SHAPE MORPHING HYDROGEL ACTUATORS AND CONSTRUCTS

BACKGROUND

Continuous changes in tissue and organ architecture, such as rearrangement/reorganization, remodeling, and morphogenesis, occur throughout development. The resulting shapes of tissues and relative positions of different cell types, extracellular matrix molecules and soluble factors ultimately contribute to their functionality. For this reason, biomaterials and tissue engineering strategies that possess characteristics enabling morphological changes that biomimic these natural processes may enhance our ability to form complex tissues that structurally resemble native ones. However, the current state of tissue engineering, which holds great potential for replacement of damaged or lost tissues/organs, generally relies on geometrically static materials that are unable to recapitulate the aforementioned critical dynamic behaviors. Given the significance of the dynamic nature of tissues and organs during their formation to achieve their mature structure, there has recently been great interest in incorporating dynamic shape changing capabilities into materials. To make the materials intrinsically maneuverable and programmable, hydrogel actuators that can change their shape have been engineered for multiple applications in biomedical engineering. In contrast to geometrically static materials, hydrogel actuators respond to external stimuli by changing their shape. The capacity to temporally manipulate the actuator spatial structure and composition positioning may be valuable in guiding development-inspired morphogenetic processes during organoid formation and engineering of tissues.

Although hydrogel actuators exhibit great potential in tissue engineering, drawbacks of the current systems heavily restrict their practical applications. For example, non-biocompatible and/or cytotoxic materials and techniques are often used for hydrogel fabrication and/or harsh conditions, such as low pH, high temperature, and toxic chemicals and solvents, are needed to activate shape responses. Hydrogel actuators designed for tissue engineering applications aimed at replicating aspects of tissue morphogenesis should meet important criteria such as: a) mechanical integrity that allows repeatable shape manipulation and high resistance to complex cell-culture environments and long-time incubation; b) cyto- and bio-compatible stimulation that empowers their spatiotemporal tunability under biological conditions; c) a simple fabrication method that enables reproducible shape change response outcomes. Some cell-laden hydrogel actuators (CHAs) fabricated by using biocompatible materials such as polyethyleneglycol (PEG), collagen, and derivatives of hyaluronic acid and alginate have been designed to undergo deformations without compromising cell viability. However, they typically present limitations such as single-stage shape change (e.g., unidirectional bending/folding) and lack of controllability and reversibility over the shape manipulation. No work to date has been reported cytocompatible CHAs that enable complex multiple and reversible shape transformations in a programmed and/or “on-demand” controllable manner for biomimicry of native tissue morphogenesis.

SUMMARY

Embodiments described herein relate to shape morphing hydrogel and/or cell condensate actuators or constructs that includes a biocompatible and/or cytocompatible polymer-based shape morphing hydrogel and/or cell condensate, which is configured to undergo multiple, reversible, and/or controllable different shape transformations over time via either pre-programmed design or user-controlled environmental condition alterations. Shape-morphing hydrogels bear promising prospects as soft actuators and for robotics. However, they are mostly restricted to applications in the abiotic domain due to the harsh physicochemical conditions typically necessary to induce shape morphing. We developed a biocompatible polymer-based shape morphing hydrogel and cell condensates using biocompatible polymers and cell condensates that permits encapsulation and maintenance of living cells and implements programmed and controlled actuations with multiple shape changes. The biocompatible polymer-based shape morphing hydrogel and cell condensates enable defined self-folding and/or user-regulated, on-demand-folding into specific 3D architectures under physiological conditions, with the capability to partially bioemulate complex developmental processes, such as branching morphogenesis. The biocompatible polymer-based shape morphing hydrogel and cell condensates can be utilized as platforms for promoting new complex tissue formation and regenerative medicine applications.

In some embodiments, a construct includes a biocompatible and/cytocompatible polymer-based shape morphing hydrogel that is configured to undergo one or more multiple, reversible, controllable and/or different shape transformations over time via either pre-programmed design or user-controlled environmental condition alterations, wherein the hydrogel is cytocompatible and, upon degradation, produces substantially non-toxic products.

In some embodiments, the shape morphing hydrogel includes at least one layer, wherein the swelling and/or degradation rate of the at least one layer actuates the shape transformations.

In some embodiments, the construct includes a first layer that includes a first hydrogel forming biocompatible polymer macromer and a second layer that includes a second hydrogel forming biocompatible polymer macromer different than the first hydrogel forming biocompatible polymer macromer.

In other embodiments, the construct includes multiple layers having similar or different swelling ratios.

In some embodiments, at least one of the layers includes hydrogel forming acrylated and/or methacrylated polymer macromers that are optionally oxidized. The acrylated and/or methacrylated polymer macromers can be reversibly and ionically crosslinkable. The acrylated and/or methacrylated polymer macromers can also be photocrosslinkable, ionically crosslinkable, physically crosslinkable, pH crosslinkable, dual crosslinkable, and/or thermally crosslinkable.

In some embodiments, the acrylated and/or methacrylated polymer macromers are acrylated and/or methacrylated polysaccharides that are optionally oxidized.

In some embodiments, at least one of the layers includes an acrylated and/or methacrylated alginate that is optionally oxidized and/or at least one of the layers includes an acrylated and/or methacrylated gelatin.

In some embodiments, at least one of the layers includes a first oxidized and acrylated and/or methacrylated natural polymer macromer and another layer includes a second oxidized and acrylated and/or methacrylated natural polymer macromer. The oxidation and/or acrylation and/or methacrylation of the second natural polymer macromer can be different from the oxidation and/or acrylation and/or methacrylation of the second polymer macromer.

In some embodiments, the shape morphing hydrogel can exhibit a repeatable and reversible shape change based on exogenous stimulation. The exogenous stimulation can include, for example, at least one of chemical, biochemical, irradiation, magnetic, biological, electric, ultrasound/sound, mechanical or a change in pH or temperature.

In other embodiments, the shape morphing hydrogel is self-morphing and/or user regulated on-demand morphing into three dimensional architectures under physiological or non-physiological conditions.

In some embodiments, the construct can include a plurality of cells dispersed in the hydrogel. For example, at least a portion of the construct can have a cell density up to 1×10¹⁰cells/ml. The plurality of cells can include progenitor cells, undifferentiated cells, differentiated cells, and/or cancer cells. In one example, the plurality of cells can include mesenchymal stem cells.

In some embodiments, the construct includes a plurality of layers of hydrogel forming polymer macromers. At least two of the layers can have different macromer concentration, acrylation and/or methacrylation, oxidation, thickness, and/or cell density.

In some embodiments, at least two layers are covalently linked at adjoining portions.

In some embodiments, the construct can include at least three layers, wherein a middle layer is covalently linked to adjoining portions of two outer layers.

In some embodiments, the construct includes the biocompatible, polymer-derived shape morphing hydrogel and a plurality of cells dispersed in at least a portion of the construct, wherein the plurality of cells has a cell density up to 1×10¹⁰cells/ml.

In some embodiments, the shape morphing hydrogel is self-morphing and/or user regulated on-demand morphing into three dimensional architectures under physiological conditions.

Other embodiments described herein relates to a method of forming a construct. The method includes adhering a first layer that includes a first hydrogel forming natural polymer macromer to a second layer that includes a second hydrogel forming polymer macromer having a different swelling ratio and/or degradation rate than the first hydrogel forming natural polymer macromer. The different swelling ratio and/or degradation rate allows the hydrogel to undergo multiple, reversible, controllable and/or different shape transformations The hydrogel is cytocompatible and, upon degradation, produces substantially non-toxic products.

In some embodiments, at least three layers of hydrogel forming natural polymer macromer are adhered. At least two of layers can have different compositions and a different swelling ratio and/or degradation rate.

In some embodiments, the method can further include adhering a third layer to the first and second layer such that the second layer is sandwiched between the first layer and the third layer. The third layer includes a third hydrogel forming polymer macromer.

In some embodiments, the at least one of the first layer, the second layer, and/or third layer can have different swelling ratios.

In some embodiments, at least one of the layers includes hydrogel forming acrylated and/or methacrylated polymer macromers that are optionally oxidized. The acrylated and/or methacrylated polymer macromers can be reversibly and ionically crosslinkable. The acrylated and/or methacrylated natural polymer macromers can also be photocrosslinkable, ionically crosslinkable, pH crosslinkable, physically crosslinkable, dual crosslinkable, and/or thermally crosslinkable.

In some embodiments, the acrylated and/or methacrylated polymer macromers are acrylated and/or methacrylated polysaccharides that are optionally oxidized.

In other embodiments, at least one layer includes a first oxidized and acrylated and/or methacrylated natural polymer macromer and another layer includes a second oxidized and acrylated and/or methacrylated natural polymer macromer. The oxidation and/or acrylation and/or methacrylation of the second natural polymer macromer can be different from the oxidation and/or acrylation and/or methacrylation of the second polymer macromer.

In other embodiments, the shape morphing hydrogel is self-morphing and/or user regulated on-demand morphing into three dimensional architectures under physiological or non-physiological conditions.

In some embodiments, the method can include dispersing a plurality of cells in at least a portion of the construct. For example, at least a portion of the construct can have a cell density up to 1×10¹⁰cells/ml. The plurality of cells can include progenitor cells, undifferentiated cells, differentiated cells, and/or cancer cells. In one example, the plurality of cells can include mesenchymal stem cells.

In some embodiments, at least two layers are covalently linked at adjoining portions.

In some embodiments, the construct can include at least three layers, wherein a middle layer is covalently linked to adjoining portions of two outer layers.

In some embodiments, the shape morphing hydrogel is self-morphing and/or user regulated on-demand morphing into three dimensional architectures under physiological conditions.

In some embodiments, the shape morphing hydrogel biomimics tissue developmental processes. For example, the tissue developmental process includes at least one of lung or kidney branching morphogenesis or budding processes.

In some embodiments, the method includes printing the first hydrogel forming polymer macromer into a self-healing, shear thinning, crosslinkable, biocompatible hydrogel support medium. The printed first hydrogel forming polymer macromer can form the first layer having a defined shape. A second hydrogel forming polymer macromer can be printed into the support medium such that the second hydrogel forming polymer macromer forms the second layer with a defined shape. At least a portion of the second layer can adjoin at least a portion of the first layer. The hydrogel support medium can maintain the defined shape of the first layer and the second layer during printing and optionally culturing.

In some embodiments, at least one of the first hydrogel forming natural polymer macromer or the second hydrogel forming natural polymer macromer includes a plurality of cells.

In some embodiments, the method further includes culturing the printed first layer and the printed second layer to form a flow-resistant or free-standing cell condensation structure with a defined shape.

Other embodiments described herein relate to a construct that includes a biocompatible polymer-based shape morphing hydrogel that is configured to undergo multiple, reversible, controllable and/or different shape transformations over time via either pre-programmed design or user-controlled environmental condition alterations. The shape morphing hydrogel includes at least one gradient in polymer concentration, polymer type, polymer swelling, polymer degradation and/or polymer cross-linking density that extends through at least one portion of the shape morphing hydrogel and allows the shape morphing hydrogel to undergo the multiple, reversible, controllable and/or different shape transformations. The hydrogel is cytocompatible and, upon degradation, produces substantially non-toxic product.

In some embodiments, the at least one gradient is provided by layers, regions, or portions of the hydrogel having differing polymer concentration, polymer type, polymer swelling, polymer degradation and/or polymer cross-linking density.

In some embodiments, the hydrogel includes one or more acrylated and/or methacrylated polymer macromers that are optionally oxidized. The acrylated and/or methacrylated polymer macromers are reversibly and ionically crosslinkable.

In some embodiments, the acrylated and/or methacrylated polymer macromers include acrylated and/or methacrylated polysaccharides that are optionally oxidized. For example, the hydrogel includes a mixture of acrylated and/or methacrylated alginate that is optionally oxidized and an acrylated and/or methacrylated gelatin.

In other embodiments, the shape morphing hydrogel exhibits a repeatable and reversible shape change based on exogenous stimulation. For example, the exogenous stimulation can include at least one of chemical, biochemical, irradiation, magnetic, biological, electric, ultrasound/sound, mechanical or a change in pH or temperature.

In some embodiments, the shape morphing hydrogel is self-morphing and/or user regulated on-demand morphing into three dimensional architectures under physiological or non-physiological conditions.

In other embodiments, the construct further includes a plurality of cells dispersed in the hydrogel. At least a portion of the construct can have a cell density up to 1×10¹⁰cells/ml. The plurality of cells can include progenitor cells, undifferentiated cells, differentiated cells, and/or cancer cells. For example, the plurality cells can include mesenchymal stem cells.

In some embodiments, the shape morphing hydrogel can include a single biocompatible polymer or copolymer.

In some embodiments, the at least one gradient can include a gradient of polymer cross-linking density that extends through at least one portion of the shape morphing hydrogel and allows the shape morphing hydrogel to undergo one or more multiple, reversible, controllable and/or different shape transformations.

In some embodiments, the shape morphing hydrogel includes a photocrosslinkable hydrogel forming polymer and a photo-absorber and a photoinitiator dispersed within the hydrogel.

In other embodiments, the shape morphing hydrogel includes multiple gradients in polymer concentration, polymer type, polymer swelling, polymer degradation and/or polymer cross-linking density that extend through portions of the shape morphing hydrogel and allows the shape morphing hydrogel to undergo multiple, reversible, controllable and/or different shape transformations.

In some embodiments, the construct includes the biocompatible, polymer-derived shape morphing hydrogel and a plurality of cells dispersed in at least a portion of the construct. The plurality of cells can have a cell density up to 1×10¹⁰cells/ml.

Other embodiments described herein relate to a method of forming a construct as described herein. The method includes providing at least one gradient in polymer concentration, polymer type, polymer swelling, polymer degradation and/or polymer cross-linking density in a biocompatible polymer-based hydrogel. The at least one gradient can extend through at least one portion of hydrogel and allows the hydrogel to undergo multiple, reversible, controllable and/or different shape transformations.

In some embodiments, the shape morphing hydrogel exhibits a repeatable and reversible shape change based on exogenous stimulation. The exogenous stimulation can include at least one of at least one of chemical, biochemical, irradiation, magnetic, biological, electric, ultrasound/sound, mechanical or a change in pH or temperature.

