Patent Application
20230285637
This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted concurrently herewith as the sequence listing text file entitled “Sequence Listing.TXT,” file size 892 bytes, created on May 20, 2021, as Appendix A. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e)(5).
The present disclosure relates to magnetically-aligned synthetic extracellular matrix (ECM) fibers within a hydrogel.
This section provides background information related to the present disclosure which is not necessarily prior art.
Stromal ECM provides manifold biophysical cues that direct both physiologic and pathologic cell behavior. A major component of stromal ECM is fibrous proteins (e.g., collagens, fibronectin, and elastin) that serve as cell-adhesive scaffolding and provide structural and mechanical support to a variety of tissues. Cells dynamically deposit, reorganize, and respond to the fibrous architecture of the ECM. Through contact guidance cues, fibrous protein structures direct a variety of morphogenetic processes including tenogenesis, branching morphogenesis, and angiogenesis. Fibrous proteins are also heavily implicated in initiating and directing invasion from primary tumors during breast cancer progression. Second harmonic generation (SHG) imaging has provided valuable insights into collagen architecture during morphogenesis and disease progression. With this insight, biomaterials recapitulating aligned fibrous architectures have been developed to model and direct such processes in vitro.
Purified biopolymers, such as type I collagen and fibrin, have been used to model stromal ECM, as both possess fibrous topography. However, polymerization of these materials under typical conditions produces hydrogels with isotropic fibrous architecture due to the stochastic nature of fibrillogenesis. To better model highly anisotropic fibrous architecture, such as that found in tendons and around primary breast tumors, several approaches to align collagen fibers have been developed. Early methods to align collagen gels take advantage of the slight negative charge of collagen to align fibers with powerful magnetic fields. More recently, a diversity of methods to align collagen gels have emerged, including flow-induced alignment, embedding of magnetic particles which are dragged through the gel with an external magnetic field, application of tensile forces via stretching, and fibroblast-mediated reorganization of fibers. These methods create highly anisotropic collagen gels and have been instrumental in investigating how aligned fibrous architecture influences cell behavior. However, purified biopolymers typically have limited orthogonal control of relevant biophysical cues. For example, increasing type I collagen gel concentration leads to commensurate increases in fiber density, stiffness, and ligand density. In contrast, synthetic hydrogels (e.g., polyethylene glycol (PEG), methacrylated gelatin, and functionalized dextran) offer enhanced orthogonal tunability of these physical properties. However, these amorphous hydrogels typically lack fibrous architecture.
Electrospinning offers a means to generate fibrous topography that closely recapitulates the geometry and length-scale of fibrous proteins found in stromal ECM. The electrospinning process uses a voltage gradient to draw solid fibers from a charged polymer solution. Previous work with polyvinyl alcohol (PVA), poly(lactic-co-glycolic acid) (PLGA), and dextran methacrylate has shown that cell migration on electrospun, synthetic fiber matrices captures key aspects of cell migration in fibrous natural ECM proteins like type I collagen. Recent work has established means to generate suspended fiber segments within amorphous synthetic hydrogels. These hydrogel composites enable cell studies within topographically complex fibrous environments in which fiber density and stiffness can be orthogonally controlled. However, such composites rely on encapsulating fiber segments within a hydrogel, resulting in a random distribution of embedded fibers. Accordingly, anisotropically-aligned synthetic ECM fibers within a hydrogel are desired.
This section provides a general summary of the disclosure, and is not a comprehensive disclosure of its full scope or all of its features.
In various aspects, the current technology provides a composite material including a hydrogel matrix having a three-dimensional geometry and fibers embedded and substantially uniformly distributed within the hydrogel matrix, wherein the fibers have a substantially circular cross-sectional geometry and are anisotropically aligned.
In one aspect, the hydrogel matrix includes a polysaccharide, a polypeptide, a synthetic polymer, or combinations thereof.
In one aspect, the hydrogel matrix includes a polysaccharide selected from the group consisting of dextran, starch, cellulose, alginate, hyaluronic acid, chitosan, chitin, pectin, derivatives thereof, and combinations thereof.
In one aspect, the hydrogel matrix includes a polypeptide selected from the group consisting of collagen, fibronectin, gelatin, derivatives thereof, and combinations thereof.
In one aspect, the hydrogel matrix includes a synthetic polymer, the synthetic polymer being PEG.
In one aspect, the fibers include a polysaccharide, a synthetic polymer, or a combination thereof.
In one aspect, the fibers include a polysaccharide selected from the group consisting of dextran, starch, cellulose, alginate, hyaluronic acid, chitosan, chitin, pectin, derivatives thereof, and combinations thereof.
In one aspect, the fibers comprise a vinyl sulfone functionalized dextran.
In one aspect, the fibers include a synthetic polymer selected from the group consisting of polyvinyl alcohol (PVA), polyethylene oxide (PEO), poly(hydroxyethyl methacrylate) (PHEMA), polyvinylpyrrolidone (PVP), polyimide (PI), polyacrylate (PA), polyurethane (PU), a polyester, and combinations thereof.
In one aspect, the composite material further includes magnetic nanoparticles at least partially embedded within the fibers.
In one aspect, the magnetic nanoparticles are coated with a biomaterial.
In one aspect, the fibers are embedded within the hydrogel matrix at a fiber density of greater than or equal to about 1 v/v % to less than or equal to about 6 v/v %.
In one aspect, the hydrogel matrix is crosslinked with a peptide crosslinker.
In one aspect, the fibers are crosslinked with a peptide crosslinker.
In one aspect, cell adhesion promoters are coupled to at least one of the hydrogel matrix or the fibers.
In one aspect, the composite material further includes cells embedded within the hydrogel matrix.
In one aspect, the fibers have a fiber length of greater than or equal to about 100 μm to less than or equal to about 150 μm.
In various aspects, the current technology also provides a method of producing a composite material, the method including preparing a suspension of electrospun fibers having a substantially circular cross-sectional geometry, where at least a portion of the electrospun fibers have at least one magnetic nanoparticle at least partially embedded therein. The method comprises combining the suspension with a hydrogel precursor solution including polymer molecules to form a composite suspension; and crosslinking the polymer molecules within a magnetic field to form the composite material. The composite material formed from such a method thus includes the electrospun fibers embedded and substantially uniformly distributed within a three-dimensional hydrogel matrix formed from the polymer molecules and the electrospun fibers are anisotropically aligned.
In one aspect, the method further includes preparing the electrospun fibers by electrospinning a fiber mat having magnetic nanoparticles embedded within a plurality of continuous fibers; disposing a photomask over the fiber mat, the photomask including a plurality of apertures; applying ultraviolet (UV) light through the plurality of apertures to crosslink the continuous fibers at region exposed by way of the apertures and form the electrospun fibers; isolating the electrospun fibers from portions of the fiber mat that were blocked from being crosslinked by the photomask; and suspending the electrospun fibers in a solvent.
