Creating and using optoelectronically active bioinks

Abstract

An optoelectronically active bioink can be created with a biomaterial precursor solution; and one or more optoelectronic materials suspended in a fluid. The one or more optoelectronic materials can be configured to convert light energy into electrical energy to sync with or modulate at least one electrically active tissue (e.g., in contact with a scaffold made of the optoelectronically active bioink). The optoelectronically active bioink can be used to form an optically stimulable tissue platform using a scaffold printed from optoelectronically active bioink into an anatomical shape. Cultured biological cells can be seeded to the scaffold and / or within the optoelectronically active bioink.

Description

BWH2025-196 NONPROVISIONAL APPLICATIONCREATING AND USING OPTOELECTRONICALLY ACTIVE BIOINKSGOVERNMENT SUPPORT

[0001] This invention was made with government support under 5R21 EB026175-02, 5R01 EB028143-04, R21 EB030257-01 , 1 R56EB034702-01 , 5R01 CA282451 -02, 5R01 HL165176-03, and 7R21 EB030257-02 awarded by the National Institutes of Health. The government has certain rights in the invention.Related Applications

[0002] This application claims priority to U.S. Provisional Application Serial No.63 / 749,052, filed January 24, 2025, entitled “OPTOELECTRONICALLY ACTIVE BIOINKS”. The entirety of this provisional application is hereby incorporated by reference for all purposes.Technical Field

[0003] This disclosure relates generally bioprinting, and more specifically to systems and methods for creating and using optoelectronically active bioinks.Background

[0004] Bioprinted tissues have been engineered to include many similarities to the naturally occurring tissues they mimic. As such, bioprinted tissues have emerged as a powerful tool for disease modeling, drug screening and testing, regenerative medicine and implants, personalized therapeutics, and organ engineering. However, traditional bioprinted tissues lack the inherent capability of generating or conducting electrical signals as is found in electroactive tissues, such as nerve tissues, heart tissues, and the like. Currently, to compensate for this lack of the ability to generate or conduct electrical signals, traditional bioprinted tissues must be connected with external electrical stimulation sources and conducting means (e.g., planar electrodes, wires, rods, or the like). These external electrical stimulation sources necessitate wired setups and invasive electrode placement, often leading to local damage at the stimulation sites and limited usefulness. Alternatively, the traditional bioprinted tissues can be genetically modified to generate an electrical stimulation.BWH2025-196 However, genetic modification is costly and complex, making genetically modified bioprinted tissues an unattractive and unattainable alternative for real-world use.Summary

[0005] Described herein are systems and methods for creating and using an optoelectronically active bioink. The optoelectronically active bioink can be used to create bioprinted tissues that can achieve electrical generation, modulation, or the like, in response to an optical signal (e.g., light signal).

[0006] In an aspect, the present disclosure can include an optoelectronically active bioink configured to be printed into a scaffold. The optoelectronically active bioink can include a biomaterial precursor solution; and one or more optoelectronic materials suspended in a fluid. The one or more optoelectronic materials can be configured to convert light energy into electrical energy to sync with or modulate at least one electrically active tissue in contact with the scaffold.

[0007] In another aspect, the present disclosure can include another optoelectronically active bioink configured to be printed into a scaffold. The other optoelectronically active bioink can include a solution, with one or more optoelectronic materials suspended in the solution and one or more cultured biological cells within the solution. The one or more optoelectronic materials can be configured to convert light energy into electrical energy to sync with or modulate at least one electrically active tissue in contact with the scaffold.

[0008] In a further aspect, the present disclosure can include a system configured to form an optically stimulable tissue platform. The system can include a scaffold printed into an anatomical shape from optoelectronically active bioink. The optoelectronically active bioink can include a biomaterial precursor solution; and one or more optoelectronic materials suspended in a fluid and configured to convert light energy into electrical energy. The system can also include cultured biological cells seeded to the scaffold.

[0009] In yet another aspect, the present disclosure can include a method for optically stimulating a scaffold seeded with cultured electroactive cells. The method can include attaching the scaffold, printed into an anatomical shape from optoelectronically active bioink and seeded with cultured electroactive cells, to an electrically active target tissue of a patient; and optically stimulating the scaffold toBWH2025-196 synchronize the scaffold with an electrical activity of the electrically active target tissue or to cause the scaffold to modulate the electrically active target tissue.Brief Description of the Drawings

[0010] The foregoing and other features of the present disclosure will become apparent to those skilled in the art to which the present disclosure relates upon reading the following description with reference to the accompanying drawings, in which:

[0011] FIGS. 1 and 2 are a block diagrams of different optoelectronically active bioinks that can be created;

[0012] FIG. 3 is a block diagram of a system that can use the optoelectronically active bioink of FIGS. 1 and / or 2 to generate electrical signals;

[0013] FIG. 4 is a block diagram showing a physical connection between the scaffold of FIG. 3 to electrically active target tissue;

[0014] FIG. 5 is a block diagram showing an electrical connection between the scaffold of FIG. 3 the electrically active tissue;

[0015] FIG. 6 is a process flow diagram of a method for using the optoelectronically active bioink of FIGS. 1 and / or 2 to generate electrical signals;

[0016] FIG. 7 is a process flow diagram of a method for constructing a scaffold;

[0017] FIG. 8 is a process flow diagram of a method for constructing the optoelectronically active bioink

[0018] FIG. 9 is a process flow diagram of a method for constructing micro-solar cells;

[0019] FIG. 10 is a process flow diagram of a method for preparing a specific optoelectronically active bioink;

[0020] FIG. 11 shows example composition, printing, and use of an optoelectronically active bioink;

[0021] FIG. 12 shows the distribution of micro solar cells in optoelectronically active bioink;

[0022] FIG. 13 shows printing characteristics of optoelectronically active bioink;

[0023] FIG. 14 shows solar cell fabrication, images, and photovoltaic characterization;

[0024] FIG. 15 shows solar cell characteristics and direct stimulation of cardiomyocytes (CMs);BWH2025-196

[0025] FIG. 16 shows CM variability and expression of cardiac markers on an optoelectronically active scaffold;

[0026] FIG. 17 shows cardiac markers expression of rat CMs (rCMs) on an optoelectronically active scaffold;

[0027] FIG. 18 shows phototoxicity of optoelectronically active scaffolds under control conditions (without light stimulation);

[0028] FIG. 19 shows an optoelectronically active scaffold evaluation in vitro; and

[0029] FIG. 20 shows an optoelectronically active tissue evaluation on a live rabbit heart.Detailed DescriptionI. Definitions

[0030] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains.

[0031] As used herein, the singular forms “a,” “an,” and “the” can also include the plural forms, unless the context clearly indicates otherwise.

[0032] As used herein, the terms “comprises” and / or “comprising,” can specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups.

[0033] As used herein, the term “and / or” can include any and all combinations of one or more of the associated listed items.

[0034] As used herein, the terms “first,” “second,” etc. should not limit the elements being described by these terms. These terms are only used to distinguish one element from another. Thus, a “first” element discussed below could also be termed a “second” element without departing from the teachings of the present disclosure. The sequence of operations (or acts / steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise.

[0035] It will be understood that when an element is referred to as being "on," "attached" to, "connected" to, "coupled" with, "contacting," etc., another element, it can be directly on, attached to, connected to, coupled with or contacting the other element or intervening elements may also be present. In contrast, when an elementBWH2025-196 is referred to as being, for example, "directly on," "directly attached" to, "directly connected" to, "directly coupled" with or "directly contacting" another element, there are no intervening elements present. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature.

[0036] As used herein, the term “optoelectronically active” refers to a material or device that can convert light signals (e.g., optical signals) to electrical signals and / or electrical signals to light signals. For example, converting a light signal to electrical signal can modulate an electrically active tissue (e.g., nerve tissue, muscle tissue, etc.).

[0037] As used herein, the term “bioink” refers to one or more cultured biological cells and / or polymeric compositions (e.g., natural and / or synthetic) that is at least partially biocompatible (also referred to as a biomaterial) and is selected for a specific application due to biocompatible components and / or rheological properties. In some instances, the bioink can be constructed from biomaterial precursors. The bioink be used in connection with and / or include one or more cultured biological cells. The bioink can aim to maintain cell viability, to promote formation of a tissuespecific extracellular environment, etc.

[0038] As used herein, the term “optoelectronically active bioink” refers to a bioink constructed for the specific application of converting light to electricity. As an example, an optoelectronically active bioink can include one or more optoelectronically active materials that can convert light to electricity to modulate an electrically active tissue.

[0039] As used herein, the term “bioprinting” refers to a technology used for biofabrication of biological construct(s) (e.g., living tissues, organs, or the like). One example of bioprinting using a printer and one or more bioinks, which can be mixed with and / or include biological cells. Bioprinting can include two dimensional (2D) bioprinting and / or three dimensional (3D) bioprinting. For example, bioprinting can refer to extrusion-based bioprinting, droplet-based bioprinting, or light-based bioprinting, among others. It should be noted that bioprinting in this context may also refer to biofabrication more generally including any conventional method, like casting, molding, etc.