In some embodiments, the shape morphing hydrogel is self-morphing and/or user regulated on-demand morphing into three dimensional architectures under physiological or non-physiological conditions.

In some embodiments, the shape morphing hydrogel biomimics tissue developmental processes. The tissue developmental process can include at least one of lung or kidney branching morphogenesis or budding processes.

In some embodiments, the hydrogel is cytocompatible and, upon degradation, produces substantially non-toxic products.

In other embodiments, the method further includes providing a plurality of cells in at least one layer of the hydrogel. The cells can be provided in at least a portion of the hydrogel at a cell density of, for example, up to 1×10⁹cells/ml. The plurality of cells can include progenitor cells, undifferentiated cells, differentiated cells, and/or cancer cells. For example, the plurality cells can include mesenchymal stem cells.

In some embodiments, the hydrogel includes a single biocompatible polymer or copolymer.

In some embodiments, the at least one gradient includes a gradient polymer cross-linking that extends through at least one portion of the shape morphing hydrogel and allows the shape morphing hydrogel to undergo multiple, reversible, controllable and/or different shape transformations.

In other embodiments, the shape morphing hydrogel includes a photocrosslinkable hydrogel forming polymer and a photo-absorber and a photoinitiator dispersed within the hydrogel.

In some embodiments, the method includes forming multiple gradients in polymer concentration, polymer type, polymer swelling, polymer degradation and/or polymer cross-linking that extend through portions of the shape morphing hydrogel and allow the shape morphing hydrogel to undergo multiple and reversible different shape transformations.

Other embodiments relate to a method of forming a construct. The method includes printing a bioink comprising a plurality of cells into a hydrogel support medium. The hydrogel support medium can include at least one gradient in polymer concentration, polymer type, polymer swelling and/or polymer cross-linking, wherein the at least one gradient extends through at least one portion of hydrogel and allows the hydrogel to undergo multiple and reversible different shape transformations.

In some embodiments, the method further includes culturing the printed plurality of cells to form a tissue construct, wherein the support medium maintains the defined shape of the printed bioink during culturing.

Other embodiments described herein relate to a method of forming a construct. The method includes providing a biocompatible polymer-based hydrogel that includes at least one gradient in polymer concentration, polymer type, polymer swelling and/or polymer cross-linking, wherein the at least one gradient extends through at least one portion of hydrogel and allows the hydrogel to undergo multiple and reversible different shape transformations, and seeding and culturing a layer of cells on a surface of the hydrogel.

In some embodiments, the hydrogel is firmly adhered on a surface of a substrate, such as a glass plate, by covalent bonding. The glass plate can include a surface that is modified with at least one molecule that facilitates binding of the hydrogel to the glass plate.

Still other embodiments relate to a construct that includes shape morphing cell condensate that is configured to undergo one or multiple, reversible, controllable and/or different shape transformations over time via either pre-programmed design or user-controlled environmental condition alterations.

In some embodiments, the cell contractile forces or exogenous stimulation allows the construct to undergo controllable different shape transformations over time.

In some embodiments, the cell to cell interactions, cell to extracellular matrix interactions, cell to aptamer interactions, and/or condensation of the cells of the condensate allow the construct to undergo controllable different shape transformations over time.

In some embodiments, the construct further includes a biocompatible polymer-based shape morphing layer that is conjugated to the cell condensate. The biocompatible polymer-based shape morphing layer includes at least one gradient in polymer concentration, polymer type, polymer swelling, polymer degradation and/or polymer cross-linking density that extends through at least one portion of the preformed biocompatible polymer-based shape morphing layer shape.

In some embodiments, the construct includes a preformed biocompatible polymer-based shape morphing layer that includes at least one gradient in polymer concentration, polymer type, polymer swelling, polymer degradation and/or polymer cross-linking density that extends through at least one portion of the preformed biocompatible polymer-based shape morphing layer shape; and a photocurable and degradable cell-supporting microgel (MG) layer that is printed with cells. The MG layer can maintain the shape of the printed cells upon printing. The degradation of the MG layer and/or differential swelling and/or degradation of the preformed biocompatible polymer-based shape morphing layer during culture in tissue-specific formation conditions allows the construct to undergo controllable different shape transformations over time.

In some embodiments, the preformed hydrogel layer includes hydrogel forming acrylated and/or methacrylated polymer macromers that are optionally oxidized. The acrylated and/or methacrylated polymer macromers can reversibly and ionically crosslinkable. The acrylated and/or methacrylated polymer macromers can also be photocrosslinkable, ionically crosslinkable, physically crosslinkable, pH crosslinkable, dual crosslinkable, and/or thermally crosslinkable.

In some embodiments, the acrylated and/or methacrylated polymer macromers include acrylated and/or methacrylated polysaccharides that are optionally oxidized.

In some embodiments, the photocurable and degradable cell-supporting microgel includes an acrylated and/or methacrylated alginate that is optionally oxidized.

In some embodiments, the preformed layer includes a mixture of an acrylated and/or methacrylated alginate that is optionally oxidized and acrylated and/or methacrylated gelatin.

In some embodiments, the printed photocurable and degradable cell-supporting microgel (MG) layer includes a plurality of printed cells. The printed cells can include progenitor cells, undifferentiated cells, differentiated cells, and/or cancer cells. For example, the plurality cells can include mesenchymal stem cells.

In some embodiments, the shape morphing hydrogel layer includes a single biocompatible polymer or copolymer.

In other embodiments, the preformed biocompatible polymer-based shape morphing layer includes a gradient of polymer cross-linking density through the thickness of the layer that allows the shape morphing hydrogel to undergo one or more multiple, reversible, and/or controllable different shape transformations.

In some embodiments, preformed biocompatible polymer-based shape morphing layer includes a photocrosslinkable hydrogel forming polymer and a photo absorber and a photoinitiator dispersed within the hydrogel.

In some embodiments, the preformed biocompatible polymer-based shape morphing layer includes one or multiple gradients in polymer concentration, polymer type, polymer swelling, polymer degradation and/or polymer cross-linking density that extend through portions of the preformed biocompatible polymer-based shape morphing layer.

In some embodiments, the preformed biocompatible polymer-based shape morphing layer includes a plurality of cells. The cells can include progenitor cells, undifferentiated cells, differentiated cells, and/or cancer cells. For example, the plurality of cells can include mesenchymal stem cells.

Other embodiments described herein relate to a method of forming a layered construct. The method includes providing a preformed biocompatible polymer-based shape morphing layer that includes at least one gradient in polymer concentration, polymer type, polymer swelling, polymer degradation and/or polymer cross-linking density that extends through at least one portion of the preformed biocompatible polymer-based shape morphing layer shape. A printed photocurable and degradable cell-supporting microgel (MG) layer that is configured to allow printing of cells inside MG layer and maintains shape initially upon printing is applied over at least a portion of the preformed biocompatible polymer-based shape morphing layer. Cells are then printed within the MG layer. The degradation of the MG layer and differential swelling and/or degradation of the preformed biocompatible polymer-based shape morphing layer during culture in specific tissue-specific formation conditions allows the construct to undergo controllable different shape transformations over time.

In some embodiments, the preformed hydrogel layer includes a hydrogel forming acrylated and/or methacrylated polymer macromers that are optionally oxidized. The acrylated and/or methacrylated polymer macromers can be reversibly and ionically crosslinkable. The acrylated and/or methacrylated polymer macromers can also be photocrosslinkable, ionically crosslinkable, physically crosslinkable, pH crosslinkable, dual crosslinkable, and/or thermally crosslinkable. For example, the acrylated and/or methacrylated polymer macromers include acrylated and/or methacrylated polysaccharides that are optionally oxidized.

In some embodiments, the photocurable and degradable cell-supporting microgel can include an acrylated and/or methacrylated alginate that is optionally oxidized.

In other embodiments, the preformed layer can include a mixture of an acrylated and/or methacrylated alginate that is optionally oxidized and acrylated and/or methacrylated gelatin.

In some embodiments, the printed cells can include progenitor cells, undifferentiated cells, differentiated cells, and/or cancer cells. For example, the printed cells can include mesenchymal stem cells.

In some embodiments, the preformed biocompatible polymer-based shape morphing layer includes a single biocompatible polymer or copolymer.

In other embodiments, the preformed biocompatible polymer-based shape morphing layer includes a gradient of polymer cross-linking density through the thickness of the layer that allows the shape morphing hydrogel to undergo one or more multiple, reversible, controllable and/or different shape transformations.

In some embodiments, the preformed biocompatible polymer-based shape morphing layer includes a photocrosslinkable hydrogel forming polymer, a photo-absorber, and a photoinitiator dispersed within the hydrogel.

In other embodiments, the preformed biocompatible polymer-based shape morphing layer includes one or multiple gradients in polymer concentration, polymer type, polymer swelling, polymer degradation and/or polymer cross-linking density that extend through portions of the preformed biocompatible polymer-based shape morphing layer.

In some embodiments, the method further includes crosslinking the MG layer printed with the cell to enhance the mechanical stability of the MG layer.

In other embodiments, the method includes culturing the layered construct in a culture medium. The culture medium can include a cell differentiation medium.

Other embodiments described herein relate to a composition that includes a plurality of polymer macromer nanoparticle and/or microparticle hydrogels (MGs) and optionally a plurality of cells. The composition is configurable into a stable 3D hydrogel (bio)construct in the absence/presence of cells and is configured to be crosslinkable to form a more robust hydrogel construct.

In some embodiments, the hydrogel construct includes at least one of an anisotropic property in crosslinking density, internal strain, and/or micro/macro-pores distribution.

In some embodiments, the MGs comprise jammed heterogenous natural or synthetic polymer macromer hydrogels. The MGs can include a photoinitiator (PI) and UV absorber.

In some embodiments, the composition is printed into 3D hydrogel (bio)constructs that are programmably reshaped into a defined shape.

In other embodiments, the composition is extrudable or printable into a defined shape.

In some embodiments, the composition is capable of being crosslinked to form a flow-resistant structure with the defined shape and with a gradient in crosslinking density that extends through at least one portion of the hydrogel. The gradient in crosslinking density can allow the 3D hydrogel (bio)construct to undergo one or multiple, reversible, controllable and/or different shape transformations.

In some embodiments, the composition is cytocompatible and, upon degradation, produces a substantially non-toxic product.

In some embodiments, the viscosity of the MGs can decrease with increased shear and/or strain on the MGs and recover after removal of the increased shear and/or strain. The increased shear and/or strain can be associated with extruding or printing the composition, and the viscosity of the composition can recover after extruding or printing the composition to provide the 3D hydrogel (bio)construct with the defined shape.

In some embodiments, the composition can include a plurality of cells. The cells can include progenitor cells, undifferentiated cells and/or differentiated cells. For example, the cells can include mesenchymal stem cells.

In some embodiments, the MGs can have a flake morphology with an average diameter of about 10 μm to about 70 μm.

Other embodiments described herein relate to a method of forming a shape-morphing construct. The method includes providing a plurality of polymer macromer nanoparticle and/or microparticle hydrogels (MGs) and optionally a plurality of cells dispersed with MGs. The MGs and optional cells are then printed into a 3D hydrogel (bio)construct having a defined shape. The 3D hydrogel (bio)construct is crosslinked to further stabilize the 3D hydrogel (bio)construct.

In some embodiments, the 3D hydrogel (bio)construct includes at least one of an anisotropic property in crosslinking density, internal strain, and/or micro/macro-pores distribution.

In some embodiments, the MGs can include a photoinitiator (PI) and UV absorber.

In some embodiments, the gradient in crosslinking density allows the 3D hydrogel (bio)construct to undergo one or multiple, reversible, controllable and/or different shape transformations.

In some embodiments, the shape-morphing construct is cytocompatible and, upon degradation, produces substantially non-toxic product.

In some embodiments, the viscosity of the MGs decreases with increased shear and/or strain on the MGs and recovers after removal of the increased shear and/or strain. The increased shear and/or strain can be associated with printing the MGs and the viscosity of the MGs recovering after printing the MGs to provide the 3D hydrogel (bio)construct with the defined shape.

In some embodiments, the cells can include progenitor cells, undifferentiated cells and/or differentiated cells. For example, the cells can include mesenchymal stem cells.

In some embodiments, the MGs can have a flake morphology with an average diameter of about 10 μm to about 70 μm.

Still other embodiments relate to a composition for forming a shape morphing cell-laden construct. The composition can include a plurality of cells and optionally at least one polymer macromer. The composition can be configurable into a stable 3D bioconstruct having an initial shape, wherein cell contractile forces of the cells of the 3D (bio)construct allows the 3D bioconstruct to undergo one or multiple, reversible, controllable and/or different shape transformations over time.

In some embodiments, the cell contractile forces are associated with at least one of cell to cell interactions, cell to extracellular matrix interactions, cell to aptamer interactions, and/or cell condensation.

In some embodiments, the composition includes a photoinitiator (PI).

In some embodiments, the composition can be printed into 3D hydrogel (bio)constructs that are programmably reshaped into a defined shape.

In other embodiments, the composition is extrudable or printable into a defined shape.

In some embodiments, the composition is capable of being crosslinked to form a flow-resistant structure with the defined shape.

In some embodiments, the shape morphing cell-laden construct can be cytocompatible and, upon degradation, producing substantially non-toxic product.

In some embodiments, the viscosity of the composition decreases with increased shear and/or strain on the composition and recovers after removal of the increased shear and/or strain. The increased shear and/or strain can be associated with extruding or printing the composition and the viscosity of the composition can recover after extruding or printing the composition to provide the 3D hydrogel construct with the defined shape.

In some embodiments, the cells can include progenitor cells, undifferentiated cells and/or differentiated cells. For example, the cells can include mesenchymal stem cells.

Other embodiments described herein relate to a construct that includes at least one degradable cell hydrogel layer or cell condensate layer whose initial shape is maintained by a support, wherein cell contractile forces of the cells of the construct allows the construct to undergo one or multiple, reversible, controllable, and/or different shape transformations over time.

In some embodiments, the support includes hydrogel and/or microgel in the hydrogel layer.

In some embodiments, the support is external to the cell condensate.

