Tissue scaffold with patterned microstructure

Abstract

This specification provides a tissue scaffold (118) which may include one or more surface microstructure patterns that can alter the physical properties of the tissue scaffold. The microstructure patterns may be cell-directed over a larger spatial range than in the prior art using chemically modified substrates. The disclosure further includes a tissue scaffold configured such that cells can infiltrate the tissue scaffold by responding to a pattern of surface energy gradients on the tissue scaffold, and the cells cannot be constrained to the pattern by physical means. As the cells proliferate, they associate and orient themselves in response to the long-range pattern to form a confluent monolayer of cells, constructing a functional macrostructure across the surface of the tissue scaffold.

Description

[Technical Field]

[0001] The present invention generally relates to tissue and cell scaffolds having relevant microstructured surfaces. [Background technology]

[0002] Living cells and tissues can exhibit high sensitivity to local micro- and nanoscale chemical topographic patterns, including those provided in vivo by the complex and distinct structure of the extracellular matrix (ECM). However, given the small scale of the underlying interactions, their effects on cellular and tissue function are not fully understood.

[0003] The study of designed cell-biomaterial interactions at the nanoscale level within cells provides some evidence for the potential importance of submicrometer cues for cell signaling, adhesion, proliferation, and differentiation. However, initial attempts based on short-range stimuli to direct functional tissue formation are often uninspired or non-biomimic and fail to replicate the multiscale long-range effects of complex ECM structures and associated chemical ligands that control integrated multicellular ensembles on a scale ranging from a few micrometers to several hundred micrometers. Recent advances in nanofabrication techniques can enable the design and fabrication of scalable scaffold materials that mimic structural and mechanical cues present in the in vivo ECM environment.

[0004] Tissue formation, wound repair, and many disease processes depend on the expression of appropriate ECM proteins and cell-mediated assembly. ECM provides the long-range organizing structures necessary for the cell-mediated assembly of functional tissues. These long-range structures can be interpreted as a type of cellular language recognized by appropriate cell types for organizing into biologically functional macroscopic structures.

[0005] Cell signaling using various cytokines is currently being used to promote cell-specific infiltration into synthetic tissue scaffolds. However, these cytokines typically provide only local signals to cells. Cytokines can promote the infiltration of specific cell types, but they do not provide information on how these cells should be macroscopically organized within and between cell types. In today's tissue scaffolds, long-distance cell signaling may generally be lacking.

[0006] Macroscopic or long-distance cell organization and the resulting tissue can be defined as any biologically functional tissue that is at least 10 times the size of the constituent cells that make up the macroscopic tissue structure.

[0007] The importance of long-distance cell signaling can be exemplified by the ECM. The orientation of ECM fibers can be essential for normal tissue development and homeostasis. However, the organization of the ECM can be disrupted at many disease and injury sites, generating disorganized collagen fibers that form in scar tissue. Scar tissue typically lacks the biological functionality of the tissue it replaces.

[0008] Therefore, the goal of regenerative medicine can be to promote the formation of new tissues that closely resemble normal tissues in terms of organization and function (compared to damaged or diseased tissues). By controlling cell growth in a long-distance spatially defined manner, it is possible to regenerate damaged or diseased tissues that have the appropriate orientation of constituent cells and / or the orientation of molecular complexes produced by the cells. This can be achieved. Typically, these long-distance spatially organized signals are often generated by surface energy gradients mediated by patterns of hydrophilic and hydrophobic sites.

[0009] In particular, the surface energy gradient guides the arrangement of ECM fibrils to correspond to cell actin filaments by using cell surface receptors that are indirectly connected to the actin cytoskeleton. Thus, a major challenge in regenerative medicine is to facilitate cells to assemble ECM fibrils such as collagen in a specific long-range orientation or alignment on a scaffold device in order to generate tissues with the required functional properties.

[0010] Muscle tissues have complex structured organizations at multiple scales from micro (1 micron to 100 microns) to macro (100 microns to several 1000 microns), but macroscale control of function has not been extensively analyzed. For example, the myocardium can be an ensemble of different cell types embedded in a complex and well-defined structure of the ECM and arranged on macroscale topography and molecular patterns. The structure of heart tissue is highly organized in vivo, but cardiomyocyte ensembles lose their native tissue and take on a random distribution when cultured in vitro by common culture techniques, potentially losing many of their physiological properties.

[0011] Various methods such as mechanical stretching, microcontact printing, and electrical stimulation have been used to manipulate more well-organized cardiomyocyte cultures. Both 2D and 3D substrates with a feature size of 10 μm were used to induce cardiomyocytes into anisotropic arrangements for electrophysiological and mechanical property evaluations. However, the structure and function of in vivo heart tissue may be regulated by much larger macroscale cues provided by the ECM, and it may be possible to perform complex multiscale control of cell and tissue function. Therefore, it is important to investigate and understand whether patterned control of the cell-material interface at the macroscale facilitates the fabrication of truly biomimetic heart tissue constructs that reproduce the structural and functional aspects of in vivo large-scale tissue phenotypes.

[0012] Furthermore, the ability to generate uniformly controlled (structurally and functionally), robust, reproducible, and precisely defined biological tissues is likely necessary for tissue scaffolds that truly promote regeneration. In this regard, the inability to specifically differentiate pluripotent progenitor cells into mature muscle cells over long distances remains a major obstacle to optimal in vivo cell generation during soft tissue repair after injury.

[0013] Furthermore, while cell-based therapeutic methods exist that utilize cells on scaffolds, their use may have limited utility unless they adequately support the long-distance proliferation, differentiation, and function of cells for functionally designed biological tissues.

[0014] Therefore, what is needed is a scaffold device that, in some aspects, provides long-distance proliferation, differentiation, and function of cells for a functionally designed biological tissue that better represents the biological tissue. [Overview of the project]

[0015] This disclosure is based, at least in part, on the discovery that the surface energy of microstructures can generate cell adhesion patterns at nanoscale and microscale dimensions by using techniques for surface-modifying solids or polymers to create devices with utility as bioscaffold materials. These scaffold materials can be used to generate functional tissue to be implanted ex vivo, or the scaffold material can be directly implanted. Directly implanted scaffold materials are functional It can be used to direct functional tissue repair or to create a tissue-compatible interface between biological tissue and non-biological implants such as prostheses, sensors, and electrodes.

[0016] Some embodiments, as disclosed herein, can generate cellular language that can be used, for example, in regenerative medicine, wound repair, and / or transplantation biology. Furthermore, these embodiments can be used in screening assays to determine the effects of a test compound on living tissues by examining the effects of the test compound on various biological responses, such as cell viability, cell proliferation, migration, differentiation, and maintenance of cellular phenotype, for example.

[0017] Accordingly, in one embodiment, several embodiments may provide micro and / or nanostructured surfaces for scaffolds for tissue generation both in vivo and ex vivo. In one embodiment, the tissue scaffold may comprise a base layer containing a first pattern of microstructure and a capping layer containing a smooth layer, or it may comprise a second pattern of microstructure.

[0018] In another embodiment, the structural scaffold may comprise a base layer having a microstructure pattern and a capping layer on each microstructure having one of a hydrophilic layer and one of a hydrophobic layer. The hydrophobicity or surface energy of the capping layer may be due to the chemical composition of the capping material, the geometric shape of the capping layer, or both.

[0019] In another embodiment, the structural scaffold may comprise a base layer having a microstructure pattern and a capping layer comprising a hydrophilic or hydrophobic layer, the hydrophilic layer having a water contact angle which may be 100° or less, and the hydrophobic layer having a water contact angle which may be greater than 100°. More generally, the difference between the hydrophobic angle and the hydrophilic angle may be at least 5 degrees. The capping layer may comprise a hydrophobic / hydrophilic material, a single material formed as a microstructure with a desired contact angle, or one or more of each capping layer type.

[0020] In another embodiment, the structural scaffold may include a base layer having a microstructure pattern and a capping layer having a hydrophilic layer and / or a hydrophobic layer, wherein, if a hydrophilic layer is present, the layer has a water contact angle that may be 100° or less, and if a hydrophobic layer is present, the layer has a water contact angle greater than 100°. More generally, the difference between the hydrophobic angle and the hydrophilic angle may be at least 10°, and the microstructure is arranged in zones that include fixing patterns of hydrophilic and hydrophobic end caps.

[0021] In certain embodiments, the microstructure may be arranged on the substrate surface in a square or triangular grid. In some embodiments, the microstructure zones may include blocks of microstructure, which may be hexagonal, triangular, or quadrilateral, and their three-dimensional analogues.

[0022] In another embodiment, the artificial tissue may include living cells attached to a tissue scaffold, and the scaffold may comprise a base layer having a pattern of microstructures arranged thereon. The base layer may further comprise a capping layer having a pattern of microstructures or materials having at least two contact angles.

[0023] In another embodiment, the artificial tissue may include living cells attached to a tissue scaffold, and the scaffold may comprise a base layer having a pattern of microstructures arranged thereon. The scaffold may further comprise a capping layer having a pattern of microstructures or materials having at least two contact angles, and the base layer and the capping layer The combination with the Ping layer forms a Wenzel-Cassie interface when it comes into contact with the liquid.

[0024] In embodiments where the liquid is a composite liquid, the liquid components may be separated into individually isolated Wenzel zones. These Wenzel zones may be connected by capillary bridges. A capillary bridge containing one component of the composite liquid may be interwoven with a capillary bridge containing another component of the composite liquid.

[0025] In another embodiment, the artificial tissue may comprise living cells attached to a tissue scaffold which may include a base layer, and the base layer further comprises a capping layer having a pattern of microstructures arranged thereon and a pattern of microstructures or material having at least two contact angles, and the combination of the base layer, pillars and capping layer forms a Wenzel-Cassie interface when in contact with a liquid. When the liquid is a composite liquid, the liquid components may separate into Wenzel zones. Also, when the composite liquid is present between the artificial tissue and the biological tissue, the Wenzel zones may form adhesive capillary bridges to the biological tissue.

[0026] In some embodiments disclosed herein, patterned tissue scaffolds may consist of polymer substrates for use in compositions and methods for generating tissue-designed soft tissues disclosed herein. Microstructures on the patterned tissue scaffolds may be spatially organized on length scales from nanometers to centimeters and can be generated by the methods described herein.

[0027] In certain embodiments, the polymer may include, for example, biopolymers (e.g., proteins, carbohydrates, glycoproteins, etc.), and may be deposited on a transition polymer surface using patterning techniques that enable nanometer-scale spatial positioning of the deposited polymer.

[0028] The patterning techniques disclosed herein may include, but are not limited to, soft lithography, self-assembly, vapor deposition, and photolithography. When placed on a surface, interpolymer interactions can attract polymers to each other so that they bond together. These interactions can be hydrophilic, hydrophobic, ionic, covalent, van der Waals, hydrogen bonding, and / or physical entanglement, depending on the specific polymers involved.

[0029] In another embodiment, the artificial tissue includes living cells that can be attached to a tissue scaffold comprising a base layer which may contain a microstructured pattern and a capping layer which contains a microstructured pattern or material having at least two contact angles. The combination of the microstructured base layer and the capping layer may form a Wenzel-Cassie interface when in contact with a liquid. Furthermore, the pillars may be arranged in a pattern that creates regions of hydrophilic / hydrophobic surface energy, and one or more regions may generate long-range structures. When the liquid is a composite liquid, the liquid components may separate into Wenzel zones. Also, when the composite liquid is present between the artificial tissue and the biological tissue, the Wenzel zones may form adhesive capillary bridges to the biological tissue.

[0030] In another embodiment, the tissue scaffold may be formed by photolithography to include a microstructure pattern on a base layer to form a substrate having a patterned base layer. The scaffold may further include a capping layer by depositing a capping layer on the patterned base layer to form a substrate having a patterned or hydrophilic / hydrophobic capping layer. The patterned capping layer may be brought into contact with the tissue layer to produce a substrate having a patterned tissue adhesive layer.

