing, particularly in the context of more severe SCI.

Enzyme-functionalized alginate microparticles enable anaerobic culture under ambient oxygen.

Methods for culturing oxygen-sensitive cells and organisms under anaerobic conditions are vital to biotechnology research. Here, we report a biomaterial-based platform for anaerobic culture that consists of glucose oxidase (GOX) functionalized alginate microparticles (ALG-GOX), which are designed to deplete dissolved [O2 ] through enzymatic activity. ALG-GOX microparticles were synthesized via a water-in-oil emulsion and had a size of 132.0 ± 51.4 µm. Despite having a low storage modulus, the microparticles remained stable under aqueous conditions due to covalent crosslinking through amide bonds. Enzyme activity was tunable based on the loaded GOX concentration, with a maximum activity of 3.6 ± 0.3 units/mg of microparticles being achieved at an initial loading concentration of 5 mg/mL of GOX in alginate precursor solution. High enzyme activity in ALG-GOX microparticles resulted in rapid oxygen depletion, producing a suitable environment for anaerobic culture. Microparticles loaded with both GOX and catalase (ALG-GOX-CAT) to reduce H2 O2 buildup exhibited sustained activity for potential long-term anaerobic culture. ALG-GOX-CAT microparticles were highly effective for the anaerobic culture of Bacteroides thetaiotaomicron, with 10 mg/mL of ALG-GOX-CAT microparticles supporting the same level of growth in an aerobic environment compared to an anaerobic chamber after 16 h (8.70 ± 0.96 and 10.03 ± 1.03 million CFU, respectively; N.S. p = 0.07). These microparticles could be a valuable tool for research and development in biotechnology.

Dynamically Cross-Linked Granular Hydrogels for 3D Printing and Therapeutic Delivery.

Granular hydrogels have recently emerged as promising biomaterials for tissue engineering and 3D-printing applications, addressing the limitations of bulk hydrogels while exhibiting desirable properties such as injectability and high porosity. However, their structural stability can be improved with post-injection interparticle cross-linking. In this study, we developed granular hydrogels with interparticle cross-linking through reversible and dynamic covalent bonds. We fragmented photo-cross-linked bulk hydrogels to produce aldehyde or hydrazide-functionalized microgels using chondroitin sulfate. Mixing these microgels facilitated interparticle cross-linking through reversible hydrazone bonds, providing shear-thinning and self-healing properties for injectability and 3D printing. The resulting granular hydrogels displayed high mechanical stability without the need for secondary cross-linking. Furthermore, the porosity and sustained release of growth factors from these hydrogels synergistically enhanced cell recruitment. Our study highlights the potential of reversible interparticle cross-linking for designing injectable and 3D printable therapeutic delivery scaffolds using granular hydrogels. Overall, our study highlights the potential of reversible interparticle cross-linking to improve the structural stability of granular hydrogels, making them an effective biomaterial for use in tissue engineering and 3D-printing applications.

Development of optimal in vitro release and permeation testing method for rectal suppositories.

Currently there are no compendial assays for testing drug release from rectal suppositories. It is therefore essential to study different in vitro release testing (IVRT) and in vitro permeation testing (IVPT) methods for identifying a suitable technique to compare in vitro drug release and to predict in vivo performance of rectal suppositories. In the present study, three different rectal suppository formulations of mesalamine (CANASA, Generic, and In-house) were studied for in vitro bioequivalence. All the different suppository products were characterized by performing weight variation, content uniformity, hardness, melting time, and pH tests. Viscoelastic behavior of the suppositories was also tested both in presence and absence of mucin. Four different IVRT techniques such as Dialysis, Horizontal Ussing Chamber, Vertical Franz cell, and USP apparatus 4. IVPT studies were performed using Horizontal Ussing chamber and Vertical Franz cell methods. Q1/Q2 equivalent products (CANASA, Generic) and a half-strength product were studied to understand the reproducibility, bio relevance, and discriminatory ability of the IVRT and IVPT methods. This study is the first of its kind where molecular docking studies were performed to determine the potential interactions of drug (mesalamine) with mucin, IVRT studies were conducted with and without the presence of mucin, and porcine rectal mucosa was used to perform IVPT tests. The USP 4 method and Horizontal Ussing chamber methods were found to be suitable IVRT and IVPT techniques, respectfully, for rectal suppositories. RLD (Reference Listed Drug) and Generic rectal suppositories were found to exhibit similar release rate and permeation profiles obtained from USP 4, and the IVPT studies, respectfully. Wilcoxon Rank Sum/Mann-Whitney rank test, conducted for the IVRT profiles obtained using USP 4 method, proved the sameness of RLD and Generic suppository products.