In some embodiments, the at least one degradable cell hydrogel layer or cell condensate layer includes a mixture of oxidized and methacrylated alginate (OMA), methacrylated gelatin, uncrosslinked gelatin microspheres, and plurality of cells as well as optionally a photoinitiator (PI).

In some embodiments, the construct is capable of being crosslinked to form a flow-resistant structure with the defined shape.

In some embodiments, the construct is cytocompatible and, upon degradation, producing substantially non-toxic product.

In some embodiments, the cells can include progenitor cells, undifferentiated cells and/or differentiated cells. For example, the cells can include mesenchymal stem cells.

In some embodiments, the construct can include a hydrogel layer conjugated to the at least one degradable cell hydrogel layer or cell condensate layer. The hydrogel layer can be a non-swelling and/or swelling hydrogel layer.

In some embodiments, condensation of the cells in the cell hydrogel layer or cell condensate layer and optionally degradation of the hydrogel layer during culture allows the construct to undergo one or multiple, reversible, controllable and/or different shape transformations over time.

In some embodiments, the hydrogel layer includes a hydrogel forming acrylated and/or methacrylated polymer macromers that are optionally oxidized. The acrylated and/or methacrylated polymer macromers can be reversibly and ionically crosslinkable. The acrylated and/or methacrylated polymer macromers can also be photocrosslinkable, ionically crosslinkable, physically crosslinkable, pH crosslinkable, dual crosslinkable, and/or thermally crosslinkable.

In some embodiments, the acrylated and/or methacrylated polymer macromers include at least one of an acrylated and/or methacrylated alginate that is optionally oxidized and/or acrylated and/or methacrylated gelatin.

In some embodiments, the construct includes a first degradable cell laden hydrogel layer, and a second degradable cell laden hydrogel layer overlying the first degradable cell laden hydrogel layer. The first degradable cell laden hydrogel layer and second degradable cell laden hydrogel layer can differ in at least one of amount of cells, cell types, or cell adhesive properties.

Other embodiments relate to a construct that includes a biocompatible polymer-based hydrogel. The hydrogel includes a first portion and a second portion separated by an intermediate portion. The first portion and second portion include a plurality cells encapsulated by hydrogel and the intermediate portion being devoid of cells. The construct is configured to undergo one or multiple, reversible, controllable, and/or different shape transformations over time via cell interactions between cells in the first portion and the second portion and/or cell to extracellular matrix interaction of cells in the first portion and/or second portion.

Still other embodiments relate to a layered construct that includes a photocurable cell-supporting microgel (MG). The MG includes a first cell condensate layer and second cell condensate layer overlying the first cell condensate layer. The second cell condensate layer is different than the first cell condensate layer. The MG is configured to allow printing of cells inside MG layer and maintains shape initially upon printing. The cell condensation in the first layer and/or second layer during culture allows the construct to undergo one or multiple, reversible, controllable, and/or different shape transformations over time.

Other embodiments relate to a layered construct that includes a first degradable cell laden hydrogel layer, and a second degradable cell laden hydrogel layer overlying the first degradable cell laden hydrogel layer. The first degradable cell laden hydrogel layer and second degradable cell laden hydrogel layer differ in at least one of amount of cells, cell types, or cell adhesive properties, and wherein cell to cell interactions, cell to extracellular matrix interactions, cell to aptamer interactions, and/or condensation of the cells of the construct allows the construct to undergo one or multiple, reversible, controllable, and/or different shape transformations over time.

Other embodiments described herein relate to a construct that includes a biocompatible polymer-based hydrogel layer. The hydrogel layer includes a first portion and a second portion separated by an intermediate portion. The first portion and second portion include a plurality cells encapsulated by hydrogel of the layer. The intermediate portion is devoid of cells. The construct is configured to undergo one or multiple, reversible, controllable and/or different shape transformations over time by degradation of the intermediate portion relative to the first portion and second portion and/or cell contractile forces

Still other embodiments relate to a layered construct that includes a first biocompatible and/or cytocompatible hydrogel layer that is non-degradable or slowly degrades, and a second biocompatible and/or cytocompatible aptamer hydrogel layer overlying the first hydrogel layer wherein aptamer interactions result in layer degradation or expansion which allows the construct to undergo one or multiple, reversible, controllable and/or different shape transformations over time.

Other embodiments relate to a bio-kirigami construct that includes a preformed hydrogel frame; and a photocurable and degradable cell-supporting hydrogel member that includes a plurality of cell inside a hydrogel of the member. The member maintains shape initially upon fabrication. Swelling of the hydrogel frame and/or cell support hydrogel member, degradation of the hydrogel member and/or condensation of the cells during culture allows the construct to undergo one or multiple, reversible, controllable, and/or different shape transformations over time.

Other embodiments relate to a construct that includes a biocompatible polymer-based hydrogel. The hydrogel includes portions having at least one of differing stiffness, thickness, and/or degradation rates. The hydrogel includes a plurality cells encapsulated by hydrogel. The construct is configured to undergo one or multiple, reversible, controllable and/or different shape transformations over time based on cell mediated contractile forces within the portions of the hydrogel.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(A-H3) illustrate (A) Schematic of the five-phase transitions of a trilayer hydrogel bar. (B) Sandwiching method to fabricate a trilayer hydrogel. (C) Linkers between OMA and GelMA chains at the layer interface. (D-F) The bending degree of the three trilayers as a function of time. (G) Cell viability in the constructs of the three groups after shape evolution: (1) O10M20A/GelMA/O10M30A, (2) O10M20A/GelMA/O10M45A, and (3) O10M30A/GelMA/O10M45A. (H) Cell-laden four-arm trilayer gripper and the programmable deformations: (H1) schematic illustrating the entire process of shape evolution, (H2) top view photomicrograph of a synthesized four-arm gripper, (H3) photographs of four-arm gripper shape changes over time. Scale bar indicates 0.5 cm. The two OMA layers were co-crosslinked with methacryloxyethyl thiocarbamoyl rhodamine B (0.005%) for visualization. Data are presented as mean±standard deviation (±SD), N=3.

FIGS. 2(A-C3) illustrate cell-laden “smart” trilayer hydrogel fabrication and the programmed deformation. (A) Overlapping parallel-strip patterns on both surfaces of a GelMA hydrogel: (a1) schematic of the sample design, (a2) photographs of top view and section view of a prepared sample, (a3) schematic illustrating the entire process of the construct shape changes over time, (a4) photographs of the construct actual shape changes over time, (a5) the speculated mechanism for the formation of an intermediate phase. (B) Parallel-strip patterns orthogonal to each other on both surfaces of a GelMA hydrogel: (b1) schematic of the sample design, (b2) top view and section view photographs of a prepared sample, (b3) schematic illustrating the entire process of the construct shape changes over time, (b4) photographs of the construct actual shape changes over time. All samples were cultured in GM at 37° C., and GM was replaced with PBS before taking images for clarity. Insets in a4 and b4 on the top left corner show the constructs from the top view, and insets on the top right corner of some images (a4, 5 min; b4, 1 h and 8 h) show the constructs from the side view; (C) Biomimicry of branching morphogenesis: (c1) schematic of branching morphogenesis of lung, step i: formation of a nascent bud, steps ii and iii: cleft formation and terminal bifurcation; (c2) photomicrograph of a typical cell-laden discrete trilayer hydrogel bar designed to undergo branching morphogenesis; (c3) photomicrographs of the 4D hydrogel system mimicking the process of lung branching morphogenesis by the discrete trilayer cultured in GM at 37° C. Images were taken after replacing the GM with PBS for clarity. The two OMA layers were co-crosslinked with methacryloxyethyl thiocarbamoyl rhodamine B (0.005%) for visualization. Scale bars indicate 4 mm.

FIGS. 3(A-E3) illustrate (A) schematic of proposed “on-demand” reversible deformations of a bilayer derived from a trilayer by switching between exposure to Ca²⁺ and EDTA solutions. (B) Cyclic reversible bending of O10M30A/GelMA bilayers due to alternating incubation in EDTA and Ca²⁺ solutions. Inset image shows the reversible bending of the hydrogel bar under alternating stimulations (transparent and red layers are the OMA layer and the GelMA layer, respectively). (C) Shape manipulation of a cell-laden bilayer by alternating Ca²⁺ and EDTA stimulation at 37° C. (D) Photomicrographs of the reversible 3D structure transitions of a quasi-four-petal flower by sequential treatment with Ca²⁺ (50 mM, 10 min) and EDTA (5 mM, 20 min) solutions. (E) (e1) Higher magnification images showing the cells in the GelMA layer and photomicrographs of live/dead stained cells (e2) prior to and (e3) after Ca²⁺ and EDTA treatment. Photos were taken after replacing the GM with PBS for clarity. Data are presented as mean±SD, N=3.

FIGS. 4(A-D) illustrate (A) Programmable shape changes of trilayer CHA (O10M20A/GelMA/O10M45A) with encapsulated cells undergoing chondrogenesis and biochemical quantification of DNA content and GAG/DNA at each time point. *p<0.05 compared to groups D1, D2, and Ctrl2; (B) Programmable shape changes of trilayer CHA with encapsulated cells undergoing osteogenesis and biochemical quantification of DNA content, ALP/DNA and calcium/DNA at each time point. ^*,#,&p<0.05 compared to all groups with a different symbol or lacking a symbol. Ctrl1 stands for GelMA only hydrogel bar cultured in chondrogenic media at D21 (in (a)) or osteogenic media at D28 (in (b)), Ctrl2 stands for trilayer hydrogel bar cultured in growth media at D21 (in (a)) and D28 (in (b)); (c) Shape changes of GelMA/O10M45A bilayer derived from the O10M20A/GelMA/O10M45A trilayer during 3-week culture in chondrogenic media: pre-programmed shape morphing (group 1), shape inversion at W1 by Ca²⁺ stimulation and subsequent culture to D21 (group 2), shape recovery at W2 by EDTA and subsequent culture to D21 (group 3); (D) DNA content and GAG/DNA ratios of all conditions at D21. Ctrl1 stands for GelMA only hydrogel bar cultured in chondrogenic media at D21. Ctrl2 stands for bilayer (GelMA/O10M45A) hydrogel bar cultured in growth media at D21. *p<0.05 compared to all other groups. GRP1, GRP2, GRP3 stand for group 1, group 2, and group 3, respectively. Black scale bars in the hydrogel photographs indicate 2 mm. Data are presented as mean±SD, N=3. Statistical tests were performed using one-way ANOVA.

FIGS. 5(A-D) illustrate a schematic illustration for patterning OMAs onto the surface of a GelMA hydrogel: (A) single GelMA surface patterning of OMA; (B) patterning OMA onto a quartz surface; formation of (C) parallel strips and (D) orthogonal strips on dual surfaces of GelMA hydrogel.

FIG. 6 illustrates a pictorial depiction of the definition of bending angle measurement for the programmable shape change of trilayer hydrogel bars. The bending angle is positive when the trilayer bends toward the slower-swelling OMA side (purple layer), and it becomes negative when the bending direction changes after the degradation of the outer faster-swelling OMA layer (red layer).

FIGS. 7(A-C) illustrate representative photomicrographs showing the actual shape changes of the trilayer constructs overtime: (A) O10M20A/GelMA/O10M30A, (B) O10M20A/GelMA/O10M45A, and (C) O10M30A/GelMA/O10M45A. Methacryloxyethyl thiocarbamoyl rhodamine B (RhB, 0.005%) was crosslinked within the OMA layer in red font to aid in visualization. The dotted outlines indicate the shape of the hydrogel strips. Scale bar: 5 mm.

FIGS. 8(A-C) illustrate representative photomicrographs of live/dead stained NIH3T3 cells encapsulated in the hydrogels after the five-phase transitions: (A) O10M20A/GelMA/O10M30A, (B) O10M20A/GelMA/O10M45A, and (C) O10M30A/GelMA/O10M45A.

FIGS. 9(A-B) illustrate (A) Swelling ratios of single layer OMA hydrogels and GelMA hydrogel in DMEM-LG at 4° C. after 10 hours, *p<0.05 compared to all other groups (one-way ANOVA); (B) Photographs of hydrogels after equilibrated swelling in DMEM-LG: (1) O10M45A, (2) O10M30A, (3) O10M20A, and (4) GelMA. Data are presented as mean±SD, N=3.

FIGS. 10(A-B) illustrate photomicrographs of (A) top and (B) side views of a discrete trilayer hydrogel bar.

FIG. 11 is a pictorial depiction of the definition of bending angle measurement for the “on-demand” shape changes of bilayer hydrogel bars. The bending angle is positive when the bilayer bends toward the GelMA side (green layer), and it becomes negative after changing its bending direction toward the OMA side (purple layer).

FIGS. 12(A-B) are (A) Schematic illustration and (B) the actual pornographic images of the reversible shape switching of a 4-arm bilayer gripper upon alternating Ca²⁺/EDTA treatment. The blue (in (A))/transparent (in (B)) and red layers are the OMA and GelMA layers, respectively.

FIG. 13 illustrates the application of the 4-arm gripper for transferring an aluminum ball (0.2 g). Scale bars indicate 2 cm.

FIG. 14 illustrates photomicrographs of live/dead stained NIH3T3 cells in bilayer hydrogel bars after treatment with Ca²⁺ (50 mM) or EDTA (10 mM) for 10 or 30 min, and subsequent 24 h culture in NIH3T3 GM.

FIGS. 15(A-B) illustrate OMA “X” patterned cell-laden GelMA layer folded into a “quasi-four-petal” flower: (A) photomicrographs of a sample and (B) a schematic showing the folding process.

FIGS. 16(A-C) illustrate swelling ratios of (A) O10M20A, (B) O10M30A, and (C) O10M45A hydrogels at different concentrations after 10 hours in DMEM-LG at 4° C., *p<0.05 compared to all other groups (one-way ANOVA). Data are presented as mean±SD, N=3.

FIG. 17 illustrates the impact of OMA concentrations (6%, 8%, or 10% in both OMA layers) on the bending angles of the O10M20A/GelMA/O10M30A trilayer hydrogel bar in DMEM-LG. Data are presented as mean±SD, N=3.