In one aspect, the apertures of the photomask have a diameter of greater than or equal to about 75 μm to less than or equal to about 250 μm and the electrospun fibers have a fiber length of greater than or equal to about 100 μm to less than or equal to about 150 μm.
In one aspect, the electrospinning is performed with a composition including a fiber precursor, a photoinitiator, and the magnetic nanoparticles at a density of greater than or equal to about 2.5 mg/mL to less than or equal to about 10 mg/mL.
In one aspect, the composite suspension is formed in, or transferred to, a sealed or water-tight container, and the method further includes periodically rotating the water-tight container about 180° to provide a substantially uniform distribution of the electrospun fibers within the composite suspension until the crosslinking is complete.
In one aspect, the method further includes generating the magnetic field between two magnets.
In one aspect, the magnetic field is characterized by a magnet flux density of greater than or equal to about 5 mT to less than or equal to about 1 T.
In one aspect, the method further includes adding a plurality of cells to the composite suspension.
In various aspects, the current technology additionally provides a method of preparing an implant that may be used for repairing a tissue having a damaged region in a subject in need thereof. The method may include growing cells of the tissue in a composite material until an artificial tissue is formed and implanting the composite material with the artificial tissue into the damaged region of the tissue. The composite material includes a hydrogel matrix having a three-dimensional geometry and fibers embedded and substantially uniformly distributed within the hydrogel matrix, wherein the fibers have a substantially circular cross-sectional geometry and a fiber length of greater than or equal to about 100 μm to less than or equal to about 150 μm and the fibers are anisotropically aligned.
In one aspect, the tissue is a tendon and the cells include tendon fibroblasts.
In one aspect, the tissue is a heart and the cells include cardiomyocytes.
In various aspects, the current technology further provides a method of modeling a cellular environment, the method including growing cells in a composite material until the cellular environment is formed, wherein the composite material includes a hydrogel matrix having a three-dimensional geometry and fibers embedded and substantially uniformly distributed within the hydrogel matrix, wherein the fibers have a substantially circular cross-sectional geometry and a fiber length of greater than or equal to about 100 μm to less than or equal to about 150 μm and the fibers are anisotropically aligned.
In one aspect, the cellular environment is breast tissue and the cells include breast tissue cells.
In one aspect, the cellular environment is vasculature and the cells include endothelial cells.
In one aspect, the cellular environment is cardiac tissue and the cells include cardiomyocytes, epicardial cells, cardiac fibroblasts, endothelial cells, endocardial cells, or combinations thereof.
In one aspect, the cellular environment is connective tissue and the cells include fibroblasts.
Further areas of applicability will become apparent from the description provided herein. The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure.
The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure.
Corresponding reference numerals indicate corresponding parts throughout the several views of the drawings.
Example embodiments are provided so that this disclosure will be thorough, and will fully convey the scope to those who are skilled in the art. Numerous specific details are set forth such as examples of specific compositions, components, devices, and methods, to provide a thorough understanding of embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, that example embodiments may be embodied in many different forms and that neither should be construed to limit the scope of the disclosure. In some example embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail.
The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” may be intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms “comprises,” “comprising,” “including,” and “having,” are inclusive and therefore specify the presence of stated features, elements, compositions, steps, integers, operations, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Although the open-ended term “comprising,” is to be understood as a non-restrictive term used to describe and claim various embodiments set forth herein, in certain aspects, the term may alternatively be understood to instead be a more limiting and restrictive term, such as “consisting of” or “consisting essentially of.” Thus, for any given embodiment reciting compositions, materials, components, elements, features, integers, operations, and/or process steps, the present disclosure also specifically includes embodiments consisting of, or consisting essentially of, such recited compositions, materials, components, elements, features, integers, operations, and/or process steps. In the case of “consisting of,” the alternative embodiment excludes any additional compositions, materials, components, elements, features, integers, operations, and/or process steps, while in the case of “consisting essentially of,” any additional compositions, materials, components, elements, features, integers, operations, and/or process steps that materially affect the basic and novel characteristics are excluded from such an embodiment, but any compositions, materials, components, elements, features, integers, operations, and/or process steps that do not materially affect the basic and novel characteristics can be included in the embodiment.
Any method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed, unless otherwise indicated.
When a component, element, or layer is referred to as being “on,” “engaged to,” “connected to,” or “coupled to” another element or layer, it may be directly on, engaged, connected or coupled to the other component, element, or layer, or intervening elements or layers may be present. In contrast, when an element is referred to as being “directly on,” “directly engaged to,” “directly connected to,” or “directly coupled to” another element or layer, there may be no intervening elements or layers present. Other words used to describe the relationship between elements should be interpreted in a like fashion (e.g., “between” versus “directly between,” “adjacent” versus “directly adjacent,” etc.). As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.
Although the terms first, second, third, etc. may be used herein to describe various steps, elements, components, regions, layers and/or sections, these steps, elements, components, regions, layers and/or sections should not be limited by these terms, unless otherwise indicated. These terms may be only used to distinguish one step, element, component, region, layer or section from another step, element, component, region, layer or section. Terms such as “first,” “second,” and other numerical terms when used herein do not imply a sequence or order unless clearly indicated by the context. Thus, a first step, element, component, region, layer or section discussed below could be termed a second step, element, component, region, layer or section without departing from the teachings of the example embodiments.
Spatially or temporally relative terms, such as “before,” “after,” “inner,” “outer,” “beneath,” “below,” “lower,” “above,” “upper,” and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. Spatially or temporally relative terms may be intended to encompass different orientations of the device or system in use or operation in addition to the orientation depicted in the figures.
Throughout this disclosure, the numerical values represent approximate measures or limits to ranges to encompass minor deviations from the given values and embodiments having about the value mentioned as well as those having exactly the value mentioned. Other than in the working examples provided at the end of the detailed description, all numerical values of parameters (e.g., of quantities or conditions) in this specification, including the appended claims, are to be understood as being modified in all instances by the term “about” whether or not “about” actually appears before the numerical value. “About” indicates that the stated numerical value allows some slight imprecision (with some approach to exactness in the value; approximately or reasonably close to the value; nearly). If the imprecision provided by “about” is not otherwise understood in the art with this ordinary meaning, then “about” as used herein indicates at least variations that may arise from ordinary methods of measuring and using such parameters. For example, “about” may comprise a variation of less than or equal to 5%, optionally less than or equal to 4%, optionally less than or equal to 3%, optionally less than or equal to 2%, optionally less than or equal to 1%, optionally less than or equal to 0.5%, and in certain aspects, optionally less than or equal to 0.1%.
In addition, disclosure of ranges includes disclosure of all values and further divided ranges within the entire range, including endpoints and sub-ranges given for the ranges. As referred to herein, ranges are, unless specified otherwise, inclusive of endpoints and include disclosure of all distinct values and further divided ranges within the entire range. Thus, for example, a range of “from A to B” or “from about A to about B” is inclusive of A and B.