[0040] As used herein, the term “scaffold” can refer to a 3D printed, biomimetic structure, made from bioink, that can provide a framework (e.g., a certain shape) forBWH2025-196 creating functional biological constructs (e.g., one or more tissues, and / or one or more organs).

[0041] As used herein, the term “electrically active tissue” can refer to one or more cells that naturally generate, propagate, respond to, etc., electrical signals. For example, electrically active tissue can include one or more cardiac muscle cells, one or more skeletal muscle cells, one or more nerve cells, etc.II. Overview

[0042] Traditional bioprinted tissues lack the inherent capability to generate or conduct electrical signals, which limits the ability to utilize bioprinted tissues in place of and / or to regenerate electrically active tissues. Traditionally bioprinted tissues are composites of cells and an extracellular matrix (ECM) that lack the intrinsic ability to generate electrical signals necessary for stimulating cellular activity or reacting to a natively generated electrical signal. To compensate, traditional bioprinted tissues need to be connected with external electrical stimulation sources and / or genetically modified. The external electrical stimulation sources can include invasive stimulators, planar electrodes, wires, rods, or the like, which necessitate wired setups and invasive electrode placement, often leading to local damage at the stimulation sites and requiring tethering to the external electrical stimulation source. Genetic modification is costly, complex, and impractical to use in real-world bioprinted applications.

[0043] By integrating optoelectronic materials into a bioink, bioprinted scaffolds and tissues can be active, electrically generative, and light-responsive. The bioprinted scaffolds and tissues can be untethered, noninvasive, and provide distributed modulation of electrically active tissues, such as cardiac tissues. Such bioprinted scaffolds and tissues can circumvent the challenges of conventional electrical stimulators and genetic modification and can enhance and / or modulate cellular activities of a host organism. Accordingly, described herein are systems and methods for creating and using an optoelectronically active bioink.III. Optoelectronically Active Bioink

[0044] An optoelectronically active bioink 10 (FIG. 1 ) can be created by integrating optoelectronic material(s) 14 into at least a base solution 12 of a bioink. The optoelectronically active bioink 10 can be used to bioprint a scaffold and / orBWH2025-196 tissue (of a predefined shape) that is itself optoelectronically active and capable of dynamically interacting with electrically active host tissues (e.g., myocardium, neural tissue, muscle tissues, etc.). The optoelectronically active bioink 10 (and tissues and / or scaffolds created thereof) can convert an optical signal (e.g., light) to an electrical signal (e.g., current). In contrast to the traditional bioinks, and printed materials thereof, that rely on additional electrode / wiring / electrical stimulator components, the optoelectronic material(s) 14 can be distributed through the optoelectronically active bioink 10 (as opposed to conductivity limited to areas with electrode contacts), untethered (from wires and / or an electrical current source), and light responsive (instead of relying on a current source). Accordingly, optoelectronically active bioink 10 can break significant barriers in the tissue regeneration and host integration capabilities for electrically active tissues such as cardiac tissues, neural tissues, muscle tissues, and the like.

[0045] The optoelectronically active bioink 10 can include the base solution 12 integrated with the optoelectronic material(s) 14. The optoelectronically active bioink 10 can also include cell(s) 16 (e.g., cultured biological cells) and / or one or more buffer solutions (buffer 18). The base solution 12 can be selected by a person skilled in the art for a specific application based on necessary biocompatible, biofunctional, mechanical, and / or rheological properties (e.g., based on the desired scaffold and / or tissue to be printed from the optoelectronically active bioink 10). The base solution 12 can include at least one polymeric composition (e.g., natural and / or synthetic) that is at least partially biocompatible and may include cell(s) 16 and / or buffer 18. The at least one polymeric composition of the base solution 12 can be one or more biomaterials engineered to interact with one or more biological systems, such as for a therapeutic or diagnostic purpose, or one or more biomaterial precursors. For example, biomaterial precursors or other components of base solutions can be based on gelatin, collagen, hyaluronic acid, decellularized extracellular matrix and their derivatives (e.g. GelMA), or their combinations, or synthetic polymers, like polyethylene glycol)-diacrylate (PEGDA), or combinations of natural and synthetic elements / polymers / etc. The inclusion of cell(s) 16 (e.g., one or more types of cultured biological cells) and / or the one or more buffer 18 can be used to further create desired biocompatible, biofunctional, mechanical, and / or rheological properties not found in a base solution 12. As an example, the base solution 12 can inherently include the cell(s) 16 (e.g., one or more cultured biological cells) and aBWH2025-196 polymeric composition (e.g., natural and / or synthetic) that is at least partially biocompatible and may include the one or more buffer 18. As another example, the base solution 12 can include the polymeric composition (e.g., natural and / or synthetic) that is at least partially biocompatible and may include the one or more buffer 18 (in this case, the cells 16 can be added during and / or after the bioprinting).

[0046] The biomaterial and / or the biomaterial precursor of the base solution 12 can be any stable biomaterial or stable biomaterial precursor commonly used for bioinks. As an example, the biomaterial precursor can include a base material and at least one crosslinker - a photo-crosslinker, a chemical crosslinker, a physical crosslinker, or the like - that can be used for the standard bioink. As another example, the biomaterial precursor can include only the base material and need not include a crosslinker for the standard bioink; instead, the crosslinker can be integrated with the optoelectronically active bioink 10 when the bioink is printed or after printing, or in some instance a crosslinker may not be necessary. One example biomaterial precursor can include gelatin methacryloyl (GelMA). It should be noted that concentrations and volumes thereof of the components of the optoelectronically active bioink 10 can depend on the tissues and / or scaffolds to be printed and can be determined by a person having ordinary skill in the art based on the tissues and / or scaffolds to be printed from the optoelectronically active bioink.

[0047] As noted, to be optoelectronically active bioink 10, at least the base solution 12 can be combined with one or more optoelectronic material(s) 14. The optoelectronic material(s) 14 can be one or more types of optoelectronic materials. The optoelectronic material(s) 14 can be suspended in a fluid (such as a buffer solution). The optoelectronic material(s) 14 can be integrated with the base solution 12, and optionally the cell(s) 16 and / or buffer 18 by mixing, centrifugation, or the like to create a distribution of the optoelectronic material(s) throughout the optoelectronically active bioink 10. The optoelectronic material(s) 14 can be uniformly distributed in the optoelectronically active bioink 10, majority uniformly distributed (e.g., greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or the like), or randomly distributed (e.g., less than 50% uniformly distributed). The optoelectronic material(s) 14 can include one or more materials that are optoelectronically active (in other words, the one or more materials can convert an optical signal, like light, into an electric signal, like a current). In some instances, the optoelectronic material(s) 14 can be stable for a time period afterBWH2025-196 combining with the solution (e.g., before degrading or the like). The optoelectronic material(s) 14 can degrade over time (e.g., after a known period of time) (e.g., for shorter term use of printed scaffolds and / or tissues such as naturally regrowing tissues where surgical removal is not desired, or the like) or can be stable and permanent (e.g., for instances where long term use of the printed tissue and / or scaffold is desired). In some instances, the one or more optoelectronic materials can be photovoltaic. As an example, the one or more optoelectronic materials can be one or more micro-solar cells (e.g., a micro-semiconductor made of silicon). The optoelectronic material(s) 14 can respond to light such as, but not limited to, gamma rays, X-rays, ultraviolet light, infrared light, and / or one or more wavelengths of visible light, or the like. The optoelectronic material(s) can convert light energy into electrical energy to sync with or modulate at least one electrically active tissue (e.g., cardiac tissue, neural tissue, skeletal muscle tissue, or the like) in contact with a scaffold and / or tissue printed of the optoelectronic bioink (as discussed in greater detail below).