FIGS. 18(A-E) illustrate (A) schematic diagram for fabricating 4D high cell density model construct based on different swelling ratios between OMA and GelMA. Bilayered high cell density (0.2-1.0±10⁸cells mL⁻¹) OMA/GelMA constructs spontaneously changed geometry into rolled structures. Scale bars indicate 1 cm. Chemical structures and NMR analysis for B) the OMA with different theoretical oxidation rates of 10% and 15% with fixed theoretical 20% methacrylation rate and C) the GelMA. Magnified OMA NMR spectra from 5.4 to 5.6 ppm is presented in a small box on upper right side of the OMA NMR graph. “M” labels indicate methacrylation peaks of the polymers. Profiles of D) swelling and e) degradation of the individual OMA and GelMA hydrogels over 21 days of culture. “*”, “#”, “†”, and “§” indicate statistical significance compared to 12% OMA10, 12% OMA15, 12% GelMA, and 15% GelMA, respectively (p=0.05).

FIGS. 19(A-D) illustrate A) photographic images and B) angle measurements of the bended or rolled model 4D constructs demonstrating the effect of OMA oxidation rate and macromer percentages of GelMA on 4D geometric changes. Diameter of the wells is 15.6 mm. C) Photographic images and D) angle measurements of the bended or rolled model 4D constructs with varied thickness ratios of OMA and GelMA layers at fixed overall construct thickness of 0.6 mm demonstrating the effect of layer thicknesses on 4D geometric changes. Diameter of the wells is 15.6 mm.

FIGS. 20(A-K) illustrate A) photographic images and B) angle measurements of the bended or rolled model 4D constructs containing NIH3T3 cells in the GelMA layer to determine the effect of cell density and OMA layer thickness on the 4D geometric change. Diameter of the wells is 15.6 mm. “*”, “#”, “†”, and “§” indicate statistical significance compared to 2.0×10⁷, 5.0×10⁷, 1.0×10⁸, and 1.0×10⁸cells mL⁻¹with 0.4 mm OMA groups, respectively. C) DNA content of the 4D high cell density constructs with 1.0×10⁸cells mL⁻¹and 0.4 mm OMA thickness at different time points. D) Live/dead stained images of the high cell density construct at 1^st- and 21^st-day of culture. Scale bar indicates 200 μm. E) Photographic images of the bent or rolled model 4D constructs containing ASCs in the GelMA layer at 1.0×10⁸cells mL⁻¹density which were cultured in growth, chondrogenic, or osteogenic differentiation media over 21 days to demonstrate differentiation capacity in the system and F) angle measurement of the bent or rolled model ASC 4D constructs. Diameter of the wells is 15.6 mm. G) DNA content of the 4D ASC constructs cultured in the different media for 21 days. H) GAG production/DNA of 4D ASC constructs cultured in chondrogenic and growth media for 21 days. I) ALP activity/DNA of 4D ASC constructs cultured in osteogenic and growth media for 21 days. “*” indicates significant statistical difference (p<0.05). Cross-sectional images of J) toluidine blue 0 stained sections of control group cultured in growth media (left) and chondrogenically differentiated 4D ASC construct demonstrating GAG production (right). K) Alizarin Red S stained sections of control group cultured in growth media (left) and osteogenically differentiated 4D ASC construct demonstrating calcium deposition (right). Scale bars in (J) and (K) indicate 1 mm.

FIGS. 21(A-G) illustrate A) photographic images over 14 days of 4D high cell density constructs composed of 0.2 mm cell-laden GelMA and 0.4 mm cell-laden OMA layers. NIH3T3 cells were incorporated at 1.0×10⁸cells mL⁻¹in both layers. The diameter of the wells in the images is 15.6 mm. B) Images exhibiting the presence of NIH3T3 cells in both the GelMA and OMA layers. Left image is a phase contrast image of the construct and right image is a fluorescence image of cells labeled with purple and green dyes in the 12% OMA15 and 12% GelMA layers, respectfully. Scale bars indicate 500 μm. C) Angle measurements of the 4D high density NIH3T3 (1.0×10⁸cells mL⁻¹) constructs during 14 days of culture and image of the construct at 14th day (scale bar=1 mm). D) Photographic image of a photomask used to generate a pattern of alternating regions of photocrosslinked OMA in the model construct. Scale bar indicates 500 μm. E) Photographic images of the bent constructs over 7 days corresponding to OMA pattern directions perpendicular or parallel to the constructs' longest axis. Diameter of the wells is 15.6 mm. F) CAD image of layered checkerboard pattern. Red and light gray indicate cell-laden OMA and GelMA regions, respectively. G) 4D high cell density layered checkerboard construct fabricated by printing NIH3T3-laden 12% OMA15 (1.0×10⁸cells mL⁻¹) and NIH3T3-laden 12% GelMA (1.0×10⁷cells mL⁻¹) inks using a CAD file. The printed construct was flat and did not exhibit curvature or angled shape at day 0, while defined 4D geometric changes occurred over the course of 7 days culture as indicated by white dotted lines. Scale bars in (F) and (G) indicate 1 cm.

FIGS. 22(A-K) illustrate (A) A typical setup for crosslinking gradient hydrogel fabrication; (B) Schematic showing gradient hydrogel cut into specific initial shape and its subsequent deformation after swelling; Curved hydrogel bars of (C) PEGDA, (D) GelMA, and (E) OMA obtained using RhB (0.03% w/v) as UV absorber; (F) Magnified image showing a clear continuous gradient in the OMA hydrogel; Curved hydrogel bars of OMA obtained using (G) FITC (0.03% w/v), (H) AAb (0.05% w/v), and (I) HMAP (0.01% w/v) as UV absorber; bilayer hydrogel bars obtained from (J) OMA/GelMA and (K) OMA(g)/GelMA demonstrating feasibility of multi-material fabrication, where OMA represents non-gradient OMA hydrogel and OMA(g) represents gradient OMA hydrogel. The dotted outlines indicate (G-J) the shape of the hydrogel bars or (J-K) the interface of two layers. Scale bars in C-E and G-K indicate 2 mm.

FIGS. 23(A-E) illustrate bending degree of OMA hydrogels as a function of (A) UV irradiation time (6% w/v polymer, 0.6 mm thickness, 0.03% w/v RhB) in H₂O, *p<0.05 compared with all other groups, (B) RhB concentration (6% w/v polymer, 0.6 mm thickness, 30 s UV) in H₂O, *p<0.05 compared with all other groups, (C) hydrogel thickness (6% w/v polymer, 0.03% w/v RhB, 30 s UV) in H₂O, *p<0.05 compared with all other groups except for “0.4”, #p<0.05 compared with all other groups, (D) polymer concentration (0.6 mm thickness, 0.03% w/v RhB, 30 s UV) in H₂O, *p<0.05 compared with all other groups, and (E) aqueous swelling solution (6% w/v polymer, 0.6 mm thickness, 0.03% w/v RhB, and 30 s UV), *p<0.05 compared with all other groups. All scale bars indicate 2 mm.

FIGS. 24(A-D) illustrate cyclic reversible bending angle of OMA hydrogel bars in water solutions (A) of alternating pH of 1 and 7 and (B) with alternating presence of chemicals EDTA and Ca2+, 0.03% w/v RhB, *p<0.05 compared with all other groups, #p<0.05 compared with “pH 1” and original groups, scale bars indicate 4 mm. (C) Bending degree of cell-free and cell-laden OMA hydrogel bars in GM, 0.02% w/v HMAP, *p<0.05 compared with all other groups, scale bars indicate 2 mm. (D) Viability of encapsulated cells inside non-gradient (without UV absorber) and gradient (with UV absorber) hydrogels in GM after 3 days culture, 0.02% w/v HMAP, *p<0.05 compared to hMSC without UV absorber group. Insets: representative images showing the deformed shapes under respective conditions.

FIGS. 25(A-E) illustrate images of 4D biofabricated cell-laden structures: (A₁) six-petal blossoms and (A₂) four-arm grippers obtained by photomask-aided biofabrication, insets illustrate the deformation process of the microfabricated hydrogels; (B₁) Schematic of a “double-faced” hydrogel bar and its deformation, and (B₂) a typical “S” shape formed by the “double-faced” hydrogel; (C) hydrogel tubes obtained by post-photopatterning of a pre-fabricated gradient hydrogel sheet or disk, (C₁) patterned regions on a hydrogel sheet with dark pink regions denoting the UV-exposed section, (C₂) top-view image obtained using an optical microscope, (C₃) side-view image taken in ambient light; ITP-generated (D₁) hydrogel spiral and (D₂) pseudo-four-petal flower, insets show the ion-printed section on a pre-formed gradient hydrogel bar or hydrogel sheet; and (E) bioprinted multiple-arm grippers and their deformed structures, insets show the as-printed hydrogel construct before photo-gelation (upper left) and the top view of deformed hydrogel constructs (lower left). All hydrogel deformations were performed in GM at 37° C. for at least 2 h to obtain ultimate stable structures, and images were taken after replacing the GM with PBS (pH 7.4) for clarity.

FIGS. 26(A-F) illustrate (A) Bending degree of cell-laden hydrogel bars as a function of culture time in osteogenic medium. Inset: representative images at respective time points showing the shapes of the cell-laden hydrogel bars. (B) Live/dead staining images of encapsulated cells inside a hydrogel bar after 4-week culture in osteogenic medium. Biochemical quantification of (C) DNA content, (D) ALP activity normalized to DNA content, and (E) calcium content normalized to DNA content in the cell-laden hydrogels at varying time points. NC (negative control): cell-laden hydrogel bar obtained in the presence of UV absorber and cultured in GM, EG (experimental group): cell-laden hydrogel bar obtained in the presence of UV absorber and cultured in osteogenic medium, PC (positive control): cell-laden hydrogel bar obtained in the absence of UV absorber and cultured in osteogenic medium. (F) Typical alizarin red stained cell-laden hydrogel bars after 4-week culture. *p<0.05 compared with all other time points within a group, #p<0.05 compared with NC group at the same time point.

FIG. 27 is a schematic illustration of 4D cell-condensate bioprinting, tissue maturation, and tissue release.

FIGS. 28(A-F) illustrate (A) Compressive elastic moduli of various hydrogels, and the change of (B) modulus and (C) viscosity of O5M20A MGs with increasing shear strain and shear rate, respectively. (D) The rapid and reversible phase transition of O5M20A MGs between elastic state and viscous state by alternating the shear strain between 1% and 100%, and the (E) degradation and (F) swelling profiles of various hydrogels. ^*,#,&p<0.05 compared to group sharing no or a different symbol.

FIGS. 29(A-F) illustrate (A) Illustration and real sample photographs of a bilayer hydrogel disc (B) before and (C) after shape morphing. Effects of varying UV time applied to (D) the upper layer and (E) the bottom layer and (F) of varying MG layer thickness on the bending behaviors of the hydrogel strips. The hydrogel strip thickness for the UV time variation study was set to 0.8/0.5 (mm/mm) and the UV time for the MG layer thickness variation study was set to 30 s/30 s.

FIG. 30(A-C) illustrate (A) Live/dead cell staining of a printed cell filament inside photocrosslinked MGs after culturing in cell growth media for 4 h. Scale bars indicate 0.5 mm. (B) Photomicrographs of a printed cell strip (18×4×1.2 mm³) from top view (left) and side view (middle) and a cell sheet (13×13×1.2 mm³, right) inside photocrosslinked MG constructs. The cell strip images were taken immediately after UV photocrosslinking, the cell sheet image was taken immediately after placing in cell growth media. Scale bars indicate 5 mm. (C) Representative photomicrographs of cell-laden (left) and cell-free (middle) bilayer strips after culturing in cell growth media for 4 h, and quantified bending angles of the cell-laden and cell-free bilayer strips (right). *p<0.05 and scale bars indicate 5 mm. The UV irradiation times for the gradient layer and the MG layer fabrications were set to 30 s and 20 s (30 s/20 s), respectively.

FIGS. 31(A-D) illustrate large cell-laden bilayer constructs with defined structures of cell filling and the corresponding deformed configurations after culturing in cell growth media for 4 h: (A) sheet, (B) disc, (C) bar grid, and (D) net. Deformed cell-free bilayer counterparts were obtained under the same conditions and included for comparison. HeLa cells were used in this study, and UV irradiation times for the actuation layer (bottom layer) and the cell-laden layer (upper layer) were set to 40 s and 20 s (40 s/20 s), respectively. Scale bars indicate 5 mm.

FIGS. 32(A-D) illustrate (A) Photomicrographs of cell-net infilled bilayer sheet at D3, D6, D9, and D12. The black arrow and blue arrow in the image at D6 show the separated gradient hydrogel layer and the cell condensate-laden layer, respectively. Arrows in images of D9 and D12 show “liberated” cell-net filaments. Scale bars indicate 10 mm. Photomicrographs showing a cell-net filament inside the hydrogel under bright-field channel, green (live) channel, red (dead) channel, and merged channel of green and red at (B) D3, (C) D7, and (D) D12. Scale bars indicate 0.5 mm. UV crosslinking time was set to bottom/top 40 s/20 s.

FIGS. 33(A-K) illustrate (A) Shapes of hMSC cell condensate-laden bilayer strips cultured in chondrogenic media at different times. Exp stands for the experimental group, Ctrl stands for the control group, and scale bar is 10 mm. The UV irradiation times for the gradient layer and the MG layer were set to 50 s and 20 s (50 s/20 s), respectively. (B) 4D engineered letter “C”-shaped cartilage-like tissue after 21 days of culture. Scale bar is 10 mm. (C) The change of bending angle as a function of the culturing time, *p<0.05 compared to control group. (D) Biochemical analysis of GAG production (normalized to DNA content) at D21. (E) Young's moduli of the ex vivo engineered cartilage-like tissues at D21. Histologic characterization of ex vivo 4D engineered cartilage-like tissues: (F) hematoxylin and eosin (H&E) staining, and GAG (G) Safranin O (SafO, pink/red) and (I) toluidine blue O (TBO, blue and purple) staining for GAG. Scale bars indicate 0.2 mm. Helix-shaped cartilage-like tissue obtained at day 21 (I) before and (J) after manual removal of outer hydrogel layer. Scale bars indicate 5 mm. The UV irradiation times for the gradient layer and the MG layer were set to 30 s and 20 s (30 s/20 s), respectively. (K) TBO stained helix-shaped cartilage-like tissue, scale bar is 5 mm.