Example embodiments will now be described more fully with reference to the accompanying drawings.
The current technology provides a composite material comprising a hydrogel that is three-dimensional and reinforced with fibers, i.e., the fibers are embedded within a three-dimensional hydrogel matrix. The fibers are uniformly or substantially uniformly distributed throughout the hydrogel matrix and are anisotropically aligned by way of embedded magnetic nanoparticles. By “substantially uniformly distributed,” it is meant that the distribution of the fibers may slightly vary from one portion of the hydrogel matrix to another by less than or equal to about 10 v/v %. The composite material mimics an ECM and is capable of directing morphogenetic processes, supporting mechanical loads, and facilitating cell migration. The current technology also provides methods of making and using the composite material.
With reference to
The fiber 30 comprises a polymer, which may be a polysaccharide, a synthetic polymer, or a combination thereof. Non-limiting examples of polysaccharides include dextran, starch, cellulose, alginate, hyaluronic acid, chitosan, chitin, pectin, derivatives thereof, and combinations thereof. Non-limiting examples of synthetic polymers include PVA, PEO (including PEG in certain variations), PHEMA, PVP, PI, PA (e.g., polyacrylic acid (PAA)), PU, polyesters (including polycaprolactone (PCL), polylactic acid (PLA), and PLGA), derivatives thereof, and combinations thereof. The derivatives of the polysaccharides and synthetic polymers include base polysaccharides that are modified, e.g., coupled, with a functional group, a cell adhesion promoter (such as cell adhesion peptides), a light-emitting marker, or combinations thereof. Non-limiting examples of functional groups include vinyl sulfone, methacrylates, acrylates, diacrylates, norbornenes, maleimides, and combinations thereof. Non-limiting examples of cell adhesion promoters include peptides, such as arginylglycylaspartic acid (RGD peptide) and/or collagen integrin-binding peptides (e.g., having the amino acid sequence GFOGER (SEQ ID NO:1)); polypeptides (which may be full length polypeptides), such as collagen, fibronectin, laminin, and/or gelatin; derivatives thereof; and combinations thereof. Non-limiting examples of light-emitting markers include cyanine dyes, coumarins, rhodamines, xanthenes, quantum dots, and combinations thereof.
Each fiber 30 has an individual and independent fiber length LF, when measured from a first end 34 to an opposing second end 36, optionally of greater than or equal to about 100 micrometers (μm) to less than or equal to about 150 μm, such as fiber lengths LF of about 100 μm, about 105 μm, about 110 μm, about 115 μm, about 120 μm, about 125 μm, about 130 μm, about 135 μm, about 140 μm, about 145 μm, and about 150 μm. Each fiber 30 also has an individual and independent fiber diameter DF of greater than or equal to about 0.5 μm to less than or equal to about 5 μm, such as fiber diameters DF of about 0.5 μm, about 0.75 μm, about 1 μm, about 1.25 μm, about 1.5 μm, about 1.75 μm, about 2 μm, about 2.25 μm, about 2.5 μm, about 2.75 μm, about 3 μm, about 3.25 μm, about 3.5 μm, about 3.75 μm, about 4 μm, about 4.25 μm, about 4.5 μm, about 4.75 μm, and about 5 μm. In some aspects, the fiber length LF approximates the size of a cell to be cultured in the composite material, where “approximates the size of a cell” means that the length LF is within about 25% of the diameter of the cell. In other aspects, the fiber lengths LF can be shorter than about 100 μm and/or longer than the about 150 μm. As shown in
The at least one magnetic nanoparticle 32 comprises Fe2O3, Fe3O4, or a combination thereof and may be a super paramagnetic iron oxide nanoparticle (SPION). However, it is understood that the magnetic nanoparticle material is not limiting and that the at least one magnetic nanoparticle 32 may include a compound that is not based on iron. The at least one magnetic nanoparticle 32 has a magnetic nanoparticle diameter DMNP of greater than or equal to about 1 nm to less than or equal to about 250 nm or greater than or equal to about 1 nm to less than or equal to about 150 nm, such as magnetic nanoparticle diameters DMNP of about 1 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 210 nm, about 220 nm, about 230 nm, about 240 nm, and about 250 nm.
Because the at least one magnetic nanoparticle 32 is at least partially embedded within the fiber 30, the cytotoxicity of the at least one magnetic nanoparticle 32 is less than the cytotoxicity of comparative magnetic nanoparticles that are not at least partially embedded within the fiber 30 or another material. Nonetheless, in some aspects, the at least one magnetic nanoparticle 32 is at least partially coated, including completely coated, with a biomaterial that further decreases cytotoxicity. Non-limiting examples of suitable biomaterials include PVP, PEG, PEI, PLGA, PLA, PCL, polypyrrole (PPy), poly(N-vinylpyrrolidine), polyanhydrides, poly(N-isopropylacrylamide) (NIPAAm), and combinations thereof.
With renewed reference to
The polymer composition is a polymer solution or a polymer melt comprising a fiber precursor, the fiber precursor being the polysaccharide, the synthetic polymer, or the combination thereof, as discussed above with reference to
The polymer composition may also comprise the magnetic nanoparticles (optionally coated with the biomaterial) at a concentration of greater than or equal to about 1 mg/mL to less than or equal to about 15 mg/mL or greater than or equal to about 2.5 mg/mL to less than or equal to about 10 mg/mL, including concentrations of about 1 mg/mL, about 1.5 mg/mL, about 2 mg/mL, about 2.5 mg/mL, about 3 mg/mL, about 3.5 mg/mL, about 4 mg/mL, about 4.5 mg/mL, about 5 mg/mL, about 5.5 mg/mL, about 6 mg/mL, about 6.5 mg/mL, about 7 mg/mL, about 7.5 mg/mL, about 8 mg/mL, about 8.5 mg/mL, about 9 mg/mL, about 9.5 mg/mL, about 10 mg/mL, about 10.5 mg/mL, about 11 mg/mL, about 11.5 mg/mL, about 12 mg/mL, about 12.5 mg/mL, about 13 mg/mL, about 13.5 mg/mL, about 14 mg/mL, about 14.5 mg/mL, and about 15 mg/mL.
The polymer composition also comprises a photoinitiator. Non-limiting examples of suitable photoinitiators include lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), 2-hydroxy-1-[4-hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure 2959), benzophenone, 2,2-dimethoxy-2-phenylacetophenone, 2,2-dimethoxy-1,2-diphenylethan-1-one, 2-hydroxy-2-methyl-1-phenylpropanone, 1-hydroxy-cyclohexylphenylketone, isopropylthioxanthone, 2-ethylhexyl-(4-N,N-dimethyl amino)benzoate, ethyl-4-(dimethylamino)benzoate, and combinations thereof. The photoinitiator is included in the polymer composition at a concentration of greater than or equal to about 1 v/v % to less than or equal to about 20 v/v %.
