Microgels for Cell Delivery in Tissue Engineering and Regenerative Medicine

Microgels prepared from natural or synthetic hydrogel materials have aroused extensive attention as multifunctional cells or drug carriers,that are promising for tissue engineering and regenerative medicine.Microgels can also be aggregated into microporous scaffolds,promoting cell infiltration and proliferation for tissue repair.This review gives an overview of recent developments in the fabrication techniques and applications of microgels.A series of conventional and novel strategies including emulsification,microfluidic,lithography,electrospray,centrifugation,gas-shearing,three-dimensional bioprinting,etc.are discussed in depth.The characteristics and applications of microgels and microgel-based scaffolds for cell culture and delivery are elaborated with an emphasis on the advantages of these carriers in cell therapy.Additionally,we expound on the ongoing and foreseeable applications and current limitations of microgels and their aggregate in the field of biomedical engineering.Through stimulating innovative ideas,the present review paves new avenues for expanding the application of microgels in cell delivery techniques.

Application of Monodisperse Thermo-Responsive Composite Microgels with Core-Shell Structure Based on Au@Ag Bimetallic Nanorod as Core in Surface Enhanced Raman Spectroscopy Substrate

The monodisperse Au@Ag bimetallic nanorod is encapsulated by crosslinked poly( N-isopropylacrylamide)( PNIPAM) to produce thermo-responsive composite microgel with well-defined core-shell structure( Au@ Ag NR@ PNIPAM microgel)by seed-precipitation polymerization method using butenoic acid modified Au @ Ag NRs as seeds. When the temperature of the aqueous medium increases from 20℃ to 50℃,the localized surface plasmon resonance( LSPR) band of the entrapped Au @ Ag NR is pronouncedly red-shifted because of the decreased spatial distances between them as a result of shrinkage of the microgels,leading to their plasmonic coupling. The temperature tunable plasmonic coupling is demonstrated by temperature dependence of the surface enhanced Raman spectroscopy( SERS) signal of 1-naphthol in aqueous solution. Different from static plasmonic coupling modes from nanostructured assembly or array system of noble metals,the proposed plasmonic coupling can be dynamically controlled by environmental temperature. Therefore, the thermo responsive hybrid microgels have potential applications in mobile LSPR or SERS microsensors for living tissues or cells.

Structured Microgels through Microfluidic Assembly and Their Biomedical Applications

Droplet based microfluidic is an effective, versatile and scalable approach which can be used to produce structured microgels with desirable features. The high degree of control endowed with microfluidics enables the formation of various functional microgels such as multicompartment encapsulations, Janus shaped particles and non spherical microgels. These microgels have aroused great interest in biological engineering aspect, since they outperform their counterparts produced from other techniques and have been applied in drug delivery, 3 Dimensional cell culture, micro tissues, single cell assay, tissue engineering and bioimaging. In this review, we will summarize the fabrication processes, technology comparisons and the usages in biomedical applications.

Application of reactive acrylate microgels in water-base coatings

Reactive acrylate microgels with different reactive groups such as carboxyl, hydroxide groups had excellent properties such as quick-dry, low viscosity, high adhesion and hardness, which made them extensively used in preparing paints or in coating-modification. Reactive acrylate microgels were prepared by emulsion co-polymerization with zwitterions surfactant, anionic surfactant and nonionic surfactant as co-emulsifier. The water-base baking paints made from reactive acrylate microgels and melamine-formaldehyde resin had excellent combination properties. The aluminium powder can be well-dispersed in the paints. The influences of monomer components on the properties of the water-base baking paints were discussed in this paper. And the baking paints were also compared with the marketing solvent acrylate baking paints. It was found that the water-base acrylate amino baking paints had better combination properties than the organic solvent acrylate baking paints, which means that the water-base baking paints had a bright marketing future.