FIG. 34 is a schematic illustration of the PI and UV absorber incorporated 4D MFH bioprinting: i) printing the jammed cell-laden MFH bioinks into a bioconstruct, ii) UV crosslinking to generate a crosslinking gradient within the 3D printed bioconstruct, iii) culturing in media to drive shape morphing.

FIGS. 35(A-K) illustrate (A) Photomicrograph of safranin O stained MFHs. (B) Schematics showing lower packing density of granular microgels (upper) and more highly packed irregular MFHs (bottom). (C) Storage (G′) and loss (G″) moduli of MFHs as a function of frequency. Material viscosity decreases while continuously increasing (D) shear rate and (E) shear strain over 10% strain. (F) Crossover of G′ and G″ with increasing shear strain indicative of shear yielding. Rapid recovery of MFHs' (G) modulus and (H) viscosity by alternating the applied strain between 1% and 100%. (I) Photomicrograph of a filament printed through a 22-gauge (22G) needle (inner diameter 413 μm). Photographs of 3D printed (J) hydrogel bar (25×4×1 mm³) and (K) hydrogel cuboid (10×8×6 mm³). (1) Fidelity of the as-printed 3D construct in j before UV crosslinking.

FIGS. 36(A-H) illustrate 4D shape-morphing behaviors of hydrogel bars in different incubation solutions or fabricated with different parameters. (A) Hydrogel bending angle kinetics in diH2O, PBS (pH 7.4), and GM at room temperature. (B) Photomicrographs of deformed hydrogel bars in diH2O, PBS (pH 7.4), and GM after swelling for 2 h. Effects of (C) infill density, (D) printing speed, (F) needle gauge, (F) UV irradiation time, (G) hydrogel bar width, and (H) hydrogel bar length on the bending angles of hydrogel bars cultured in PBS (pH 7.4) for 2 h at room temperature. UV absorber: 0.02% 4′-hydroxy-3′-methylacetophenone (HMAP), 0.005% methacryloxyethyl thiocarbamoyl rhodamine B (RhB) was incorporated to impart the hydrogel with red color for better clarity.

FIG. 37 illustrates snapshots showing the shape changes of a hydrogel bar in response to different pH treatments at room temperature.

FIGS. 38(A-I) illustrate (A) Bending behaviors of MFH gradient hydrogel bars with and without embedded cells. Insets show representative photomicrographs of the bent hydrogel bars. (B) Photomicrograph of a NIH3T3-laden MFH-based construct. (C) Representative live/dead image of NIH3T3 fibroblasts in the gradient hydrogel bar. (D) Photomicrograph of a cell-laden “biohelix” structure. (E) Photograph of a cell-laden “bioS” structure. Photographs of (F) a cell-laden “pseudo-four-petal” and (G) a cell-laden “pseudo-six-petal” flower. Inset photographs show the corresponding as-printed structures. Kirigami-based structures and the deformed configurations: hydrogels in bar-grid patterns (H) without and (I) with inner horizontal bars. For cell-laden gradient hydrogel bars (insets in a), only 0.02% HMAP was used as the UV absorber, they were cultured in GM overnight at 37° C., and then imaged and live/dead stained. For complex cell-laden hydrogel configuration formation, a mixture of 0.02% HMAP and 0.005% RhB UV absorbers was used. These bioconstructs were obtained after culturing in GM at 37° C. overnight, and images were immediately taken after replacing the GM with FBS for clarity.

FIG. 39(A-F) illustrate demonstration of 3D-to-3D shape morphing. A pillar gripper (A) before being subject to deformation, after being submerged in media (B) less than 10 s and (C) for 60 s. A shark-fin sheet: (D) as-printed shape, (E) front-view image of deformed shape, and (F) image of deformed shape with construct on its side. Scale bars indicate 5 mm.

FIGS. 40(A-F) illustrate MFH based 4D bioprinting for application in tissue engineering. (A) The bending angles of hydrogel bars in EG and the corresponding photomicrographs depicting the shape changes of 4D bioprinted cell-laden hydrogel bars in CM over time. (B) Photomicrographs depicting cell morphology and distribution and live/dead stained cells within the EG hydrogel bars in CM on D1 and D21. (C) Biochemical quantification of GAG production normalized to DNA, *p<0.05 compared to NC at the same time point, #p<0.05 compared to D7 within a group. TBO stained (D) hydrogel bars and (E) four- and (F) six-petal flower-shaped hydrogels after chondrogenesis for 21 days in CM. NC: negative control, 4D bioprinted cell-laden hydrogel bars cultured in GM; EG: experimental group, 4D bioprinted cell-laden hydrogel bars cultured in CM; PC: positive control, 3D bioprinted (without incorporation of UV absorber) cell-laden hydrogel bars cultured in CM.

FIG. 41 illustrates 1H NMR spectrum of O1M30A (D₂O, 2%).

FIGS. 42(A-D) illustrate photographs of (A) as-prepared MFHs in 70% EtOH (B) and (C, D) reconstituted jammed MFHs (bioink).

FIG. 43 illustrates photomicrographs of safranin O stained MFHs.

FIG. 44 illustrates photograph of a MFH bioink filament extruded through a 22G needle.

FIG. 45 illustrate Young's moduli of as-prepared MFH, UV-crosslinked non-gradient MFH, and crosslinked gradient MFH, *p<0.05 compared to other groups.

FIG. 46 illustrates determination of the bending angle (θ).

FIG. 47 illustrates Swelling ratio of UV crosslinked MFHs in different media and pH, *p<0.05 compared to other groups.

FIGS. 48(A-B) is an illustration of Illustration of the strain distribution on the deformed hydrogel bar. The strain varies along the radical direction (er) but keeps relatively constant along the tangential direction (eθ).

FIG. 49 illustrates reversible bending behavior of hydrogel bars by switching the pH of the PBS incubation solution between 2.0 and 7.4 at room temperature.

FIG. 50 illustrates the viability of hMSCs cultured with 0.02% HMAP (w/v) for different culture times and subsequent culture in fresh GM to reach a total 24 h culture as measured using MTT assay.

FIG. 51 illustrates photomicrographs of printed cell-free MFH hydrogels (i) before and (ii) after UV crosslinking and UV-crosslinked cell-laden MFH hydrogels and live/dead stained images: (iii, v) hMSC and (iv, vi) and HeLa cells.

FIG. 52 is a schematic showing the formation of a discretely patterned gradient hydrogel.

FIG. 53 is a schematic shows the formation of a dual-orientated gradient hydrogel.

FIGS. 54(A-D) illustrate top views of the (A) four- and (B) six-petal flowers, (C) the “rib cage” like structure, and (D) the “net tube”.

FIGS. 55(A-B) illustrate schematics illustrating the gradient creation in two 3D models and their deformations: (A) “pillar bar”, (B) “shark-fin sheet”.

FIGS. 56(A-E) illustrate a demonstration of more complex 3D-to-3D shape morphing: (A) A “double sharkfin” sheet and its (B) morphed structure, (C) a “double pillar gripper” and (D, E) its morphed structure (upside down) with bend pillars pointing horizontally to the left and right, (D) side view and (E) top view. Scale bars indicate 5 mm.

FIG. 57 illustrates the change in DNA levels over 21 days of culture. NC: negative control, 4D bioprinted cell-laden hydrogel bar cultured in GM; EG: experimental group, 4D bioprinted cell-laden hydrogel bar cultured in CM; PC: positive control, 3D bioprinted cell-laden hydrogel bar (without incorporation of UV absorber) cultured in CM.

FIG. 58 illustrates a Change in GAG levels over 21 days of culture. NC: negative control, 4D bioprinted cell-laden hydrogel bar cultured in GM; EG: experimental group, 4D bioprinted cell-laden hydrogel bar cultured in CM; PC: positive control, 3D bioprinted cell-laden hydrogel bar (without incorporation of UV absorber) cultured in CM. *p<0.05 compared to NC at the same time point, #p<0.05 compared to D7 group within a group.

FIGS. 59A-F illustrate rheology of composite OMA/GelMA bioink for 3D printing. (A) Schematic of the overall strategy for CTF-mediated 4D biomaterials. The composite bioink exhibits shear thinning properties, where viscosity decreases as shear rate increases (B). Similarly, the storage modulus is greater than the loss modulus at low shear strains and less than the loss modulus at high shear strains (C). The composite bioink also exhibits self-healing properties, with the storage and loss moduli maintaining their initial values after several oscillations of strain (D). Adding gelatin microspheres at concentrations of 25, 50, and 100 mg/ml increases the bulk modulus (E). After one day of culture, the gelatin microspheres have liquified, causing the G′ to decrease dramatically (F).

FIGS. 60(A-E) illustrate shrinkage of 3D-printed cell-laden constructs. A schematic outlines the 3D printing process (A). OMA microgels are combined with 3% w/v GelMA and varying concentrations of gelatin microspheres. 1 mL of this solution is added to 100 million cells to form the cell-laden bioink. Here, the bioink was used to print 10 mm×10 mm×0.6 mm squares, which were monitored for 14 days to evaluate shrinkage. A selection of images shows the progression from initial to final shape (B). Quantifying the area reveals that increasing gelatin microsphere concentration leads to an increase in shrinkage (C). Scale bar=10 mm. “*”, “†”, “#”, and “$” indicate statistical significance at day 14 compared to 0 mg/ml, 25 mg/ml, 50 mg/ml, and 100 mg/ml, respectively (p<0.05).

FIGS. 61(A-D) illustrate 4D shape morphing in bilayer constructs. (A) Schematic illustration of the printing process. A thin hydrogel layer is printed followed by printing of the cell-laden layer. (B) To demonstrate the role of cellular forces in the 4D process, printed constructs were cultured in media containing 5 μM Cytochalasin D. Constructs were also cultured in media containing 0.1% DMSO as a vehicle control. Constructs in normal growth media and media with DMSO conditions followed similar 4D bending (C), demonstrating that Cytochalasin D is responsible for the lack of bending seen in this condition. The average bending angles in the GM and DMSO conditions are not significantly different from each other at all timepoints (p<0.05). (D) Histological analysis and staining with H&E at day 14 corroborate these observations, with cells in growth medium and DMSO conditions forming fibrous borders while cells in Cyto D are unable to do so. White scale bar: 5 mm. Black scale bar: 200 μm. Red scale bar: 50 μm.

FIGS. 62(A-D) illustrate chondrogenesis in 4D bilayer constructs. hMSCs were printed in bilayer constructs and cultured in normal growth media (GM) or chondrogenic pellet medium (CPM) and cultured for 21 days. (A) Macroscopic images of constructs over 21 days. (B) Quantification of bending angle over time (n=4). (C) Quantification of GAG production relative to DNA content for both GM and CPM conditions. Constructs cultured in CPM produced significantly more GAG (p<0.05). These results are qualitatively confirmed by histological staining at day 21 (D). Samples were stained with H&E to visualize cell condensation. Safranin O and Fast Green were used to visualize GAG production. Since the alginate in these constructs also stains positively with Safranin O, constructs were also stained with Alcian Blue at a pH of 0.2, revealing much greater positive staining in CPM samples. White scale bar=5 mm. Black scale bar=200 μm. “*” indicates statistical significance (p<0.05).

FIGS. 63(A-C) illustrates complex patterning of the cell-laden and hydrogel layers. (A) Complex patterning of the cell-laden layer. Schematics of the printed constructs followed by photomicrographs at days 0, 3, and 14. (B) Complex patterning of the hydrogel layer. Rectangular hydrogel patterns were printed either vertically or horizontally onto a cell-laden rectangle. The direction of the hydrogel pattern influenced the bending direction of the constructs. (C) Complex patterning of both the cell-laden and hydrogel layers. Layers were printed sequentially according to the schematic, resulting in bending around two separate, non-parallel axes.

FIG. 64 illustrates frequency sweeps of all composite bioinks with varying gelatin microsphere concentration. All bioink compositions show dominating solid-like properties at low frequencies. After one day of culture, the moduli of the 50 mg/ml condition have decreased but still maintain the same trend.

FIG. 65 illustrates the viability of printed constructs with varying concentrations of gelatin microspheres at days 0, 7, and 14.

FIGS. 66(A-B) illustrate the viability of GM, DMSO, and Cyto D conditions at day 14. (A) Composite live/dead images reveal that cell morphology appears similar in GM and DMSO conditions, while cells appear more rounded in the Cyto D condition. (B) Live/dead image of a construct in the GM group, centered at an inflection point. Differences in cell morphology can be observed by imaging through the cell-free layer (indicated with *) versus imaging through the cell-laden layer (indicated with #). Scale bar=200 μm.

FIG. 67(A-D) illustrates the description of bending angle measurement. Here, the Day 5 GM photomicrograph from FIG. 66B is used as an example. First, the image is uploaded into PowerPoint (A). Then, a circle with crosshairs is fitted to the contours of the printed construct (B). This overlay is then copied into ImageJ and the angle tool is used to find the angle between the ends of the construct. The middle of the crosshairs is used as the vertex (C). The bending angle is then measured and recorded (D).

FIG. 68 illustrates histological evidence of gelatin microsphere liquefaction. At day 0, gelatin microspheres appear pink under H&E staining. After one day of culture, the gelatin has liquefied and diffused out of the constructs, leaving macroscale pores (black arrows). Scale bar=200 μm.

FIG. 69 illustrates cell-cell interaction generated force mediated 4D system.

FIG. 70 illustrates cell generated force mediated 4D system.

FIG. 71 illustrates cell-matrix interaction force mediated 4D system.

FIG. 72 illustrates cell-generated force mediated 4D systems based on photolithography-produced hydrogel thickness differences/changes.

FIG. 73 illustrates cell-generated force mediated 4D systems based on degradable joints.

FIG. 74 illustrates 4D aptamer hydrogels.

FIG. 75 illustrates 4D cell condensate formation based on a specifically designed bilayer system.

FIG. 76 illustrates fully coat with a thin non-deformable OMA layer and a gradient OMA layer.

FIG. 77 illustrates an alternative strategy.