[0031] In another embodiment, the method includes providing a tissue scaffold having a microstructure pattern on a base layer by photolithography to form a substrate having a patterned base layer; depositing a capping layer on the patterned base layer to form a substrate having a patterned or hydrophilic / hydrophobic capping layer; contacting the substrate having the patterned tissue adhesion layer with cells and culturing them under conditions suitable for the production of extracellular matrix components; and removing the cells from the substrate to provide a biological tissue scaffold containing extracellular matrix components.

[0032] In another embodiment, a method of using a tissue scaffold may include depositing a capping layer on a patterned base layer to form a pattern of pillars on a base layer comprising a substrate having a patterned base layer and a substrate having a patterned or hydrophilic / hydrophobic capping layer, contacting the substrate having a patterned tissue adhesion layer with cells and culturing them under conditions suitable for the production of extracellular matrix components, and then contacting the tissue scaffold having extracellular matrix components with biological tissue.

[0033] In another embodiment, a method for identifying compounds that modulate tissue function may include providing a patterned scaffold for tissue as described herein, contacting the tissue with a test compound, and measuring the effect of the test compound on tissue function in the presence and absence of the test compound, thereby identifying a compound that modulates tissue function by demonstrating that the test compound modulates tissue function compared to the tissue function in the absence of the test compound.

[0034] In another embodiment, a method for identifying long-range microstructure patterns that modulate tissue function may include providing a patterned scaffold for tissue according to this specification, comprising patterned regions that can be placed on the long-range microstructure pattern; bringing the tissue into contact with a test long-range microstructure pattern; and measuring the effect of the test long-range microstructure pattern on tissue function in the presence and absence of the test long-range microstructure pattern, thereby identifying a long-range microstructure pattern that modulates tissue function by demonstrating that the test long-range microstructure pattern modulates tissue function compared to tissue function in the absence of the test long-range microstructure pattern.

[0035] In another embodiment, a method is disclosed for identifying long-range microstructure patterns useful for treating tissue defects, the method comprising providing a patterned scaffold for tissue as described herein, and a method for identifying long-range microstructure patterns modulating tissue function comprising providing a patterned scaffold for tissue as described herein, comprising a patterned region that can be placed on a long-range microstructure pattern, bringing the tissue into contact with a test long-range microstructure pattern, and measuring the effect of the test long-range microstructure pattern on tissue function in the presence and absence of the test long-range microstructure pattern, thereby identifying long-range microstructure patterns useful for treating or preventing tissue diseases by demonstrating that the test long-range microstructure pattern modulates tissue function compared to tissue function in the absence of the test long-range microstructure pattern.

[0036] In another embodiment, a tissue scaffold that may be used to treat a tissue defect is disclosed. [Brief explanation of the drawing]

[0037] [Figure 1] This is a diagram of an embodiment of the tissue scaffold disclosed herein. [Figure 2A]This is a diagram of the coding zone of the microstructure disclosed herein. [Figure 2B] This is a diagram of the coding zone of the microstructure disclosed herein. [Figure 2C] This is a diagram of the coding zone of the microstructure disclosed herein. [Figure 2D] This is a diagram of the coding zone of the microstructure disclosed herein. [Figure 3A] This is a diagram of an encoding region of a fine structure that can act as a cell-connecting region as disclosed herein. [Figure 3B] This is a diagram of an encoding region of a fine structure that can act as a cell-connecting region as disclosed herein. [Figure 3C] This is a diagram of an encoding region of a fine structure that can act as a cell-connecting region as disclosed herein. [Figure 3D] This is a diagram of an encoding region of a fine structure that can act as a cell-connecting region as disclosed herein. [Modes for carrying out the invention]

[0038] This disclosure is based, at least in part, on the discovery of nanoscale and microscale cellular signaling long-range patterns using techniques for surface-modifying solids or polymers to prepare devices having utility as tissue scaffold materials. The patterned surface modification generates a surface energy gradient when the tissue scaffold containing the long-range pattern comes into contact with biological tissue or cells in a culture medium.

[0039] The term "contact angle" can be understood as a quantitative measure of wetting a solid by a liquid. Geometrically, the contact angle may be defined as the angle formed by a droplet at the three-phase boundary where the liquid, gas, and solid intersect, after the droplet has been in contact with the gas and solid for a given time of at least 1 second at 20°C. Unless otherwise specified, in certain embodiments the gas may be air and the liquid may be water.

[0040] The terms "compound fluid" and "compound liquid" can be understood to refer to a liquid composed of at least two substances in liquid form. For example, a mixture of water and alcohol may be a compound liquid, also known as a binary solution.

[0041] The term "long-range pattern" can be understood as referring to any pattern of microstructure features that have spatial periodicity greater than the dimensions of the microstructure. Typically, the Fourier transform of a long-range microstructure pattern can be understood to exhibit multiple periodicities.

[0042] The term "reabsorbable" can be understood as referring to polymers that can be absorbed during circulation in cells or tissues when transplanted.

[0043] As used herein, the term “deposit” may be understood to mean the process of placing or coating an article or substance onto another article or substance (which may be identical, similar, or dissimilar to the first article or substance). Deposit may include, but is not limited to, methods using spraying, dip casting, spin coating, evaporation, sputtering, immersion, extractive deposition, or other methods for associating the item or substance. The term deposit may also include methods of coating an article or substance substantially over an entire surface, as well as methods of coating an article or substance onto a portion of a surface.

[0044] As used herein, the term “continuous layer” can be understood to mean a layer that may be formed by a matrix of individual molecules that are chemically or mechanically bonded to one another.

[0045] As used herein, the various forms of the term “modulate” are intended to include stimulation (e.g., increasing or upregulating a particular response or activity) and inhibition (e.g., decreasing or downregulating a particular response or activity).

[0046] As used herein, the term “to bring into contact” (e.g., to bring tissue into contact with the test compound) should be understood to include any form of interaction between the test compound and the tissue (e.g., direct or indirect interaction). The term “to bring into contact” includes incubating the compound and the tissue (e.g., adding the test compound to the tissue).

[0047] As used herein, the term “comprise” can be understood to include the enumerated elements as well as other elements that are not specifically stated but may similarly be present. The use of “comprise” indicates inclusion, not limitation. In other words, the term “comprise” means “primarily, but not necessarily limited to.” Furthermore, variations of the word “comprising,” such as “comprise” and “comprises,” correspond to the same meaning. In one respect, the techniques described herein may include ("comprise") unspecified elements, whether essential or not, relating to the compositions, methods, and their respective components described herein as essential to the disclosure.

[0048] As used herein, the term “sign” can be understood to refer to a microstructure comprising a juxtaposition of surface energy gradients in a defined surface area. In some embodiments, the surface energy gradient may be defined by microstructure surfaces of different surface energies. For example, low surface energy (hydrophobic) microstructures may be juxtaposed with high surface energy (hydrophilic) microstructures. If these microstructures are labeled as 0 for hydrophobic microstructures and 1 for hydrophilic microstructures, the sign can be described using an array of 0s and 1s. The array may be two-dimensional, or more dimensional when two-dimensional microstructured surfaces are stacked, and may be hierarchical, for example, but not limited to these.

[0049] When considering two-dimensional codes, the zone defining a code may consist of lines of 0s and 1s. Considering a first line, adjacent lines can be considered as permutations of the first line. Consequently, a code may be specified as a group of permutations acting on the first lines of 0s and 1s and a specified first line. Choosing different first lines generates different code zones for the same group operation. Therefore, the zone specified by a code can represent elements of a mathematical group.

[0050] As used herein, the term “substrate” may be understood to refer to any suitable carrier material. In some embodiments, the carrier material may be a material to which cells can adhere or adhere in order to form a corresponding cell complex, such as a tissue-engineered smooth muscle composition. In some embodiments, the matrix or carrier material may already exist in a three-dimensional form desirable for later use. For example, bovine pericardial tissue can be used as a matrix that can be crosslinked with collagen, decellularized, and photofixed.

[0051] As used herein, the term “pattern” may be understood to mean a given arrangement or design, generally a substantially microscale design of encoded microstructures within a surface, as described herein.

[0052] As disclosed herein, the inventors have found that a long-range patterned polymer platform, fabricated to have an array of encoding zones and referred to herein as “encoded microstructured scaffold” or “CMS”, can anisotropically organize immature cells, such as muscle cells, and that CMS can promote a mature phenotype in anisotropically arranged tissues compared to the same cells cultured on a substrate lacking encoding zones. Therefore, the inventors have discovered that the microstructural code may have the capability for large-scale functionality and can produce functional tissue-engineered cells that have significantly superior functionality compared to existing tissue-engineered tissues of the prior art.

[0053] Compared to the aforementioned methods and devices of the prior art, the devices and methods of this disclosure may be found to induce cell signaling that can follow and organize cells to a macroscale structure defined by long-range energy gradients generated on the surface of a tissue scaffold. These long-range energy gradients can mimic an in vivo environment, appropriately enriching and oriented cell types to form functional tissue.

[0054] Accordingly, the present disclosure may be a synthetic tissue scaffold, in whole or in part, that can be embedded in living tissue to repair soft tissue defects. The resulting tissue repair may be characterized by appropriate cellular organization and biological activity.

[0055] Certain embodiments disclosed herein do not necessarily rely on short-range chemical treatment of tissue scaffolds. For example, avoiding the use of biological cell adhesion molecules to promote cell invasion on tissue scaffolds may help prevent damage to cell receptors that may be involved in innate cell diffusion and extracellular interactions that affect cell organization and tissue alignment.

[0056] In particular, embodiments disclosed herein may include tissue scaffolds that modify the physical properties of a substrate using microstructure patterns. These embodiments may be more durable than the aforementioned methods and devices that rely on treatment of the tissue scaffold surface with unstable and environmentally sensitive biofunctional molecules.

[0057] In some embodiments disclosed herein, the microstructure patterns may be cell-oriented over a larger spatial range than in the prior art using chemically modified substrates. In one embodiment, cells may infiltrate the tissue scaffold by responding to a pattern of surface energy gradients on the tissue scaffold disclosed herein, and the cells may not be constrained to the pattern by physical means. As the cells proliferate, they associate and orient themselves in response to the long-range pattern to form a confluent monolayer of cells, constructing a functional macrostructure across the surface of the tissue scaffold.

[0058] In some embodiments, the CMS disclosed herein can be patterned to modify the cellular environment at multiple spatial scales in order to induce the maturation of in vitro differentiated cells and subsequently organize the in vitro differentiated cells into two-dimensional and three-dimensional tissue structures, and can therefore be used to generate functional muscle tissue, such as functional artificial cardiac muscle. In some embodiments, the CMS can be coated with active substances such as differentiation factors that promote cell differentiation.

[0059] Some embodiments disclosed herein may have aspects that represent significant advances in strategies for large-scale manipulation of functional tissues from stem cell sources. In particular, since conventional efforts to produce mature cells from precursor or progenitor cells are highly inefficient and do not produce functional tissues that resemble mature adult tissues in vivo, one important aspect may be the ability to mature differentiated cells into functional tissues in vitro. In some cases, this may be because functional tissues are ensembles of different cell types embedded in a complex structure of an extracellular matrix (ECM) arranged in topographic and chemical patterns.

[0060] In some embodiments, the pattern may be characterized by a combination of surface energy gradients. In some embodiments, the sign of CMA is the spatial relationship of the surface energy gradient. Distribution may also be a factor. In common culture techniques, this sign may be absent, and the resulting in vitro differentiated cells lack their native composition, resulting in a random distribution when cultured in vitro and impairing many of their physiological properties.

[0061] In some embodiments, the cellular environment of the CMS may be designed on a micrometer scale to encompass patterned zones of macroscopic length, providing precise micro and long-range cues to enable the organization of differentiated cells to adopt more mature organization. The patterned zones may be contained on microstructures having multiple surface energy gradients. In an aqueous environment, these surface energy gradients may manifest as hydrophilic and / or hydrophobic regions. Thus, the patterned zones or regions may consist of microstructures corresponding to specific combinations of hydrophilic and hydrophobic regions. These zones or regions may then be arranged in a pattern to provide long-range structures. Structural features within a zone or region can be scaled up in combinations of zones or regions to construct self-similar patterns.