Swellable and Thermally Responsive Hydrogel/Shape Memory Polymer Foam Composites for Sealing Lung Biopsy Tracts.

Lung tissue biopsies can result in a leakage of blood (hemothorax) and air (pneumothorax) from the biopsy tract, which threatens the patient with a collapsed lung and other complications. We have developed a lung biopsy tract sealant based on a thiol-ene-crosslinked PEG hydrogel and polyurethane shape memory polymer (SMP) foam composite. After insertion into biopsy tracts, the PEG hydrogel component contributes to sealing through water-driven swelling, whereas the SMP foam contributes to sealing via thermal actuation. The gelation kinetics, swelling properties, and rheological properties of various hydrogel formulations were studied to determine the optimal formulation for composite fabrication. Composites were then fabricated via vacuum infiltration of the PEG hydrogel precursors into the SMP foam followed by thermal curing. After drying, the composites were crimped to enable insertion into biopsy tracts. Characterization revealed that the composites exhibited a slight delay in shape recovery compared to control SMP foams. However, the composites were still able to recover their shape in a matter of minutes. Cytocompatibility testing showed that leachable byproducts can be easily removed by washing and washed composites were not cytotoxic to mouse lung fibroblasts (L929s). Benchtop testing demonstrated that the composites can be easily deployed through a cannula, and the working time for deployment after exposure to water was 2 min. Furthermore, testing in an in vitro lung model demonstrated that the composites were able to effectively seal a lung biopsy tract and prevent air leakage. Collectively, these results show that the PEG hydrogel/SMP foam composites have the potential to be used as lung biopsy tract sealants to prevent pneumothorax post-lung biopsy.

Glucose biosensors based on Michael addition crosslinked poly(ethylene glycol) hydrogels with chemo-optical sensing microdomains.

Continuous glucose monitoring (CGM) devices have the potential to lead to better disease management and improved outcomes in patients with diabetes. Chemo-optical glucose sensors offer a promising, accurate, long-term alternative to the current CGMs that require frequent calibration and replacement. Recently, we have proposed glucose sensor designs using phosphorescence lifetime-based measurement of chemo-optical glucose sensing microdomains embedded within alginate hydrogels. Due to the poor long-term stability of calcium-crosslinked alginate, we propose poly(ethylene glycol) (PEG) hydrogels synthesized via thiol-Michael addition chemistry as an alternative hydrogel carrier. The objective of this study was to evaluate the suitability of Michael addition crosslinked PEG hydrogels compared to calcium crosslinked alginate hydrogels for encapsulating glucose-sensing microdomains. PEG hydrogels crosslinked via thiol-vinyl sulfone addition achieved gelation in under 5 minutes, resulting in an even distribution of sensing microdomains. The shear storage modulus of the PEG hydrogels was tunable from 2.2 ± 0.1 kPa to 9.5 ± 1.8 kPa, which was comparable to the alginate hydrogels (10.5 ± 0.8 kPa), and the inclusion of microdomains did not significantly impact stiffness. The high water content of PEG hydrogels resulted in high glucose permeability that closely corresponded to the glucose permeability of alginate (D = 0.09 and 0.12 cm2 s-1, respectively; p = 0.47), but the PEG hydrogels exhibited superior stability. Both PEG and alginate-embedded sensors exhibited a sensing range up to ∼200 mg dL-1 glucose. The lower limits of detection (LOD) for PEG and alginate-based glucose sensors were 19.8 and 20.6 mg dL-1 with a difference of just 4.2% variation. The small difference between PEG and alginate embedded sensors indicates that their sensing properties are primarily determined by the glucose sensing microdomains rather than the hydrogel matrix. Overall, the results of this study indicate that Michael addition-crosslinked PEG hydrogels are a promising platform for encapsulation of chemo-optical glucose sensing microdomains.

Microparticle Hydrogel Material Properties Emerge from Mixing-Induced Homogenization in a Poly(ethylene glycol) and Dextran Aqueous Two-Phase System.