STABILITY OF POLY(N,N'-METHYLENEBISACRYLAMIDE-co-4-VINYLPYRIDINE)MICROGEL DISPERSION AND ADJUSTMENT OF THE HYDROPHOBICITY IN THE MICROGELS

In the UV-Vis spectra of pure light-scattering systems, there is an exponential relationship between absorbance and wavelength （A = Kλ^-n）. Here, the exponent n is named as flocculation-coagulation parameter. In the present paper, the effects of different additives on the stability of poly（N,N＇-methylenebisacrylamide-co-4-vinylpyridine） （poly（Bis-co-4-VP）） microgel dispersion were studied in detail via this parameter. The results showed that the stability of the dispersion mainly comes from the ionization of pyridine groups, making the microgel positively charged on its surface. This was confirmed by the measurement of Zeta potential and the result of conductometric titration. The result of fluorescence analysis indicated that the hydrophobicity in the microgels is enhanced with the increase in total 4-VP unit content.

PARAMETERS AFFECTING PARTICLE SIZE OF POLYBUTYL-ACRYLATE MICROGELS

The factors affecting particle size of reactive microgels formed during the self-emulsifying copolymerization of unsaturated polyester (UP)with butyl acrylate (BA)have been studied. The parameters discussed are: the proportion of the UP in the monomer mixture, the molecular weight and the carboxyl value of the UP, the phase ratio, the electrolyte concentration and the polar solvent additive. The seeding emulsion polymerization is discussed as well.It turned out that the particle size of the reactive microgels can be controlled in a definite range by changing the experimental conditions. However the particle size distribution becomes broader as the average diameter increases. It is suggested that the agglomeration of primary particles plays an important role during the growth of microgel particle.

Fluorescent Nile blue-functionalized poly(N-isopropylacrylamide)microgels responsive to temperature and polyamines

Fluorescent poly(N-isopropylacrylamide-co-Nile blue)(pNIPAm-co-NB)microgels were synthesized that exhibited fluorescence intensity changes in a water temperature-dependent fashion.NB is well known to exhibit fluorescence intensity that depends on the hydrophobicity of the environment,while pNIPAm-based microgels are well known to transition from swollen(hydrophilic)to collapsed(relatively hydrophobic)at temperatures greater than 32℃;hence,we attribute the above behavior to the hydrophobicity changes of the microgels with increasing temperature.This phenomenon is ultimately due to NB dimers(relatively quenched fluorescence)being broken in the hydrophobic environment of the microgels leading to relatively enhanced fluorescence.We went on to show that the introduction of cucurbit[7]uril(CB[7])into the pNIPAm-co-NB microgels enhanced their fluorescence allowing them to be used for polyamine(e.g.,spermine[SPM])detection.Specifically,CB[7]forms a host–guest interaction with NB in the microgels,which prevents NB dimerization and enhances their fluorescence.When SPM is present,it forms a host–guest complex that is favored over the CB[7]-NB host–guest interaction,which frees the NB for dimerization and leads to fluorescence quenching.As a result,we could generate an SPM sensor capable of SPM detection down to~0.5µmol/L in complicated matrixes such as serum and urine.

Bidirectionally aligned MXene hybrid aerogels assembled with MXene nanosheets and microgels for supercapacitors

MXene nanomaterials are one of the most promising electrode material candidates for supercapacitors owing to their high conductivity,abundant surface functional groups and large surface area.However,electrodes based on MXene may result in low ion-accessible surface area and blocked ion transport pathways because of the self-restacking of MXene nanosheets.It is essential to suppress the self-res tacking of nanosheets and increase the electrochemical active sites in order to optimize the electrode.In this work,bidirectionally aligned MXene hybrid aerogel(A-MHA)assembled with MXene nanosheets and microgels is prepared using a facile bidirectional freeze casting and freeze-drying method.The bidirectionally aligned structure together with the three-dimensional structured microgels in the A-MHAs,can improve the ionaccessible surface area and provide more barrier-free channels by exposing more active sites and ensuring electrolyte transport freely.The A-MHA with MXene microgels content of 40 wt%exhibits a high specific capacitance of 760 F·g^(-1)at 1 A·g^(-1)and a remarkable cyclic performance of 97%after 10,000 cycles at100 mV·s^(-1)in 1 mol·L^(-1)H_(2)SO_(4)electrolyte.A-MHAs show remarkable electrochemical properties and are of potential application in energy storage.

