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.

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.

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.

Assembly of PEG Microgels into Porous Cell-Instructive 3D Scaffolds via Thiol-Ene Click Chemistry.

The assembly of microgel building blocks into 3D scaffolds is an emerging strategy for tissue engineering. A key advantage is that the inherent microporosity of these scaffolds provides cells with a more permissive environment than conventional nanoporous hydrogels. Here, norbornene-bearing poly(ethylene glycol) (PEG) based microgels are assembled into 3D cell-instructive scaffolds using a PEG-dithiol linker and thiol-ene click photopolymerization. The bulk modulus of these materials depends primarily on the crosslink density of the microgel building blocks. However, the linker and initiator concentrations used during assembly have significant effects on cell spreading and proliferation when human mesenchymal stem cells (hMSCs) are incorporated in the scaffolds. The cell response is also affected by the properties of the modular microgel building blocks, as hMSCs growing in scaffolds assembled from stiff but not soft microgels activate Yes-associated protein signaling. These results indicate that PEG microgel scaffolds assembled via thiol-ene click chemistry can be engineered to provide a cell-instructive 3D milieu, making them a promising 3D platform for tissue engineering.

Creation of an injectable in situ gelling native extracellular matrix for nucleus pulposus tissue engineering.

BACKGROUND CONTEXT: Disc degeneration is the leading cause of low back pain and is often characterized by a loss of disc height, resulting from cleavage of chondroitin sulfate proteoglycans (CSPGs) present in the nucleus pulposus. Intact CSPGs are critical to water retention and maintenance of the nucleus osmotic pressure. Decellularization of healthy nucleus pulposus tissue has the potential to serve as an ideal matrix for tissue engineering of the disc because of the presence of native disc proteins and CSPGs. Injectable in situ gelling matrices are the most viable therapeutic option to prevent damage to the anulus fibrosus and future disc degeneration. PURPOSE: The purpose of this research was to create a gentle decellularization method for use on healthy nucleus pulposus tissue explants and to develop an injectable formulation of this matrix to enable therapeutic use without substantial tissue disruption. STUDY DESIGN: Porcine nuclei pulposi were isolated, decellularized, and solubilized. Samples were assessed to determine the degree of cell removal, matrix maintenance, gelation ability, cytotoxic residuals, and native cell viability. METHODS: Nuclei pulposi were decellularized using serial detergent, buffer, and enzyme treatments. Decellularized nuclei pulposi were solubilized, neutralized, and buffered. The efficacy of decellularization was assessed by quantifying DNA removal and matrix preservation. An elution study was performed to confirm removal of cytotoxic residuals. Gelation kinetics and injectability were quantified. Long-term in vitro experiments were performed with nucleus pulposus cells to ensure cell viability and native matrix production within the injectable decellularized nucleus pulposus matrices. RESULTS: This work resulted in the creation of a robust acellular matrix (>96% DNA removal) with highly preserved sulfated glycosaminoglycans (>47%), and collagen content and microstructure similar to native nucleus pulposus, indicating preservation of disc components. Furthermore, it was possible to create an injectable formulation that gelled in situ within 45 minutes and formed fibrillar collagen with similar diameters to native nucleus pulposus. The processing did not result in any remaining cytotoxic residuals. Solubilized decellularized nucleus pulposus samples seeded with nucleus pulposus cells maintained robust viability (>89%) up to 21 days of culture in vitro, with morphology similar to native nucleus pulposus cells, and exhibited significantly enhanced sulfated glycosaminoglycans production over 21 days. CONCLUSIONS: A gentle decellularization of porcine nucleus pulposus followed by solubilization enabled the creation of an injectable tissue-specific matrix that is well tolerated in vitro by nucleus pulposus cells. These matrices have the potential to be used as a minimally invasive nucleus pulposus therapeutic to restore disc height.

Recyclable Saccharomyces cerevisiae loaded nanofibrous mats with sandwich structure constructing via bio-electrospraying for heavy metal removal.