[0062] In some embodiments, factors designed or manipulated to provide one or more benefits may include, but are not limited to, the mechanical properties of the material, the solubility of the material, spatial patterning of topological features such as ridges and pillars, soluble bioactive compounds, mechanical perturbations (such as periodic or static strain, stress, shear), electrical stimulation, and / or thermal perturbations.

[0063] Accordingly, as described herein, the methods and devices can be used in a wide range of applications, for example, in regenerative medicine, wound repair, transplantation biology, drug delivery, testing of the effects of substances on cells, tissue formation, cell action, and developmental biology.

[0064] Accordingly, in one embodiment, the present disclosure provides an embodiment having a patterned scaffold for a tissue. In one embodiment, the tissue scaffold may include a base layer having a microstructure pattern. The base layer may further include a capping layer having a chemical surface energy modification composition or a second surface energy modification microstructure pattern.

[0065] In another embodiment, the tissue scaffold may comprise a base layer containing a microstructure pattern and a capping layer microstructure on each base layer microstructure comprising one of a hydrophilic material and one of a hydrophobic material, and the first and second microstructures form a Wenzel-Cassie interface when placed in contact with a moist target surface. The Wenzel-Cassie interface can generate a non-biological cell adhesion layer placed on the tissue scaffold.

[0066] In some embodiments disclosed herein, the base layer may be a solid polymer surface, a rigid polymer surface, or a hard polymer surface, a semi-rigid polymer surface, a flexible polymer surface, a hard nonpolymer surface, a semi-rigid nonpolymer surface, or a flexible nonpolymer surface, or a combination thereof.

[0067] In some embodiments, the base layer may be biologically absorbable and / or chemically inert.

[0068] In some embodiments, the base layer may include one or more surfaces or components arranged hierarchically. For example, the base layer may comprise soft polymer pillars, each including an upper surface on which smaller pillars may be placed. In such a base layer, a surface energy gradient may exist between the base pillars, and this may be different from the surface gradient between the capping layer pillars.

[0069] In some embodiments, the base layer for use in the compositions and methods of the present disclosure may have a Young's modulus of about 0.000001 to 0.01, 0.005 to 0.2, 0.005 to 0.5, 0.05 to 1.0, 0.075 to 1.0, 0.1 to 2.0, 1.0 to 2.0, 1.5 to 5.0, 2.0 to 5.0, 3.0 to 7.0, 3.0 to 10, 5.0 to 15, 5.0 to 20, 10 to 20, 15 to 30, 20 to 30, 25 to 50, 30 to 50, 50 to 75, 50 to 100, 75 to 125, 100 to 150, 125 to 150, 150 to 200, 175 to 200, 200 to 250, or about 250 to 300 gigapascals (GPa).

[0070] Intermediate ranges and values ​​within the above ranges and values ​​are also considered to be part of this disclosure. For example, though not an exclusive example, Young's moduli of approximately 3.5 to 9.5, or 4.0 to 5.0 GPa, may be included in this disclosure for applications where minimizing distortion of the microstructure pattern is desirable. However, the Young's modulus of natural tissues ranges from approximately 1 to 1000 kilopascals (kPa). As with the examples provided above, other examples of other ranges for each of the values ​​listed above are also conceivable in the previous paragraph.

[0071] In embodiments of this disclosure intended to repair dynamic soft tissue, such as the heart, a Young's modulus of about 2.2 to 10.0 kPa or 4.0 to 5.0 kPa may be desirable.

[0072] In other embodiments, the base layer for use in the compositions and methods of the present disclosure may have a Young's modulus of about 0.001-0.1, 0.005-0.2, 0.005-0.5, 0.05-1.0, 0.075-1.0, 0.1-2.0, 1.0-2.0, 1.5-5.0, 2.0-5.0, 3.0-7.0, 3.0-10, 5.0-15, 5.0-20, 10-20, 15-30, 20-30, 25-50, 30-50, 50-75, 50-100, 75-125, 100-150, 125-150, 150-200, 175-200, 200-250, or about 250-300 kilopascals (kPa). Intermediate ranges and values ​​within the above ranges and values ​​are also considered part of this disclosure. For example, Young's moduli of approximately 3.5 to 9.5 or 8.0 to 9.0 kPa may be intended to be included in this disclosure. As with the examples provided above, other examples of other ranges for each of the values ​​listed above are also possible in the paragraph.

[0073] In one embodiment, the base layer may be selected from the group consisting of a rigid polymer surface, a semi-rigid polymer surface, and / or a flexible polymer surface, a rigid nonpolymer surface, a semi-rigid nonpolymer surface, and / or a flexible nonpolymer surface, and combinations thereof.

[0074] In one embodiment, the base layer is made of polyamide, polyurethane, polyurea, polyester, polyketone, polyimide, polysulfide, polysulfoxide, polysulfone, polythiophene, polypyridine, polypyrrole, polyether, silicone (polysiloxane), polysaccharide, fluoropolymer, epoxy, aramid, amide, polypeptide, polyethylene, polystyrene, polypropylene, glass-reinforced epoxy, liquid crystal polymer, thermoplastic resin, bismaleimidotriazine (BT) resin, benzocyclobutene ABFGx13, glass and epoxy low coefficient of thermal expansion (CTE) films, polyvinyl, polyacrylic, poly acrylate, polycarbonate, polytetrafluoroethylene (PTFE), polyethylene terephthalate (PET), quartz, silicon (e.g., silicon wafers), glass, ceramics, and metals and metal alloys including titanium, titanium alloys, tantalum, zirconium, stainless steel and cobalt-chromium alloys, and metal oxides, poly(vinylpyrrolidone), poly(2-hydroxyethyl methacrylate), poly(N-vinylpyrrolidone), poly(methyl methacrylate), poly(vinyl alcohol), poly(acrylic acid), polyacrylamide, polyreethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylate) The material may include poly(lylic acid), polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), polyanhydride, polyphosphazene, polygermane, polyorthoester, polyolefin, polycarbonate, biopolymers such as silk and collagen, copolymers and derivatives thereof, and composite materials containing these polymers. In one preferred embodiment, the base layer may be silicon, polyetheretherketone (PEEK), nylon including nylon 6,6 or PET, and combinations thereof.

[0075] In some embodiments, the CMS may be a microstructure patterned polymer, such as a biopolymer, and in certain embodiments, it can be created by providing a transition polymer on a substrate, depositing a biopolymer on the transition polymer, shaping the polymer into a structure having a selected pattern on the transition polymer (e.g., poly(N-isopropylacrylamide)), and releasing the biopolymer from the transition polymer without compromising its structure and integrity.

[0076] In some embodiments, the microstructure patterns and polymers may be based on, for example, extracellular matrix proteins, biologically active carbohydrates, biologically derived homopolymers, silk, polyproteins (e.g., poly(lysine)), or combinations thereof. For example, polymers can be selected from the group consisting of fibronectin, vitronectin, laminin, collagen, fibrinogen, silk, or silk fibrin. The polymer components of the structure may comprise a combination of two or more ECM proteins, such as fibronectin, vitronectin, laminin, collagen, fibrinogen, and structurally related proteins (e.g., fibrin).

[0077] In another embodiment, the base layer may be absorbent polylactic acid or poly(lactic acid-coglycolic acid). In another embodiment, the base layer may be absorbent polyurethane. In another embodiment, the base layer may be non-absorbent polytetrafluoroethylene.

[0078] In certain embodiments, a medical device incorporating one or more embodiments disclosed herein may be used, and such medical devices include, but are not limited to, diagnostic implant devices, biosensors, stimulators, nerve stimulators, nerve activity recorders, diabetes implants such as glucose monitoring devices, external fixation devices, external fixation implants, orthopedic trauma implants, and plates, screws, rods, plugs, cages, scaffolds, artificial joints (e.g., hands, wrists, elbows, shoulders, spines, hips, knees, ankles), wires, etc., used for joint and spinal injury / reconstruction. This includes implants, tumor-related bone and soft tissue replacement devices, dental and oral / maxillofacial devices, cardiovascular implants such as stents, catheters, valves, rings, and implantable cardioverter-defibrillators, contact lenses, ocular implants, corneal prostheses, dermatological implants, cosmetic implants, implantable drug delivery pumps, and, but not limited to, common surgical devices and implants such as drainage catheters, shunts, tapes, meshes, ropes, cables, wires, sutures, skin staples, burn sheets, and vascular patches, as well as temporary / non-permanent implants.

[0079] In a particular embodiment, the base layer may have a microstructure pattern having at least two microstructure elements, with adjacent microstructure elements separated by space. In one embodiment, the width and spacing of the microstructure elements may be about 0.1 μm to about 1000 μm. In another embodiment, the microstructure elements are selected from pillars having circular, square, triangular, or hexagonal cross-sections. The sides of the pillars may be straight and perpendicular to the base layer surface, or they may be tapered distally and proximally. Long pillars include ridges, pillars, and the like.

[0080] In some embodiments, the width and spacing of the pillars are approximately 0.1 μm to 1000 μm, approximately 1 μm to 500 μm, approximately 1 μm to 250 μm, approximately 5 μm to 250 μm, approximately 1 μm to 100 μm, approximately 1 μm to 90 μm, approximately 1 μm to 80 μm, approximately 1 μm to 70 μm, approximately 1 μm to 60 μm, approximately 1 μm to 50 μm, approximately 1 μm to 40 μm, approximately 1 μm to 30 μm, approximately 1 μm to 20 μm, approximately 1 μm to 10 μm, approximately 2 μm to 100 μm, approximately 2 μm to 90 μm, approximately 2 μm to 80 μm, and approximately 2 μm to 70 μm. m may vary over the ranges of approximately 2 μm to 60 μm, approximately 2 μm to 50 μm, approximately 2 μm to 40 μm, approximately 2 μm to 30 μm, approximately 2 μm to 20 μm, approximately 2 μm to 10 μm, approximately 1 μm to 100 μm, approximately 5 μm to approximately 160 μm, approximately 5 μm to approximately 100 μm, approximately 5 μm to approximately 90 μm, approximately 5 μm to approximately 80 μm, approximately 5 μm to approximately 70 μm, approximately 5 μm to approximately 60 μm, approximately 5 μm to approximately 50 μm, approximately 5 μm to approximately 40 μm, approximately 5 μm to approximately 30 μm, approximately 5 μm to approximately 20 μm, and approximately 5 μm to approximately 10 μm. Intermediate ranges and values ​​of the above ranges and values ​​are also considered to be part of this disclosure. As an example, widths and spacings of approximately 10–200 μm, or 50–200 μm, are intended to be included in this disclosure. Based on the above ranges and values, other examples are also envisioned by this disclosure.

[0081] The width and spacing of the pillars may be equal or different. In the art, the pitch of the pillars may be the sum of the width and spacing. For example, both the width and spacing may be about 0.1, about 0.2, about 0.25, about 0.5, about 0.75, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 5, about 16, about 17, about 18, about 19, or about 20 μm. In other embodiments, the widths are approximately 0.1, 0.2, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μm, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 , or can be about 40 μm, and the spacing can be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 μm. Intermediate values ​​and ranges of the values ​​listed above are also considered to be part of this disclosure. For example, widths and spacings of about 5.0 and 30.0 are intended to be included in this disclosure. Based on the above ranges and values, other examples are also assumed by this disclosure.

[0082] In one embodiment, the pillars may have a width of approximately 5 μm to 100 μm and be spaced approximately 5 μm to 100 μm apart. In another embodiment, the pillars may have a width of approximately 5 μm and be spaced approximately 5 μm apart. In yet another embodiment, the pillars may have a width of approximately 10 μm and be spaced approximately 10 μm apart. In yet another embodiment, the pillars may have a width of approximately 20 μm and be spaced approximately 20 μm apart. In yet another embodiment, the pillars may have a width of approximately 30 μm and be spaced approximately 30 μm apart. In yet another embodiment, the pillars may have a width of approximately 20 μm and be spaced approximately 10 μm apart. Based on the above ranges and values, other examples are also envisioned by this disclosure.