[0048] FIG. 2 shows a specific example of the optoelectronically active bioink 10. In this example, the base solution 12 can be a biomaterial precursor solution that includes base material(s) 22 and one or more photoinitiator materials (e.g., photoinitiator material(s) 24). The base material 22 can be one or more base materials as described above and depend on the desired scaffold and / or tissue to be bioprinted (e.g., cardiac tissue, skeletal muscle, neural tissue, or the like). For example, gelatin methacryloyl (GelMA). The photoinitiator material(s) 24 can be one or more chemical compounds that can absorb one or more wavelengths of light and convert energy from the light into one or more reactive species (e.g., free radicals or ions) to trigger a polymerization reaction in the optoelectronically active bioink 10 (e.g., can facilitate the optoelectronically active bioink 10 hardening during and / or after the printing process, like in response UV curing). For example, the photoinitiator material can be lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), tris(bipyridine)ruthenium(l I) chloride (Ru) / sodium persulfate (SPS), and / or eosin Y. The optoelectronic material(s) 14 can include one or more micro-solar cell(s) 26 (e.g., which can be a micro-semiconductor made of silicon), which can optionally be suspended in a buffer solution 28 (which can be the same as the buffer 18 or another buffer solution or fluid) or another fluid. The optoelectronic material(s) 14 (e.g., the micro-solar cell(s) 26) can be mixed with the biomaterial precursor baseBWH2025-196 solution 12 to form the optoelectronically active bioink 10. In some instances, the optoelectronically active bioink 10 can include one or more cell(s) 16 (e.g., cultured biological cells) and / or one or more additional or alternative buffer 18. For example, cells can be from 105to 108cells / mL. Solar cells, for example, can be in the 0.1-10% range.IV. Systems

[0049] As shown in FIG. 3, an optoelectronically active bioink 10 can be used to bioprint a scaffold and / or tissue (represented as scaffold 34) that retains the optoelectronic properties of the optoelectronically active bioink (e.g., via the optoelectronic material transferred to the scaffold) in a functional shape. A bioprinter 32 can use the optoelectronically active bioink 10 to print a scaffold 34 in a predetermined anatomical shape (e.g., tissue, at least part of an organ, at least part of a nerve, or the like). In some instances, the printer can print a scaffold 34 with the optoelectronically active bioink 10 and one or more other bioinks (traditional or another type of optoelectronically active bioink) to print more complex scaffolds and / or tissues. The optoelectronically active bioink 10 and / or the scaffold 34 can be embedded and / or seeded with cultured biological cells that can facilitate generation of the scaffold or attach to the scaffold. As an example, the optoelectronically active bioink 10 can provide the one or more optoelectronic material(s) to the scaffold in a distributed pattern (e.g., uniformly, majority uniformly, randomly, or the like). As noted, in some instances, the one or more optoelectronic materials can be photovoltaic, (e.g., one or more micro-solar cells). The scaffold 34 can be printed into an anatomical shape (e.g., shaped similarly to a target portion of a patient’s body, shaped to interface with the target portion of the patient’s body, etc.). The scaffold 34 can be used to create a stimulable tissue platform to stimulate and / or work with cells, tissue, and / or organs within electrically active target tissue (e.g., from portion of a patient’s body). The scaffold 34 and / or the optically stimulable tissue platform can be used for in vitro drug discovery, tissue replacement, and / or in vivo tissue regeneration.

[0050] The bioprinted scaffold 34 can be positioned in proximity to, in contact with, integrated to, surgically attached to, or the like, one or more electrically active target tissue 42 (also referred to as electrically active tissue), as shown in FIG. 4. The electrically active target tissue 42 can include cardiac cells / tissue, skeletalBWH2025-196 muscle cells / tissue, neural cel Is / tissue, or the like that traditionally conduct and / or respond to native electrical signals (e.g., from a brain). As used herein, in proximity to can be used to mean close enough so the electrically active target tissue 42 can receive an electrical stimulation from the scaffold 34. In some instances, the scaffold 34 can be in contact with the electrically active target tissue 42. As an example, at least a portion of the scaffold 34 can be bonded to the electrically active target tissue 42. The bonding can include laminating the scaffold to the electrically active target tissue, as an example. In other instances, the scaffold 34 can be positioned within 1 mm of the electrically active target tissue 42. In further instances, the scaffold 34 can be within 500 pm of the electrically active target tissue 42. In still other instances, the scaffold 34 can be within 100 pm of the electrically active target tissue 42.

[0051] The optoelectronic material(s) cause the scaffold 34 to be responsive to an optical signal (also referred to as light responsive) to become conductive and / or to generate and output an electrical signal. For example, the optical signal can include one or more pulses (or continuous) of light that can be at one or more intensities and / or wavelengths. The electrical response (e.g., current generation, voltage generation, conductivity, or the like) can be based at least in part on the amount and / or type of the optoelectronic material, the wavelength(s), the intensities, or one or more other parameters of the light signal. In some instances, optoelectronic material(s) can be selected to respond to one or more different wavelengths or a range of wavelengths of light (e.g., visible, infrared, ultraviolet, or the like). The scaffold 34 comprising the distributed optoelectronic materials can better represent natural tissue and does not require tethering to electrodes and / or electrical signal generators / stimulators.

[0052] As shown in the example 50 of FIG. 5, the scaffold 34 can receive an optical signal from one or more light source(s) 52. The one or more light source(s) 52 can be light(s), laser(s), LED(s), the sun, or the like. For example, an amount of light at an intensity can be shined on the scaffold 34 for a time period (continuously or pulsed at a frequency). The optoelectronic material in the scaffold 34 can convert the optical signal input into an electrical signal (e.g., electrical current). The scaffold 34 can output the electrical signal (e.g., electrical current) to stimulate the electrically active target tissue 42. Based on the optical stimulation, the electrically active targe tissue 42 can be synced with the electrically active target tissue, provide an electricalBWH2025-196 current to the electrically active target tissue, and / or modulate an electrically active target tissue (e.g., like a living pacemaker), or the like. The conductivity and / or electrical generation may depend on the intensity of the optical signal). For example, the scaffold 34 can produce a same or a different electrical current in response to a same or a different light shining on the scaffold 34. The amount of electrical current that can be produced by the scaffold 34 can depend on the wavelength, intensity, timing, or the like of the light that the scaffold 34 is exposed to.V. Methods

[0053] Another aspect of the present disclosure can include methods 60-100 (FIGS. 6-10) for creating and using an optoelectronically active bioink. Creation of the optoelectroncally active bioink is shown, for example, in FIGS. 1 and 2. Uses of the optoelectroncally active bioink are shown, for example, in FIGS. 3-5.

[0054] Unless otherwise stated, the methods described herein generally follow the usual practices widely known in related fields. Moreover, examples of how elements of the methods can operate and / or what the elements can be are described in the Systems section above. Moreover, additional steps may be required to perform the methods and these steps will be obvious to a person having ordinary skill in the art.

[0055] Referring now to FIG. 6, illustrated is a method 60 for using an optoelectronically active bioink to generate electrical signals. Optionally, at 62, the optoelectronically active bioink can be composed / constructed (as shown in FIGS, 1 and 2). The optoelectronically active bioink, in some instances, can include one or more optoelectronic materials, which can be photovoltaic (e.g., one or more microsemiconductors made of silicon). Also optionally, at 63 a scaffold (e.g., scaffold 34) can be constructed (e.g., printed). Both as described in more detail below. In some instances, the scaffold can already be created from the optoelectronically active bioink ahead of the use. At 64, the scaffold can be attached to an electrically active target tissue of a patient (e.g., electrically active target tissue 42, such as a cardiac tissue, skeletal tissue, neural tissue, or the like). In some instances, the scaffold can be in contact with the electrically active target tissue. As an example, at least a portion of the scaffold can be bonded to the electrically active target tissue. In other instances, the scaffold can be within 10 cm of the electrically active target tissue. In further instances, the scaffold can be within 2 cm of the electrically active targetBWH2025-196 tissue. In still other instances, the scaffold can be within 500 mm of the electrically active target tissue. At 65, the scaffold can be optically stimulated (e.g., with an optical signal, like light, applied to at least a portion of the scaffold). The scaffold can convert the optical signal to an electrical signal. In some instances, the scaffold can generate an electrical signal in response to the optical signal. In another instance, the optical signal can cause the scaffold to become at least partially conductive. Optionally, at 66, the optically stimulated scaffold can be synchronized with an electrical activity of the electrically active target tissue (e.g., can beat with native cardiac tissue, react to muscle contractions like native skeletal muscle, conduct a neural impulse, or the like). Alternatively, optionally, at 67, the optically stimulated scaffold can modulate the electrically active target tissue (e.g., effect the native electrical signal in the electrically active target tissue, such as increasing or decreasing a heart rate, increasing or decreasing a strength of a muscle contraction, or the like) .

[0056] Referring now to FIG. 7, illustrated is a method 70 for constructing a scaffold. At 72, the optoelectronically active bioink (e.g., optoelectronically active bioink 10) can be bioprinted (e.g., by printer 32) into an anatomical shape (e.g., at least a portion of a tissue, organ, nerve, or the like). The optoelectronically active bioink can be created by integrating optoelectronic material(s) into a bioink base solution as described herein. The bioprinting can be a layer-by-layer method that can be performed with an inkjet printer, a light-based printer, an extrusion printer, or the like. At 74, a scaffold can be crosslinked to form chemical bonds between the polymer chains of the bioink to give the scaffold strength and stability. The crosslinking can be, for example, chemical (e.g., enzymes, aldehydes, vulcanizations, peroxides, etc.), physical (e.g., using borax, calcium ions, temperature, or the like), radiation (e.g., UV, electron beam, or the like). At 76, optionally, cultured electroactive cells can be seeded onto the scaffold if not already part of the bioink mixture. Also optionally, at 78, the seeded scaffold can be incubated. The type and / or amount of cultured electroactive cells can be based on the desired tissue.