FIG. 78 illustrates 4D bio-kirigami system.

DETAILED DESCRIPTION

Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises, such as Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Commonly understood definitions of molecular biology terms can be found in, for example, Rieger et al., Glossary of Genetics: Classical and Molecular, 5th Ed., Springer-Verlag: New York, 1991, and Lewin, Genes V, Oxford University Press: New York, 1994. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present invention.

Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers, but not the exclusion of any other step or element or integer or group of elements or integers. Thus, in the context of this specification, the term “comprising” means “including principally, but not necessarily solely”.

The term “bioactive agent” can refer to any agent capable of promoting tissue formation, destruction, and/or targeting a specific disease state. Examples of bioactive agents can include, but are not limited to, chemotactic agents, various proteins (e.g., short term peptides, bone morphogenic proteins, collagen, glycoproteins, and lipoprotein), cell attachment mediators, biologically active ligands, integrin binding sequence, various growth and/or differentiation agents and fragments thereof (e.g., epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factors (VEGF), fibroblast growth factors (e.g., bFGF), platelet derived growth factors (PDGF), insulin-like growth factor (e.g., IGF-I, IGF-II) and transforming growth factors (e.g., TGF-β I-III), parathyroid hormone, parathyroid hormone related peptide, bone morphogenic proteins (e.g., BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-13, BMP-14), transcription factors, such as sonic hedgehog, growth differentiation factors (e.g., GDF5, GDF6, GDF8), recombinant human growth factors (e.g., MP52 and the MP-52 variant rhGDF-5), cartilage-derived morphogenic proteins (CDMP-1, CDMP-2, CDMP-3), small molecules that affect the upregulation of specific growth factors, tenascin-C, hyaluronic acid, chondroitin sulfate, fibronectin, decorin, thromboelastin, thrombin-derived peptides, heparin-binding domains, heparin, heparan sulfate, polynucleotides, DNA fragments, DNA plasmids, MMPs, TIMPs, interfering RNA molecules, such as siRNAs, oligonucleotides, proteoglycans, glycoproteins, glycosaminoglycans, and DNA encoding for shRNA.

The term “biomaterial” refers to any naturally occurring, naturally derived, or synthetic material or substance which is compatible with biological systems.

The terms “biodegradable” and “bioresorbable” may be used interchangeably and refer to the ability of a material (e.g., a natural polymer or macromer) to be fully resorbed in vivo. “Full” can mean that no significant extracellular fragments remain. The resorption process can involve elimination of the original implant material(s) through the action of body fluids, enzymes, cells, and the like.

The term “gel” includes gels and hydrogels.

The term “microgel” refers to hydrogels having a diameter less than about 1000 μm, less than about 400 μm, or less than about 300 μm, for example, about 1 μm to about 1000 μm.

The term “function and/or characteristic of a cell” can refer to the modulation, growth, and/or proliferation of at least one cell, such as a progenitor cell and/or differentiated cell, the modulation of the state of differentiation of at least one cell, and/or the induction of a pathway in at least one cell, which directs the cell to grow, proliferate, and/or differentiate along a desired pathway, e.g., leading to a desired cell phenotype, cell migration, angiogenesis, apoptosis, etc.

The term “macromer” can refer to any natural polymer or oligomer or their derivatives.

The term “polynucleotide” can refer to oligonucleotides, nucleotides, or to a fragment of any of these, to DNA or RNA (e.g., mRNA, rRNA, siRNA, tRNA) of genomic or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acids, or to any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g., iRNPs). The term can also encompass nucleic acids (i.e., oligonucleotides) containing known analogues of natural nucleotides, as well as nucleic acid-like structures with synthetic backbones.

The term “polypeptide” can refer to an oligopeptide, peptide, polypeptide, or protein sequence, or to a fragment, portion, or subunit of any of these, and to naturally occurring or synthetic molecules. The term “polypeptide” can also include amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain any type of modified amino acids. The term “polypeptide” can also include peptides and polypeptide fragments, motifs and the like, glycosylated polypeptides, and all “mimetic” and “peptidomimetic” polypeptide forms.

The term “cell” can refer to any progenitor cell, such as totipotent stem cells, pluripotent stem cells, and multipotent stem cells, as well as any of their lineage descendant cells, including more differentiated cells. The terms “stem cell” and “progenitor cell” are used interchangeably herein. The cells can derive from embryonic, fetal, or adult tissues. Examples of progenitor cells can include totipotent stem cells, multipotent stem cells, mesenchymal stem cells (MSCs), hematopoietic stem cells, neuronal stem cells, hematopoietic stem cells, pancreatic stem cells, cardiac stem cells, embryonic stem cells, embryonic germ cells, neural crest stem cells, kidney stem cells, hepatic stem cells, lung stem cells, hemangioblast cells, and endothelial progenitor cells. Additional exemplary progenitor cells can include de-differentiated chondrogenic cells, chondrogenic cells, cord blood stem cells, multi-potent adult progenitor cells, myogenic cells, osteogenic cells, tendogenic cells, ligamentogenic cells, adipogenic cells, and dermatogenic cells.

The terms “inhibit,” “silencing”, and “attenuating” can refer to a measurable reduction in expression of a target mRNA (or the corresponding polypeptide or protein) as compared with the expression of the target mRNA (or the corresponding polypeptide or protein) in the absence of an interfering RNA molecule of the present invention. The reduction in expression of the target mRNA (or the corresponding polypeptide or protein) is commonly referred to as “knock-down” and is reported relative to levels present following administration or expression of a non-targeting control RNA.

The term “subject” can refer to any animal, including, but not limited to, humans and non-human animals (e.g., rodents, arthropods, insects, fish (e.g., zebrafish)), non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.), which is to be the recipient of a particular treatment. Typically, the terms “patient” and “subject” are used interchangeably herein in reference to a human subject.

The term “tissue” can refer to an aggregate of cells having substantially the same function and/or form in a multicellular organism. “Tissue” is typically an aggregate of cells of the same origin but may be an aggregate of cells of different origins. The cells can have the substantially same or substantially different function and may be of the same or different type. “Tissue” can include, but is not limited to, an organ, a part of an organ, bone, cartilage, skin, neuron, axon, blood vessel, cornea, muscle, fascia, brain, prostate, breast, endometrium, lung, pancreas, small intestine, blood, liver, testes, ovaries, cervix, colon, stomach, esophagus, spleen, lymph node, bone marrow, kidney, peripheral blood, embryonic, or ascite tissue.

DETAILED DESCRIPTION

Embodiments described herein relate to shape morphing hydrogel and/or cell condensate actuators or constructs that include a biocompatible and/or cytocompatible polymer-based shape morphing hydrogel and/or cell condensate, which is configured to undergo multiple, reversible, and/or controllable different shape transformations over time via either pre-programmed design or user-controlled environmental condition alterations. Shape-morphing hydrogels bear promising prospects as soft actuators and for robotics. However, they are mostly restricted to applications in the abiotic domain due to the harsh physicochemical conditions typically necessary to induce shape morphing. We developed a biocompatible polymer-based shape morphing hydrogel and cell condensates using biocompatible polymers and cell condensates that permits encapsulation and maintenance of living cells and implements programmed and controlled actuations with multiple shape changes. The biocompatible polymer-based shape morphing hydrogel and cell condensates enable defined self-folding and/or user-regulated, on-demand-folding into specific 3D architectures under physiological conditions, with the capability to partially bioemulate complex developmental processes, such as branching morphogenesis. The biocompatible polymer-based shape morphing hydrogel and cell condensates can be utilized as platforms for promoting new complex tissue formation and regenerative medicine applications.

In some embodiments, the shape morphing hydrogel includes at least one layer, wherein the swelling and/or degradation rate of the at least one layer actuates the shape transformations.

In other embodiments, the construct includes multiple layers having similar or different swelling ratios.

In some embodiments, at least one of the layers includes hydrogel forming acrylated and/or methacrylated polymer macromers that are optionally oxidized. Acrylated and/or methacrylated natural polymer macromers can include saccharides (e.g., mono-, di-, oligo-, and poly-saccharides), such as glucose, galactose, fructose, lactose and sucrose, collagen, gelatin, glycosaminoglycans, poly(hyaluronic acid), poly(sodium alginate), hyaluronan, alginate, heparin and agarose that can be readily oxidized to form free aldehyde units.

In some embodiments, the acrylated and/or methacrylated polymer macromers are acrylated and/or methacrylated polysaccharides that are optionally oxidized.

The acrylated or methacrylated, alginates can be optionally oxidized so that up to about 50% of the saccharide units therein are converted to aldehyde saccharide units. Natural source alginates, for example, from seaweed or bacteria, are useful and can be selected to provide side chains with appropriate M (mannuronate) and G (guluronate) units for the ultimate use of the polymer. Alginate materials can be selected with high guluronate content since the guluronate units, as opposed to the mannuronate units, more readily provide sites for oxidation and crosslinking. Isolation of alginate chains from natural sources can be conducted by conventional methods. See Biomaterials: Novel Materials from Biological Sources, ed. Byrum, Alginates chapter (ed. Sutherland), p. 309-331 (1991). Alternatively, synthetically prepared alginates having a selected M and G unit proportion and distribution prepared by synthetic routes, such as those analogous to methods known in the art, can be used. Further, either natural or synthetic source alginates may be modified to provide M and G units with a modified structure. The M and/or G units may also be modified, for example, with polyalkylene oxide units of varied molecular weight such as shown for modification of polysaccharides in Spaltro (U.S. Pat. No. 5,490,978) with other alcohols such as glycols. Such modification generally will make the polymer more soluble, which generally will result in a less viscous material. Such modifying groups can also enhance the stability of the polymer. Further, modification to provide alkali resistance, for example, as shown by U.S. Pat. No. 2,536,893, can be conducted.

The oxidation of the natural polymer macromers (e.g., alginate material) can be performed using a periodate oxidation agent, such as sodium periodate, to provide at least some of the saccharide units of the natural polymer macromer with aldehyde groups. The degree of oxidation is controllable by the mole equivalent of oxidation agent, e.g., periodate, to saccharide unit. For example, using sodium periodate in an equivalent % of from 2% to 100%, preferably 1% to 50%, a resulting degree of oxidation, i.e., % if saccharide units converted to aldehyde saccharide units, from about 2% to 50% can be obtained. The aldehyde groups provide functional sites for crosslinking and for bonding tissue, cells, prosthetics, grafts, and other material that is desired to be adhered. Further, oxidation of the natural polymer macromer facilitates their degradation in vivo, even if they are not lowered in molecular weight. Thus, high molecular weight alginates, e.g., of up to 300,000 daltons, may be degradable in vivo, when sufficiently oxidized, i.e., preferably at least 5% of the saccharide units are oxidized.

In some embodiments, the natural polymer macromer (e.g., alginate) can be acrylated or methacrylated by reacting an acryl group or methacryl with a natural polymer or oligomer to form the oxidized, acrylated or methacrylated natural polymer macromer (e.g., alginate). For example, oxidized alginate can be dissolved in a solution chemically functionalized with N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride to activate the carboxylic acids of alginate and then reacted with 2-aminoethylmethacrylate to provide a plurality of methacrylate groups on the alginate.

The acrylated and/or methacrylated gelatin can be formed by reacting an acryl group and/or methacryl with gelatin. For example, bovine type-B gelatin can be dissolved in a phosphate buffered solution and then reacted with methacrylic anhydride to provide a plurality of methacrylate groups on the gelatin.

The degree of acrylation or methacrylation can be controlled to control the degree of subsequent crosslinking of the acrylate and methacrylates as well as the mechanical properties, and biodegradation rate of the composition. The degree of acrylation or methacrylation can be about 1% to about 50%, although this ratio can vary more or less depending on the end use of the composition.

The acrylated and/or methacrylated polymer macromers can be reversibly and ionically crosslinkable. The acrylated and/or methacrylated polymer macromers can also be photocrosslinkable, ionically crosslinkable, physically crosslinkable, pH crosslinkable, dual crosslinkable, and/or thermally crosslinkable to adjust the mechanical properties of the hydrogel. The mechanical properties that can be adjusted include, for example, stiffness, Young's modulus, tensile strength, viscosity, resistance to shear or tensile loading and excessive swelling, as well as biodegradation rate.

In some embodiments, at least one of the layers includes a first oxidized and acrylated and/or methacrylated natural polymer macromer and another layer includes a second oxidized and acrylated and/or methacrylated natural polymer macromer, the oxidation and/or acrylation and/or methacrylation of the second natural polymer macromer different from the oxidation and/or acrylation and/or methacrylation of the second polymer macromer.

In some embodiments, the shape morphing hydrogel is ionically cross-linkable and the shape transformation is actuated by increasing or decreasing the concentration of ionic cross-linker in the shape morphing hydrogel. For example, gluronic acids of different alginates in alginate hydrogel can form ionic crosslinks with Ca²⁺ provide by an aqueous solution of CaCl₂resulting in a crosslinked hydrogel network. As the concentration of the ionic crosslinker (e.g., Ca²⁺) in the hydrogel increases the secondary crosslinking and elastic moduli increase resulting in an increase in stiffness of the hydrogel. Subsequent decrease in the concentration of the ionic crosslinker by, for example, incubation of the hydrogel in a buffer solution decreases the ionic crosslinking and the elastic moduli of the hydrogel resulting in softening of the hydrogel.

In other embodiments, the shape morphing hydrogel is self-morphing and/or user regulated on-demand morphing into three dimensional architectures under physiological or non-physiological conditions.

In some embodiments, the construct can include a plurality of cells dispersed in the hydrogel. For example, at least a portion of the construct has a cell density up to 1×10¹⁰cells/ml. The plurality of cells can include progenitor cells, undifferentiated cells, differentiated cells, and/or cancer cells. In one example, the plurality of cells can include mesenchymal stem cells.

The cells provided in the hydrogel can be autologous, xenogeneic, allogeneic, and/or syngeneic. Where the cells are not autologous, it may be desirable to administer immunosuppressive agents in order to minimize immunorejection. The cells employed may be primary cells, expanded cells, or cell lines, and may be dividing or non-dividing cells. Cells may be expanded ex vivo prior to introduction into or onto the hydrogel. For example, autologous cells can be expanded in this manner if a sufficient number of viable cells cannot be harvested from the host subject. Alternatively or additionally, the cells may be pieces of tissue, including tissue that has some internal structure. The cells may be primary tissue explants and preparations thereof, cell lines (including transformed cells), or host cells.