Polymer-polymer aqueous two-phase systems (ATPSs) are attractive for microgel synthesis, but given the complexity of phase separation, predicting microgel material properties from ATPS formulations is not trivial. The objective of this study was to determine how the phase diagram of a poly(ethylene glycol) (PEG) and dextran ATPS is related to the material properties of PEG microgel products. PEG-dextran ATPSs were prepared from four-arm 20 kDa PEG-norbornene and 40 kDa dextran in phosphate buffered saline (PBS), and the phase diagram was constructed. PEG microgels were synthesized from five ATPS formulations using an oligopeptide cross-linker and thiol-norbornene photochemistry. Thermogravimetric analysis (TGA) revealed that the polymer concentration of microgel pellets linearly correlates with the average concentration of PEG in the ATPS rather than the separated phase compositions, as determined from the phase diagram. Atomic force microscopy (AFM) and bulk rheology studies demonstrated that the mechanical properties of microgels rely on both the average concentration of PEG in the ATPS and the ATPS volume ratio as determined from the phase diagram. These findings suggest that PEG-dextran ATPSs undergo homogenization upon mixing, which principally determines the material properties of the microgels upon gelation.

Generalizing hydrogel microparticles into a new class of bioinks for extrusion bioprinting.

Hydrogel microparticles (HMPs) are an emerging bioink that can allow three-dimensional (3D) printing of most soft biomaterials by improving physical support and maintaining biological functions. However, the mechanisms of HMP jamming within printing nozzles and yielding to flow remain underexplored. Here, we present an in-depth investigation via both experimental and computational methods on the HMP dissipation process during printing as a result of (i) external resistance from the printing apparatus and (ii) internal physicochemical properties of HMPs. In general, a small syringe opening, large or polydisperse size of HMPs, and less deformable HMPs induce high resistance and closer HMP packing, which improves printing fidelity and stability due to increased interparticle adhesion. However, smooth extrusion and preserving viability of encapsulated cells require low resistance during printing, which is associated with less shear stress. These findings can be used to improve printability of HMPs and facilitate their broader use in 3D bioprinting.

Supramolecular Click Product Interactions Induce Dynamic Stiffening of Extracellular Matrix-Mimetic Hydrogels.

Progressive stiffening of the extracellular matrix (ECM) is observed in tissue development as well as in pathologies such as cancer, cardiovascular disease, and fibrotic disease. However, methods to recapitulate this phenomenon in vitro face critical limitations. Here, we present a poly(ethylene glycol)-based peptide-functionalized ECM-mimetic hydrogel platform capable of facile, user-controlled dynamic stiffening. This platform leverages supramolecular interactions between inverse-electron demand Diels-Alder tetrazine-norbornene click products (TNCP) to create pendant moieties that undergo non-covalent crosslinking, stiffening a pre-existing network formed via thiol-ene click chemistry over the course of 6 h. Pendant TNCP moieties have a concentration-dependent effect on gel stiffness while still being cytocompatible and permissive of cell-mediated gel degradation. The robustness of this approach as well as its simplicity and ease of translation give it broad potential utility.

Polymer-Coated Extracellular Vesicles for Selective Codelivery of Chemotherapeutics and siRNA to Cancer Cells.

Combination therapies involving small-interfering RNA (siRNA)-mediated gene silencing and small-molecule drugs are of high interest for cancer treatment. Among the current gene delivery carriers, cell-derived extracellular vesicles (EVs) are particularly promising candidates due to their high biocompatibility, low immunogenicity, in vivo stability, and inherent targeting ability. Here, we developed a multifunctional EV platform capable of selective codelivery of siRNA and doxorubicin (DOX) to cancer cells. siRNA was first loaded into engineered lipid-hybridized EVs (eEVs) to serve as a core. Subsequently, DOX was incorporated into a polyelectrolyte shell surrounding eEVs, which was deposited by layer-by-layer (LbL) assembly. This approach resulted in the production of a stable EV-polymer complex (LbL-eEV) with a diameter of 140.2 ± 9.0 nm and zeta potential of +22.1 ± 0.5 mV. Experiments were performed to assess cellular uptake, cytotoxicity, and gene silencing efficacy in lung adenocarcinoma cells (A549), with noncancerous fibroblast cells (CCL-210) used as a control. The results demonstrated that the LbL-eEV complex can traffic through cells and release siRNA in the cytoplasm, while delivered DOX enters nuclei to induce programmed cell death. Moreover, the inherent selectivity of the particles for cancer cells resulted in effective gene silencing and cancer killing efficiency with reduced cytotoxicity to normal cells. Synchronous delivery of siRNA and DOX was also verified by flow cytometry analysis of single cells. In summary, these data provide a proof of concept for engineering EVs to deliver multiple therapeutics and suggest that LbL-eEVs are a promising drug delivery platform for targeting cancer.