The polymer composition can also comprise a visual marker, such as at least one of the light-emitting markers described herein. The visual marker may particularly be included when the polymer is not modified with a light-emitting marker. The visual marker is included in the polymer composition at a concentration of greater than or equal to about 1 v/v % to less than or equal to about 10 v/v %.
After the fiber mat is formed, the method of preparing the fibers comprises disposing a photomask over the fiber mat. The photomask is a UV light-blocking sheet or plate defining a plurality of apertures that are transparent to UV light. Each aperture of the plurality has a diameter of greater than or equal to about 25 μm to less than or equal to about 500 μm or greater than or equal to about 75 μm to less than or equal to about 250 μm, including diameters of about 25 μm, about 50 μm, about 75 μm, about 100 μm, about 125 μm, about 150 μm, about 175 μm, about 200 μm, about 225 μm, about 250 μm, about 275 μm, about 300 μm, about 325 μm, about 350 μm, about 375 μm, about 400 μm, about 425 μm, about 450 μm, about 475 μm, and about 500 μm. The apertures can have a geometrical shape selected from the group consisting of a square, a rectangle, a circle, an oval, a triangle, a pentagon, a hexagon, and combinations thereof. However, it is understood that the geometrical shape of the apertures is not limited. An exemplary photomask is described in the below example with reference to
The method then comprises crosslinking portions of the fiber mat to form fiber segments, which are also referred to as “electrospun fibers” or “fibers,” by applying a UV light through the apertures of the photomask onto portions of the fiber mat that are exposed to the UV light by way of the apertures. Due to the presence of the photomask, only the portions of the fiber mat that are exposed to the UV light are crosslinked, resulting in the formation of a photopatterned, crosslinked fiber mat having segmented continuous fibers produced by the electrospinning. Accordingly, the method next comprises removing portions of the continuous fibers that were blocked from being crosslinked by way of the photomask to form the fibers (as described with reference to
In an alternative method, the continuous fibers are processed into the electrospun fibers by fracturing the continuous fibers using shear forces.
With continued reference to
As shown in block 16, the method 10 then comprises combining the suspension with a hydrogel precursor solution to form a composite suspension. The hydrogel precursor solution comprises a hydrogel precursor, a crosslinking agent, and a hydrogel solvent. The hydrogel precursor comprises polymer molecules, such as polysaccharides, synthetic polymers, polypeptides, or combinations thereof. Non-limiting examples of polysaccharides include dextran, starch, cellulose, alginate, hyaluronic acid, chitosan, chitin, pectin, derivatives thereof, and combinations thereof. Non-limiting examples of synthetic polymers include PEO and PEG. However, in alternative aspects, the synthetic polymer can include PVA, PHEMA, PVA, PVP, PI, PA (e.g., PAA), PU, polyesters (including PCL, PLA, and PLGA), derivatives thereof, and combinations thereof, which can be cast into a polymer matrix (instead of a hydrogel matrix) using an organic solvent. Non-limiting examples of polypeptides include collagen, fibronectin, gelatin, derivatives thereof, and combinations thereof. The derivatives of the polysaccharides, synthetic polymers, and polypeptides include base polymers (polysaccharides, synthetic polymers, and polypeptides) that are modified with a functional group which facilitates crosslinking and hydrogel formation. Non-limiting examples of functional groups include vinyl sulfone, methacrylates, acrylates, diacrylates, norbornenes, maleimides, and combinations thereof. In some aspects, the hydrogel precursor is coupled to the cell adhesion promoter, as described above with references to the fibers. Accordingly, the cell adhesion promoter can be coupled to at least one of the fibers or the hydrogel precursor. Non-limiting examples of the crosslinking agent include peptide crosslinkers, e.g., VPMS crosslinker at a concentration of greater than or equal to about 2 mM to less than or equal to about 30 mM, dithiolated or diacrylated PEG chains, or dithiothreitol (DTT). The hydrogel precursor solution can also include at least one additive, such as an additional functional group, e.g., heparin binding peptide at a concentration of greater than or equal to about 750 μM to less than or equal to about 20 mM, as a non-limiting example. Non-limiting examples of the solvent include water, PBS, cell culture medium, and combinations thereof. The composite suspension is prepared in, or transferred to, a sealable (e.g., with a lid, cap, or the like) water-tight container that can be inverted without leaking.
The amount of the suspension added to the hydrogel precursor solution depends on the fiber concentration in the suspension and the level of fibers desired to be included in the composite material. Therefore, the fiber density of the composite material is tunable by adding a predetermined amount of the fiber suspension to the hydrogel precursor solution, such that a desired fiber density in the composite material is achieved. Alternatively, the fiber concentration of the fiber solution can be adjusted to a predetermined level so that a precalculated amount of the fiber solution can be added to the hydrogel precursor solution, such that a desired fiber density in the composite material is achieved. In some aspects, the fiber density in the composite suspension prepared by combining the suspension with the hydrogel precursor solution is greater than or equal to about 0.5 v/v % to less than or equal to about 10 v/v % or greater than or equal to about 1 v/v % to less than or equal to about 6 v/v %, including fiber densities of about 0.5 v/v %, about 1 v/v %, about 1.5 v/v %, about 2 v/v %, about 2.5 v/v %, about 3 v/v %, about 3.5 v/v %, about 4 v/v %, about 4.5 v/v %, about 5 v/v %, about 5.5 v/v %, about 6 v/v %, about 6.5 v/v %, about 7 v/v %, about 7.5 v/v %, about 8 v/v %, about 8.5 v/v %, about 9 v/v %, about 9.5 v/v %, and about 10 v/v %.
In some aspects, the composite suspension further comprises a plurality of cells to be cultured and grown in the composite material. The cells can be pluripotent, differentiated, or undifferentiated cells, such as cancer cells. Moreover, the cells can be primary cells obtained from a subject or immortalized cells, including immortalized cells of established cell lines that are known in the art. Exemplary pluripotent cells include stem cells, embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, and epithelial stem cells. Differentiated cells include epidermal cells, endothelial cells, fibroblasts, tissue specific cells, and organ specific cells, such as cells normally found in a breast, lung, liver, heart (e.g., cardiomyocytes, epicardial cells, cardiac fibroblasts, endothelial cells, and endocardial cells), brain, pancreas, prostate, cervix, ovary, bladder, gall bladder, kidney, colon, stomach, oral cavity, skin, tendon (i.e., tenocytes, also referred to as tendon fibroblasts), cancer cells thereof, and combinations thereof, as non-limiting examples. The cells can be added to the composite material in at least one of the suspension or the hydrogel precursor.
In additional aspects, the composite suspension further comprises an adjunct agent, such as a growth factor, serum, antimicrobial (e.g., antibiotic, antiviral, antifungal), amino acid, and combinations thereof, as non-limiting examples.