[0083] The capping material for use in the capping layer of a tissue scaffold may include portions that, upon contact with the patterned base layer, form a continuous layer on the pillars of the patterned base layer. In some embodiments, the capping material may be a polymer. In some embodiments, the polymer may be formed from PEG or a silicone precursor. In some embodiments, the polymer may be an alkoxide.

[0084] In embodiments having polymer alkoxides, the polymer may have 3 to 6 alkoxide groups or a mixture of oxo groups and alkoxide groups. In certain embodiments, The lucooxide group may have 2 to 4 carbon atoms and may include, for example, ethoxide, propoxide, isopropoxide, butoxide, isobutoxide, tert-butoxide, and fluorinated alkoxide.

[0085] In some embodiments described herein, the polymer of the capping layer may contain cell adhesion groups. The cell adhesion chemical compound may be an organic compound containing a phosphone group, a carboxylic acid group, a sulfone group, a phosphine group, a phosphate group, a sulfin group, or a hydroxam group. In some embodiments, the cell adhesion layer may contain a phosphonate.

[0086] In certain embodiments, the tissue scaffold may comprise living cells. In some embodiments, it has been found that cells can adhere to a patterned tissue scaffold in the absence of a cell adhesion layer positioned on the capping layer. Thus, in some embodiments, the artificial tissue may comprise living cells attached to a tissue scaffold comprising a base layer, the base layer having a microstructure pattern. In some embodiments, the base layer may comprise a capping layer having a second microstructure pattern, or a capping layer of hydrophilic and hydrophobic polymers. In another embodiment, the artificial tissue may comprise living cells attached to a tissue scaffold comprising a base layer having a microstructure pattern and a capping layer having a long-range pattern of hydrophilic and hydrophobic caps.

[0087] The types of cells disclosed herein in some embodiments may include, but are not limited to, fibroblasts, endothelial cells, keratinocytes, osteoblasts, chondrocytes and chondrocytes, hepatocytes, macrophages, cardiomyocytes, smooth muscle cells, skeletal muscle cells, tendinocytes, ligament cells, nerve cells, epithelial cells, and stem cells. Stem cells may be understood to include, but are not limited to, embryonic stem cells, adult stem cells, and induced pluripotent stem cells. In one embodiment, the cells may be mesenchymal stem cells. In another embodiment, the cells may be human cells.

[0088] In another embodiment, the tissue scaffold may include a base layer having a pillar pattern and a capping layer having a pillar pattern, the base pillars may be arranged in regions of a microstructured pattern, the regions may be arranged in a pattern, and the long-range energy gradient on the surface of the tissue scaffold may be biomimetic to the extracellular matrix.

[0089] In another embodiment, the tissue scaffold comprises a base layer including a pillar pattern and a capping layer including hydrophilic and hydrophobic polymer caps, wherein the capping layer comprises regions of hydrophilic and hydrophobic caps that may be arranged in a pattern and regions arranged in a long-range pattern, and when the tissue scaffold is placed in a humid environment, the surface may include a capping layer and components that can generate an extracellular matrix associated with the long-range pattern of microstructure zones, forming a non-biological cell adhesion layer placed on the capping layer. The scaffold comprising components for generating an extracellular matrix may further comprise living cells attached to the tissue scaffold.

[0090] Therefore, in another embodiment, the artificial tissue may comprise living cells attached to a tissue scaffold having a base layer with a pillar pattern, a capping layer with a hydrophilic / hydrophobic capped pillar pattern, and extracellular matrix components.

[0091] In another embodiment, the artificial tissue is a tissue scaffold comprising a base layer having a pillar pattern and a capping layer having a hierarchically microstructured pillar pattern. The artificial tissue may contain living cells attached to it, and when placed in a humid environment, it may induce a biological cell adhesion effect that can be generated at least partially by the Wenzel-Cassie interface with the target tissue surface.

[0092] In another embodiment, a method is provided for fabricating a tissue scaffold, which may include generating a pattern of pillars on a base layer by photolithography to form a patterned base layer, and depositing a capping layer on the patterned base layer to form a pattern of pillars individually capped with high-surface-energy and low-surface-energy polymers.

[0093] A patterned base layer may be formed using photolithography methods known in the art. For example, the method may include generating a photoresist layer by depositing a photoresist onto a base layer, placing a mask on top of the photoresist layer, and generating a patterned base layer by exposing the photoresist layer to ultraviolet light. A patterned base layer fabricated by photolithography can function as a mold for mass production of patterned base layers.

[0094] In one embodiment, a photoresist layer may be applied to a base layer using spin coating. Spin coating is a process in which the base layer may be mounted on a chuck, for example under vacuum, or rotated to spin the base layer around its axis of symmetry, or a liquid or semi-liquid substance, such as photoresist, may be dropped onto the base layer, and the centrifugal force generated by the spin causes the liquid or semi-liquid substance to spread substantially uniformly across the surface of the base layer. Variations of this process, such as coating and subsequent spinning, or spinning and subsequent dropping, may also be used.

[0095] As used herein, the term “photoresist” may be understood to refer to any material that may be sensitive to ultraviolet light, for example, ultraviolet light or light with a shorter spectrum (<400 μm). It will be understood that photoresists may be positive or negative.

[0096] In some embodiments, a base layer comprising a photoresist may be patterned by providing a mask having a desired shape and / or pattern, i.e., a dot pattern. The mask may be a solid mask, such as a photolithography mask. The mask may be provided and placed on the photoresist layer. Subsequently, a portion of the photoresist layer (i.e., the portion of the photoresist not covered by the mask) may be exposed to ultraviolet light.

[0097] In some embodiments, the mask placed on the photoresist layer may typically be manufactured by a standard photolithography procedure, such as electron beam lithography. Other methods for fabricating such a mask can be understood to include focusing energy for ablation (micromachining), including lasers, electron beams, and focused ion beams. Similarly, chemical etchants may be used to erode the material through the photoresist when using alternative mask materials.

[0098] Examples of chemical etching agents include, but are not limited to, hydrofluoric acid and hydrochloric acid. Photolithography masks are also commercially available.

[0099] In some embodiments, as disclosed herein, the microstructures on the patterned scaffold substrate may include features having one or more dimensions less than 1 micrometer. Polymers containing these microstructures can be processed, for example, by soft lithography. They can be deposited. In some embodiments, the polymer can be printed onto the transition polymer using a polydimethylsiloxane stamp. Optionally, the process involves printing multiple polymer structures with different surface energies in a sequential layered printing process. For example, if each polymer could be a protein, different proteins may be printed in different printing processes.

[0100] In some embodiments, polymers may be deposited on the transition polymer by self-assembly. Exemplary self-assembly processes include, but are not limited to, the assembly of collagen into protofibrils, actin into filaments, and DNA into double helixes.

[0101] In some embodiments, the polymer may be deposited by vaporization of the polymer and / or by deposition of the polymer on a transition polymer via a mask. For example, in some embodiments, the polymer may be deposited via patterned photocrosslinking on the transition polymer, where patterned light photocrosslinks the polymer in a selected pattern. The method may optionally include, in certain embodiments, a step of dissolving a non-crosslinked polymer outside the selected pattern. The patterned light may alter the reactivity of the polymer via the release of photodegradable groups or secondary photosensitive compounds in the selected pattern.

[0102] In some embodiments, the method for capping the microstructure may include the step of binding polymers together by forces selected from hydrophilic, hydrophobic, ionic, covalent, van der Waals, hydrogen bonding, and / or physical entanglement, and combinations thereof. The polymer structure may be released in some embodiments by coating the transition polymer with a solvent to dissolve the transition polymer or to change the surface energy of the transition polymer, or the polymer structure may be released into the solvent as a self-supporting structure. For example, the polymer may be released by applying a positive charge bias to the transition polymer, by hydrolyzing the transition polymer, or by subjecting the transition polymer to enzymatic action. The polymer may be constructed in a pattern, such as on an array of pillars. In some embodiments, multiple structures may be generated, and for example, the method may include the step of stacking multiple polymer structures to generate a multilayer hierarchical microstructured tissue scaffold.

[0103] In some embodiments, the microstructure may include at least one dimension on the scale of 10 nanometers. In certain embodiments, capillary force lithography or nanoimprint techniques may be used to produce highly uniform nanopatterned substrates over large areas. This robust technique can be used for a number of polymers having a variety of chemical, physical, and electrical properties. The technique is scalable, and the scaffold size (surface area) can vary, depending on the embodiment used, from very small or individual cell cultures to large constructs of macroscopic tissues.

[0104] In some embodiments, nanostructures may be used to vary the surface energy of the microstructure surface present on the soft tissue scaffold. In certain embodiments, the nanostructures may be between 10 nm and 10 μm, or between about 50 nm and 500 nm, or at least about 10 nm, or at least about 20 nm, or at least about 30 nm, or at least about 40 nm, or at least about 50 nm, or at least about 75 nm, or at least about 100 nm, or at least about 150 nm, or at least about 200 nm, or at least about 250 nm, or at least about 500 nm, or at least about 1000 nm, or at least about 2000 nm, or at least about 3000 nm, or at least about 4000 nm, or at least about 5000 nm, or at least about 6 μm, or at least about 7 μm, or at least about 8 μm, or at least about 9 μm It can be a ridge or pillar having a depth of m or at least about 10 μm.

[0105] In some embodiments, the depth may be between approximately 300 nm, but may be less than 1000 nm. In some embodiments, the groove depth may be between 5 nm and 1000 nm (1 μm), for example, at least approximately 5 nm, at least approximately 10 nm, at least approximately 20 nm, at least approximately 30 nm, at least approximately 40 nm, at least approximately 50 nm, at least approximately 60 nm, at least approximately 70 nm, or at least approximately 80 nm, or at least approximately 90 nm, at least approximately 100 nm, at least approximately 200 nm, at least approximately 300 nm, at least approximately 400 nm, or at least approximately 500 nm, or at least approximately 600 nm, or at least approximately 700 nm, or at least approximately 800 nm, or at least approximately 900 nm, or at least approximately 1000 nm (1 μm), but may be less than or equal to 1000 nm (1 μm).

[0106] In some embodiments, the groove depth may be between approximately 200 and 800 nm, or between approximately 100 and 200 nm, or between approximately 200 and 400 nm, or between approximately 400 and 600 nm, or between approximately 600 and 800 nm, or between approximately 800 and 1000 nm (1 μm).

[0107] In some embodiments, the groove depth may be 200 nm. In some embodiments, the groove depth may be between 20 and 100 nm, or between approximately 20 and 50 nm, or between approximately 40 and 60 nm, or between approximately 50 and 75 nm, or between approximately 75 and 100 nm.

[0108] In some embodiments, the groove width may be between 50 nm and 10 μm, or between approximately 200 nm and 1000 nm, or at least approximately 50 nm, or at least approximately 75 nm, or at least approximately 100 nm, or at least approximately 150 nm, or at least approximately 200 nm, or at least approximately 250 nm, or at least approximately 500 nm, or at least approximately 1000 nm, or at least approximately 2000 nm, or at least approximately 3000 nm, or at least approximately 4000 nm, or at least approximately 5000 nm, or at least approximately 6 μm, or at least approximately 7 μm, or at least approximately 8 μm, or at least approximately 9 μm, or at least approximately 10 μm.

[0109] In some embodiments, the depth may be between approximately 300 nm and less than 1000 nm. In some embodiments, the groove width of mature cardiomyocytes may be between approximately 200 nm and 1000 nm.

[0110] In some embodiments, the groove width may be between 10 and 100 nm, for example, at least about 10 nm, or at least about 20 nm, or at least about 30 nm, or at least about 40 nm, or at least about 50 nm, or at least about 60 nm, or at least about 70 nm, or at least about 80 nm, or at least about 90 nm, or at least about 100 nm, or greater than 100 nm.