Poly(N-isopropylmethacrylamide-co-4-acrylamidobenzo-18-crown-6)microgels with expanded networks for excellent adsorption of lead(Ⅱ)ions

Lead(Ⅱ)(Pb2+)ions are toxic heavy metal ions that can accumulate in the human body through water and cause severe health problems,including neurotoxicity,nephrotoxicity,hematological toxicity and even genotoxicity effects.To remove Pb2+selectively and effectively from aqueous solutions,we develop a novel type of Pb2+-recognizable microgels with excellent adsorption capacity to Pb2+,which are fabri-cated from 4-acrylamidobenzo-18-crown-6(B18C6Am)and N-isopropylmethacrylamide(NIPMAM)monomers by precipitation copolymerization method.The prepared poly(N-isopropylmethacrylamide-co-4-acrylamidobenzo-18-crown-6)(PNMB)microgels exhibit expanded structures,because the electron-donating methyl groups atα-carbon could descend the polarity of C=O in the NIPMAM monomers and thus weaken the polymer segment…segment interactions.The expanded structures of PNMB microgels are beneficial for adsorption of Pb2+due to the low steric hindrance in the polymeric networks.The Pb2+adsorption isotherms of PNMB microgels are consistent with the Langmuir model for monolayer adsorption.The results indicate that the prepared Pb2+-recognizable PNMB microgels are highly promising for the selective removal of lead(Ⅱ)ions from aqueous solutions.

Synovial fluid-inspired biomimetic lubricating microspheres:Zwitterionic polyelectrolyte brushes-grafted microgels

Synovial fluid is made up of various biomacromolecules,including hyaluronic acid,aggrecans,lubricins,and phosphatidylcholine lipid,which are assembled onto the surface of articular cartilage in a gel state.Among them,brush-like biomacromolecules or assemblies have a vital effect on human joint lubrication.Inspired by this,the combination of brush-like molecular structures and gel-like assembly may be an efficient approach for the synthesis of biomimetic lubricating matters.Learning from the lubrication system of human joints,poly(2-methacryloyloxyethyl phosphorylcholine)(PMPC)brushes grafted poly(N-isopropylacrylamide-co-acrylic acid)(poly(NIPAAm-co-AA))microgels,abbreviated as MBs-g-MGs,were synthesized as one kind of biomimetic lubricating additives.It is worth noting that this bionic strategy considered both molecular structure and assembled form,which enabled this hairy microgel to achieve low friction in aqueous medium.Meanwhile,the effective lubrication was still achieved when using MBs-g-MGs at high temperature,indicating that this microgel maintains a good lubricating effect over a wide range of temperature.In addition,this kind of microgel possessed good biocompatibility,which laid the foundation for potential biomedical applications.Looking beyond,these biomimetic microgels may provide an effective lubricating effect for water-based sliding interfaces,especially in biomedical systems.

Responsive Hybrid Poly(vinyl alcohol)Hydrogel Membranes with Embedded Microgels as Valves

The research and application of responsive materials have long been hampered by their complicated designs and tedious construction processes.Besides,many current responsive materials show retard or weak responsiveness.In this study,responsive hybrid poly(vinyl alcohol)hydrogel membranes with embedded poly(N-isopropylacrylamide-acrylic acid)microgels as valves were constructed by simple mixing and subsequent freezing-thawing process.In the structure of the membranes,the matrix poly(vinyl alcohol)chains thread through and entangle with the microgels,and the microgels are firmly constrained within the hybrid hydrogel network.The fast and sharp temperature responsiveness of the embedded microgels was largely retained and endowed the hydrogel membrane with excellent temperature and pH responsiveness.Moreover,the hydrogel membrane showed excellent fatigue resistance in both temperature and pH-responsive flux examination.This study presented the great potential of these hybrid hydrogel membranes in biomedical applications and provided a new strategy for the future design and construction of responsive biomaterials.