An exemplary composite suspension 40 is shown in
Referring back to
The magnetic field is generated by positioning a first magnet and a second magnet apart from each other. The first and second magnets magnetically communicate to form the magnetic field, which is characterized by a magnetic flux density of greater than or equal to about 1 mT to less than or equal to about 1 T, greater than or equal to about 1 mT to less than or equal to about 500 mT, or greater than or equal to about 5 mT to less than or equal to about 150 mT, such as about 1 mT, about 5 mT, about 10 mT, about 15 mT, about 20 mT, about 25 mT, about 30 mT, about 35 mT, about 40 mT, about 45 mT, about 50 mT, about 55 mT, about 60 mT, about 65 mT, about 70 mT, about 75 mT, about 80 mT, about 85 mT, about 90 mT, about 95 mT, about 100 mT, about 105 mT, about 110 mT, about 115 mT, about 120 mT, about 125 mT, about 130 mT, about 135 mT, about 140 mT, about 145 mT, about 150 mT, about 200 mT, about 250 mT, about 300 mT, about 350 mT, about 400 mT, about 450 mT, about 500 mT, about 550 mT, about 600 mT, about 650 mT, about 700 mT, about 750 mT, about 800 mT, about 850 mT, about 900 mT, about 950 mT, or about 1 T. A predetermined magnetic flux density can be achieved by adjusting the distance between the magnets. For example, the magnetic flux density increases as the magnets are brought closer together and decreases as the magnets are moved apart from each other. The strength of the magnet also may influence the distance provided between the magnets. As a non-limiting example, the magnets can be neodymium magnets.
The composite suspension is disposed between the first and second magnets in order to crosslink the polymer molecules in the presence of the magnetic field. When in the presence of the magnetic field, the magnetic nanoparticles embedded within the fibers are pulled in the direction of the magnetic field. As the magnetic nanoparticles are pulled under the magnetic field, they apply aligning forces to their corresponding fibers. As a result, the fibers align in the direction of the magnetic field. Therefore, the fibers transition from an isotropic orientation to an anisotropic orientation.
A system for applying the magnetic field and periodically rotating the composite suspension is described in the below example with reference to
Referring again to
In some variations, the crosslinking of the polymer molecules in the composite suspension in the presence of the magnetic field (as shown in block 18) and the forming the composition material (as shown in block 22) are performed in vivo, and the method 10 does not include periodically rotating the composite suspension (as shown in block 20). In these variations, the magnetic field is provided by a magnetic resonance imaging (MRI) machine or other clinically available magnetic field generator.
A composite material 50 formed from the method 10 is shown in
The current technology also provides a method of preparing a tissue implant to repair a tissue having a damaged region or defect in a subject in need thereof, the subject being a human or non-human mammal, a fish, a bird, a reptile, or an amphibian. The method comprises growing cells of the tissue in the composite material described herein until an artificial tissue is formed. The method then comprises implanting the composite material including the artificial tissue into or on the damaged region of the tissue. The tissue is non-limiting and can be, for example, a tendon (wherein the cells comprise tendon fibroblasts), a heart (wherein the cells comprise cardiomyocytes), or a pancreas (wherein the cells comprise islet cells (alphas cells, beta cells, or a combination thereof), delta cells, pancreatic polypeptide cells (PP cells), or combinations thereof). In some aspects, the damaged region or defect is a hole or a tear that is congenial, a result of an accidental injury, or an aspect of a medical procedure or surgery. The implanting can be performed by creating an incision in the subject and manually disposing the composite material onto the damaged region of the tissue as a patch or by injecting the composite material onto the damaged region of the tissue by way of a syringe.
The current technology also provides a method of modeling a cellular environment, for example, for pathophysiologic studies. The method comprises growing cells in the composite material described herein until the cellular environment is formed. In some aspects, the cellular environment is normal breast tissue (and the cells comprises normal breast tissue cells), neoplastic tissue (and the cells comprise breast cancer cells), vasculature (and the cells comprise endothelial cells), cardiac tissue (and the cells comprise cardiomyocytes, epicardial cells, cardiac fibroblasts, endothelial cells, endocardial cells, or combinations thereof), or connective tissue (and the cells comprise fibroblasts). In certain aspects, the cellular environment is propagated until an artificial organ or organoid is formed.
The current technology yet further provides a method of repairing a tissue having a damaged region or defect in a subject in need thereof, the subject being a human or non-human mammal, a fish, a bird, a reptile, or an amphibian. The method comprises implanting the composite material into or on the damaged region of the tissue, wherein the composite material does not include cells prior to implantation, i.e., the composite material is cell-free. The tissue is non-limiting and can be, for example, a tendon or ligament or a portion of an organ, such as a heart, breast, lung, liver, brain, pancreas, prostate, cervix, ovary, bladder, gall bladder, kidney, colon, stomach, oral cavity, or skin, as non-limiting examples. In some aspects, the damaged region or defect is a hole or a tear that is congenial, a result of an accidental injury, or an aspect of a medical procedure or surgery. The implanting can be performed by surgically or endovascularly disposing the anisotropically-aligned composite material onto the damaged region of the tissue (e.g., as a patch). Alternatively, the implanting can be performed by injecting the hydrogel composite precursor solution onto or into the damaged region of the tissue as a gel or hydrogel via a syringe. Given the non-contact nature of magnetic alignment, the hydrogel composite precursor solution, e.g., the composite suspension, can be injected onto or into the damaged region and then aligned, for example, with the use of a MRI machine or any other clinically available magnetic field generator. In some aspects, cells from the subject's tissue adjacent to the damaged region or defect enter and infiltrate the patch or hydrogel composite precursor solution.
Embodiments of the present technology are further illustrated through the following non-limiting example.
Fibrous ECM proteins provide mechanical structure and adhesive scaffolding to resident cells within stromal tissues. Aligned ECM fibers play an important role in directing morphogenetic processes, supporting mechanical loads, and facilitating cell migration. Various methods have been developed to align matrix fibers in purified biopolymer hydrogels, such as type I collagen, including flow-induced alignment, uniaxial tensile deformation, and magnetic particles. However, purified biopolymers have limited orthogonal tunability of biophysical cues including stiffness, fiber density, and fiber alignment. Here, synthetic, cell-adhesive fiber segments of the same length-scale as stromal fibrous proteins are generated through electrospinning. SPIONs embedded in synthetic fiber segments enable magnetic field-induced alignment of fibers within an amorphous bulk hydrogel. It is found that SPION density and magnetic field strength jointly influence fiber alignment and identify conditions to control the degree of alignment. Tuning fiber length allows the alignment of dense fibrous hydrogel composites without fiber entanglement or regional variation in the degree of alignment. Functionalization of fiber segments with cell adhesive peptides induces tendon fibroblasts to adopt a uniaxial morphology akin to within native tendon. Furthermore, the utility of this hydrogel composite to direct multicellular migration from MCF10A spheroids is demonstrated, and it is found that fiber alignment prompts invading multicellular strands to separate into disconnected single cells and multicellular clusters. These magnetic fiber segments can be readily incorporated into other natural and synthetic hydrogels and aligned with inexpensive and easily accessible rare earth magnets without the need for specialized equipment. Three-dimensional hydrogel composites where stiffness/crosslinking, fiber density, and fiber alignment can be orthogonally tuned provide insights into morphogenetic and pathogenic processes that involve matrix fiber alignment and enable systematic investigation of the individual contribution of each biophysical cue to cell behavior.