Biosorbents, such as algae and yeast, have been applied in heavy metal adsorption due to their low cost and efficacy. However, they cannot be recycled and reused after direct application, which may cause a secondary pollution. In this study, we used bio-electrospraying technique to immobilize Saccharomyces cerevisiae (a byproduct from food fermentation) onto the surface of poly(ε-caprolactone)/chitosan/rectorite ternary composites based nanofibrous mats. This technique not only combined the advantages of both S. cerevisiae (cheap) and nanofibers (large surface area) in heavy metal removal, but also made biosorbents easy to recollect and reuse. Layer-by-layer structured nanofibrous mats were also fabricated by alternating electrospinning and bio-electrospraying for a couple of times and loaded more S. cerevisiae for enhancing heavy metal biosorption. The morphology of S. cerevisiae loaded nanofibrous mats with different numbers of layers was observed. Biosorption assay was performed on PbNO3 solution under different pH values, contact time, initial concentrations of Pb2+ and biosorbents weights, at last the elemental composition was measured before and after biosorption. The results showed that S. cerevisiae loaded nanofibrous mats had a biosorption capacity of Pb2+ up to 238mg/g. Desorption assay indicated that these mats were reusable and maintained high biosorption capacity after three biosorption-desorption cycles.

Localized and sustained release of brain-derived neurotrophic factor from injectable hydrogel/microparticle composites fosters spinal learning after spinal cord injury.

Damaged axons in the adult mammalian central nervous system (CNS), including those of the spinal cord, have extremely limited endogenous capacity to regenerate. This is the result of both the intrinsic and extrinsic inhibitory factors that limit the regeneration of adult neurons. Despite attempts to limit or eliminate the extrinsic inhibitory components, regeneration of adult neurons in the CNS is still limited. Therefore, additional factors that can further enhance the intrinsic plasticity of adult neurons need to be considered. Herein, we examine the effects of brain-derived neurotrophic factor (BDNF), a known growth factor for neuronal survival and plasticity, using an in vivo delivery method for a localized and sustained delivery to the spinal cord. A highly versatile injectable biomaterial platform for the sustained delivery of BDNF was developed using a physical blend of hyaluronic acid (HA) and methylcellulose (MC), in combination with poly-lactic-co-glycolic acid (PLGA) microparticles. Contemporary studies examining the plasticity of the CNS suggest that the spinal cord is an important site for activity-dependent learning that can mediate motor function after injury or disease. Here we utilized such a learning paradigm in combination with local and sustained BDNF application (at L3-S2) to foster spinal learning after complete spinal cord injury in rodents. Our data suggest that composite biomaterial systems such as the one described herein can be utilized for the sustained and localized delivery of therapeutics following damage to the spinal cord.

Into the groove: instructive silk-polypyrrole films with topographical guidance cues direct DRG neurite outgrowth.

Instructive biomaterials capable of controlling the behaviour of the cells are particularly interesting scaffolds for tissue engineering and regenerative medicine. Novel biomaterials are particularly important in societies with rapidly aging populations, where demand for organ/tissue donations is greater than their supply. Herein we describe the preparation of electrically conductive silk film-based nerve tissue scaffolds that are manufactured using all aqueous processing. Aqueous solutions of Bombyx mori silk were cast on flexible polydimethylsiloxane substrates with micrometer-scale grooves on their surfaces, allowed to dry, and annealed to impart ß-sheets to the silk which assures that the materials are stable for further processing in water. The silk films were rendered conductive by generating an interpenetrating network of polypyrrole and polystyrenesulfonate in the silk matrix. Films were incubated in an aqueous solution of pyrrole (monomer), polystyrenesulfonate (dopant) and iron chloride (initiator), after which they were thoroughly washed to remove low molecular weight components (monomers, initiators, and oligomers) and dried, yielding conductive films with sheet resistances of 124 ± 23 kΩ square(-1). The micrometer-scale grooves that are present on the surface of the films are analogous to the natural topography in the extracellular matrix of various tissues (bone, muscle, nerve, skin) to which cells respond. Dorsal root ganglions (DRG) adhere to the films and the grooves in the surface of the films instruct the aligned growth of processes extending from the DRG. Such materials potentially enable the electrical stimulation (ES) of cells cultured on them, and future in vitro studies will focus on understanding the interplay between electrical and topographical cues on the behaviour of cells cultured on them.

Novel layer-by-layer structured nanofibrous mats coated by protein films for dermal regeneration.