[0057] Referring now to FIG. 8, illustrated is a method 90 for constructing the optoelectronically active bioink (e.g., optoelectronically active bioink 10). At 82, a precursor solution can be formed. At 84, one or more optoelectronic materials can be suspended in a fluid. At 86, the precursor solution can be combined with the one orBWH2025-196 more optoelectronic materials suspended in the fluid. Optionally, at 88, cultured biological cells suspended in another solution can be mixed into the combination. The cultured biological cells can alternatively be present in the precursor solution and / or seeded on the scaffold during the printing process.

[0058] Referring now to FIG. 9, illustrated is an example method 90 for constructing micro-solar cells. At 92, a silicon-on-insulator wafer can be selectively doped to define a P-l-N junction. At 93, a top layer of the doped silicon can be isolated into micro-solar cells. This can be done with a photolithography process to form individual micrometer-sized particles. The sizes of the micro-solar cells can depend on the photomask design. At 94, a bulk substrate of the silicon-on-insulator wafer can be released. For instance, by removing the buried oxide layer in the wafer with hydrofluoric acid. At 95, one or more of the micro-solar cells can be picked up with dissolvable tape. At 96, the dissolvable tape can be dissolved in a buffer or water. At 97, the one or more micro-solar cells can be collected in a fluid to form a suspension of the one or more micro-solar cells to be added to the optoelectronically active bioink.

[0059] Referring now to FIG. 10, illustrated is an example method 100 for preparing a specific optoelectronically active bioink. This specific optoelectronically active bioink includes gelatin methacryloyl (GelMA) and micro-solar cells. It will be understood that other materials can be used in a similar method. For example, other biomaterial precursors can be based on gelatin, collagen, hyaluronic acid, decellularized extracellular matrix and their derivatives (e.g. GelMA), for the base material, or their combinations, or synthetic polymers like polyethylene glycol)-diacrylate (PEGDA). Examples of photoiniators that can be part of the optoelectronically active bioink can include, but are not limited to lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), tris(bipyridine)ruthenium(ll) chloride (Ru) / sodium persulfate (SPS), and eosin Y.

[0060] At 102, a suspension of micro-solar cells can be created. The micro-solar cells can be suspended in a buffer solution (e.g., DPBS). For example, a volume of the micro-solar cells can be from 1000 micro-solar cells / mL to 1500 micro-solar cells / mL. At 104, a GelMA precursor solution can be created. As an example, the GelMA precursor can include the GelMA, lithium-phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and a volume of a buffer (e.g., DPBS). The GelMA precursor solution can be, tor example, from 3 wt% to 20 wt% GelMABWH2025-196 precursor solution with 0.1 wt% to 1 wt% LAP. At 106, the suspension of one or more micro-solar cells can be combined with the GelMA precursor solution and an additional volume of the buffer. The buffer can be from 0% to 10% of the solution. At 108, the combination can be mixed to form the optoelectronically active ink. For example, the combination can be centrifuged at 300 g for 5 minutes.VI. Experimental

[0061] This experiment demonstrates how a printed gelatin methacryloyl (bioink) embedded with micro-solar cells (also referred to as p-solar cells) and seeded with cardiomyocytes (CMs) (a type of cultured biological cell) can be used to generate a viable optically controllable tissue. This optically controllable tissue can enable untethered, noninvasive, and damage-free optoelectronic stimulation-induced modulation of cardiac beating behaviors only requiring light without needing wires or genetic modifications to the tissue.Materials and Methods

[0062] Fabrication of p-solar cells

[0063] Ultrathin Si p-solar cells were fabricated by selectively doping a silicon-on-insulator wafer (1250-nm-thicktop Si layer, Soitec, Isere, France) to define planar P-l-N junctions. The top device layer was then isolated into squares using photolithography processes to form individual micrometer-sized particles. The sizes of the p-solar cells were determined by a photomask designed using a maskless mask aligner (pMLA Maskless Aligner, Heidelberg Instruments Mikrotechnik GmbH, Heidelberg, Germany) and the AutoCAD software (Autodesk Inc., San Francisco, CA, USA). After releasing from the bulk substrate by removing the buried oxide layer in hydrofluoric acid, the p-solar cells were picked up with water-dissolvable tape. Finally, the p-solar cells were collected for further studies after dissolving the tape in DPBS (Sigma-Aldrich, St. Louis, MO, USA).

[0064] Preparation of the optoelectronically active ink

[0065] GelMA was synthesized; 10 g of type-A gelatin from the porcine skin was dissolved in 100 ml of DPBS at 50°C under constant stirring for 30 min. Next, 5.0 ml of methacrylic anhydride was then added to the gelatin solution dropwise and the reaction was carried out at 50°C. After 3 hours, the reaction was quenched by adding 100 ml of warm DPBS, followed by the dialysis of the product against distilled water at 40°C for 5 days using a dialysis membrane (molecular weight cutoff: 12,000BWH2025-196 to 14,000 Da). The dialyzed GelMA solution was then filtered through a 0.2-pm filter and lyophilized to obtain porous white foam. GelMA was stored at ~20°C until use. The optoelectronically active ink was prepared by first making a solution of a GelMA-precursor solution, which also consisted of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP; Sigma-Aldrich, St. Louis, MO, USA) and DPBS. A 7 wt % GelMA-precursor solution with 0.25 wt % LAP was prepared. The appropriate volume of DPBS was added after the p-solar cell suspension (1300 p-solar cells / ml) was mixed with the GelMA-precursor solution. The solution was then centrifuged in a conical tube at 300 g for 5 min, followed by loading into a 3-ml syringe.

[0066] Rheological measurements

[0067] Rheology tests of the optoelectronically active ink and GelMA only (as a control) were performed using an HR-20 rheometer (TA Instruments, New Castle, DE, USA) with a 20-mm-diameter sand-blasted parallel plate, a Peltier steel plate, and a 500-pm plate-to-plate distance. The uncross-linked solutions for printing the GelMA and optoelectronically active ink were prepared as described earlier (n = 3). The solutions were stirred and transferred immediately to the plate of rheometer. First, the temperature dependence of G' and G" was observed by conducting oscillation temperature sweeps at a constant shear strain of 5% in the frequency range of 0.1 to 100 rad / s. The temperature was decreased from 37°C to 2°C at a cooling rate of 5°C / min, and 180 s of soaking time was set to keep temperature homogeneous throughout the hydrogels, which better reflected the physically crosslinked hydrogels that underwent a liquid to solid transition. The measurement of the viscosity of the hydrogels as a function of shear rate (0 to 100 s-1) was conducted at 24°C after cooling the hydrogels on the plate at 4°C for 5 min and recovery at 24°C for another 5 min.

[0068] Compression tests

[0069] Compression tests were performed using a 5966 series advanced electromechanical testing system with a 1-kN load cell (Instron, Norwood, MA, USA). Briefly, cross-linked cylinder-shaped samples (GelMA only and optoelectronically active ink) made using an EcoFlex mold with a 6-mm diameter and 2.5-mm thickness (n = 5) were compressed at a rate of 1.3 mm / min to 40% strain. The initial modulus (kPa) was calculated from the first 10% compression strain of the resulting curves.

[0070] Swelling and degradationBWH2025-196

[0071] For both tests, cross-linked cylinder-shaped samples (GelMA only and optoelectronically active ink) with a 6-mm diameter and 2.5-mm thickness were made using an EcoFlex mold (n = 4). The samples for the degradation test were immediately immersed in DPBS at 37°C after cross-linking, but the samples for the swelling test were lyophilized overnight before being immersed. To accelerate the degradation of the samples, collagenase type 4 (Worthington Biochemical Corporation, Lakewood, NJ, USA) was added to DPBS at a concentration of 190 collagen digestion units (CDU) / mL Afterward, at 30 min and 1, 2, 4, and 6 hours, the samples were taken out, rinsed, and lyophilized, and sample dry weight was measured. For the swelling test, at the same time points as the degradation test, the samples were taken out and their wet weight was measured.

[0072] Absorption and impedance

[0073] For the absorption test, uncross-linked and cross-linked solutions of GelMA only and the optoelectronically active ink were used. Each uncross-linked solution was filled into a cuvette, and a UH4150 spectrometer (Hitachi High-Tech America Inc., Tokyo, Japan) was used to perform the ultraviolet-visible absorption measurement before and after cross-linking. The impedance of the cross-linked GelMA only and optoelectronically active ink was measured using a Multi / Autolab M204 electrochemical impedance analyzer (Metrohm AG, Riverview, FL, USA). A two-electrode configuration was used for each measurement.