In this example, SPIONs are embedded within synthetic fiber segments during the electrospinning process to enable fiber alignment under an externally applied magnet field. Degree of fiber alignment within a three-dimensional amorphous hydrogel proves sensitive to SPION density as well as the strength of the imposed magnetic field. Computational modeling of the magnetic field reveals dependence upon magnet placement, where hydrogels appropriately positioned during crosslinking can achieve homogeneous alignment of constituent fibers. It is found that fiber length influences the frequency of fiber entanglement as a function of fiber density during magnetic alignment. Finally, the use of magnetic fiber alignment within the hydrogel composite system to control encapsulated tendon fibroblast (tenocyte) alignment and elicit directional migration of a breast epithelial cell line is demonstrated. Interestingly, it is found that fiber alignment not only biases epithelial cell migration direction, but also promotes cell-cell breakage events leading to a switch in three-dimensional migration phenotype.
Reagents: All reagents are purchased from Sigma Aldrich and used as received, unless otherwise stated.
Synthesis of modified DVS: Dextran is functionalized with vinyl sulfone pendant groups using a previously described protocol. Briefly, linear high molecular weight dextran (MW 86,000 Da; MP Biomedicals) is reacted with pure divinyl sulfone (Fisher) under basic conditions (pH 13.0). Functionalization is terminated through pH adjustment to 5.0 with hydrochloric acid. Reaction products are dialyzed against milli-Q water for 3 days, with water changed twice daily. Purified products are then lyophilized for 3 days and reconstituted at 100 mg mL−1 in a Michael-type addition buffer (MTAB; 1 N NaOH, 1 M HEPES, 1 mg mL−1 phenol red in milli-Q water).
DVS fiber segment fabrication: DVS is dissolved at 0.6 g ml−1 in a 1:1 mixture of milli-Q water and DMF. SPIONs with or without PVP coating (US Research Nanomaterials) are added at 2.5, 5, or 10 w/v %. LAP photoinitiator (10 v/v %), and methacrylated rhodamine (2.5 v/v %) (Polysciences, Inc.) are added to the solution to facilitate photoinitiated crosslinking and fluorescent visualization, respectively. Polymer solutions are electrospun in a humidity-controlled glove box held at 21° C. and 30-35% relative humidity. Electrospinning is performed at 0.25 ml hr−1 flow rate, 7 cm gap distance, and −9.0 kV voltage onto a grounded copper collective surface. Fibers are collected on glass cover slides and crosslinked under ultraviolet light (100 mW cm−2) for 20 seconds. Custom-fabricated chrome photomasks are placed over fiber mats during UV photocrosslinking to control fiber length. Fiber mats are detached from cover slides into milli-Q water and broken into individual fiber segments. Fiber segments are purified through a series of centrifugation steps to remove uncrosslinked polymer and entangled fibers before resuspension in MTAB at 10 v/v %. Prior to encapsulation within bulk hydrogels, fibers are coupled with 2.0 mM RGD (CGRGDS (SEQ ID NO:2); CPC Scientific) via Michael-type addition to enable eventual cell adhesion.
Magnetic gelation chamber fabrication: The magnet housing apparatus is created with solid ½″ aluminum and 1.00″×2.00″ T-slotted aluminum. Two N52 neodymium magnets (K&J Magnetics) are housed in carriages made from solid aluminum, securing the magnets on either side with a hole removed at the face of the magnet. The carriages are attached to the T-slotted aluminum rails on the base of the setup, which aligns the carriages and allows them to controllably slide along the rails with a crankshaft. A three-dimensional printed clamp is designed to hold a petri dish containing fiber-reinforced hydrogel composites within the center of the magnet axis. To prevent encapsulated fibers and cells from settling during hydrogel crosslinking, an Arduino-controlled stepper motor with a three-dimensional-printed grip is programmed to flip the petri dish 180° every 20 seconds.
Computation visualization of magnet field lines: COMSOL Multiphysics software is used to quantify magnetic flux densities and visualize field lines between magnets. A three-dimensional stationary study modeling magnetic fields generated without current (permanent magnet) is performed. Two cylinders (1.905×3.81 cm) modeling the two magnets are placed within a 15-25 cm radius sphere to model air impedance. Surface flux density of the two cylinders is set to 661.9 mT, the innate surface field of N52 neodymium magnets. A single slice in the X-Z plane is generated to visualize magnetic flux density around and between the magnets. White arrows are overlaid to visualize magnetic field lines with the arrow size logarithmically proportional to strength of magnet flux along the field line. To generate one-dimensional plots of magnetic flux density in the Z-direction across various magnet spacings, flux density values are extracted from a path along the magnet axis.
Hydrogel formation and fiber alignment: DVS gels are formed via an analogous click reaction at 3.5 w/v % with 9.64 mM VPMS crosslinker and heparin binding peptide (2 mM). All hydrogel precursor solutions are made in PBS. To create fibrous hydrogels, a defined stock solution (10 v/v %) of suspended fiber segments in MTAB is mixed into hydrogel precursor solutions prior to gelation. Via controlling the dilution of the fiber suspension, fiber density is tuned at a constant hydrogel weight percentage and bulk stiffness. Hydrogel precursor solutions are injected into 5 mm in diameter polydimethylsiloxane gaskets and crosslinked at 37° C. for 1 hour. To align encapsulated fibers, hydrogels are crosslinked between the two magnets of magnetic gelation chamber.
Cell lines and culture: Mouse resident tenocytes were harvested via primary tenocyte harvesting with a previously established protocol. Briefly, mouse tail tendons are encapsulated in 2 mg ml−1 collagen I (Advanced Biomatrix), allowing cells to proliferate into the gel for 11 days. Gels are then digested with 0.25 mg ml−1 collagenase from C. histolyticum, and cells are centrifuged out. Tenocytes are cultured in DMEM supplemented with 10 v/v % fetal bovine serum (Fisher) and 1 v/v % penicillin/streptomycin/amphotericin B. Tenocytes are passaged near confluency at a 1:2 ratio and used for studies until passage 3. For three-dimensional hydrogel encapsulation studies, media is additionally supplemented with 50 ng ml−1 L-ascorbic acid-2-phosphate and transforming growth factor-β3 (Peprotech). Human mammary epithelial cells MCF10A (ATCC) are cultured in DMEM/F12 (1:1) supplemented with 5 v/v % horse serum (Fisher), 20 ng mL−1 rhEGF (Peprotech), 0.5 mg mL−1 hydrocortisone, 100 ng mL−1 cholera toxin, and 10 μg mL−1 insulin (Fisher). MCF10As are passaged at confluency at a 1:4 ratio and used for studies until passage 8. Then, MCF10As are detached with 0.25% trypsin-EDTA (Life Technologies), counted, and formed into 200 cell-sized spheroids overnight in inverse pyramidal PDMS microwells (AggreWell™, Stem Cell Technologies) treated with 0.5% Pluronic F-127 to prevent cell adhesion. All cells are cultured at 37° C. and 5% CO2.