Layer-by-layer coating technique is effective in modifying the surface of nanofibrous mats, but overmuch film-coating makes the mats less porous to hardly suit the condition for tissue engineering. We developed novel nanofibrous mats layer-by-layer coated by silk fibroin and lysozyme on the cellulose electrospun template via electrostatic interaction. The film-coating assembled on the mats was not excessive because the charge of the proteins varied in the coating process due to different pH value. In addition, pure nature materials made the mats nontoxic, biodegradable and low-cost. The morphology and composition variation during layer-by-layer coating process was investigated and the results showed that the structure and thickness of film-coatings could be well-controlled. The antibacterial assay and in vitro cell experiments indicated that the mats could actively inhibit bacteria and exhibit excellent biocompatibility. In vivo implant assay further verified the mats cultured with human epidermal cells could promote wound healing and avoid wound infection. Therefore, these mats showed promising prospects when performed for dermal reconstruction.

Hydroxypropyl chitosan/organic rectorite-based nanofibrous mats with intercalated structure for bacterial inhibition.

This paper reported antibacterial hydroxypropyl chitosan (HPCS)/organic rectorite (OREC)-based nanofibrous mats with intercalated structure fabricated via solution intercalation method and electrospinning. Field emission scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectra, Energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and inhibition zone surrounding circular mats disks measurement were performed to characterize the morphology, intercalation structure, elements analysis, and the antibacterial properties of the as-spun nanofibrous mats. The results showed that the nanofibrous mats were with better fiber shape with the addition of OREC, the polymer chains were successfully intercalated into the interlayer of OREC, and nanofibrous mats containing HPCS exhibited good antibacterial activities against Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus. In addition, the bacterial inhibition ability of the nanofibrous mats was enhanced when OREC was added.

Cytotoxicity and antibacterial ability of scaffolds immobilized by polysaccharide/layered silicate composites.

Chitosan and pectin/organic rectorite (OREC) were initially deposited on the surface of cellulose acetate electrospun nanofibers by a layer-by-layer (LBL) technique to fabricate scaffolds for bacterial inhibition, and the cytotoxicity of the LBL structured scaffolds was also investigated. A couple of opposite charged material, pectin and OREC, were firstly used to fabricate the intercalated composites. The intercalated structure was determined by selected area electron diffraction. Field-emission scanning electron microscope, X-ray diffraction and X-ray photoelectron spectroscopy were applied for the characterization of LBL structured nanofibrous scaffolds. Antibacterial assay results showed that the diameters of the inhibition zone increased from 7.6 to 15.8 mm for Escherichia coli, as well as from 7.4 to 14.2 mm for Staphylococcus aureus. Finally, human epidermal (EP) cells grew well on the LBL films coating. These novel scaffolds could be an ideal candidate for wound dressings and food packaging.

Nanofibrous mats coated by homocharged biopolymer-layered silicate nanoparticles and their antitumor activity.

Quaternized chitosan (QC)-organic rectorite (OREC) intercalated composites based nanoparticles were immobilized on cellulose acetate nanofibrous mats by layer-by-layer deposition technique via hydrogen bonds. The key design of those mats was the utilization of all negatively charged materials to reduce the quantity of nanoparticles immobilized on the fibers and then the cytotoxicity of the mats. The intercalated structure in nanoparticles was confirmed by selected area electron diffraction. The morphology and composition of NPs-assembled nanofibrous mats were studied by field emission scanning electron microscopy and X-ray photoelectron spectroscopy. The results trended to give clues as how the assembly process varied by adding OREC. MTT assay and cell culture experiments showed that NPs-assembled nanofibrous mats were commendably compatible with normal cells and could selectively kill human lung carcinoma epithelial cells. Hemolysis test indicated these mats had excellent blood compatibility. As a result, QC-OREC NPs-assembled nanofibrous mats by homocharged deposition process can be considered as a novel alternative method for cancer therapy.

Novel polymer-layered silicate intercalated composite beads for drug delivery.