[0074] Photocurrent characterization of p-solar cells

[0075] Photocurrent characterization was performed using a patch-clamp setup (HEKA EPC-10 amplifier with the Patchmaster Next software, HEKA Elektronik, Lambrecht, Germany) integrated with an inverted microscope. A focused light spot was delivered using an M530L4-C5 collimated light-emitting diode (LED; Thorlabs Inc., Newton, NJ, USA) through the light path typically used for the eyepieces. The glass pipette was pulled in a P-97 micropipette puller (Sutter Instruments, Novato, GA, USA) with a resistance around from 1 megohms to 5 megohms. Voltage clamp mode was used to measure the photocurrent, which was extracted using a silver chloride electrode loaded in the glass pipette filled with DPBS mixed with a few p-solar cells, uncross-linked GelMA only, or uncross-linked optoelectronically active bioink. After filling with any solutions containing p-solar cells, the pipette tip typically had one p-solar cell inside near the tip. During measurement, the pipette tip was first lowered into a DPBS bath, and the light was illuminated onto the tip (100-ms lightBWH2025-196 pulses, 532 nm, and 2.8 mW / mm2) through a 10x objective. For pipettes containing GelMA or the optoelectronically active bioink, the photocurrent measurements were taken before and after cross-linking using the parameters mentioned earlier. The data were analyzed and plotted using the MATLAB software (The MathWorks Inc., Natick, MA, USA).

[0076] Expansion and cardiac differentiation

[0077] The hiPSCs (SCVI20) used in this study were donated by the Stanford University Cardiovascular Institute Biobank, and the CMs generated in this study were obtained by differentiating the hiPSCs using the STEMdiff Cardiomyocyte Differentiation Kit (StemCell Technologies Inc., Vancouver, BC, Canada). The cells were cultured and maintained in a feeder-free system of human embryonic stem cell (hESC) -qualified Matrigel (Corning, Corning, NY, USA) and TeSR1 E8 (StemCell Technologies Inc., Vancouver, BC, Canada) under standard culture conditions (37°C at 5% CO2). Briefly, 1 x 105 cells were plated in the TeSR E8 medium supplemented with Y-27632 (10 pM, StemCell Technologies Inc., Vancouver, BC, Canada). The medium was changed daily, and the cells were passaged using the cell dissociation recombinant enzymatic solution TrypLE Express (Gibco, Waltham, MA, USA). After the end of the differentiation protocol (day 15), the cells were harvested using the STEMdiff Cardiomyocyte Dissociation Kit (StemCell Technologies Inc., Vancouver, BC, Canada). The cells were washed two times with DPBS, and 1 ml of the Cardiomyocyte Dissociation Medium (37°C) was added per well. Culture plates were incubated for 15 min at 37°C and 5% CO2. Afterward, the cells were dislodged by adding the Cardiomyocyte Support Medium and pipetting up and down 5 to 10 times. The cells were centrifuged at 300 g for 5 min, and the pellet was resuspended in the Cardiomyocyte Support Medium to obtain hiPSC-CMs. Approximately 8 x 106 hiPSC-CMs / ml were used for each stimulation study. In addition to the hiPSC-CMs generated in this study, hiPSC-CMs (FUJIFILM Cellular Dynamics Inc., Madison, Wl, USA) and rat neonatal CMs (Lonza, Basel, Switzerland) were also used to assess viabilities and morphological characteristics of CMs on the printed optoelectronically active scaffolds (described later).

[0078] Stimulation of hiPSC-CMs / rCMs with p-solar cells

[0079] First, a small droplet of the p-solar cell suspension was placed on the center of each MEA plate (60EcoMEA-Glass, Multichannel Systems MCS GmbH, Reutlingen, Germany) pretreated with fibronectin (1 mg / ml; Sigma-Aldrich, St. Louis,BWH2025-196 MO, USA). Next, the CMs were seeded onto the center of each MEA plate on top of the p-solar cell layer. MEA plates with CMs in the absence of the p-solar cells served as the control group. Last, the plates were incubated and the media were changed daily until experimentation.

[0080] CMs cultured for 4 or 5 days were used to validate the p-solar cells. The cardiac field potentials of the CMs were monitored by the multichannel system (MEA2100 Lite, MultiChannel Systems MCS GmbH, Reutlingen, Germany). An M530L4-C5 green LED light (Thorlabs Inc., Newton, NJ, USA) was passed through an amplifying lens to focus on the small spot atop the area containing the p-solar cells to accelerate the beating of the CMs toward the targeted beating rate. The light and recording equipment were kept in an incubator to maintain the temperature and pH level throughout the entire experiment. Before stimulation, the CMs were allowed to stabilize for 10 min; afterward, the CM’s signals were recorded, and the spontaneous initial beating rate was determined. The targeted beating rates were chosen to be slightly higher than the spontaneous beating rate. A 4-min break was added after every 10 min of light stimulation to avoid any long-term light toxicity. The signals were recorded before stimulation (1 min), during stimulation (10 min), and after stimulation (1 min). This constituted one round of stimulation and was repeated until the target frequency was reached. This cyclic light stimulation ensured the successful stimulation of the CM beating without introducing any side effects. Unless otherwise stated, all recorded signals from the MEAs were processed in MATLAB using a third-order Butterworth filter with a bandpass from 1 to 60 Hz. R-peak detection was performed using the R-DECO plugin to determine the BPM over time.

[0081] Preparation of optoelectronically active cardiac scaffolds

[0082] After loading the optoelectronically active ink into syringes, the loaded syringes were stored in the fridge (4°C) for at least 20 min prior to use with the CELLINK BIO X (CELLINK, Gothenburg, Sweden) or the Allevi 2 (Allevi Inc., Philadelphia, PA, USA) printer. Conical nozzles of varying sizes (22, 25, and 27 Ga) were attached to the syringe although the final printing designs used 25-Ga nozzles. The printing speed was set to from 1 mm / s to 2 mm / s, the nozzle temperature was 24°C to 25°C, the bed temperature was 14°C, and the pressure was 5 kPa to 20 kPa. Scaffolds for the viability and morphological assessments were made with dimensions of 10 mm by 10 mm by 0.5 mm (L x Wx H). Scaffolds for the stimulation procedures were made with dimensions of 5 mm by 4.4 mm by 0.5 mm (L x Wx H).BWH2025-196 After printing, the scaffolds were cross-linked using a 385-nm CS20K3 UV Curing LED System (Thorlabs Inc., Newton, NJ, USA) for 30 seconds with the light source placed -7 cm (-45.8 W / cm2) above each sample.

[0083] Viability of optoelectronically active cardiac scaffolds

[0084] For viability and morphological assessments, frozen hiPSC-CMs (FUJIFILM Cellular Dynamics Inc., Madison, Wl, USA) were thawed at 37°C, added dropwise to 5 ml of an iCell cardiomyocyte plating medium (FUJIFILM Cellular Dynamics Inc., Madison, Wl, USA) according to the instructions provided. The hiPSC-CMs suspension was centrifuged at 1000 rpm for 5 min and resuspended in 200 pl of the iCell cardiomyocyte plating medium to obtain a cell suspension with 5 x 106 hiPSC-CMs. Each of the printed optoelectronically active scaffolds was seeded with from 60 pl to 80 pl of the cell suspension and incubated at 37°C, in a cell incubator with 5% CO2 and 95% humidity. After 24 hours of incubation, the plating medium was replaced with the iCell cardiomyocyte maintenance medium. The maintenance medium was refreshed every 48 hours.

[0085] Similarly, rCMs at -80% confluency were washed with DPBS, trypsinized, and resuspended in rat cardiomyocyte growth medium (RCGM) to obtain an rCM-cell suspension at a concentration of 8 x 106 cells / ml. Next, from 50 pl to 60 pl of the rCM-cell suspension was seeded onto the scaffold. The scaffolds were incubated at 37°C, in a cell incubator with 5% CO2 and 95% humidity. After 12 hours, RCGM was replaced with RCGM containing 200 pM BrdU (5-bromo-2'-deoxyuridine) and was refreshed every 48 hours. Viability, morphology, and synchronized beating of both optoelectronically active cardiac scaffolds seeded with hiPSC-CMs or rCMs were evaluated

[0086] Viabilities of CMs on their respective optoelectronically active cardiac scaffolds were measured by the Live / Dead viability / cytotoxicity kit according to the manufacturer's instructions. Briefly, the cardiac scaffolds were washed twice with DPBS and placed in the wells of a 12-well plate, followed by the addition of live / dead staining solution containing 4 mM calcein acetoxymethyl (Calcein-AM) (1 pl / ml) and 2 mM ethidium homodimer-1 (2 pl / ml) in DPBS. After incubation at 37°C for 30 min, the scaffolds were washed three times with DPBS and imaged using an Eclipse Ti2 inverted microscope (Nikon, Melville, NY, USA). Percentages of viable CMs were determined using the ImageJ software.