Cytotoxicity screens: SPIONs with or without PVP coating are suspended in complete MCF10A media over a range of densities. To create SPION conditioned media, SPIONs are incubated in complete MCF10A media for 48 hours and then centrifuged out at 20,000 rcf for 30 minutes. To conduct a two-dimensional monolayer assay, MCF10A cells plated on glass coverslips are exposed to SPION containing media or SPION conditioned media for 12 hours, then incubated in serum free MCF10A media with Hoechst stain (1 μg/ml) and propidium iodide (1 μg/ml) for 20 minutes prior to fixing. To conduct a three-dimensional hydrogel assay, DVS fiber segments containing SPIONs with or without PVP coating are coencapsulated with single MCF10A cells (1000000 cells mL−1) in DVS hydrogels. After 12 hours in culture, hydrogels are incubated in serum free MCF10A media with Hoechst stain (2 μg/ml) and propidium iodide (2 μg/ml) and incubated on a rocker plate at 0.33 Hz for 1 hour to enhance diffusive transport prior to fixing.
Single cell spreading studies: Primary-derived tenocytes (5000000 cells mL−1) and fiber segments (3 v/v %) are coencapsulated in DVS hydrogels. Studies are maintained in complete tenocyte media for 7 days, with media replenished every other day.
Spheroid migration studies: MCF10A spheroids are harvested and centrifuged to remove residual single cells. Spheroids (6000 per mL of gel) and fiber segments (3 v/v %) are simultaneously encapsulated in DVS hydrogels. Studies are cultured in complete MCF10A media for 6 days, with media replenished every other day.
Fluorescence, staining, and microscopy: Samples are fixed with 4% paraformaldehyde for 1 hour at room temperature. To visualize the actin cytoskeleton and nuclei, samples are stained with phalloidin and DAPI for 1 hour at room temperature. For immunostaining, gels are additionally permeabilized in PBS containing Triton X-100 (5 v/v %), sucrose (10 w/v %), and magnesium chloride (0.6 w/v %) and blocked in 4% BSA. Fluorescent imaging is performed with a Zeiss LSM 800 laser scanning confocal microscope. For migration analysis, Z-stacks are acquired with a 10× objective. High-resolution images are acquired with a 40× objective. All images are presented as maximum intensity projections.
Cell migration analysis: A previously established custom MATLAB image analysis code is used to extract morphometric data from spheroid migration studies. Briefly, max intensity projections of spheroid nuclei and F-actin channels are separately thresholded and object size filtered to remove background. A user-drawn ellipsoidal ROI covering the spheroid body is used to separate the spheroid body from migratory cells within outgrowths. The code segments F-actin structures into individual outgrowths, which are defined as either contiguous or noncontiguous based on contiguity with the spheroid body. A separate function segments overlapping nuclei to identify all nuclei within outgrowths. Individual outgrowth F-actin masks are used to determine migration distance into the surrounding hydrogel utilizing a separate custom function. Corresponding individual nuclei masks are used to determine nuclei locations, nuclear counts, and mark noncontiguous outgrowths as either multicellular clusters or single cells. All individual outgrowth nuclei and F-actin masks are then summed to produce final images of nuclei and F-actin channels. Individual outgrowth nuclei and F-actin masks are saved with counted nuclei or plotted lengths, respectively, and assigned an index to address discrepancies or outliers within final quantified data. Resulting data are stratified by contiguity with the spheroid body and exported to a spreadsheet containing individual outgrowth indices, number of migratory cells, outgrowth areas, and migration distances. Finally, spheroid body and outgrowth masks are summed across all analyzed spheroids to produce heatmap overlays.
Statistics: Statistical significance is determined by one-way analysis of variance (ANOVA) with post-hoc analysis (Tukey test), with significance indicated by p<0.05. All data are presented as mean±SD.
Fabrication of Magnetically Responsive Electrospun Fiber Segments.
To create fiber segments on the same length-scale as fibrous proteins found in stromal ECM, DVS polymer solution is electrospun to produce fibers approximately 2 μm in diameter. Electrospun fiber mats are processed into suspensions of fiber segments, which can then be encapsulated in three-dimensional hydrogels and aligned by an externally applied magnetic field, as shown in
Computational Visualization of Magnetic Field Lines and Field Strength.
Magnetic field lines produced by a single permanent magnet resemble concentric ellipses radiating from the magnet's north to south pole. When opposite poles of two juxtaposed permanent magnets are aligned, field lines combine and densify as a function of spacing between the magnets. To determine the strength of the magnetic field produced within the magnetic gelation chamber, magnetic flux density is modeled using COMSOL. A three-dimensional model of two permanent magnets is created by placing two cylinders of equivalent geometry within a sphere to model air impedance, as shown in
Degree of Fiber Alignment is Jointly Regulated by SPION Density and Magnet Spacing.
To optimize DVS fiber alignment, the density at which SPIONs are incorporated into the DVS electrospinning solution is first modulated. A slight decrease in fiber segment yield occurs with increasing SPION density (data not shown), likely due to the SPIONs interfering with the electrospinning process. Fiber segments are encapsulated in three-dimensional DVS gels at 1 v/v % and aligned at a magnet spacing of 6 cm, as shown in
Decreasing Fiber Length Prevents Entanglement at High Fiber Density.
Previous reports on approaches to align type I collagen gels have noted collagen fiber entanglement. As a high fiber density is key to modeling fibrous tissues, such as tendons and the stroma of breast tissue, during cancer progression, it is next determined whether increases in fiber density lead to entanglement. Fiber density is modulated through the input fiber volume fraction of the hydrogel precursor solution over a range of 1-5 v/v % and gels are crosslinked at a magnet spacing of 6 cm. At fiber densities at or below 3 v/v %, highly anisotropic fiber alignment is achieved with minimal evidence of entanglement, as shown in
SPION Encapsulation within Fiber Segments Prevents Cytotoxic Interaction with Cells.