Harnessing decellularised extracellular matrix microgels into modular bioinks for extrusion-based bioprinting with good printability and high post-printing cell viability

The printability of bioink and post-printing cell viability is crucial for extrusion-based bioprinting.A proper bioink not only provides mechanical support for structural fidelity,but also serves as suitable three-dimensional(3D)microenvironment for cell encapsulation and protection.In this study,a hydrogel-based composite bioink was developed consisting of gelatin methacryloyl(GelMA)as the continuous phase and decellularised extracellular matrix microgels(DMs)as the discrete phase.A flow-focusing microfluidic system was employed for the fabrication of cell-laden DMs in a high-throughput manner.After gentle mixing of the DMs and GelMA,both rheological characterisations and 3D printing tests showed that the resulting DM-GelMA hydrogel preserved the shear-thinning nature,mechanical properties,and good printability from GelMA.The integration of DMs not only provided an extracellular matrix-like microenvironment for cell encapsulation,but also considerable shear-resistance for high post-printing cell viability.The DM sizes and inner diameters of the 3D printer needles were correlated and optimised for nozzle-based extrusion.Furthermore,a proof-of-concept bioink composedg of RSC96 Schwann cells encapsulated DMs and human umbilical vein endothelial cell-laden GelMA was successfully bioprinted into 3D constructs,resulting in a modular co-culture system with distinct cells/materials distribution.Overall,the modular DM-GelMA bioink provides a springboard for future precision biofabrication and will serve in numerous biomedical applications such as tissue engineering and drug screening.

Upconverting nanoparticle-containing erythrocyte-sized hemoglobin microgels that generate heat,oxygen and reactive oxygen species for suppressing hypoxic tumors

Inspired by erythrocytes that contain oxygen-carrying hemoglobin(Hb)and that exhibit photo-driven activity,we introduce homogenous-sized erythrocyte-like Hb microgel(μGel)systems(5-6μm)that can(i)emit heat,(ii)supply oxygen,and(iii)generate reactive oxygen species(ROS;1O2)in response to near-infrared(NIR)laser irradiation.Hb μGels consist of Hb,bovine serum albumin(BSA),chlorin e6(Ce6)and erbium@lutetium upconverting nanoparticles(UCNPs;~35 nm)that effectively convert 808 nm NIR light to 660 nm visible light.These Hb μGels are capable of releasing oxygen to help generate sufficient reactive oxygen species(^(1)O_(2))from UCNPs/Ce6 under severely hypoxic condition upon NIR stimulation for efficient photodynamic activity.Moreover,the Hb μGels emit heat and increase surface temperature due to NIR light absorption by heme(iron protoporphyrin IX)and display photothermal activity.By changing the Hb/UCNP/Ce6 ratio and controlling the amount of NIR laser irradiation,it is possible to formulate bespoke Hb μGels with either photothermal or photodynamic activity or both in the context of combined therapeutic effect.These Hb μGels effectively suppress highly hypoxic 4T1 cell spheroid growth and xenograft mice tumors in vivo.