[0087] Viability and staining assaysBWH2025-196

[0088] Using 1 ml of Accutase, the hiPSC-CMs were divided into single cells for from 10 min to 15 min. FlowBuffer-1 [DPBS with 0.5% bovine serum albumin (BSA)] was used to resuspend the hiPSC-CMs after which the hiPSC-CMs were stained using the appropriate conjugated primary antibodies. A BD Accuri C6 Plus flow cytometer was used to acquire the data, and the FlowJo software was used to process the data. For immunophenotyping by immunofluorescence, differentiated CMs attached to the RBOES were fixed with 4% paraformaldehyde and stained with 4',6-diamidino-2-phenylindole (DAPI).

[0089] Morphological analyses and immunostaining of optoelectronically active cardiac scaffolds

[0090] For morphological analyses, Alexa Fluor 488-phalloidin or Alexa Fluor 549-phalloidin was used for F-actin staining. The optoelectronically active cardiac scaffolds were washed twice with DPBS and fixed with paraformaldehyde (4 vol % in DPBS) for from 15 min to 20 min. The scaffolds were then treated with Triton X-100 (0.2 vol % in DPBS) for 1 hour at room temperature. After gentle washing three times with DPBS, Alexa Fluor 488-phalloidin or Alexa Fluor 549-phalloidin (1 :200 in 0.1 vol % BSA) was added to the scaffolds and incubated for 1 to 2 hours at room temperature. The samples were washed again with DPBS, and then nuclei were stained with DAPI for 5 min at room temperature. After washing three times with DPBS, fluorescence images were taken using a Zeiss LSM 880 confocal microscope (Carl Zeiss AG, NY, USA).

[0091] Moreover, the optoelectronically active cardiac scaffolds were assessed for expression of the cardiac cell specific markers by immunostaining. For this, the scaffolds were fixed with paraformaldehyde (4 vol % in DPBS) for from 15 min to 20 min at room temperature, permeabilized with Triton X-100 (0.2 vol % in DPBS) for 1 hour at room temperature, and then blocked with goat serum (5 vol % in DPBS) for 2 hours at room temperature. The scaffolds were then incubated overnight at 4°C with the primary antibodies, including sarcomeric a-actinin, connexin 43, cardiac troponin T, and cardiac troponin I antibodies (Abeam Limited, Waltham, MA, USA) at 1 :200 dilution in a blocking buffer. The scaffolds were washed three times with DPBS and incubated overnight at 4°C with the relevant secondary antibody (Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody or Alexa Fluor 488-conjugated goat anti-mouse secondary antibody) at 1 :200 dilution in a blocking buffer. After washingBWH2025-196 with the scaffolds with DPBS, the nuclei were counterstained with DAPI and examined under the Zeiss LSM880 confocal microscope.

[0092] Stimulation of optoelectronically active scaffolds

[0093] The optoelectronically active scaffolds were first printed directly on the electrode areas of the MEA plates. The hiPSC-CMs were seeded at a concentration of 8 x 106 cells / ml onto the scaffolds in 25 pl to 30 pl droplets. The optoelectronically active scaffolds were left in the incubator until consistent beating was observed (after 3 days to 5 days of incubation). The media were changed daily until experimentation. The same general stimulation protocol (round by round) was followed, and the MEA 2100 Lite system was used to collect data. The beating rates from the recorded ECG were determined using the R-DECO plugin, and the activation maps were created using MATLAB. The pre- and poststimulation BPMs were determined from a 10 sec period in the recordings before and after each stimulation round.

[0094] Phototoxicity assay of the optoelectronically active scaffolds

[0095] The scaffolds used for this assay were stimulated for 7 days, ~1 hour / day using the stimulation protocol as described earlier (four rounds). The optoelectronically active scaffolds seeded with hiPSC-CMs were first placed in a collagenase type 4 (1 mg / ml; Worthington Biochemical Corporation, Lakewood, NJ, USA) solution to digest the hydrogel. The hiPSC-CMs were dissociated into single cells with 1 ml of Accutase for from 10 min to 15 min. The cells were resuspended in FlowBuffer-1 (DPBS with 0.5% BSA), and a drop of TO-PRO3 Ready Flow Reagent live / dead cell stain (Invitrogen, Waltham, MA, USA) was added. After incubating the samples in the dark for from 5 min to 10 min, results were obtained with a BD Accuri C6 Plus flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and processed using the FlowJo software.

[0096] ECG waveform parameter calculations and statistical analysis

[0097] To analyze the ECG signal, the raw data were first preprocessed and segmented into individual beats. The mean ECG signal was calculated across all channels, and R-peaks were detected with a minimum peak height threshold set at 2 standard deviations (SDs) above the mean and a minimum peak distance of 0.6 sec to avoid detecting multiple peaks within a single heartbeat. Each beat was extracted into segments using a window centered on the R-peak location, with a total segment length of 2000 data points (1 sec at a sampling frequency of 2000 Hz). For each beat and channel, the extracted signal was filtered using a 50th-order low-pass finiteBWH2025-196 impulse response (FIR) filter with a cutoff frequency of 100 Hz, designed using a Hamming window. This filtering step removed high-frequency noise without phase distortion by applying the filter in both forward and reverse directions. A moving average filter with a window size of 100 data points was subsequently applied to further smooth the signal to clearly identify the QRS locations. QRS durations were calculated by identifying the Q and S points surrounding each R-peak. A search window of 0.1 sec before the R-peak was used to detect the Q point, and a 0.2 sec window after the R-peak was used to detect the S point. The QRS duration was computed as the time difference between the identified Q and S points. This process was repeated for each detected R-peak across all beats and channels. For the calculation of QR and RS slopes, the difference in signal amplitude between the R-peak and Q point, and the R-peak and S point, was computed. The QR slope was defined as the change in amplitude between the Q point and the R-peak, divided by the time between these two points. Similarly, the RS slope was computed as the change in amplitude between the R-peak and the S point, divided by the time between them. The slopes were averaged across all detected beats for each channel. The peak-to-peak amplitude for each beat was computed by taking the difference between the maximum and minimum values of the signal within each segment. This calculation was performed for each beat and across all channels, with the peak-to-peak amplitudes averaged per channel.

[0098] A statistical analysis was conducted to compare the RS slope, QR slope, QRS duration, and peak-to-peak amplitude features before and after stimulation using paired t tests for each channel. The mean Rvalues for RS slope, QR slope, QRS duration, and peak-to-peak amplitude were 0.5485, 0.6406, 0.5867, and 0.6018, respectively. In all cases, the Rvalues were greater than 0.05, indicating that the differences between the two groups were not statistically significant. The standard error of the mean (SEM) was also calculated, with values ranging from 0.0074 to 4.0504, further supporting the lack of significant variation between the groups.

[0099] Stimulation of rat heart with the optoelectronically active scaffolds

[0100] The optoelectronically active scaffolds (same dimensions as used for in vitro stimulation) were printed onto tissue culture-treated petri dishes and then seeded with rCMs (8 x 106 cells / ml). After 5 days of seeding, the scaffolds were then used for the in vivo experiment. After the rat was placed under anesthesia, the chestBWH2025-196 was opened to expose the heart. The scaffold was laminated on the epicardial surface of the rat heart after sprinkling a small amount of Transglutaminase (Modernist Pantry LLC, Eliot, ME, USA) powder onto the side that would contact the heart. A green LED light (Thorlabs Inc., Newton, NJ, USA) was passed through an amplifying lens to focus on the optoelectronically active scaffold. The target stimulation rate was set to ~5 Hz (40 ms pulse duration) with a function generator. The stimulation period began after 1 min of recording the spontaneous beating rate and was stopped when the target beating rate was reached. The Mouse Monitor (Indus Instruments, Webster, TX, USA) was used to capture the ECG signals from the body of the rat. The beating rates from the recorded ECG were determined using the R-DECO plugin in MATLAB. Temperature measurements were captured using an FLIR ONE Gen 3 Professional Thermal Camera (Teledyne FLIR LLC, Wilsonville, OR, USA). The animals in this study were handled and maintained per the requirements of the Laboratory Animal Welfare Act (P.L. 89-544) and its 1970 (P.L.91-579), 1976 (P.L. 94-279), and 1985 (P.L. 99-198) amendments and within the specifications indicated below. Compliance was accomplished by conforming to the standards in the Guide for the Care and the Use of Laboratory Animals, ILAR, National Academy Press, revised 2011 , and all procedures were approved by the Texas Heart Institute Institutional Animal Care and Use Committee (#2023-02). Results

[0101] Optoelectronically active ink

[0102] The optoelectronically active ink (a composite of p-solar cells and GelMA) was printed into scaffolds with different structures and then the printed scaffolds were seeded with CMs (FIGS. 11, 12, elements A. B). The optoelectronically active ink was prepared as described in more detail in the Materials and Methods section. Briefly, the preparation involved mixing a photocross-linkable precursor solution of GelMA (7 wt%) with a suspension of p-solar cells. The p-solar cells were prepared by typical microfabrication and lithography procedures as described in Materials and Methods. Images of exemplary printed structures demonstrating the distribution of p-solar cells in the GelMA are shown in FIG. 11 , element B. In FIG. 11 , element B , an example of a printed heart using the optoelectronically active ink consisting of the atria and ventricles is presented. As a potential application, this optoelectronically active scaffold could be used to replace damaged scar tissue (FIG. 11 , element C) in the native myocardium and beBWH2025-196 modulated to restore normal beating to the heart. Studies of the printing nozzle size and p-solar cell size effects on the p-solar cell distribution were performed to optimize the printing parameters and appropriate p-solar cell size. The density of the p-solar cells solution was fixed to -1300 p-solar cells / ml as this density was sufficient to modulate the beating rate of CMs, as evidenced in in vitro evaluations. From these studies, the optimal printing conditions (line speed, bed / printhead temperatures, and extrusion pressure) using a 25-gauge (Ga) nozzle and 80-pm p-solar cells were determined, as detailed in the Materials and Methods section.