Generally, cells can be introduced into the hydrogels in vitro, although in vivo seeding approaches can optionally or additionally be employed. Cells may be mixed with the macromers used to form the hydrogels and cultured in an adequate growth (or storage) medium to ensure cell viability. If the hydrogel is to be implanted for use in vivo after in vitro seeding, for example, sufficient growth medium may be supplied to ensure cell viability during in vitro culture prior to in vivo application. Once the hydrogels have been implanted, the nutritional requirements of the cells can be met by the circulating fluids of the host subject.

Any available method may be employed to introduce the cells into the hydrogels. For example, cells may be injected into the hydrogels (e.g., in combination with growth medium) or may be introduced by other means, such as pressure, vacuum, osmosis, or manual mixing. Alternatively or additionally, cells may be layered on the hydrogels, or the hydrogels may be dipped into a cell suspension and allowed to remain there under conditions and for a time sufficient for the cells to incorporate within or attach to the hydrogel. Generally, it is desirable to avoid excessive manual manipulation of the cells in order to minimize cell death during the impregnation procedure. For example, in some situations it may not be desirable to manually mix or knead the cells with the hydrogels; however, such an approach may be useful in those cases in which a sufficient number of cells will survive the procedure. Cells can also be introduced into the hydrogels in vivo simply by placing the hydrogel in the subject adjacent a source of desired cells.

As those of ordinary skill in the art will appreciate, the number of cells to be introduced into the hydrogels will vary based on the intended application of the hydrogel and on the type of cell used. Where dividing autologous cells are being introduced by injection or mixing into the hydrogel, for example, a lower number of cells can be used. Alternatively, where non-dividing cells are being introduced by injection or mixing into the hydrogel, a larger number of cells may be required. It should also be appreciated that the hydrogel can be in either a hydrated or lyophilized state prior to the addition of cells. For example, the hydrogel can be in a lyophilized state before the addition of cells is done to re-hydrate and populate the scaffold with cells.

In other embodiments, the hydrogels can include at least one attachment molecule to facilitate attachment of at least one cell thereto. The attachment molecule can include a polypeptide or small molecule, for example, and may be chemically immobilized onto the hydrogel to facilitate cell attachment. Examples of attachment molecules can include fibronectin or a portion thereof, collagen or a portion thereof, polypeptides or proteins containing a peptide attachment sequence (e.g., arginine-glycine-aspartate sequence) (or other attachment sequence), enzymatically degradable peptide linkages, cell adhesion ligands, growth factors, degradable amino acid sequences, and/or protein-sequestering peptide sequences.

In other embodiments, the construct can include at least one bioactive agent. Advantageously, the release of the bioactive agent from the hydrogel can be controlled by dynamically adjusting the mechanical properties of the hydrogel. The at least one bioactive agent can include any agent capable of modulating a function and/or characteristic of a cell that is dispersed on or within the hydrogel. Alternatively or additionally, the bioactive agent may be capable of modulating a function and/or characteristic of an endogenous cell surrounding the hydrogel implanted in a tissue defect, for example, and guide the cell into the defect.

Examples of bioactive agents include chemotactic agents, various proteins (e.g., short term peptides, bone morphogenic proteins, collagen, glycoproteins, and lipoprotein), cell attachment mediators, biologically active ligands, integrin binding sequence, various growth and/or differentiation agents and fragments thereof (e.g., EGF), HGF, VEGF, fibroblast growth factors (e.g., bFGF), PDGF, insulin-like growth factor (e.g., IGF-I, IGF-II) and transforming growth factors (e.g., TGF-β I-III), parathyroid hormone, parathyroid hormone related peptide, bone morphogenic proteins (e.g., BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-13, BMP-14), sonic hedgehog, growth differentiation factors (e.g., GDF5, GDF6, GDF8), recombinant human growth factors (e.g., MP-52 and the MP-52 variant rhGDF-5), cartilage-derived morphogenic proteins (CDMP-1, CDMP-2, CDMP-3), small molecules that affect the upregulation of specific growth factors, polynucleotides, DNA fragments, DNA plasmids, MMPs, TIMPs, interfering RNA molecules, such as siRNAs, DNA encoding for an shRNA of interest, oligonucleotides, proteoglycans, glycoproteins, and glycosaminoglycans.

In some embodiments, at least two layers are covalently linked at adjoining portions.

In some embodiments, the construct can include at least three layers, wherein a middle layer is covalently linked to adjoining portions of two outer layers.

In some embodiments, the shape morphing hydrogel is self-morphing and/or user regulated on-demand morphing into three dimensional architectures under physiological conditions.

Other embodiments described herein relates to a method of forming a construct. The method includes adhering a first layer that includes a first hydrogel forming natural polymer macromer to a second layer that includes a second hydrogel forming polymer macromer having a different swelling ratio and/or degradation rate than the first hydrogel forming natural polymer macromer. The different swelling ratio and/or degradation rate allows the hydrogel to undergo multiple, reversible, controllable and/or different shape transformations. The hydrogel is cytocompatible and, upon degradation, produces substantially non-toxic products.

In some embodiments, the method can further include adhering a third layer to the first and second layer such that the second layer is sandwiched between the first layer and the third layer. The third layer can include a third hydrogel forming polymer macromer.

In some embodiments, the at least one of the first layer, the second layer, and/or third layer can have different swelling ratios.

In some embodiments, at least one of the layers includes a hydrogel forming acrylated and/or methacrylated polymer macromers that are optionally oxidized. The acrylated and/or methacrylated polymer macromers can be reversibly and ionically crosslinkable. The acrylated and/or methacrylated natural polymer macromers can also be photocrosslinkable, ionically crosslinkable, pH crosslinkable, physically crosslinkable, dual crosslinkable, and/or thermally crosslinkable.

In some embodiments, the acrylated and/or methacrylated polymer macromers are acrylated and/or methacrylated polysaccharides that are optionally oxidized.

In other embodiments, the shape morphing hydrogel is self-morphing and/or user regulated on-demand morphing into three dimensional architectures under physiological or non-physiological conditions.

In some embodiments, at least two layers are covalently linked at adjoining portions.

In some embodiments, the construct can include at least three layers, wherein a middle layer is covalently linked to adjoining portions of two outer layers.

In some embodiments, the shape morphing hydrogel is self-morphing and/or user regulated on-demand morphing into three dimensional architectures under physiological conditions.

The self-healing, shear thinning, crosslinkable, biocompatible hydrogel support medium can maintain the first hydrogel forming polymer macromer and the second hydrogel forming macromer in a defined shape during printing of the first hydrogel forming polymer macromer and the second hydrogel forming macromer. The hydrogel support medium can be resistant to flow at a first shear stress and behave as a viscous fluid at a second higher shear stress. This allows the hydrogel support medium to behave as a viscous fluid during printing and be resistant to flow before and after printing. For example, initially, the hydrogel support medium is in a flow-resistant or solid-like state before being printed with the first hydrogel forming polymer macromer and the second hydrogel forming macromer. The hydrogel support medium becomes fluidized under the increased shear stress caused by printing the first hydrogel forming polymer macromer and the second hydrogel forming macromer into the hydrogel support medium. Then, after the printing is finished and the increased shear stress is removed, the hydrogel support medium can self-heal and form a flow-resistant or solid-like stable support medium. The hydrogel support medium can be crosslinked after printing to maintain the defined shape of the printed the first hydrogel forming polymer macromer and the second hydrogel forming macromer during culturing in the hydrogel support medium.

In some embodiments, the self-healing, shear thinning, crosslinkable, biocompatible hydrogel support medium can include a plurality of crosslinkable hydrogel particles that are provided in a container. The plurality of crosslinkable hydrogel particles are in contact with each other in the container such that interstitial spaces are provided between individual hydrogel particles. The interstitial spaces between individual particles form pores in the hydrogel support medium in which a culture medium can be provided and/or flow to the printed bioink during culturing of the cells. The sizes of the pores can be dependent on the sizes of the individual hydrogel particles. For example, smaller pores can result from smaller spaces between the smaller hydrogel particles, and, conversely, larger pores can result from larger spaces between the larger hydrogel particles.

In some embodiments, the hydrogel particles can have average diameter of about 10 nm to about 10 mm, for example, about 100 nm to about 1000, about 1 μm to about 500 μm, about 25 μm to about 400 μm, or about 50 μm to 200 μm. The plurality of hydrogel particles can have substantially homogenous diameters or include particles of varying diameters to provide a heterogenous mixture of the hydrogel particles.

The hydrogel particles can be cytocompatible and, upon degradation, produce substantially non-toxic products. In some embodiments, the hydrogel particles can include a plurality of crosslinkable biodegradable natural polymer macromers. The crosslinkable natural polymer macromers can be any crosslinkable natural polymer or oligomer that includes a functional group (e.g., a carboxylic group) that can be further polymerized. Examples of natural polymers or oligomers are saccharides (e.g., mono-, di-, oligo-, and poly-saccharides), such as glucose, galactose, fructose, lactose and sucrose, collagen, gelatin, glycosaminoglycans, poly(hyaluronic acid), poly(sodium alginate), hyaluronan, alginate, heparin and agarose.

The natural polymer macromers can optionally be at least partially crosslinked with a first agent and further crosslinkable with the first agent or crosslinkable with a second agent. The crosslinkable natural polymer macromer can include dual crosslinkable natural polymer macromers, such as an acrylated and/or methacrylated natural polymer macromers. Acrylated and/or methacrylated natural polymer macromers can include saccharides (e.g., mono-, di-, oligo-, and poly-saccharides), such as glucose, galactose, fructose, lactose and sucrose, collagen, gelatin, glycosaminoglycans, poly(hyaluronic acid), poly(sodium alginate), hyaluronan, alginate, heparin and agarose that can be readily oxidized to form free aldehyde units.

In some embodiments, the acrylated or methacrylated, natural polymer macromers are polysaccharides, which are optionally oxidized so that up to about 50% of the saccharide units therein are converted to aldehyde saccharide units. Control over the degree of oxidation of the natural polymer macromers permits regulation of the gelling time used to form the hydrogel as well as the mechanical properties, which allows for tailoring of these mechanical properties depending on the clinical application.

In other embodiments, acrylated and/or methacrylated, natural polymer macromers can include oxidized, acrylated or methacrylated, alginates, which are optionally oxidized so that, for example, up to about 50% of the saccharide units therein are converted to aldehyde saccharide units. Natural source alginates, for example, from seaweed or bacteria, are useful and can be selected to provide side chains with appropriate M (mannuronate) and G (guluronate) units for the ultimate use of the polymer. Alginate materials can be selected with high guluronate content since the guluronate units, as opposed to the mannuronate units, more readily provide sites for oxidation and crosslinking. Isolation of alginate chains from natural sources can be conducted by conventional methods. See Biomaterials: Novel Materials from Biological Sources, ed. Byrum, Alginates chapter (ed. Sutherland), p. 309-331 (1991). Alternatively, synthetically prepared alginates having a selected M and G unit proportion and distribution prepared by synthetic routes, such as those analogous to methods known in the art, can be used. Further, either natural or synthetic source alginates may be modified to provide M and G units with a modified structure. The M and/or G units may also be modified, for example, with polyalkylene oxide units of varied molecular weight such as shown for modification of polysaccharides in Spaltro (U.S. Pat. No. 5,490,978) with other alcohols such as glycols. Such modification generally will make the polymer more soluble, which generally will result in a less viscous material. Such modifying groups can also enhance the stability of the polymer. Further, modification to provide alkali resistance, for example, as shown by U.S. Pat. No. 2,536,893, can be conducted.

In some embodiments, the natural polymer macromer (e.g., alginate) can be acrylated or methacrylated by reacting an acryl group or methacryl with a natural polymer or oligomer to form the oxidized, acrylated or methacrylated natural polymer macromer (e.g., alginate). For example, oxidized alginate can be dissolved in a solution chemically functionalized with N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride to activate the carboxylic acids of alginate and then reacted with 2-amionethylmethacrylate to provide a plurality of methacrylate groups on the alginate.

In some embodiments, a solution of natural polymer macromers can be ionically crosslinked and/or chemically crosslinked with a first agent to form a plurality of hydrogel particles. The ionically crosslinked hydrogel can be in the form of a plurality of hydrogel particles. By way of example, a solution of natural polymer macromers can be dispensed as microdroplets into an aqueous solution of CaCl₂) and ionically crosslinked to form the plurality of microgels. The extent of crosslinking can be controlled by the concentration of CaCl₂). The higher concentration can correspond to a higher extent of crosslinking. The extent of crosslinking alters the mechanical properties of the microgel and can be controlled as desired for the particular application. In general, a higher degree of crosslinking results in a stiffer gel.

In some embodiments, the hydrogel particles can be crosslinked with a second agent to form dual crosslinked hydrogel. A plurality of second crosslink networks can be formed by crosslinking acrylate and/or methacrylate groups of the acrylated or methacrylated natural polymer macromer. The second crosslinking networks formed by crosslinking the acrylate groups or methacrylate groups of the acrylated and/or methacrylated natural polymer macromer can provide improved mechanical properties, such as resistance to excessive swelling, as well as delayed biodegradation rate.

In some embodiments, the acrylate or methacrylate groups of the acrylated and/or methacrylated natural polymer macromer of the hydrogel can be crosslinked by photocrosslinking using UV light in the presence of photoinitiators. For example, acrylated and/or methacrylated natural polymer macromers of the hydrogel particles can be photocrosslinked with a photoinitiator that is provided in the hydrogel support medium. The hydrogel particles can be exposed to a light source at a wavelength and for a time to promote crosslinking of the acrylate groups of the polymers and form the photocrosslinked biodegradable hydrogel particles.

A photoinitiator can include any photo-initiator that can initiate or induce polymerization of the acrylate or methacrylate macromer. Examples of the photoinitiator can include camphorquinone, benzoin methyl ether, 2-hydroxy-2-methyl-1-phenyl-1-propanone, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, benzoin ethyl ether, benzophenone, 9,10-anthraquinone, ethyl-4-N,N-dimethylaminobenzoate, diphenyliodonium chloride and derivatives thereof.