[0111] In some embodiments, the groove width may be between 5 nm and 1 μm, for example, at least about 5 nm, at least about 10 nm, at least about 20 nm, at least about 30 nm, at least about 40 nm, at least about 50 nm, at least about 60 nm, at least about 70 nm, or at least about 80 nm, or at least about 90 nm, at least about 100 nm, at least about 200 nm, at least about 300 nm, at least about 400 nm, or at least about 500 nm, or at least about 600 nm, or at least about 700 nm, or at least about 800 nm, or at least about 900 nm, or at least about 1000 nm (1 μm), but may also be less than or equal to 1000 nm (1 μm). .

[0112] In some embodiments, the groove width may be between approximately 200 and 800 nm, or between approximately 100 and 200 nm, or between approximately 200 and 400 nm, or between approximately 400 and 600 nm, or between approximately 600 and 800 nm, or between approximately 800 and 1000 nm (1 μm).

[0113] In some embodiments, the width of the ridge may be between 50 nm and 10 μm, or between approximately 200 nm and 1000 nm, or at least approximately 50 nm, or at least approximately 75 nm, or at least approximately 100 nm, or at least approximately 150 nm, or at least approximately 200 nm, or at least approximately 250 nm, or at least approximately 500 nm, or at least approximately 1000 nm, or at least approximately 2000 nm, or at least approximately 3000 nm, or at least approximately 4000 nm, or at least approximately 5000 nm, or at least approximately 6 μm, or at least approximately 7 μm, or at least approximately 8 μm, or at least approximately 9 μm, or at least approximately 10 μm. In some embodiments, the depth may be between approximately 300 nm, but may be less than 1000 nm.

[0114] In some embodiments, the ridges between grooves have a width of approximately 50 nm to 1 μm (1000 nm), for example, at least about 50 nm, or at least about 60 nm, or at least about 70 nm, or at least about 80 nm, or at least about 90 nm, or at least about 100 nm, or at least about 125 nm, or at least about 150 nm, or at least about 175 nm, or at least about 200 nm, or at least about 250 nm, or at least about 300 nm, or at least about 400 nm, or at least about 500 nm, or at least about 600 nm, or at least about 700 nm, or at least about 800 nm, or at least about 900 nm, or at least about 50 nm, or any integer width between 50 nm and 1 μm (1000 nm), but less than or equal to 1000 nm.

[0115] In some embodiments, the width of the ridge may be 150 nm. In some embodiments, the width of the ridge may be between 5 nm and 1 μm, for example, at least about 5 nm, at least about 10 nm, at least about 20 nm, at least about 30 nm, at least about 40 nm, at least about 50 nm, at least about 60 nm, at least about 70 nm, or at least about 80 nm, or at least about 90 nm, at least about 100 nm, at least about 200 nm, at least about 300 nm, at least about 400 nm, or at least about 500 nm, or at least about 600 nm, or at least about 700 nm, or at least about 800 nm, or at least about 900 nm, or at least about 1000 nm (1 μm), but may be less than or equal to 1000 nm (1 μm). In some embodiments, the groove width may be between approximately 200 and 800 nm, or between approximately 100 and 200 nm, or between approximately 200 and 400 nm, or between approximately 400 and 600 nm, or between approximately 600 and 800 nm, or between approximately 800 and 1000 nm (1 μm).

[0116] Any suitable material, such as a material with a flat surface, such as metals (gold, silver, platinum, tantalum, or aluminum), or ceramics (alumina, titanium oxide, silica, or silicon nitride), may be used to fabricate the mask.

[0117] In some embodiments, a combination of positive and negative photoresists can be used. For example, a positive photoresist may be deposited on a base layer in a specific pattern, followed by coating with a negative photoresist in a complementary pattern. This results in a patterned scaffold containing a pattern in which regions of a specific surface energy are adjacent to regions of a different surface energy.

[0118] Once the photoresist layer is exposed to ultraviolet light and a patterned base layer is formed, the mask may be removed, or a capping material may be deposited on the patterned base layer to form an inter-microstructure patterned capping layer. The capping material binds directly to the base layer and does not depend on the introduction of reactive side-chain-containing species into the base layer.

[0119] In one embodiment, a thin capping layer, such as a silicone polymer, may be deposited on the patterned base layer as a continuous layer on each microstructure of the patterned base layer.

[0120] In one embodiment, capping molecules, such as hydrophobic molecules, may bond together to at least a portion of the microstructure of the patterned base layer to form a continuous layer. In another embodiment, the capping layer, such as hydrophobic molecules, may be deposited on the microstructure of the patterned base layer as a discontinuous layer, i.e., a pattern of individual molecules covering the surface.

[0121] The capping material may be deposited on the microstructure of the patterned base layer under conditions suitable for forming a capping layer on the patterned base layer. This may be achieved using any suitable technique known to those skilled in the art, including, for example, an xy translation device for spot printing the capping material individually onto the microstructure. In some embodiments, the step of forming the patterned capping layer may include subjecting the continuous capping layer to thermal decomposition, microwave irradiation, complete hydrolysis, or partial hydrolysis.

[0122] In one embodiment, the patterned capping layer may be approximately 0.1 to approximately 100 μm, 0.1 to approximately 70 μm, approximately 0.1 to approximately 50 μm, approximately 0.1 to approximately 30 μm, 0.1 to approximately 20 μm, approximately 0.1 to approximately 10 μm, or approximately 0.1 to approximately 10 μm, 0.1 to approximately 7 μm, approximately 0.1 to approximately 5 μm, approximately 0.1 to approximately 3 μm, 0.1 to approximately 2 μm, approximately 0.1 to approximately 1 μm, 0.5 to approximately 2 μm, approximately 1 to approximately 2 μm, approximately 1 to approximately 1.5 μm, approximately 1.5 to approximately 2 μm, or about 0.1um, 0.5um, 1um, 1.1um, 1.2um, 1.3um, 1.4um, 1.5um, 1.6um, 1.7um, 1.8um, 1.9um, 2um, 2.1um, 2.2um, 2.3um , 2.4um, 2.5um, 2.6um, 2.7um, 2.8um, 2.9um, 3.0um, 3,1um, 3.2um, 3.3um, 3.4um, 3.5um, 3.6um, 3.7um, 3.8um, 3,9um , 4.0um, 4.1um, 4.2um, 4.3um, 4.4um, 4.5um, 4.6um, 4.7um, 4.8um, 4.9um, 5,0um, 5.1um, 5.2um, 5.3um, 5.4um, 5.5u m, 5.6um, 5.7um, 5.8um, 5.9um, 6.0um, 6.1um, 6.2um, 6.3um, 6.4um, 6.5um, 6.6um, 6.7um, 6.8um, 6.9um, 7.0um, 7.1u The thickness may be m, 7.2um, 7.3um, 7.4um, 7.5um, 7.6um, 7.7um, 7.8um, 7.9um, 8.0um, 8.1um, 8.2um, 8.3um, 8.4um, 8.5um, 8.6um, 8.7um, 8.8um, 8.9um, 9.0um, 9.1um, 9.2um, 9.3um, 9.4um, 9.5um, 3.6um, 9.7um, 9.8um, 9.9um, or approximately 10.0um.

[0123] In another embodiment, the patterned chemical layer may have a thickness of 2 μm or less. In one embodiment, the patterned chemical layer may have a thickness of about 1 to about 1.5 μm. In one embodiment, the patterned chemical layer may have a thickness of about 10 to about 70 nm. In another embodiment, multiple layers of semi-rigid or flexible polymer are coated on the base layer.

[0124] In some embodiments, the patterned layers are approximately 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 2 to 50, 2 to 45, 2 to 40, 2 to 35, and 2 The single-layer thickness can be approximately 30, 2-25, 2-20, 2-15, 2-10, 2-5, 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 20-50, 25-50, 30-50, 35-50, 40-50, or approximately 45-50 nm. Intermediate ranges and values ​​within the above ranges and values ​​are also considered to be part of this disclosure. For example, the ranges of single-layer thicknesses from 1 to 5 nm and 7 to 25 nm are intended to be covered by this disclosure. Based on the above ranges and values, other examples are also assumed by this disclosure.

[0125] In some embodiments, the tissue scaffold may further comprise a cytokine layer arranged around the capping layer. In certain embodiments, cell signaling compounds may be deposited on the patterned capping layer to form a patterned cell signaling layer.

[0126] In some embodiments, the avoidance layer may be deposited on all or part of the patterned capping layer. In certain embodiments, the avoidance layer may be deposited in a pattern that may be complementary to the cellular cytokine layer. In certain embodiments, the avoidance layer may be a layer that inhibits the adhesion of cells, bacteria, or viruses.

[0127] Compounds that may be considered suitable for use as a microbial repellent layer include, but are not limited to, compounds having terminal pegylated groups and compounds containing alkyl terminal groups.

[0128] In certain embodiments, the tissue scaffold may further comprise living cells. Thus, in certain embodiments, a method for producing an artificial tissue may include living cells attached to a tissue scaffold, and the method may include bringing the tissue scaffold into contact with the cells and culturing them under conditions suitable for cell proliferation and / or differentiation.

[0129] Suitable cell types for these embodiments include, but are not limited to, fibroblasts, endothelial cells, keratinocytes, osteoblasts, chondrocytes and chondrocytes, hepatocytes, macrophages, cardiomyocytes, smooth muscle cells, skeletal muscle cells, tendinocytes, ligament cells, nerve cells, epithelial cells, and stem cells. Stem cells may include embryonic stem cells, adult stem cells, and induced pluripotent stem cells. In one embodiment, the cells are mesenchymal stem cells.

[0130] In another embodiment, the cells are human cells. Culture conditions for cell proliferation and / or differentiation are known to those skilled in the art.

[0131] In yet another embodiment, a method for producing a tissue scaffold may include generating a microstructure pattern on a base layer by photolithography to form a substrate having a patterned base layer; depositing a capping layer on the patterned base layer to form a substrate having a patterned capping layer; contacting the synthetic tissue scaffold with cells and culturing it under conditions suitable for the production of extracellular matrix components; and removing the cells from the substrate to provide a tissue scaffold containing extracellular matrix components produced by the long-range structure of the microstructure.

[0132] In some embodiments, the capping layer may be the result of nanotopography, as well as any conformation and geometric shape of parallel grooves and ridges that allow for anisotropic and polarized cell arrangement in the direction of the nanotexture. In particular, the polarization of the capping microstructure may correspond to the long-range structure expected as a result of coding zones.

[0133] In some embodiments, the top or surface of the ridge may be substantially flat, in some embodiments convex, and in some embodiments concave. In some embodiments, the ridge may be pointed or angular.

[0134] In some embodiments, the hollow portion or bottom of the microstructure may be substantially planar, in some embodiments concave, and in some embodiments convex. Any combination of planar, convex, or concave grooves and ridges is possible and conceivable, but it is generally preferable that the nanotextured area has repeating units of the same geometric shape.

[0135] In some embodiments, the surfaces of the ridges and / or grooves all have the same geometric shape, for example, they are all substantially planar. In alternative embodiments, the ridges and / or grooves have various combinations of convex, concave, or substantially planar surfaces. In some embodiments, the ridges and grooves are convex and concave, respectively, providing a corrugated cross-sectional appearance.

[0136] In some embodiments, after the production of extracellular matrix components, the cells may be removed from the synthetic tissue scaffold comprising the substrate, i.e., the substrate may be decellularized.

[0137] Methods for decellularization are known in the art and include, for example, methods for loosening cell adhesion from the extracellular matrix, subsequently lysing the cell membrane, and solubilizing intracellular components under conditions that maintain the integrity and activity of the matrix. For example, decellularization may be achieved by removing calcium by chelation to loosen cell adhesion, followed by lysing the cell membrane and solubilizing intracellular components by incubation with a nonionic surfactant in an alkaline pH hypotonic buffer.

[0138] A scaffold containing extracellular matrix components may further comprise live cells attached to the matrix components. Thus, in another embodiment, a method for creating an artificial tissue comprising live cells attached to a tissue scaffold may include contacting the tissue scaffold with cells and culturing under conditions suitable for cell growth and / or differentiation.