Cryo-shocked cancer cell microgels for tumor postoperative combination immunotherapy and tissue regeneration

Prevention of recurrence/metastasis and tissue regeneration are critical for post-surgery treatment of malignant tumors. Here, to address these needs, a novel type of microgel co-loading cryo-shocked cancer cells, immunoadjuvant, and immune checkpoint inhibitor is presented by microfluidic electrospray technology and liquid nitrogen treatment. Owing to the encapsulation of cryo-shocked cancer cells and immunoadjuvant, the microgels can recruit dendritic cells and activate them in situ, and evoke a robust immune response. Moreover, with the combination of the immune checkpoint inhibitor, the antitumor immune response is further enhanced by inhibiting the interaction of PD1 and PDL1. With this, the excellent anti-recurrence and anti-metastasis efficacy of the microgels are demonstrated in an orthotopic breast cancer mouse model. Besides, because of the excellent biocompatibility and appropriate degradation performance, the microgels can provide support for normal cell adhesion and growth, which is beneficial to tissue reconstruction. These properties indicate the great value of the cryo-shocked cancer cell microgels for efficient tumor postoperative combination immunotherapy and tissue regeneration.

Tribological behavior of thermal- and pH-sensitive microgels under steel/CoCrMo alloy contacts

The tribological behavior of 316L stainless steel/CoCrMo alloy contacts under aqueous lubrication was investigated in this work.Three types of microgels including poly(N-isopropylacrylamide)(PNIPAM),polyacrylic acid(PAA),and poly(N-isopropylacrylamide-co-acrylic acid)(PNIPAM-co-PAA)were prepared and used as lubricant additives in aqueous solutions.Tribological tests were conducted using a ball-on-disk reciprocating tribometer,over a range of temperatures,pH,and concentrations of the microgels.The PNIPAM-co-PAA microgels were proven to have excellent anti-friction and anti-wear properties,depending on the temperature and pH values of the solutions.The maximum friction coefficient coincided with the lower critical solution temperature of the PNIPAM-co-PAA microgels at 32°C.At the same time,the friction coefficient increased with the increase in pH value of the lubricant solution with the PNIPAM-co-PAA microgels.The unique thermal-and pH-sensitive properties of the PNIPAM-co-PAA microgels provided a new strategy for controlling the friction and wear of steel/CoCrMo alloy contacts under aqueous lubrication.

A novel bioartificial pancreas fabricated via islets microencapsulation in anti-adhesive core-shell microgels and macroencapsulation in a hydrogel scaffold prevascularized in vivo

Islets transplantation is a promising treatment for type 1 diabetes mellitus. However, severe host immune rejection and poor oxygen/nutrients supply due to the lack of surrounding capillary network often lead to transplantation failure. Herein, a novel bioartificial pancreas is constructed via islets microencapsulation in core-shell microgels and macroencapsulation in a hydrogel scaffold prevascularized in vivo. Specifically, a hydrogel scaffold containing methacrylated gelatin (GelMA), methacrylated heparin (HepMA) and vascular endothelial growth factor (VEGF) is fabricated, which can delivery VEGF in a sustained style and thus induce subcutaneous angiogenesis. In addition, islets-laden core-shell microgels using methacrylated hyaluronic acid (HAMA) as microgel core and poly(ethylene glycol) diacrylate (PEGDA)/carboxybetaine methacrylate (CBMA) as shell layer are prepared, which provide a favorable microenvironment for islets and simultaneously the inhibition of host immune rejection via anti-adhesion of proteins and immunocytes. As a result of the synergistic effect between anti-adhesive core-shell microgels and prevascularized hydrogel scaffold, the bioartificial pancreas can reverse the blood glucose levels of diabetic mice from hyperglycemia to normoglycemia for at least 90 days. We believe this bioartificial pancreas and relevant fabrication method provide a new strategy to treat type 1 diabetes, and also has broad potential applications in other cell therapies.