Hydrogel Synthesis and Stabilization via Tetrazine Click-Induced Secondary Interactions.

The discovery of tetrazine click-induced secondary interactions is reported as a promising new tool for polymeric biomaterial synthesis. This phenomenon is first demonstrated as a tool for poly(ethylene glycol) (PEG) hydrogel assembly via purely non-covalent interactions and is shown to yield robust gels with storage moduli one to two orders of magnitude higher than other non-covalent crosslinking methods. In addition, tetrazine click-induced secondary interactions also enhance the properties of covalently crosslinked hydrogels. A head-to-head comparison of PEG hydrogels crosslinked with tetrazine-norbornene and thiol-norbornene click chemistry reveals an approximately sixfold increase in storage modulus and unprecedented resistance to hydrolytic degradation in tetrazine click-crosslinked gels without substantial differences in gel fraction. Molecular dynamic simulations attribute these differences to the presence of secondary interactions between the tetrazine-norbornene cycloaddition products, which are absent in the thiol-norbornene crosslinked gels.

Bioprinting 101: Design, Fabrication, and Evaluation of Cell-Laden 3D Bioprinted Scaffolds.

3D bioprinting is an additive manufacturing technique that recapitulates the native architecture of tissues. This is accomplished through the precise deposition of cell-containing bioinks. The spatiotemporal control over bioink deposition permits for improved communication between cells and the extracellular matrix, facilitates fabrication of anatomically and physiologically relevant structures. The physiochemical properties of bioinks, before and after crosslinking, are crucial for bioprinting complex tissue structures. Specifically, the rheological properties of bioinks determines printability, structural fidelity, and cell viability during the printing process, whereas postcrosslinking of bioinks are critical for their mechanical integrity, physiological stability, cell survival, and cell functions. In this review, we critically evaluate bioink design criteria, specifically for extrusion-based 3D bioprinting techniques, to fabricate complex constructs. The effects of various processing parameters on the biophysical and biochemical characteristics of bioinks are discussed. Furthermore, emerging trends and future directions in the area of bioinks and bioprinting are also highlighted. Graphical abstract [Figure: see text] Impact statement Extrusion-based 3D bioprinting is an emerging additive manufacturing approach for fabricating cell-laden tissue engineered constructs. This review critically evaluates bioink design criteria to fabricate complex tissue constructs. Specifically, pre- and post-printing evaluation approaches are described, as well as new research directions in the field of bioink development and functional bioprinting are highlighted.

Comparison of Photo Cross Linkable Gelatin Derivatives and Initiators for Three-Dimensional Extrusion Bioprinting.

The objective of this study was to evaluate the utility of gelatin-norbornene (GelNB), which is cross-linkable via thiol-ene click chemistry, and the photoinitiator lithium phenyl-2,4,6 trimethylbenzoylphosphinate (LAP) for 3D bioprinting. These materials were compared to two widely used materials, gelatin-methacryloyl (GelMA) and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959). Characterization of photocuring kinetics revealed that LAP markedly improved the kinetics compared to I2959, which improved stability and print fidelity. Additionally, GelNB exhibited improved photocuring kinetics, improved stability, and decreased filament spreading compared to GelMA. However, inks containing GelMA yielded at lower stress, were more easily extruded, and produced smoother filaments. NIH 3T3 fibroblasts exhibited high viability in printed constructs, regardless of the gelatin derivative or photoinitiator used. Overall, these results support the selection of LAP over I2959 and suggest that GelNB could be a useful alternative to GelMA, although further work is needed to optimize GelNB extrusion.

Interplay between degradability and integrin signaling on mesenchymal stem cell function within poly(ethylene glycol) based microporous annealed particle hydrogels.