[0139] In some embodiments, the cells may be attached to the tissue scaffold substrate by placing the scaffold in a culture with a cell suspension and allowing the cells to sediment and attach to the surface. The cells may respond to patterning of the scaffold surface, with respect to adhesion, and with respect to constructing ECM proteins in a pattern on the scaffold surface. The cells may also respond to patterning with respect to maturation, proliferation, and function. The cells on the scaffold may be cultured in an incubator under physiological conditions (e.g., at 37°C) until the cells form a two-dimensional tissue, and their orientation may be determined by the pattern provided on the tissue scaffold.

[0140] Tissues may be established on tissue scaffolds generally known in the art using appropriate cell culture methods. The seeding density of the cells varies depending on the cell size and cell type, but can be readily determined by methods known in the art. In one embodiment, the cells are at a density between about 1×103 and about 6×105 cells / cm 2 or about 1×103 cells / cm 2 about 2×103 cells / cm 2 about 3×103 cells / cm 2 about 4×103 cells / cm 2 about 5×103 cells / cm 2 about 6×103 cells / cm 2 about 7×103 cells / cm 2 about 8×103 cells / cm 2 about 9×103 cells / cm 2 about 1×104 cells / cm 2 about 2×104 cells / cm 2 about 3×104 cells / cm2 Approximately 4 × 10⁴ cells / c m 2 Approximately 5 × 10⁴ cells / cm² 2 Approximately 6 × 10⁴ cells / cm² 2 Approximately 7 × 10⁴ cells / cm² 2 Approximately 8 × 10⁴ cells / cm² 2 Approximately 9 × 10⁴ cells / cm² 2 Approximately 1×10⁵ cells / cm² 2 Approximately 1.5 × 10⁵ cells / cm² 2 Approximately 2 × 10⁵ cells / cm² 2 Approximately 2.5 × 10⁵ cells / cm² 2 Approximately 3 × 10⁵ cells / cm² 2 Approximately 3.5 × 10⁵ cells / cm² 2 Approximately 4 × 10⁵ cells / cm² 2 Approximately 4.5 × 10⁵ cells / cm² 2 Approximately 5 × 10⁵ cells / cm² 2 Approximately 5.5 × 10⁵ cells / cm² 2 Approximately 6 × 10⁵ cells / cm² 2 Approximately 6.5 × 10⁵ cells / cm² 2 Approximately 7 × 10⁵ cells / cm² 2 Approximately 7.5 × 10⁵ cells / cm² 2 Approximately 8 × 10⁵ cells / cm² 2 Approximately 8.5 × 10⁵ cells / cm² 2 Approximately 9 × 10⁵ cells / cm² 2 Approximately 9.5 × 10⁵ cells / cm² 2 Approximately 1×10⁶ cells / cm² 2 Approximately 1.5 × 10⁶ cells / cm² 2 Approximately 2 × 10⁶ cells / cm² 2 Approximately 2.5 × 10⁶ cells / cm² 2 Approximately 3 × 10⁶ cells / cm² 2 Approximately 3.5 × 10⁶ cells / cm² 2 Approximately 4 × 10⁶ cells / cm² 2 Approximately 4.5 × 10⁶ cells / cm² 2 Approximately 5 × 10⁶ cells / cm² 2 Approximately 5.5 × 10⁶ cells / cm² 2 Approximately 6 × 10⁶ cells / cm² 2 Approximately 6.5 × 10⁶ cells / cm² 2 Approximately 7 × 10⁶ cells / cm² 2 Approximately 7.5 × 10⁶ cells / cm²2 , about 8×106 cells / cm 2 , about 8.5×106 cells / cm 2 , about 9×106 cells / cm 2 , or approximately 9.5 × 10⁶ cells / cm² 2 Seeds are sown at the following density. Intermediate values ​​and ranges between the values ​​and ranges listed above are also assumed.

[0141] In one embodiment, a patterned tissue scaffold may be brought into contact with a plurality of cells and cultured to produce a biological tissue, such as a tissue having at least partial in vivo biological activity. In one embodiment, the biological tissue may be removed from the tissue scaffold.

[0142] In some embodiments, CMS polymer substrates useful for compositions according to the methods described herein can be sterilized using conventional disinfection / sterilization techniques, including glutaraldehyde tanning, formaldehyde tanning at an acidic pH, propylene oxide treatment, ethylene oxide treatment, gas plasma sterilization, gamma irradiation or electron beam treatment, and peracetic acid (PAA) disinfection.

[0143] While sterilization techniques that do not adversely affect the mechanical strength, structure, and biotropic properties of polymer substrates are sometimes preferred, other techniques may also be useful and sufficient. For example, strong gamma irradiation can cause a loss of strength in polymer substrate sheets. Sterilization techniques include exposing polymer substrates to peracetic acid, 1-4 Mrads of gamma irradiation (more preferably 1-2.5 Mrads of gamma irradiation), or gas plasma sterilization. In some examples, polymer substrates may undergo two or more sterilization processes. After the polymer substrate is treated in the first disinfection step, for example by treatment with peracetic acid, the polymer substrate may be wrapped in plastic or foil wrap and sterilized again using electron beam or gamma irradiation sterilization techniques.

[0144] Patterned tissue scaffolds (and / or biological tissue prepared on and removed from the tissue scaffolds) may be used in a wide range of applications, including, but not limited to, devices for tissue repair and support such as sutures, surgical and orthopedic screws, and surgical and orthopedic plates; natural coatings or components for synthetic implants; cosmetic implants and supports; organ or tissue repair or structural support; substance delivery; bioengineering platforms; platforms for testing the effects of substances on cells; cell culture; wound healing; and numerous other applications.

[0145] In one embodiment, biological tissue may be removed from the scaffold before use. In another embodiment, it is not necessary to remove biological tissue from the scaffold before use.

[0146] The base layer of the patterned tissue scaffold is prepared on top of the tissue scaffold. Medical devices such as orthopedic screws or plates may contain tissue or cells from the same tissue in which the device is used. Non-limited examples of medical devices suitable for use include diagnostic implant devices, diabetic implants such as biosensors, stimulators, and glucose monitoring devices, external fixation devices, external fixation implants, and orthopedic trauma implants; implants used for joint and spinal injury / reconstruction such as plates, screws, rods, plugs, cages, scaffolds, artificial joints (e.g., hands, wrists, elbows, shoulders, spines, hips, knees, ankles), and wires; tumor-related bone and soft tissue replacement devices; dental and oral / maxillofacial devices; cardiovascular implants such as stents, catheters, valves, rings, and implantable cardioverter-defibrillators; contact lenses, ocular implants, corneal prostheses, dermatological implants, cosmetic implants, and implantable drug delivery pumps; and general surgical devices and implants such as drainage catheters, shunts, tapes, meshes, ropes, cables, wires, sutures, skin staples, burn sheets, and vascular patches, as well as temporary / non-permanent implants.

[0147] Furthermore, since the methods described herein are applicable to flexible polymer substrate layers, flexible membranes including patterned tissue scaffolds may be used to support or connect tissue or structures that have undergone injury, surgery, or degradation. For example, a patterned tissue scaffold containing a flexible polymer may be used as a graft to connect and / or bind tissue and provide an internal or external platform for tissue regeneration. In such cases, the flexible polymer may be biodegradable or non-biodegradable.

[0148] Another use of patterned soft tissue scaffolds may be as a barrier to prevent postoperatively induced adhesions. For example, since adhesions are a result of disordered extracellular matrix (ECM), patterned tissue scaffolds may be used to organize ECM depositions and prevent adhesion formation.

[0149] Another embodiment of the patterned tissue scaffold may be used as a template for nerve proliferation. For example, the patterned tissue scaffold may be used to culture nerve cells in a pattern that mimics the in vivo environment, so that proper neural connections are formed, rather than in the unorganized sequence of nerve cells that would be produced without using the patterned scaffold.

[0150] Therefore, in one embodiment, a method for tissue repair and regeneration may include implanting artificial tissue into a subject that requires such tissue repair or regeneration.

[0151] In some embodiments, patterned tissue scaffolds in contact with or seeded with living cells may be combined with drugs to improve the function of the implant or graft. For example, antibiotics, anti-inflammatory drugs, local anesthetics, or combinations thereof may be added to cell-treated patterned tissue scaffolds to positively influence the healing process.

[0152] In one embodiment, tissue scaffolds may be used to study the functional differentiation of stem cells (e.g., pluripotent stem cells, multipotent stem cells, induced pluripotent stem cells, and progenitor cells of embryonic, fetal, neonatal, juvenile, and adult origin). For example, patterned tissue scaffolds may be brought into contact with undifferentiated cells, such as stem cells, and differentiation may be observed.

[0153] Patterned tissue scaffolds, cultured by seeding cells to form tissues, are useful for measuring tissue activity or function, investigating histogenesis and disease pathology, and for drug discovery.

[0154] Accordingly, the Disclosure also provides a method for identifying compounds that modulate tissue function. The method may include providing a tissue scaffold comprising tissue produced according to the method of the Disclosure, contacting the tissue with a test compound, and measuring the effect of the test compound on tissue function in the presence and absence of the test compound, or identifying a compound that modulates tissue function by showing that the test compound modulates tissue function compared to tissue function in the absence of the test compound.

[0155] In another embodiment, the Disclosure also provides a method for identifying compounds that may be useful in treating or preventing diseases. In certain embodiments, the method may include providing a tissue scaffold comprising tissue produced according to any one of the methods of the Disclosure, contacting the tissue with a test compound, and measuring the effect of the test compound on tissue function in the presence and absence of the test compound, thereby identifying a compound useful in treating or preventing diseases by showing that the test compound modulates tissue function compared to tissue function in the absence of the test compound.

[0156] In some embodiments, one or more test compounds used may be any agent, including chemical agents (such as toxins), small molecules, pharmaceuticals, peptides, proteins (e.g., antibodies, cytokines, enzymes), as well as nucleic acids, including gene therapies and transgenes, that may encode therapeutic agents such as proteins, antisense agents (i.e., nucleic acids containing sequences complementary to target RNA, e.g., RNAi or siRNA, expressed in a target cell type), ribozymes, and combinations thereof.

[0157] In certain embodiments, the test compound may be added to the tissue by any suitable means. For example, the test compound may be added by dropping onto the surface of a tissue scaffold and diffusing into the tissue or otherwise entering the tissue, or it may be added to a nutrient medium and diffusing through the medium.

[0158] The following embodiments are useful for further illustrating the various embodiments disclosed herein and should not be construed as limiting or exhausting the disclosure.

[0159] Example 1. Surface energy gradient of a microstructured scaffold having a microstructure pattern.

[0160] In one embodiment, the structural scaffold includes a microstructured surface and chemical modifications of the microstructured surface to generate a pattern of surface energy gradients. The surface energy of a liquid may be reported as surface tension, which can be energy per unit surface area. In the case of a solid, the surface energy in a given planar projection area is proportional to the surface area of ​​the solid in the planar projection area. The change in energy per unit area as a function of distance is the surface energy gradient. Referring to Figure 1, the pillar-on-pillar microstructured surface 100 comprises a first pillar 102 and a second pillar 104 and is in contact with a binary fluid consisting of a high surface tension liquid 106 and a low surface tension liquid 108. Liquids 106 and 108 may be immiscible in some embodiments. The surface energy gradient 110 may arise from high surface energy 112 and low surface energy 114, and continuity requires that the surface energy changes smoothly from the high surface energy area 112 to the low surface energy area 114, thus resulting in a surface energy gradient. Liquids 106 and 108 map this energy gradient by developing interfaces 116. These interfaces result in the formation of capillary bridges 118 that form both within the tissue scaffold and between the tissue scaffold and the tissue surface. Capillary bridges 122 may also be present within the tissue scaffold capillary bridges. Capillary bridges 124 separate tissue from the tissue scaffold 118. Capillary crosslinking to 120 is also possible. Zone 126 comprises a mixture of liquids 106 and 108. In zone 126, the energy gradient is not large enough to cause separation of liquids 106 and 108. Depending on environmental conditions, zone 126 can dissolve into separation of liquids 106 and 108. As a result, the two capillary crosslinking types 122 and 124 share interface 116. This structure of interface 116 is capable of self-stabilization against thermal disruption and acts as a guide surface for inducing cells into a topological arrangement. Different types of cells may follow different pathways along surface 116.