In other embodiments, the hydrogel support medium can further include at least one bioactive agent that is provided in the hydrogel particles or potentially a culture medium that can be added to the hydrogel support medium during culturing of the printed bioink. The bioactive agent can include polynucleotides and/or polypeptides encoding or comprising, for example, transcription factors, differentiation factors, growth factors, and combinations thereof. The at least one bioactive agent can also include any agent capable of promoting tissue formation (e.g., bone and/or cartilage), destruction, and/or targeting a specific disease state (e.g., cancer). Examples of bioactive agents include chemotactic agents, various proteins (e.g., short term peptides, bone morphogenic proteins, collagen, glycoproteins, and lipoprotein), cell attachment mediators, biologically active ligands, integrin binding sequence, various growth and/or differentiation agents and fragments thereof (e.g., EGF), HGF, VEGF, fibroblast growth factors (e.g., bFGF), PDGF, insulin-like growth factor (e.g., IGF-I, IGF-II) and transforming growth factors (e.g., TGF-β I-III), parathyroid hormone, parathyroid hormone related peptide, bone morphogenic proteins (e.g., BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-13, BMP-14), sonic hedgehog, growth differentiation factors (e.g., GDF5, GDF6, GDF8), recombinant human growth factors (e.g., MP-52 and the MP-52 variant rhGDF-5), cartilage-derived morphogenic proteins (CDMP-1, CDMP-2, CDMP-3), small molecules that affect the upregulation of specific growth factors, tenascin-C, hyaluronic acid, chondroitin sulfate, fibronectin, decorin, thromboelastin, thrombin-derived peptides, heparin-binding domains, heparin, heparin sulfate, polynucleotides, DNA fragments, DNA plasmids, MMPs, TIMPs, interfering RNA molecules, such as siRNAs, miRNAs, DNA encoding for an shRNA of interest, oligonucleotides, proteoglycans, glycoproteins, and glycosaminoglycans.

In some embodiments, a bioactive agent can comprise an interfering RNA or miRNA molecule incorporated on or within insoluble native collagen fibers or dispersed on or within the cell aggregate. The interfering RNA or miRNA molecule can include any RNA molecule that is capable of silencing an mRNA and thereby reducing or inhibiting expression of a polypeptide encoded by the target mRNA. Alternatively, the interfering RNA molecule can include a DNA molecule encoding for a shRNA of interest. For example, the interfering RNA molecule can comprise a short interfering RNA (siRNA) or microRNA molecule capable of silencing a target mRNA that encodes any one or combination of the polypeptides or proteins described above.

In some embodiments, at least one of the first hydrogel forming natural polymer macromer or the second hydrogel forming natural polymer macromer includes a plurality of cells. The cells provided in the first hydrogel forming natural polymer macromer or the second hydrogel forming natural polymer macromer can be autologous, xenogeneic, allogeneic, and/or syngeneic. Where the cells are not autologous, it may be desirable to administer immunosuppressive agents in order to minimize immunorejection. The cells employed may be primary cells, expanded cells, or cell lines, and may be dividing or non-dividing cells. Cells may be expanded ex vivo prior to introduction into or onto the hydrogel. For example, autologous cells can be expanded in this manner if a sufficient number of viable cells cannot be harvested from the host subject. Alternatively or additionally, the cells may be pieces of tissue, including tissue that has some internal structure. The cells may be primary tissue explants and preparations thereof, cell lines (including transformed cells), or host cells.

In some embodiments, the acrylated and/or methacrylated polymer macromers are photocrosslinkable, ionically crosslinkable, physically crosslinkable, pH crosslinkable, dual crosslinkable, and/or thermally crosslinkable. For example, the acrylated and/or methacrylated polymer macromers include acrylated and/or methacrylated polysaccharides that are optionally oxidized.

In some embodiments, the hydrogel includes a mixture of acrylated and/or methacrylated alginate that is optionally oxidized and an acrylated and/or methacrylated gelatin.

In some embodiments, the shape morphing hydrogel is self-morphing and/or user regulated on-demand morphing into three dimensional architectures under physiological or non-physiological conditions.

In some embodiments, the shape morphing hydrogel can include a single biocompatible polymer or copolymer.

In some embodiments, the shape morphing hydrogel includes a photocrosslinkable hydrogel forming polymer and a photo-absorber and a photoinitiator dispersed within the hydrogel.

In some embodiments, the construct includes the biocompatible, polymer-derived shape morphing hydrogel and a plurality of cells dispersed in at least a portion of the construct. The plurality of cells can have a cell density up to 1×10¹⁰cells/ml.

In some embodiments, the shape morphing hydrogel exhibits a repeatable and reversible shape change based on exogenous stimulation, the exogenous stimulation can include at least one of at least one of chemical, biochemical, irradiation, magnetic, biological, electric, ultrasound/sound, mechanical or a change in pH or temperature.

In some embodiments, the shape morphing hydrogel is self-morphing and/or user regulated on-demand morphing into three dimensional architectures under physiological or non-physiological conditions.

In some embodiments, the hydrogel is cytocompatible and, upon degradation, produces substantially non-toxic products.

In some embodiments, the hydrogel includes a single biocompatible polymer or copolymer.

In other embodiments, the shape morphing hydrogel includes a photocrosslinkable hydrogel forming polymer and a photo-absorber and a photoinitiator dispersed within the hydrogel.

In some embodiments, the method includes forming multiple gradients in polymer concentration, polymer type, polymer swelling, polymer degradation and/or polymer cross-linking that extend through portions of the shape morphing hydrogel and allows the shape morphing hydrogel to undergo multiple and reversible different shape transformations.

Other embodiments described herein relate to a method of forming a construct. The method includes providing a biocompatible polymer-based hydrogel that includes at least one gradient in polymer concentration, polymer type, polymer swelling and/or polymer cross-linking, wherein the at least one gradient extends through at least one portion of hydrogel and allows the hydrogel to undergo multiple and reversible different shape transformations and seeding and culturing a layer of cells on a surface of the hydrogel.

In some embodiments, the hydrogel is firmly adhered on a surface of a glass plate by covalent bonding. The glass plate can include a surface that is modified with at least one molecule that facilitates binding hydrogel to the glass plate.

In some embodiments, the cell contractile forces or exogenous stimulation allows the construct to undergo controllable different shape transformations over time.

In some embodiments, the construct further includes a biocompatible polymer-based shape morphing layer that is conjugated to the cell condensate. The biocompatible polymer-based shape morphing layer including at least one gradient in polymer concentration, polymer type, polymer swelling, polymer degradation and/or polymer cross-linking density that extends through at least one portion of the preformed biocompatible polymer-based shape morphing layer shape.

In some embodiments, the construct includes a preformed biocompatible polymer-based shape morphing layer that includes at least one gradient in polymer concentration, polymer type, polymer swelling, polymer degradation and/or polymer cross-linking density that extends through at least one portion of the preformed biocompatible polymer-based shape morphing layer shape; and a photocurable and degradable cell-supporting microgel (MG) layer that is printed with cells. The MG layer can maintain the shape of the printed cells upon printing. The degradation of the MG layer and/or differential swelling and/or degradation preformed biocompatible polymer-based shape morphing layer during culture in tissue-specific formation conditions allows the construct to undergo controllable different shape transformations over time.

In some embodiments, ach microgel includes a plurality dual crosslinkable biodegradable natural polymer macromers crosslinked with a first agent. The microgels can be capable of being crosslinked with a second agent that is different than the first cross-linking agent. The microgels cross-linked with the second agent can form a free-standing structure, such as a tissue construct, with a defined shape.

The dual cross-linkable natural polymer macromers can be any natural polymer or oligomer that includes a functional group (e.g., a carboxylic group) that can be further polymerized. Examples of natural polymers or oligomers are saccharides (e.g., mono-, di-, oligo-, and poly-saccharides), such as glucose, galactose, fructose, lactose and sucrose, collagen, gelatin, glycosaminoglycans, poly(hyaluronic acid), poly(sodium alginate), hyaluronan, alginate, heparin and agarose.

In some embodiments, the dual cross-linkable natural polymer macromer can include an acrylated and/or methacrylated natural polymer macromer. Acrylated and/or methacrylated natural polymer macromers can include saccharides (e.g., mono-, di-, oligo-, and poly-saccharides), such as glucose, galactose, fructose, lactose and sucrose, collagen, gelatin, glycosaminoglycans, poly(hyaluronic acid), poly(sodium alginate), hyaluronan, alginate, heparin and agarose that can be readily oxidized to form free aldehyde units.

The natural polymer macromer (e.g., alginate) can be acrylated or methacrylated by reacting an acryl group or methacryl with a natural polymer or oligomer to form the oxidized, acrylated or methacrylated natural polymer macromer (e.g., alginate). For example, oxidized alginate can be dissolved in a solution chemically functionalized with N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride to activate the carboxylic acids of alginate and then reacted with 2-amionethylmethacrylate to provide a plurality of methacrylate groups on the alginate.

The microgels can have a diameter less than about 500 μm, less than about 400 μm, or less than about 300 μm and include, for example, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 150,000, or 200,000 cells per microgel.

In some embodiments, the preformed hydrogel layer includes a hydrogel forming acrylated and/or methacrylated polymer macromers that are optionally oxidized. The acrylated and/or methacrylated polymer macromers can reversibly and ionically crosslinkable. The acrylated and/or methacrylated polymer macromers can also be photocrosslinkable, ionically crosslinkable, physically crosslinkable, pH crosslinkable, dual crosslinkable, and/or thermally crosslinkable.

In some embodiments, the acrylated and/or methacrylated polymer macromers include acrylated and/or methacrylated polysaccharides that are optionally oxidized.

In some embodiments, the photocurable and degradable cell-supporting microgel includes an acrylated and/or methacrylated alginate that is optionally oxidized.

In some embodiments, the preformed layer includes a mixture of an acrylated and/or methacrylated alginate that is optionally oxidized and acrylated and/or methacrylated gelatin.

In some embodiments, the printed photocurable and degradable cell-supporting microgel (MG) layer includes a plurality of printed cells. The cells can include any cells, such as, undifferentiated stem cells or progenitor cells with a cell lineage potential that corresponds to the desired tissue being engineered. The cells can be unipotent, oligopotent, multipotent, or pluripotent. In some embodiments, the cells are adult stem cells. The cells can be allogeneic or autologous. In particular embodiments, the cells include mesenchymal stem cells (MSCs).

In some embodiments, the shape morphing hydrogel layer includes a single biocompatible polymer or copolymer.

In some embodiments, preformed biocompatible polymer-based shape morphing layer includes a photocrosslinkable hydrogel forming polymer and a photo-absorber and a photoinitiator dispersed within the hydrogel.

In some embodiments, the preformed biocompatible polymer-based shape morphing layer includes a plurality of cells, the cells comprising progenitor cells, undifferentiated cells, differentiated cells, and/or cancer cells. For example, the plurality cells can include mesenchymal stem cells.

In some embodiments, the photocurable and degradable cell-supporting microgel can include an acrylated and/or methacrylated alginate that is optionally oxidized.

In other embodiments, the preformed layer can include a mixture of an acrylated and/or methacrylated alginate that is optionally oxidized and acrylated and/or methacrylated gelatin.

In some embodiments, the preformed biocompatible polymer-based shape morphing layer includes a single biocompatible polymer or copolymer.

In other embodiments, the preformed biocompatible polymer-based shape morphing layer includes gradient of polymer cross-linking density through the thickness of the layer that allows the shape morphing hydrogel to undergo one or more multiple, reversible, controllable and/or different shape transformations.

In some embodiments, the preformed biocompatible polymer-based shape morphing layer includes a photocrosslinkable hydrogel forming polymer and a photo-absorber and a photoinitiator dispersed within the hydrogel.

In some embodiments, the method further includes crosslinking the MG layer printed with the cell to enhance the mechanical stability of the MG layer.

In other embodiments, the method includes culturing the layered construct in a culture medium. The culture medium can include a cell differentiation medium.

In some embodiments, the hydrogel construct includes at least one an anisotropic property in crosslinking density, internal strain, and/or micro/macro-pores distribution.

In some embodiments, the MGs comprise jammed heterogenous natural or synthetic polymer macromer hydrogels. The MGs can include a photoinitiator (PI) and UV absorber.

In some embodiments, the composition is printed into 3D hydrogel (bio)constructs that are programmably reshaped into a defined shape.

In other embodiments, the composition is extrudable or printable into a defined shape.

In some embodiments, the composition is capable of being crosslinked to form a flow-resistant structure with the defined shape and with a gradient in crosslinking density that extends through at least one portion of the hydrogel. The gradient in crosslinking density can allowing the 3D hydrogel (bio)construct to undergo one or multiple, reversible, controllable and/or different shape transformations.

In some embodiments, the composition is cytocompatible and, upon degradation, produces a substantially non-toxic product.

In some embodiments, the MGs can have a flake morphology with an average diameter of about 10 μm to about 70 μm.

In some embodiments, the 3D hydrogel (bio)construct includes at least one an anisotropic property in crosslinking density, internal strain, and/or micro/macro-pores distribution.

In some embodiments, the MGs can include a photoinitiator (PI) and UV absorber.

In some embodiments, he gradient in crosslinking density allows the 3D hydrogel (bio)construct to undergo one or multiple, reversible, controllable and/or different shape transformations.

In some embodiments, the shape-morphing construct is cytocompatible and, upon degradation, producing substantially non-toxic product.

In some embodiments, the viscosity of the MGs decreases with increased shear and/or strain on the MGs and recovers after removal of the increased shear and/or strain. The increased shear and/or strain can be associated with printing the MFHs and the viscosity of the MGs recovering after printing the MGs to provide the 3D hydrogel (bio)construct with the defined shape.

In some embodiments, the cells can include progenitor cells, undifferentiated cells and/or differentiated cells. For example, the cells can include mesenchymal stem cells.

In some embodiments, the MGs can have a flake morphology with an average diameter of about 10 μm to about 70 μm.

In some embodiments, the composition includes a photoinitiator (PT).