Microporous annealed particle (MAP) hydrogels are promising materials for delivering therapeutic cells. It has previously been shown that spreading and mechanosensing activation of human mesenchymal stem cells (hMSCs) incorporated in these materials can be modulated by tuning the modulus of the microgel particle building blocks. However, the effects of degradability and functionalization with different integrin-binding peptides on cellular responses has not been explored. In this work, RGDS functionalized and enzymatically degradable poly(ethylene glycol) (PEG) microgels were annealed into MAP hydrogels via thiol-ene click chemistry and photopolymerization. During cell-mediated degradation, the microgel surfaces were remodeled to wrinkles or ridges, but the scaffold integrity was maintained. Moreover, cell spreading, proliferation, and secretion of extracellular matrix proteins were significantly enhanced in faster matrix metalloproteinase degrading (KCGPQGIWGQCK) MAP hydrogels compared to non-degradable controls after 8 days of culture. We subsequently evaluated paracrine activity by hMSCs seeded in the MAP hydrogels functionalized with either RGDS or c(RRETAWA), which is specific for α5ß1 integrins, and evaluated the interplay between degradability and integrin-mediated signaling. Importantly, c(RRETAWA) functionalization upregulated secretion of bone morphogenetic protein-2 overall and on a per cell basis, but this effect was critically dependent on microgel degradability. In contrast, RGDS functionalization led to higher overall vascular endothelial growth factor secretion in degradable scaffolds due to the high cell number. These results demonstrate that integrin-binding peptides can modulate hMSC behavior in PEG-based MAP hydrogels, but the results strongly depend on the susceptibility of the microgel building blocks to cell-mediated matrix remodeling. This relationship should be considered in future studies aiming to further develop these materials for stem cell delivery and tissue engineering applications. STATEMENT OF SIGNIFICANCE: Microporous annealed particle (MAP) hydrogels are attracting increasing interest for tissue repair and regeneration and have shown superior results compared to conventional hydrogels in multiple applications. Here, we studied the impact of MAP hydrogel degradability and functionalization with different integrin-binding peptides on human mesenchymal stem cells (hMSCs) that were incorporated during particle annealing. Degradability was found to improve cell growth, spreading, and extracellular matrix production regardless of the integrin-binding peptide. Moreover, in degradable MAP hydrogels the integrin-binding peptide c(RRETAWA) was found to increase osteogenic protein expression by hMSCs compared to RGDS-functionalized MAP hydrogels. These results have important implications for the development of a MAP hydrogel-based hMSC delivery system for bone tissue engineering.

Bio-orthogonal, Site-Selective Conjugation of Recombinant Proteins to Microporous Annealed Particle Hydrogels for Tissue Engineering.

Protein conjugation to biomaterial scaffolds is a powerful approach for tissue engineering. However, typical chemical conjugation methods lack site-selectivity and can negatively impact protein bioactivity. To overcome this problem, a site-selective strategy is reported here for installing tetrazine groups on terminal poly-histidines (His-tags) of recombinant proteins. These tetrazine groups are then leveraged for bio-orthogonal conjugation to poly(ethylene glycol) (PEG) hydrogel microparticles, which are subsequently assembled into microporous annealed particle (MAP) hydrogels. Efficacy of the strategy is demonstrated using recombinant, green fluorescent protein with a His tag (His-GFP), which enhanced fluorescence of the MAP hydrogels compared to control protein lacking tetrazine groups. Subsequently, to demonstrate efficacy with a therapeutic protein, recombinant human bone morphogenetic protein-2 (His-BMP2) was conjugated. Human mesenchymal stem cells growing in the MAP hydrogels responded to the conjugated BMP2 and significantly increased mineralization after 21 days compared to controls. Thus, this site-selective protein modification strategy coupled with bio-orthogonal click chemistry is expected to be useful for bone defect repair and regeneration therapies. Broader application to the integration of protein therapeutics with biomaterials is also envisioned.

Creating Physicochemical Gradients in Modular Microporous Annealed Particle Hydrogels via a Microfluidic Method.