[0161] In some embodiments disclosed herein, the tissue scaffold may guide cells into a configuration that acts as a precursor structure for long-range tissue formation. These guiding surfaces 116 can guide cells to generate functional tissue within the scaffold 100 and between the scaffold and the surrounding tissue 120, thus providing a means for inducing neovascularization from biological tissue 120 to scaffold tissue. Conventional tissue scaffolds are often encased in dense fibrous tissue, separating the tissue formed on the tissue scaffold from nutrient and oxygen sources.

[0162] Example 2. Encoding zone of a tissue scaffold having a microstructure pattern.

[0163] Referring to Figure 2a, a diagram of symbols is shown. The term “symbol” can be understood as referring to a microstructure pattern comprising a juxtaposition of surface energy gradients in a defined surface area. In a simple embodiment of the microstructure surface 200, the surface energy gradient of the microstructure pattern may be defined by microstructure surfaces of different surface energies. For example, low surface energy (hydrophobic) microstructures may be juxtaposed with high surface energy (hydrophilic) microstructures. If these microstructures are labeled as 0 for hydrophobic microstructures and 1 for hydrophilic microstructures, then the sequence of 0s and 1s can be understood as describing the symbols. The sequence can be two-dimensional, or higher-dimensional when two-dimensional microstructured surfaces are stacked.

[0164] When considering two-dimensional codes, the zone defining a code may consist of lines of 0s and 1s. Considering a first line, adjacent lines can be considered as permutations of the first line. Consequently, a code may be specified as a group of permutations acting on the first lines of 0s and 1s and a specified first line. Choosing different first lines generates different code zones for the same group operation. Therefore, the zone specified by a code can represent elements of a mathematical group.

[0165] For a given first line, a set of coding zones can be generated by a first mathematical group operation. These first zones can be treated as elements of another second group, and by applying the second group operation to the first zones, larger second zones can be constructed. There are many variations on this theme. When the same group is repeatedly applied to first, second, and so on zones, self-similar structures can be obtained. These self-similar structures are macroscopic and can mimic many natural organizational structures.

[0166] In certain embodiments, mathematical groups include Zn: a cyclic group of order n (also denoted as Cn, and potentially isomorphic to an additive group), and a dihedral group of order 2n (often D n Or D 2n (The notation used is as follows), K4: a 4-group of Klein of order 4, Sn: a symmetric group of degree n containing n! permutations of n elements, A n Q is an alternating group of degree n, containing even permutations of n elements, with order 1 for n=0,1 and order n! / 2 otherwise. 4n : may include a doubly cyclic group of order 4n, and Q8: a quaternion group of order 8.

[0167] Referring to Figures 2a to 2d, the microstructure surface zones 200, 206, 208, and 210 One example of a set includes zones of fine features that can embody group operations that function as a long-range organizing language for cells. When these zones are juxtaposed, the language encodes different cell arrangements.

[0168] For example, Figure 2a shows an embodiment in which the microstructured surface zone 200 may include microstructured lines 212. The microstructured surface zone 200 may consist of nine microstructured lines 212. Each microstructured line 212 may consist of microstructured elements 214, and each line 212 may contain up to eight microstructured elements 214. For example, the microstructured elements 214 may be of a hydrophobic type or a hydrophilic type. One simple embodiment includes microstructured elements 214 which are circular pillars capped in one case with a hydrophilic material and in the other case with a hydrophobic material. When constructing a secondary microstructure, the two zones 200, 210 of the microstructured surface can be considered hydrophilic and hydrophobic, respectively. The surface zones 200, 210 can then be juxtaposed to form a larger zone by arranging the surfaces in surface zone 206 according to the reference numerals. The construction involves replacing the hydrophilic and hydrophobic microstructure of surface zone 206 with hydrophilic and hydrophobic zones 200 and 210 of the surface. Thus, the 9 × 8 zones 206 of the microstructure become 64 × 81 zones of the larger zone microstructure. It should be understood that different combinations of these operations contain different cellular instructions that exist within a group-defined cellular signaling language.

[0169] Example 3. Two-dimensional Fourier transform of zones in a patterned tissue scaffold.

[0170] The two-dimensional Fourier transform of the image plots the spatial frequency distribution of black pixels in a grayscale image. The Fourier transform provides a visual representation of the sensing of periodic structure cells as they proliferate on a patterned scaffold containing zones, as shown in Figure 2.

[0171] Referring to Figures 3a to 3d, a language containing Fourier-transformed words may be useful in this organization's scaffold. The FFT image in Figure 3 displays the absolute values ​​(or complex amplitudes) of spatial frequencies found in the image. The amplitude of the frequency coefficient is shown as intensity, and the wavelength or frequency is shown as the radial distance from the center of the image, with low-frequency pixels at the center and high-frequency pixels at the edges of the image. The orientation of the frequency can also be seen in a 2D FFT perpendicular or orthogonal to the image features of the real image; for example, a horizontal line is observed as a vertical feature in a 2D FFT.

[0172] It should be noted that the microstructure surface zones 300, 302, 304, and 306 are macroscopically similar but differ in detail. The macroscopic similarity between microstructure surface zones 300, 302, 304, and 306 may allow cells to grow directionally across large spatial dimensions. The differences in detail between zones 300, 302, 304, and 306 may direct cells to create different topological structures that act as precursors to mature functional tissues.

[0173] Example 4. Two-dimensional Fourier transform of cells grown on a patterned tissue scaffold.

[0174] To generate secondary tissue, fast Fourier transforms were performed on cell cultures of endothelial cells that had been immobilized on ATC GaYaTri blocks and stained blue.

[0175] An FFT image displays the absolute values ​​(or complex amplitudes) of spatial frequencies found in the image. The amplitude of the frequency coefficient is displayed as intensity, and the wavelength or frequency is displayed as the radial distance from the center of the image, with low-frequency pixels at the center and high-frequency pixels at the edges of the image. The direction of the wavenumber can also be seen in a 2D FFT, where it is perpendicular or orthogonal to the image features of the real image; for example, a horizontal line is observed as a vertical feature in a 2D FFT.

[0176] Example 5.3-dimensional tissue scaffold.

[0177] In some embodiments, the tissue scaffold is a layered film biocompatible (bioreabsorbable or nonabsorbable) scaffold, at least a portion of which is porous (macroporous or microporous), and can provide controlled morphological guidance as well as a surface energy gradient based on the material and microstructure.

[0178] The film used in certain embodiments of the patterned tissue scaffold may have long-range microstructures that provide an organizing energy gradient at the microstructural level, which can ultimately lead to the regeneration of fully or partially functional tissue. The tissue scaffold film may have patterns of surface energy gradients and microstructures that enable cell-based studies for tissue endoproliferation, tissue repair, tissue regeneration, and therapeutic drug discovery. The scaffold may provide a layered film having openings that provide interconnections between living cells organized on the patterned film in order to control proliferation in a predictable manner.

[0179] The features of certain embodiments of patterned scaffolds may be controlled to suit a desired application by selecting appropriate conditions for forming a layered film structure having openings in selected regions of each film. These patterned scaffolds may be arranged in layers, which offers clear advantages over the prior art in which the scaffolds have an isotropic or random structure.

[0180] The tissue scaffolds described herein may include cellular openings of varying sizes and shapes (e.g., cellular openings defining pores in one or more films). Whether regular or irregular in shape, the diameter of the cellular openings can range from about 1 to about 10,000 microns. For example, the cellular openings can range from about 5 to 95,000 microns, about 10 to 10,000 microns, about 25 to about 7,500 microns, about 50 to 5,000 microns, about 100 to about 2,500 microns, about 100 to about 5,000 microns, about 250 to about 2,500 microns, about 250 to about 1,000 microns, about 500 to about 1,000 microns, about 750 to about 1,000 microns, or in between.

[0181] In some embodiments, cell openings can provide pathways for cell entry and nutrient diffusion. Porosity can be controlled and can range from about 10% to 95%. Since cell openings and / or channels can have diameters in the micron range, useful films and scaffolds can be described as microporous in some embodiments. They can also be nonporous and reabsorbable in certain embodiments.

[0182] Features of several embodiments of tissue scaffolds include: selecting features to obtain a surface energy gradient along three axes for preferred cell culture; channels penetrating the scaffold to enhance cell invasion, angiogenesis, and nutrient diffusion; micropatterning of the membrane on the surface to improve cell organization; adjustability of pore size and shape; anisotropic mechanical properties; composite layered structures with polymer composition gradients to modify cellular responses to different materials; blends of different polymer compositions to create structures with parts that degrade or reabsorb at different rates; films blended or coated with bioactive agents including but not limited to biological factors and growth factors; the ability to create three-dimensional structures with controlled microstructures; and the ability to provide composite structures to other medical devices. Alternatively, by combining it with other drugs, it can be controlled to suit the desired application.

[0183] In some embodiments, the biocompatible scaffold may include substantially controllable pore structures designed to facilitate intercellular connectivity between layers of the patterned tissue scaffold, so as to reflect long-range patterns present on each patterned tissue scaffold layer, including a three-dimensional tissue scaffold assembly. Properties selected from the group including composition, stiffness, pore structure, and bioabsorption rate can be controlled.

[0184] Scaffolds can be fabricated from absorbable or non-absorbable polymers. Blends of polymers can be applied to create compositional and surface energy gradients layer by layer. In applications where one composition may suffice, the scaffold provides a biocompatible scaffold that may have structural variations across one or more layers that can direct cell invasion and association on a long-range scale. Structural variations can result in variations in the degradation of the entire scaffold.

[0185] In some embodiments, the patterned three-dimensional tissue scaffold includes interconnection pores and channels to facilitate the transport of nutrients into the layers of the patterned tissue scaffold and / or cell invasion. Some channels may be fabricated to facilitate the delivery of drugs, compounds, or cells to the scaffold using delivery means. Drugs, compounds, or cells can be delivered using positive or negative pressure methods.

[0186] In one embodiment, a method for tissue repair or regeneration may include bringing a first tissue into contact with a scaffold pore gradient at a location on a scaffold having appropriate properties that enable tissue growth. The concept of controlled transitions of physical and chemical properties, and / or microstructural features within the scaffold can facilitate tissue growth or regeneration.

[0187] Scaffolds are particularly useful for creating tissue junctions between two or more layers of tissue. In multicellular systems, one type of cell may reside in one area of ​​the scaffold, and a second type of cell may reside in a separate area of ​​the scaffold. Delivery channels can be used to position drugs, compounds, or cells in specific areas of the scaffold. Channels can also be used to generate a controlled flow of a medium using positive or negative pressure means. External sources can be used to generate flow through the channels.

[0188] In some embodiments, a gradient of absorbent polymers in different layers can be created, forming a compositional gradient from one polymer material to a second polymer material. In situations where one composition may be sufficient for application, the scaffold may provide a biocompatible film scaffold that may have microstructural variations in its structure over one or more dimensions that can mimic the anatomical features of tissue. The cross-sectional area of ​​the implant may vary in this example. As the scaffold degrades due to surface erosion or bulk degradation, areas with increased cross-sectional area may degrade at a slower rate.

[0189] In some embodiments, films can be laminated and bonded to each other. The films can be bonded using ionic or covalent bonds. Photoinitiated bonds can be generated using suitable materials such as benzophenone. In certain embodiments, biocompatible adhesives can be used. Alternatively, in certain embodiments, heat and pressure can be used.