Core-shell structured beads were fabricated from chitosan (CS)/organic rectorite (OREC) composites and alginate (ALG) in Ca(2+) aqueous solutions with different mixing ratios by a cross-linking process. The mechanical properties, surface and internal morphology, intercalation structure between CS and OREC, porosity and pore size distribution, bovine serum albumin (BSA) encapsulation efficiency and its controllable release ability were investigated. Optical microscopy, scanning electron microscopy and transmission electron microscopy showed that the core-shell structure was generated in the beads. The Fourier transform infrared spectra results implied the presence of electrostatic and hydrogen-bonding interaction between CS and OREC. The energy-dispersive X-ray and X-ray photoelectron spectroscopy results verified the existence of OREC in the beads. Small-angle X-ray diffraction results confirmed that the interlayer of OREC was intercalated by CS chains successfully, and the interlayer distance increased from 2.42 to 2.60 nm. The BSA encapsulation and release test indicated that the beads released the drug continuously. OREC could not only avoid the burst release phenomenon in the first period but also improve the utilization efficacy of the drug. When the ratio of CS/OREC was 6:1 and CS-OREC/ALG was 2:1, the beads were better for drug released in stomach, and when CS/OREC was 12:1 and CS-OREC/ALG was 2:1, the beads were better for drug released in stomach than in intestine.

Quaternized chitosan-organic rectorite intercalated composites based nanoparticles for protein controlled release.

Organic rectorite (OREC) was added in the quaternized chitosan (QC)/alginate (ALG) nanoparticles using an ionic gelation method to fabricate a controllable release system for proteins for the first time. The morphology of nanoparticles, the intercalated structure of OREC, bovine serum albumin encapsulation efficiency and in vitro release properties were investigated. Fourier transform infrared spectra, energy dispersive X-ray, X-ray photoelectron spectroscopy, small angle X-ray diffraction and size distribution analysis were performed to characterize the composite nanoparticles. With the addition of OREC, the encapsulation efficiency and the loading capacity of nanoparticles had increased from 21.2% to 44.9% and from 13.7% to 25.0%, respectively. In addition, the rapid initial release was inhibited successfully from 20.15% to 11.07% in stimulated gastric fluid and from 14.69% to 4.52% in stimulated intestinal fluid. The results verified that the addition of OREC could make these nanoparticles effective carriers to encapsulate drug and slow the drug controlled release of nanoparticles.

Carboxymethyl chitin/organic rectorite composites based nanofibrous mats and their cell compatibility.

In this study, carboxymethyl chitin (CMC) - organic rectorite (OREC)/poly (vinyl alcohol) (PVA) composite nanofibrous mats were successfully prepared via electrospinning. SAXRD pattern showed that the interlayer distance of OREC was increased from 3.68 to 4.08nm, which verified that polymer chains were intercalated into the interlayer of OREC. Field emission scanning electron microscopy, Fourier transform infrared spectra and energy-dispersive X-ray spectroscopy were used to characterize the morphology and microcosmic structure of nanofibrous mats. Thermal properties of mats were determined by differential scanning calorimetry. To evaluate the cell compatibility of mats, mouse lung fibroblast (L929) was chosen for cell attachment and spreading assay. The results shows that nanofibrous mats contained OREC have better thermal properties. Besides, the addition of OREC has little effect on the cell compatibility of nanofibrous mats.

Quaternized chitosan-layered silicate intercalated composites based nanofibrous mats and their antibacterial activity.

Quaternized chitosan (HTCC)-organic rectorite (OREC) intercalated composites based electrospun nanofibrous mats were fabricated from HTCC-OREC/polyvinyl alcohol (PVA) solutions for the first time. The morphology, intercalated structure, and antibacterial activity of the as-spun mats were investigated. The transmission electron microscopy images taken from HTCC-OREC composites showed that HTCC chains were inside the OREC interlayer. Scanning electron microscopy results verified that more typical fibrous structure would be generated by adding OREC. Fourier transform infrared spectra and energy-dispersive X-ray spectroscopy results indicated that OREC existed in the HTCC-OREC/PVA nanofibrous mats. The intercalation structure in nanofibrous mats was confirmed by X-ray diffraction results, which confirmed that HTCC and PVA chains could intercalate into the interlayer of OREC. The antibacterial activity of the electrospun mats was enhanced when the amount of the OREC increased. Therefore, the novel ternary nanofibrous mats could be used in the field of food packaging and biomedical applications.