Microporous annealed particle (MAP) hydrogels are an attractive platform for engineering biomaterials with controlled heterogeneity. Here, we introduce a microfluidic method to create physicochemical gradients within poly(ethylene glycol) based MAP hydrogels. By combining microfluidic mixing and droplet generator modules, microgels with varying properties were produced by adjusting the relative flow rates between two precursor solutions and collected layer-by-layer in a syringe. Subsequently, the microgels were injected out of the syringe and then annealed with thiol-ene click chemistry. Fluorescence intensity measurements of constructs annealed in vitro and after mock implantation into a tissue defect showed that a continuous gradient profile was achieved and maintained after injection, indicating utility for in situ hydrogel formation. The effects of physicochemical property gradients on human mesenchymal stem cells (hMSCs) were also studied. Microgel stiffness was studied first, and the hMSCs exhibited increased spreading and proliferation as stiffness increased along the gradient. Microgel degradability was also studied, revealing a critical degradability threshold above which the hMSCs spread robustly and below which they were isolated and exhibited reduced spreading. This method of generating spatial gradients in MAP hydrogels could be further used to gain new insights into cell-material interactions, which could be leveraged for tissue engineering applications. A new droplet microfluidic approach to obtain microporous annealed particle hydrogels with physicochemical gradients is presented. Gradient formation is achieved by precisely controlling the mixing of two precursor solutions, and the gradient can be maintained after injection. This approach can be leveraged to produce new materials for tissue repair and to gain unique insights on cell-material interactions.

Clickable PEG hydrogel microspheres as building blocks for 3D bioprinting.

Three-dimensional (3D) bioprinting is important in the development of complex tissue structures for tissue engineering and regenerative medicine. However, the materials used for bioprinting, referred to as bioinks, must have a balance between a high viscosity for rapid solidification after extrusion and low shear force for cytocompatibility, which is difficult to achieve. Here, a novel bioink consisting of poly(ethylene glycol) (PEG) microgels prepared via off-stoichiometry thiol-ene click chemistry is introduced. Importantly, the microgel bioink is easily extruded, exhibits excellent stability after printing due to interparticle adhesion forces, and can be photochemically annealed with a second thiol-ene click reaction to confer long-term stability to printed constructs. The modularity of the bioink is also an advantage, as the PEG microgels have highly tunable physicochemical properties. The low force required for extrusion and cytocompatibility of the thiol-ene annealing reaction also permit cell incorporation during printing with high viability, and cells are able to spread and proliferate in the interstitial spaces between the microgels after the constructs have been annealed. Overall, these results indicate that our microgel bioink is a promising and versatile platform that could be leveraged for bioprinting and regenerative manufacturing.

Microporous Bio-orthogonally Annealed Particle Hydrogels for Tissue Engineering and Regenerative Medicine.

Microporous annealed particle (MAP) hydrogels are an emerging class of biomaterials with the potential to improve outcomes in tissue repair and regeneration. Here, a new MAP hydrogel platform comprising poly(ethylene) glycol (PEG) hydrogel microparticles that are annealed in situ using bio-orthogonal tetrazine click chemistry is reported (i.e., TzMAP hydrogels). Briefly, clickable PEG-peptide hydrogel microparticles with extracellular matrix mimetic peptides to permit cell adhesion and enzymatic degradation were fabricated via submerged electrospraying and stoichiometrically controlled thiol-norbornene click chemistry. Subsequently, unreacted norbornene groups in the microparticles were leveraged for functionalization with bioactive proteins as well as annealing into TzMAP hydrogels via the tetrazine-norbornene click reaction, which is highly selective and proceeds spontaneously without requiring an initiator or catalyst. The results demonstrate that the clickable particles can be easily applied to a tissue-like defect and then annealed into an inherently microporous structure in situ. In addition, the ability to produce TzMAP hydrogels with heterogeneous properties by incorporating multiple types of hydrogel microspheres is demonstrated, first with fluorophore-functionalized hydrogel microparticles and then with protein-functionalized hydrogel microparticles. For the latter, tetrazine-modified alkaline phosphatase was conjugated to PEG hydrogel microparticles, which were mixed with nonfunctionalized microparticles and used to produce TzMAP hydrogels. A biomimetic mineralized/nonmineralized interface was then produced upon incubation in calcium glycerophosphate. Finally, platelet-derived growth factor-BB (PDGF-BB) and human periodontal ligament stem cells (PDLSC) were incorporated into the TzMAP hydrogels during the annealing step to demonstrate their potential for delivering regenerative therapeutics, specifically for periodontal tissue regeneration. In vitro characterization revealed excellent PDGF-BB retention as well as PDLSC growth and spreading. Moreover, PDGF-BB loading increased PDLSC proliferation within hydrogels by 90% and more than doubled the average volume per cell. Overall, these results demonstrate that TzMAP hydrogels are a versatile new platform for the delivery of stem cells and regenerative factors.