[0190] In some cases, the material used to manufacture the patterned scaffold is, It may include a sheet. The sheet may be substantially planar. In some embodiments, the material may have a layered structure at least partially. In one embodiment, the material may include a first layer and a second layer, the first layer may have a higher absorption rate than the second layer. The first layer may be positioned adjacent to the second layer. The second layer may be configured to be located closer to the tissue structure than the first layer.

[0191] In one embodiment, the material may be at least partially porous to promote internal growth of the tissue. The first layer may have a higher pore density than the second layer. The first layer may have a smaller pore diameter than the second layer. In one example, at least a portion of the pores form at least a partial gradient with varying densities.

[0192] In another embodiment, the material may be at least partially porous to promote internal growth of the tissue. The layer may have a higher pore density in a selected region. The central region may have a higher pore density than the outer regions. In some cases, at least a portion of the pores form at least a partial gradient from one region to the next.

[0193] The material may include an anti-adhesion filler that fills at least a portion of the pores. The material may include an anti-adhesion coating along at least a portion of the surface of the material. Alternatively, a material used to promote tissue adhesion and bonding may be used in conjunction with the scaffold.

[0194] As used herein, “biocompatible substrates” may be understood to include materials suitable for implantation into a subject. Biocompatible substrates may reduce or prevent toxicity or adverse effects after implantation into a subject. In one embodiment, a biocompatible substrate may be a polymer having a surface that can be molded into a desired structure requiring repair or replacement. The polymer may also be molded into a part of a structure requiring repair or replacement. A biocompatible substrate provides a support framework that allows cells to adhere to it and grow thereon. A cultured cell population can then be grown on a biocompatible substrate containing a microstructured substrate that provides appropriate gap distances necessary for intercellular interactions.

[0195] In some embodiments, the CMS substrate comprises a polymer hydrogel containing biocompatible extracellular matrix proteins, synthetic or designed matrix polypeptides, or other designed polypeptides within the matrix of the polymer substrate. In some embodiments, the designed matrix polypeptides include poly-L-lysine, poly-D-lysine, poly-ornithine, vitronectin, or erythronectin. In some embodiments, the designed polypeptides include domains of extracellular matrix proteins that bind to integrin receptors, domains of extracellular matrix proteins that bind to integrin receptors, and others well known to those skilled in the art.

[0196] Biocompatible materials useful for the film layer may include non-absorbent polymers such as polypropylene, polyethylene, polyethylene terephthalate, polytetrafluoroethylene, polyarylether ketone, nylon, fluorinated ethylene propylene, polybutester, and silicone, or copolymers thereof (e.g., a copolymer of polypropylene and polyethylene); absorbent polymers such as polyglycolic acid (PGA), polylactic acid (PLA), polycaprolactone, and polyhydroxyalkanoates, or copolymers thereof (e.g., a copolymer of PGA and PLA); or tissue-based materials (e.g., collagen or other biological materials or tissues obtained from a patient receiving a scaffold or from another person). The polymers may be D-isoforms, L-isoforms, or mixtures of both.

[0197] In some embodiments, other materials can be selected to be used as the base material, which can be chosen from the group consisting of hydroxyapatite (HAP), tricalcium phosphate (TCP), tetracalcium phosphate (TTCP), anhydrous dicalcium phosphate (DCPA), dicalcium phosphate dihydrate (DCPD), octacalcium phosphate (OCP), calcium pyrophosphate (CPP), collagen, gelatin, hyaluronic acid, chitin, and poly(ethylene glycol). In alternative embodiments, the base material may also include additional materials, for example, but are not limited to, calcium alginate, agarose, type I, type II, type IV or other collagen isoforms, fibrin, hyaluronic acid derivatives or other materials.

[0198] In some embodiments, the CMS may contain additional components in or on the substrate selected from the group including extracellular matrix proteins, growth factors, lipids, fatty acids, steroids, sugars and other bioactive carbohydrates, biologically derived homopolymers, nucleic acids, hormones, enzymes, pharmaceuticals, cell surface ligands and receptors, cytoskeletal filaments, motor proteins, and combinations thereof. Alternatively or additionally, the structure may include at least one conductive polymer selected from poly(pyrrole), poly(acetylene), poly(thiophene), poly(aniline), poly(fluorene), poly(3-hexylthiophene), polynaphthalene, poly(p-phenylene sulfide), poly(N-isopropylacrylamide) (PIPAAm), and poly(para-phenylene vinylene). In some cases, the polymer structure includes an integrated pattern of polymers and a molecular residual trace of poly(N-isopropylacrylamide).

[0199] In some embodiments, the polymer structure comprises or includes at least one biological hydrogel selected from fibrin, collagen, gelatin, elastin, and other protein and / or carbohydrate-derived gels, or polyethylene glycol, polyvinyl alcohol, polyacrylamide, poly(N-isopropylacrylamide), poly(hydroxyethyl methacrylate), and other synthetic hydrogels, as well as combinations thereof.

[0200] In one embodiment, the scaffold material has a plurality of patterned zones. The scaffold material may have a plurality of patterned zones, and one or more of the zones within the plurality of zones have a diameter of about 100 to about 10,000 micrometers, measured along the longest axis of the zone. The scaffold material may have a plurality of zones, and one or more of the zones within the plurality of zones are essentially square, rectangular, circular, elliptical, sinusoidal, or rhombic.

[0201] In one embodiment, the thickness of one or more of the films within the scaffold may be about 0.25 cm or less. For example, the scaffold can be formed from two or more films that may be the same or different in thickness. For example, the film can be approximately 0.20 cm or less, approximately 0.18 cm or less, approximately 0.16 cm or less, approximately 0.14 cm or less, approximately 0.12 cm or less, approximately 0.10 cm or less, approximately 0.05 cm or less, approximately 0.025 cm or less, approximately 0.020 cm or less, approximately 0.015 cm or less, approximately 0.014 cm or less, approximately 0.013 cm or less, approximately 0.012 cm or less, approximately 0.011 cm or less, approximately 0.010 cm or less, approximately 0.009 cm or less, approximately 0.008 cm or less, approximately 0.007 cm or less, approximately 0.006 cm or less, approximately 0.005 cm or less, approximately 0.004 cm or less, approximately 0.003 cm or less, approximately 0.002 cm or less, or approximately 0.001 cm.

[0202] In some embodiments, CMS is constructed using a polymer structure that mimics the stiffness of tissue, such as normal smooth muscle. The stiffness of normal muscle typically varies from 5 kPa to 40 kPa. In some embodiments, CMS typically varies from 30 kPa to 200 kPa. The CMS is constructed using a polymer structure that mimics affected or aged muscle, having stiffness that varies between 5 and 200 kPa. Thus, the CMS includes a polymer substrate having stiffness in the range of 5 to 200 kPa, for example, at least about 5 kPa, or at least about 10 kPa, or at least about 20 kPa, or at least about 30 kPa, or at least about 40 kPa, or at least about 50 kPa, or at least about 60 kPa, or at least about 70 kPa, or at least about 80 kPa, or at least about 90 kPa, or at least about 100 kPa, or at least about 120 kPa, or at least about 140 kPa, or at least about 160 kPa, or at least about 180 kPa, or at least about 200 kPa or greater than 200 kPa, or any integer stiffness between 5 and 200 kPa.

[0203] Therefore, while specific embodiments of the present invention of novel and useful tissue scaffolds having patterned microstructures have been described, such references are not intended to be construed as limitations on the scope of the invention, except as set forth in the following claims.

Claims

1. A patterned structure scaffold comprising a microstructured base layer and a capping layer deposited thereon, The capping layer on each microstructure is continuous or microstructured. The microstructure pattern comprises at least two types of capping layers deposited on at least two microstructures of the base layer, The microstructure of the capping layer of at least two types is deposited on the base layer in a pattern for forming a tissue growth surface, and the microstructure of the capping layer is a tissue scaffold with a width of about 1 μm to about 1000 μm.

2. The combination of the base microstructure features and the at least two capping layers forms at least two types of hierarchical microstructures, each type having a unique surface energy, according to claim 1.

3. The structural scaffold according to claim 1, wherein the material of the base layer is capped with at least one smooth layer of a material different from the material of the base layer.

4. The microstructure of the base layer is capped by at least one microstructure having a surface energy separate from the surface energy of the base layer material, according to claim 1.

5. A patterned structure scaffold comprising at least one microstructure comprising a base layer and at least one capping layer deposited on at least one microstructure having the base layer, Each combination of at least one base layer microstructure and at least one capping layer forms at least two hierarchical microstructure types. The tissue scaffold microstructure pattern comprises at least two hierarchical microstructure types arranged in a geometric pattern on the substrate of the tissue scaffold, The aforementioned microstructure scaffold microstructure pattern presents a surface with patterned variations in surface energy to form a surface energy pattern. A patterned tissue scaffold in which the surface energy pattern guides at least one cell type that comes into contact with the patterned tissue scaffold to form at least one tissue structure that is at least 10 times the size of the constituent cells.

6. The aforementioned hierarchical microstructure type guides a first type of cell to form a first type of tissue structure, and guides a second type of cell to form a second type of tissue structure. The patterned tissue scaffold according to claim 5, wherein the first and second tissue structures form a tissue having a biological function.

7. The patterned tissue scaffold according to claim 1, further comprising a plurality of cells deposited on the tissue proliferation surface, wherein the cells are selected from cell types consisting of endothelial cells, smooth muscle cells, fibroblasts, tendinocytes, mesenchymal stem cells, skeletal muscle cells, chondrocytes, and epithelial cells.

8. The patterned tissue scaffold according to claim 7, wherein the endothelial cells are vascular endothelial cells, the smooth muscle cells are vascular smooth muscle cells, and the mesenchymal stem cells are bone marrow-derived human mesenchymal stem cells.

9. a) A step of applying a photoresist to the base layer in order to produce a photoresist-coated base layer, b) A step of covering the photoresist-coated base layer with a photomask characterized by a microstructure pattern in order to generate a masked ensemble, c) Exposing the masked ensemble with UV radiation to provide an exposed ensemble, d) The step of removing the photomask from the exposed ensemble in order to provide a coated base layer having a UV-exposed microstructure pattern, e) Developing the coated base layer to remove the UV-exposed portion of the photoresist, thereby exposing the surface of the base layer in a pattern corresponding to the microstructure pattern of the photomask; f) A step of applying a polymer to the surface of the exposed microstructured base layer, g) In order to provide a patterned polymer layer corresponding to the microstructure pattern of the photomask, the step of removing residual photoresist so as to expose the microstructure of the patterned polymer layer on the base layer. A method for producing the patterned structure scaffold described in claim 1, including the method described in claim 1.

10. The patterned tissue scaffold according to claim 5, wherein the cells form a confluent monolayer over the ECM layer, including alignment of the cells over the patterned tissue scaffold to form a biologically functional tissue structure at least 10 times the size of the constituent cells, while maintaining alignment with the pattern of alternating high-surface-energy and low-surface-energy microstructures and with the ECM.

11. A method for producing an artificial tissue comprising living cells attached to a patterned tissue scaffold, comprising bringing the tissue scaffold described in claim 1 into contact with cells and culturing the cells under conditions suitable for cell proliferation and / or differentiation.

12. The patterned structure scaffold according to claim 2, wherein the water droplet contact angle of the first microstructure layer having a first hierarchical microstructure base layer microstructure is less than 100 degrees, and the water droplet contact angle of the second microstructure layer having a second hierarchical microstructure base layer microstructure is 100 degrees or more.

13. The patterned structure scaffold according to claim 12, wherein the hysteresis of the water droplet contact angle is 5 degrees or more.

14. To form a substrate having a patterned base layer, the patterned tissue scaffold, which has a microstructure pattern on the base layer, is brought into contact with cells by photolithography. Depositing a capping layer on a patterned base layer to form a substrate having a patterned or hydrophilic / hydrophobic capping layer, The substrate having the patterned tissue adhesion layer is brought into contact with cells and cultured under conditions suitable for the production of extracellular matrix components. To provide a biological tissue scaffold containing extracellular matrix components, the cells are removed from the substrate. The patterned structure scaffold according to claim 1, further comprising:

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