[0103] Investigations of the rheological properties (shown in FIG. 13) revealed that the uncross-linked optoelectronically active ink had a slightly higher storage / loss modulus (over different temperatures and oscillatory shear strains) and viscosity compared to pure GelMA. Considering postprocessing analysis and eventual longterm evaluation, the in vitro swelling and degradation of the optoelectronically active ink were characterized. After 6 hours, the swelling of the cross-linked GelMA and optoelectronically active ink increased by -1000%. Both the cross-linked GelMA and optoelectronically active ink showed substantial degradation under an accelerated degradation test after 6 hours, reaching -98 and -88%, respectively. It is expected that the p-solar cells would also degrade over a longer period and their degradation could be controlled by modifying their thickness. The by-products of the p-solar cells when degraded (silicic acid) are known to be biocompatible. In addition, the absorbance and impedance of the optoelectronically active ink were determined to ensure that GelMA (before and after cross-linking) did not substantially hinder light penetration and the electrical potential distribution produced by the p-solar cells. Although cross-linking the optoelectronically active ink did decrease the ink’s transmittance, the ink was still relatively transparent. In addition, the addition of p-solar cells had little effect on the impedance of the cross-linked ink. This finding verifies that the electric field generated by the p-solar cells upon illumination would be minimally hindered.

[0104] p-Solar cell characterization

[0105] Details of the p-solar cells fabrication are in Materials and Methods and shown in FIG. 14, element A. Briefly, the freestanding p-solar cells were manufactured on a silicon wafer with a fabrication yield of -100% (FIG. 14, element B) and then collected by water-dissolvable tape (FIG. 14, element C), followed by storing in a Dulbecco's phosphate-buffered saline (DPBS) solution in a vial afterBWH2025-196 dissolving the tape (FIG. 14, element D). The fabricated p-solar cells exhibited a typical photovoltaic characteristic with an open-circuit voltage of -0.5 V (FIG. 14, element E). The photocurrent generated by a single p-solar cell was then investigated using a patch-clamp setup. Upon illumination with multiple 100-ms light pulses (532 nm), the p-solar cell consistently produced ~3 nA in DPBS (FIG. 15, elements A-C). As a control, the pipette was illuminated with the same parameters and did not produce any artifacts.

[0106] Next, the effect of light stimulation, specifically subthreshold stimulation, in vitro was studied. Subthreshold stimulation, which have been shown to be effective for modulating the activity of neural and cardiac cells, improves functional recovery after stroke and prevents / treats ventricular tachycardia. To study the effect of subthreshold optoelectronic stimulation, human-induced pluripotent stem cell- derived CMs (hiPSC-CMs) were cocultured with the p-solar cells. First a multielectrode array (MEA) was coated with fibronectin and then the p-solar cell suspension was dispensed onto the coated MEA. Afterward, hiPSC-CMs were seeded on top of the p-solar cells. In FIG. 15, element D, an optical image of hiPSC-CMs cultured with p-solar cells on an MEA is shown and the corresponding real-time beating rate of hiPSC-CMs under optoelectronic stimulation is plotted in FIG. 15, element E. The hiPSC-CMs had a spontaneous beating rate around 57 beats per minute (BPM) prior to any stimulation.

[0107] The stimulation protocol using light (532 nm, 100-ms pulse duration, 1.2 Hz, and -2.8 mW / mm2) is described below and illustrated in FIG. 15, element E. It is noted that, although this wavelength may not be sufficient to penetrate tissue as is, wavelengths in the near-infrared spectrum could be used or the tissue transparent window could be modified with dyes as well. The cardiac field potentials across the MEA were recorded during the prestimulation, stimulation, and poststimulation periods. It should be noted that no stimulation was applied during the pre- and poststimulation periods. In total, three rounds of the stimulation protocol were performed and each round consisted of 1 min of recording during prestimulation, 10 min of stimulation, 1 min of recording during poststimulation, and 2 min of break. The target beating rate of 72 BPM was chosen based on the spontaneous beating rate and could be adjusted to the condition of the cultured cells. As evidenced by the BPM plot in FIG. 15, element E and the cardiac field potentials in FIG. 15, element F, the light stimulation protocol took ~40 min to accelerate the hiPSC-CMs to the target rateBWH2025-196 (26% increase from the initial beating rate). These results clearly demonstrated that the p-solar cells and the appropriate untethered light stimulation protocol can be used to successfully accelerate the beating of the hiPSC-CMs.

[0108] In vitro evaluation of the optoelectronically active scaffold

[0109] In subsequent experiments, the biocompatibility of the optoelectronically active ink was evaluated after printing the ink into a scaffold and seeding the scaffold with cells (FIG. 16, element A). The bright-field and fluorescence microscope images shown in FIG. 16, elements B and C for hiPSC-CMs and in FIG. 17, elements A and B for rCMs seeded on the optoelectronically active scaffolds reveal the cytocompatibility of the materials. The cell viability remained high through the 2 weeks of maintenance (FIG. 16, element D). The confocal fluorescence microscopic images of hiPSC-CMs immunostained for sarcomeric a-actinin, connexin 43, cardiac troponin T, and cardiac troponin I on day 15 (FIG. 16, elements E and F) demonstrated the expression of these markers, along with the sarcomere patterns, indicating the structural maturation of hiPSC-CMs on the optoelectronically active scaffolds. The hiPSC-CMs showed expected beating rates on days 7 and 15 (FIG.16, element G). Similarly, the aforementioned markers were also expressed by rCMs seeded on optoelectronically active scaffolds (FIG. 17, elements C and D). To ensure that light stimulation induced little to no harmful effect on the cells, a phototoxicity assay was performed. The results shown in FIG. 16, elements H-L and FIG. 18, elements A-C reveal a negligible difference in cell viability (both >96%) between the stimulation-treated and untreated optoelectronically active scaffolds.

[0110] The modulation capability of the optoelectronically active scaffold was also evaluated. To verify the function of an individual p-solar cell in the optoelectronically active ink, again we used our patch-clamp setup. To evaluate the performance of many p-solar cells in the optoelectronically active scaffolds, the scaffolds were prepared, seeded with hiPSC-CMs, and then positioned on MEA plates. The experimental setup is depicted in FIG. 19, element A. A photograph of the printed scaffold, embedded with p-solar cells and seeded with hiPSC-CMs, is depicted in FIG. 19, element B along with its placement on an MEA plate (n = 59 channels) in FIG. 19, element C. A closer microscopic view is presented in FIG. 19, element D. First hiPSC-CMs on the scaffold were stimulated at a frequency of 1 Hz across four rounds. The resultant data revealed a BPM increase from an initial -50 to -60 BPM (20% increase). In the fourth round, the RR interval values are ~1 second.BWH2025-196 Transitioning to a stimulation frequency of 1.2 Hz for two subsequent rounds led to another increase in the beating rate, rising from -60 to -72 BPM. The CMs increased their beating rates by -44% from the original -50 BPM. This trend in beating rates across various channels, both pre- and poststimulation, is graphically represented in FIG. 19, element E.

[0111] To emphasize the capability of the optoelectronically active scaffold for synchronizing and modulating the beating rate, activation heatmaps were created. As multiple rounds of stimulation progressed, the heatmaps showed shortened activation times, symbolized by a predominant blue hue across most of the channels. This observation is outlined in FIG. 19, element F, where the propagation time across the MEA decreased with each stimulation round. The changes in BPM from before to after stimulation (for each round) show a highly negative correlation with the RR intervals, especially in later rounds. One representative channel showing the changes in beating rate over time is shown in FIG. 19, element G. In the fourth round of stimulation, the CMs near this channel beat near the target rate of 60 BPM.

