| Tissue engineering combined with materials,cells and signaling molecules has shown great potential for tissue/organ repair,improvement or treatment of diseases.Stem cells with a variety of differentiation abilities are often selected as seed cells.Some challenges exsit in translation of stem cell-based tissue engineering technology from laboratory research to clinical application,such as the preservation and functionalization of cells in damaged or diseased areas,the protection of cells in the process of cell delivery,and the limitation of cell carrier materials.Hydrogels are water-rich polymer networks that can be used to simulate the extracellular matrix(ECM)for the construction of engineered tissues.Micrometer sized hydrogels,termed microgels,can be able to accurately recreate complex cell three-dimensional microenvironment,maintain cell viability and function,protect cells from environmental stresses,and become a powerful tool for guiding cell fate in vivo and in vitro.Compared with the traditional bulk gels,the microgel has the advantages of large specific surface area,short internal and external mass transfer distance,and injectable properties.Droplet microfluidics allows for fine control of a variety of fluids on a micro scale.It can precisely create microgels with adjustable structure and composition,and effectively encapsulate cells in microgels.Cell-laden microgel:used as cell carrier for cell culture and cell transplantation;As an injectable filling material for tissue regeneration;As a modular unit,it shows great potential in the field of regenerative medicine through bottom-up modular tissue engineering to construct bionic tissue and other applications.In this paper,a continuous microfluidic process was first developed for stem cells(MSCs)encapsulation,enabling continuous and rapid fabrication of cell-laden microgels.This method integrated droplet formation,polymer gelation and droplet demulsification into one-step manufacturing process to achieve high throughput cell encapsulation at single cell level,while maintaining the survival and function of encapsulated cells.It is noteworthy that after osteoinduction of 7 days,MSCs in alginate microgel significantly enhanced osteogenic differentiation and accelerated microgel mineralization at the single cell level.In addition,in the rat tibia ablation model,compared with the control group,the new bone formation in the alginate microgel treatment group was significantly enhanced(The RBV of trabecular-tissue volume ratio was 0.34±0.056).This study showed that alginate microgel can provide threedimensional microenvironment for stem cells,induce osteogenic differentiation,and promote bone regeneration as a bone filling material.Continuous microfluidic controlled preparation process can be further used for the encapsulation of bioactive substances to achieve continuous and efficient preparation of biomedical microcarriers.Droplet microfluidic technology has recently become a powerful platform for a variety of biomedical applications,including microreactors,encapsulation of bioactive compounds,single-cell culture and analysis,all of which require droplet long-term stability.However,this also makes it difficult to recover samples after droplet demulsification.To this end,we developed a new type of thermo-responsive fluorosurfactant that can control droplet stability simply by temperature.A novel thermo-responsive surfactant was synthesized by coupling perfluoropolyether(PFPE)with hydrophilic blocks of poly(N-isopropylacrylamide)(pNIPAM).By tuning the low critical solution temperature(LCST,32)below or above the ambient temperature of pNIPAM,the hydrophilicity and hydrophobicity of pNIPAM can be changed,and then the properties of the surfactant can be changed,so that the droplet stability or aggregation can be achieved.These thermo-responsive surfactants have been shown to have good biocompatibility.This method provides convenience for microencapsulation and ondemand recovery of reaction products in droplet,which opens up a new way for the wide application of droplet template microfluidic.Photo-crosslinked hydrogels are the most widely used biomaterials,which can be used to encapsulate cells with microfabrication technology.However,due to the inhibition of oxygen on free radical polymerization,the manufacturing accuracy of photocross-linked hydrogels is limited,thus affecting the uniformity of the gel networ.For this reason,we developed a phototriggered imine crosslinking hydrogel composed of gelatin and hyaluronic acid,and the crosslinking time was less than 5s.This new polymerization strategy significantly alleviated oxygen inhibition,improves the network uniformity and mechanical strength of the microgels,and makes the engineered cell microenvironment more accurate.This photocrosslinked hydrogel combined with integrated microfluidic technology can improve the efficiency of microgel generation(processing flux was 3.2 mL/hr).Granular hydrogels formed by these microgels have strong cohesive strength(yield strain up to 200%),shear thinning and selfhealing rheological properties,and can be used as injectable and moldeable cellular tissue engineering scaffolds.After 8 weeks,the cell carrier gel was injected into the bone defect in the femoral condyle,where the highest amount of new bone formation was observed.Modular tissue engineering refers to the bottom-up fabrication of functional tissues with specific microstructural characteristics by means of modular units.As a basic module unit,microgels can be designed and processed at the single cell level,providing a more accurate extracellular three-dimensional microenvironment.Based on our previous work,a scaleup microfluidic integrated chip was developed(processing flux was 6.4 mL/hr),which overcame problem of large-scale preparation of cell-laden microgel and could produce cell-laden microgel ink for 3D printing in a short time.The bioink enabled to manufacture a variety of biofunctional structures and further cross-linking to achieve structural stability(compression modulus 14.11±1.28 kPa).In addition,the printed structure was used in bone regeneration.In vitro studies showed that the stem cells had obvious osteogenic differentiation after 3 days of induction,and highly expressed osteopontin;In vivo,it can significantly promote the repair of calvarial bone defect.Using cell microgel as bioink,3D printing technology is expected to achieve precise control of the spatial distribution of functional tissue.In summary,a new droplet microfluidic technology for continuous preparation of cell microcarriers was developed in this paper.With a new thermo-responsive fluorosurfactant,a new method of recovery on demand was proposed.What’s more,a scaleup microfluidic integrated chip was designed and developed to further improve the production efficiency of cell microcarriers.In addition,a photo-triggered imine cross-linked double-network hydrogel was developed to make the engineered cell microenvironment more accurate.The results showed that the cell-laden microgels were densely packed to form granular gels with shear thinning and self-healing properties,which could be used as injectable filling materials as well as used as bioink for 3D printing.It is verified that the cell-laden microgel improved the therapeutic effect of bone repair,and provides a new solution for cell therapy in regenerative medicine. |
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