Large-sized bone defect repair by combining a decalcified bone matrix framework and bone regeneration units based on photo-crosslinkable osteogenic microgels

Physiological repair of large-sized bone defects is great challenging in clinic due to a lack of ideal grafts suitable for bone regeneration.Decalcified bone matrix(DBM)is considered as an ideal bone regeneration scaffold,but low cell seeding efficiency and a poor osteoinductive microenvironment greatly restrict its application in large-sized bone regeneration.To address these problems,we proposed a novel strategy of bone regeneration units(BRUs)based on microgels produced by photo-crosslinkable and microfluidic techniques,containing both the osteogenic ingredient DBM and vascular endothelial growth factor(VEGF)for accurate biomimic of an osteoinductive microenvironment.The physicochemical properties of microgels could be precisely controlled and the microgels effectively promoted adhesion,proliferation,and osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)in vitro.BRUs were successfully constructed by seeding BMSCs onto microgels,which achieved reliable bone regeneration in vivo.Finally,by integrating the advantages of BRUs in bone regeneration and the advantages of DBM scaffolds in 3D morphology and mechanical strength,a BRU-loaded DBM framework successfully regenerated bone tissue with the desired 3D morphology and effectively repaired a large-sized bone defect of rabbit tibia.The current study developed an ideal bone biomimetic microcarrier and provided a novel strategy for bone regeneration and large-sized bone defect repair.

Thermo-sensitive Poly(DEGMMA-co-MEA) Microgels: Synthesis, Characterization and Interfacial Interaction with Adsorbed Protein Layer

The novel microgels, poly[di（ethylene glycol） methyl ether methacrylate-co-2-methoxyethyl acrylate] poly（DEGMMA-co-MEA） microgels, were synthesized. The poly（DEGMMA-co-MEA） microgels were thermo-sensitive and exhibited a volume phase transitive temperature（VPTT） of 14–22 ℃. The incorporation of hydrophobic comonomer MEA shifted the VPTT of poly（DEGMMA-co-MEA） microgels to lower temperatures. The interfacial interaction of poly（DEGMMA-co-MEA） microgels and three model proteins, namely fibrinogen, bovine serum albumin and lysozyme, was investigated by quartz crystal microbalance（QCM）. An injection sequence of ＂microgel-after-protein＂ was then established for the real-time study of the interaction of proteins and the microgels at their swollen and collapsed states by using QCM technique. The results indicated that the interfacial interaction of poly（DEGMMA-co-MEA） microgels and adsorbed protein layers was mainly determined by the electrostatic interaction. Because poly（DEGMMA-co-MEA） microgels were negatively charged in Tris-HCl buffer solution（pH = 7.4）, the microgels did not adsorb on negatively charged fibrinogen and bovine serum albumin layers but strongly adsorbed on positively charged lysozyme layer. Stronger interaction between lysozyme and the microgels at collapsed state（i.e. at 37 ℃） was observed. Furthermore, the incorporation of MEA might weaken the interaction between poly（DEGMMA-co-MEA） microgels and proteins.

Assembly of Preformed Gold Nanoparticles onto Thermore- sponsive Poly（N-isopropylacrylamide）-Based Microgels on the Basis of Au-thiol Chemistry

The assembly of preformed gold nanoparticles （AuNPs） onto the thermoresponsive poly（N-isopropylacrylamide） （PNIPAM）-based microgels was achieved on the basis of the driving force of Au-thiol chemistry. The loading amount of AuNPs can be controlled by varying the ratio of AuNPs relative to PNIPAM-based microgels. The as-prepared PNIPAM/Au hybrid microgels showed well-defined reversible swelling/deswelling transition in re- sponse to temperature, which can be employed to tune the plasmonic property of hybrid microgels. As the tempera- ture was increased, the position of localized surface plasmon resonance （LSPR） band red-shifted to some extent mainly due to the increase in the local refractive index around AuNPs.

Preparation of CuS-P（NIPAM-co-MAA） Hybrid Microgels with Controlled Surface Structures

Copper sulfide-poly（isopropylacrylamide-co-methacrylic acid） [CuS-P（NIPAM-co-MAA）] hybrid microgels with patterned surface structures have been synthesized by means of the polymer microgel template technique. The results showed that the surface morphology of the hybrid microgels could be regulated by controlling the decomposition of thioacetamide （TAA） in an acidic medium. The rate of precipitation and the amount of metal sulfide significantly affect the surface structures of the hybrid microgels.