[0112] The morphology of electrocardiogram (ECG) signals under subthreshold stimulation were also assed, focusing on parameters such as QRS duration, QR slope, RS slope, and peak-to-peak amplitude. The results demonstrated that subthreshold stimulation did not induce significant changes in ECG wave morphology. The analysis shows that subthreshold stimulation does not significantly affect the QRS complex or other morphological features of ECG waves as it only mildly influences the electrophysiological behavior of CMs without inducing full action potentials. This finding is valuable as it underscores the potential of subthreshold stimulation for minimally invasive modulation of cardiac tissue without compromising the integrity of intrinsic electrical signaling. By maintaining the ECG morphology, the subthreshold approach also offers an advantage in therapies aimed at subtle cardiac modulation, supporting their role in applications where precise control without full excitation is desirable. In an additional experiment (n = 59 channels), the upper limit at which the hiPSC-CMs could sustain the beating rate were tested.

[0113] In vivo evaluation of optoelectronically active tissue

[0114] Integrating bioengineered cardiac constructs with the host tissue, specifically aligning their beating rates, is essential for successful host integration. Stimulating the engineered tissues to synchronize their beating with the host allows for the assembly of functional myocardium and reduces the potential for arrhythmiaBWH2025-196 upon implantation. In a proof-of-concept demonstration, the capability of the optoelectronically active tissue to accelerate the beating heart of a rat directly in vivo was assessed (FIG. 20, element A) to determine its potential for synchronization. Initially, the scaffold was constructed as outlined earlier and subsequently cultured with rCMs to form the optoelectronically active tissue. The rat was prepared in accordance with the methodologies detailed in the Materials and Methods section. Upon heart exposure and stabilization, the optoelectronically active tissue was delicately positioned and adhered on the epicardial surface above the right atrium, depicted in FIG. 20, element B. The initial heart rate was recorded at -280 BPM, prompting selection of a target stimulation rate of -5 Hz (-300 BPM). During the in vivo implantation, continuous light stimulation was applied until the target beating rate recorded from the rat heart was unambiguously observed. This heart rate progression is detailed in FIG. 20, element C. Over the stimulation interval, the heart rate gradually converged toward the target. The acquired ECG traces for each period (prestimulation, stimulation, and poststimulation) are shown in FIG. 20, element D. Before initiating stimulation, the heart rate stood at around 280 BPM. Roughly 12 min after the stimulation at -5 Hz began, the ECG signals showed a heart rate nearing 302 BPM (7.8% increase from the initial beating rate). Once this target rate was sustained for a brief duration, the light stimulation was halted. Shortly, within -3 min, the heart rate began to recede, settling close to 290 BPM. These findings underscore the capability of the printed optoelectronically active tissue for remote, light-driven, in situ modulation of the beating characteristics of a heart. Control conditions revealed that without the presence of p-solar cells in the tissue (printed GelMA only, seeded with rCMs), the heart rate of another rat heart (initial rate of -380 BPM) did not increase when stimulated at -6.5 Hz (390 BPM). It was further verified that change in temperature on the tissue surface during light stimulation did not play a substantial role in the successful modulation. It is also noted that, although the current experiments were performed in an acute, open-chest surgery setting, using red or near-infrared light could enable penetration through the tissue for closed-chest, chronic modulation. The optoelectronically active tissues provide a dynamic, controllable platform that allows for light-stimulated modulation of beating rates. This approach not only enables potential matching of the beating rate of the implanted tissue with the host heart rate but could also offer noninvasive, bidirectional control, which is critical for both acute and long-term integration.BWH2025-196

[0115] From the above description, those skilled in the art will perceive improvements, changes, and modifications. Such improvements, changes and modifications are within the skill of one in the art and are intended to be covered by the appended claims.

Claims

BWH2025-196 The following is claimed:

1. An optoelectronically active bioink configured to be printed into a scaffold, the optoelectronically active bioink comprising:a biomaterial precursor solution; andone or more optoelectronic materials suspended in a fluid and configured to convert light energy into electrical energy to sync with or modulate at least one electrically active tissue in contact with the scaffold.

2. The optoelectronically active bioink of claim 1 , wherein the biomaterial precursor solution is stable and photo-cross-linkable.

3. The optoelectronically active bioink of claim 1 , wherein the biomaterial precursor solution comprises at least one base material and at least one photoinitiator material.

4. The optoelectronically active bioink of claim 1 , wherein the electrically active tissue comprises cardiac tissue, skeletal muscle tissue, and / or neural tissue.

5. The optoelectronically active bioink of claim 1 , further comprising a buffer.

6. The optoelectronically active bioink of claim 1 , wherein the one or more optoelectronic materials comprise one or more micro-solar cells, wherein the one or more micro-solar cells are photovoltaic.

7. The optoelectronically active bioink of claim 1 , further comprising cultured biological cells.

8. The optoelectronically active bioink of claim 1 , wherein one or more of the optoelectronic materials degrades over time.

9. A system configured to form an optically stimulable tissue platform, the system comprising:a scaffold printed into an anatomical shape from optoelectronically active bioink, the optoelectronically active bioink comprising:BWH2025-196 a biomaterial precursor solution; andone or more optoelectronic materials suspended in a fluid and configured to convert light energy into electrical energy; andcultured biological cells seeded to the scaffold.

10. The system of claim 9, wherein the system is configured to be bonded to an electrically active target tissue of a patient.11 . The system of claim 10, wherein the system is configured to synchronize with an electrical activity of the bonded electrically active target tissue of the patient.

12. The system of claim 9, wherein the scaffold is configured to produce an electrical current in response to an optical stimulation.

13. The system of claim 12, wherein the optical stimulation comprises pulses of light.

14. The system of claim 12, wherein the electrical current is configured to modulate activity of an electrically active target tissue of a patient, wherein the electrically active target tissue of the patient comprises cardiac tissue, skeletal muscle tissue, and / or neural tissue.

15. The system of claim 9, wherein the scaffold and / or the optically stimulable tissue platform is used for in vitro drug discovery and / or in vivo regeneration.

16. The system of claim 9, wherein the biomaterial precursor comprises gelatin methacryloyl (GelMA) and the one or more optoelectronic materials comprise one or more micro-solar cells.

17. A method comprising:attaching a scaffold, printed into an anatomical shape from optoelectronically active bioink and seeded with cultured electroactive cells, to an electrically active target tissue of a patient; andBWH2025-196 optically stimulating the scaffold to synchronize the scaffold with an electrical activity of the electrically active target tissue or to cause the scaffold to modulate the electrically active target tissue.

18. The method of claim 17, further comprising constructing the optoelectronically active bioink by combining a biomaterial precursor solution and one or more optoelectronic materials suspended in a fluid and configured to convert light energy into electrical energy.

19. The method of claim 18, further comprising:forming the biomaterial precursor solution;suspending the one or more optoelectronic materials in the fluid;forming the optoelectronically active bioink by combining the biomaterial precursor solution and the one or more optoelectronic materials in the fluid; and forming the scaffold by the bioink, wherein the scaffold is seeded with the cultured electroactive cells.

20. The method of claim 17, wherein the one or more optoelectronic materials comprise one or more micro-solar cells.21 . The method of claim 20, further comprising fabricating the one or more microsolar cells by:selectively doping a silicon-on-insulator wafer to define planar P-l-N junctions, wherein the silicon-on-insulator wafer comprises a top silicon layer, a middle silicone oxide layer, and a bottom silicon layer;isolating the top layer of the doped silicon-on-insulator wafer into the one or more micro-solar cells;releasing a bulk substrate of the silicon-on-insulator wafer by removing the middle silicone oxide layer with an acid;picking up the one or more micro-solar cells with a water-dissolvable tape from the silicon-on-insulator wafer;dissolving the water-dissolvable tape in a buffer; andcollecting the one or more micro-solar cells in the fluid to form a suspension of the one or more micro-solar cells.BWH2025-19622. The method of claim 21 , further comprising preparing the optoelectronically active ink by:synthesizing gelatin methacryloyl (GelMA);creating a GelMA precursor solution comprising: the GelMA, lithium-phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and a buffer;combining the suspension of the one or more micro-solar cells in the fluid with the GelMa precursor solution and an additional volume of the buffer; and mixing the combination to form the optoelectronically active ink.

23. The method of claim 22, further comprising forming the scaffold by:printing the optoelectronically active ink in an anatomical shape;cross-linking the scaffold; andseeding and incubating the scaffold with the cultured electroactive cells.

24. The method of claim 23, wherein the bonding comprises laminating the scaffold to the electrically active target tissue.

25. An optoelectronically active bioink configured to be printed into a scaffold, the optoelectronically active bioink comprising:a solution; andone or more optoelectronic materials suspended in the solution and configured to convert light energy into electrical energy to sync with or modulate at least one electrically active tissue in contact with the scaffold; andcultured biological cells within the solution.

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