The presence of charged SPIONs has previously been reported to be cytotoxic. To determine if the SPIONs used here are cytotoxic and to test if cytotoxicity results from direct interaction of SPIONs with cells versus changes in media ion concentrations due to the addition of SPIONs, SPIONs or SPION-conditioned media is added to MCF10A mammary epithelial cell monolayers. Cell death, assessed via staining with membrane-impermeable propidium iodide, is SPION dose-dependent with 1 and 0.5 mg mL−1 SPION concentration in media resulting in significant increases in cell death relative to controls, as shown in
Fiber Alignment Directs Uniaxial Spreading in Primary-Derived Mouse Tenocytes.
Alignment of fibrous ECM architecture is known to influence fibroblast spreading and polarization. For applications in tendon tissue engineering, alignment of tendon fibroblasts (tenocytes) within three-dimensional hydrogels may be critical to mechanosensing and ECM deposition. To enable fibroblast adhesion to magnetic fibers, residual VS groups are functionalized with the cell-adhesive peptide, CGRGDS (SEQ ID NO:2), via Michael-type addition. Primary tenocytes harvested from mouse tendons are coencapsulated along with SPION-containing fibers in a bulk MMP-degradable DVS hydrogel to determine the influence of fiber alignment on tenocyte spreading and orientation. Magnet spacing is modulated to produce aligned (6 cm), partially-aligned (12 cm), or nonaligned (no magnetic field) hydrogel composites. Tenocyte spreading in nonaligned gels includes both stellate morphologies in which filopodia extend in all directions (
Fiber Alignment Directs Multicellular Migration and Induces Migration Phenotype Switching.
ECM fiber alignment has also been heavily implicated in epithelial cell migration during transtromal escape from primary tumors. To examine the effect of fiber alignment on epithelial cell migration, MCF10A spheroids and SPION-containing fibers are coencapsulated within MMP-degradable DVS gels. Degree of fiber alignment is again modulated by magnetic field strength. As shown in
Here, a means to align magnetic electrospun fibers within a three-dimensional hydrogel composite that models stromal ECM is described. SPIONs are stably incorporated into DVS fiber segments, enabling control over the density and alignment of fibrous architecture via an externally applied magnetic field. SPION density and magnetic field strength jointly contribute to fiber alignment, enabling fine control over fiber alignment. Fiber entanglement due to the length and density of fiber segments impairs alignment, but shortening fibers via photomasking prevents fiber entanglement during magnetic alignment, thereby increasing the range of achievable fiber densities in three-dimensional hydrogel composites. Both the spreading of individually encapsulated cells and orientation of multicellular migratory structures from spheroids are influenced by the degree of fiber alignment. Aligned fibrous architecture directs uniaxial spreading of primary-derived mouse tenocytes in lieu of stellate morphologies. Fiber alignment also biases the direction of multicellular migration from MCF10A spheroids and increases the number of cell-cell breakage events, leading to the emergence of invading single cells and multicellular clusters. While previous methods have been developed to aligned fibers within purified biopolymer hydrogel, such as type I collagen, the synthetic fiber-reinforced hydrogel composite system presented provides more facile orthogonal tuning of fibrous architecture parameters, including the degree of alignment, fiber length, and fiber density.
The custom-designed magnetic gelation chamber holds two small N52 neodymium magnets that produce a surface field of 661.9 mT. In comparison to previous methods utilizing Tesla-range magnetic fields to align type I collagen gels, alignment of SPION-containing DVS fiber segments requires a significantly lower magnetic field strength achievable with small rare earth magnets without the need for specialized or expensive equipment (see
As the stroma possesses a high density of fibrous ECM proteins, fiber density within the hydrogel composites was modulated and fiber entanglement and reduced alignment is observed when the density of fibers exceeds 3 v/v %. To prevent entanglement, chrome photomasks were utilized to shorten fibers, as shown in
Functionalization of fiber segments with cell-adhesive RGD allows cells to engage and spread along fiber segments and respond to matrix alignment. Following the encapsulation of primary-derived mouse tenocytes into aligned hydrogel composites, cell spreading along fiber segments and a morphologic transition from stellate to uniaxial morphologies oriented in the direction of fiber alignment is observed, as shown in
Similar to single tenocyte spreading, multicellular migration from MCF10A spheroids biased in the direction of fiber alignment is observed, as shown in
While RGD is used to enable cell adhesion to fiber segments here, other ECM peptides can be used to model full length proteins, such as the GXOGER sequence (SEQ ID NO:3) of type I collagen. As DVS fiber segments are not hydrolytically or proteolytically degradable, they can also be used to study and model cell force-mediated reorganization of fibrous architecture. The magnetic electrospun fiber segments developed here can be easily integrated within other natural and synthetic biomaterials. An MMP-cleavable DVS hydrogel is selected as the bulk material here due to its tunability of bulk stiffness and crosslinking via Michael-type addition. For integration with other hydrogels, crosslinking kinetics should be carefully taken into account. Fiber segments are immobilized after 8 minutes of DVS hydrogel crosslinking via Michael-type addition. The post-gelation degree of fiber alignment is likely a function of magnetic field strength in conjunction with hydrogel precursor solution viscosity as a function of crosslinking. As such, stronger magnets may be required to achieve the same degree of fiber alignment if hydrogel crosslinking kinetics are significantly faster than the DVS hydrogels employed here.
A hydrogel composite system including SPION-containing electrospun fiber segments which can be aligned within an externally applied magnetic field is presented. Orthogonal tunability is demonstrated for key fibrous matrix attributes, including fiber length, fiber density, and degree of fiber alignment. The ability to align magnetic fibers proves insensitive to boundary conditions, allowing homogeneous fiber alignment throughout a millimeter-scale hydrogel. With this system, the ability to align single encapsulated primary mouse tenocytes is demonstrated, which may have utility as an injectable biomaterial therapy to mediate tendon repair. Furthermore, control over directional multicellular migration from MCF10A spheroids is shown, and it is found that fiber alignment induces breakage events, leading to migration phenotype switching from collective strands to single cells and multicellular clusters. The tunability of fibrous architecture within this hydrogel composite and the ability to integrate magnetic fibers with other biomaterials enables modeling of stromal tissue architectures in connective tissue repair and disease processes.
The animal was transferred to the magnetic field device, positioning the hind limb in axis with the field direction. A composite hydrogel precursor solution containing magnetic fibers (SPION-laden vinyl sulfone functionalized dextran (DexVS) fibers) was prepared and injected into the gap spanning tendon stubs. Hydrogel gelation proceeded in the presence of the magnetic field for 10 minutes with a moistened segment of gauze placed above the wound site to prevent dehydration.
The foregoing description of the embodiments has been provided for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure. Individual elements or features of a particular embodiment are generally not limited to that particular embodiment, but, where applicable, are interchangeable and can be used in a selected embodiment, even if not specifically shown or described. The same may also be varied in many ways. Such variations are not to be regarded as a departure from the disclosure, and all such modifications are intended to be included within the scope of the disclosure.
This application claims the benefit of U.S. Provisional Application No. 63/191,253, filed on May 20, 2021. The entire disclosure of the above application is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63191253 | May 2021 | US |
