Three-dimensional printing of artificial bone marrow niches using mesenchymal stem cells encapsulated in hydrogels

Abstract

Methods, systems, apparatuses, and computer program products are described herein for printing artificial constructs or tissue models from a bioink using digital light processing (DLP). Bioinks can include polyethylene glycol diacrylate (PEGDA), gelatin methacrylate (GelMA), hyaluronic acid methacrylate (HAMA), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), tartrazine, arginine-glycerine-aspartic acid (RGD), isoleucine-lysine-valine-alanine-valine (IKVAV), and Dulbecco's Phosphate-Buffered Saline (dPBS) pH buffered with calcium and magnesium. Such bioinks can result in printed constructs that exhibit improved vascularity / perfusability and improved stem cell growth and regulation. Bioink can be formed by vortexing a solution of PEGDA, GelMA, HAMA, LAP, tartrazine, RGD, IKVAV, and / or dPBS at a temperature of, e.g., about 60°C or greater. DLP bioprinting can be carried out layer-by-layer with an average thickness of each layer being between about 25 µm and about 100 µm. Average light intensity emitted during DLP bioprinting can be between about 10 mW / cm2 and about 30 mW / cm2.

Description

Attorney Docket No.: 049648 / 643192 THREE-DIMENSIONAL PRINTING OF ARTIFICIAL BONE MARROW NICHES USING MESENCHYMAL STEM CELLS ENCAPSULATED IN HYDROGELSCross-Reference to Related Applications

[0001] This application claims the benefit of priority to U. S. Provisional Patent Application Serial No. 63 / 742.695, filed January 07, 2025 and entitled “Three-Dimensional Printing of Artificial Bone Marrow Niches Using Mesenchymal Stem Cells Encapsulated in Hydrogels,” the entire disclosure of which is hereby incorporated herein by reference in its entirety for all purposes.Statement of Government Support

[0002] This invention was made with government support under Grant No. DA056172, awarded by National Institute on Drug Abuse of the National Institutes of Health. The government has certain rights in the invention.Technical Field

[0003] This disclosure relates generally to biomaterials engineering, and more specifically to three-dimensional printing of artificial bone marrow niches.Background

[0004] In the human body, organ systems (including structural organs such as the skeletal system) contains highly specialized and complex microenvironments. The advent of tissue engineering tools offers numerous advantages for recreating these human-like multi-physiological systems (MPS). MPS are favorable for understanding disease pathology, developing therapeutics, testing regenerative approaches and identifying toxic conditions.

[0005] Additive manufacturing, also commonly known as three-dimensional (3D) printing, encompasses a range of technologies used to fabricate parts by adding material to build up the part rather than by subtracting unwanted material away from a bulk starting workpiece. For freeform 3D printing of functional structures, liquid extrusion, sometimes known as direct ink writing, can be used due to its ease of implementation, high efficiency, and wide range of printable materials. However, conventional direct ink writing methods are typically not appropriate for cell-laden or cellularly viable structures.

[0006] In the field of MPS, niches are local tissue microenvironments for maintaining and regulating stem cells. Haematopoiesis provides a model for understanding stem cells in a stem cell niche. Current niche constructs are either not sufficiently physiologically relevant or do not sufficiently support haematopioietic stem cells (HSCs) viability, are also not sufficiently perivascular, and / or are not sufficiently perfusable.Attorney Docket No.: 049648 / 643192Brief Summary

[0007] Described herein are systems, apparatuses, methods, and computer program products for developing a humanized, physiologically relevant MPS of niches, such as bone marrow niches. The development of a humanized MPS of human bone marrow as described herein provides for, among other things, a bone marrow niche (BMN) construct / model that exhibits improved physiological relevance with regard to the human BMN, improved stem cell growth and regulation therein, and improved perfusability. The BMN construct / model described herein further provides for improved physiological, medical, and pharmaceutical research, making the described BMN construct / model a groundbreaking translational tool. Current stem cell niche models lack the structure, heterogeneity, cellular composition, and / or cellular viability to be sufficiently physiologically relevant with regard to the human bone marrow and the stem cell niche therein. Moreover, animal models for the study of bone marrow are limited given the significant differences between BMNs and differences in stem cell regulation between humans and other species.

[0008] Tissue engineered MPS device bioprinted constructs described herein incorporate key aspects of the human bone marrow, such as relevant vascular geometries and composition (human continuous and sinusoidal forming endothelial cells), human hematopoietic cells, bioprinted 3D gel matrices that support cell polarization and differentiation, and perfusion of vascular spaces. Using tissue engineering techniques and material science, a 3D bioprinted device was developed that contains various (e.g., three) different compartments (e.g.. a porous gel matrix, a sac-like reservoir, and interconnected vascular compartment) that mimics the characteristics of the human BMN. According to some embodiments, this device, called MarrowPrint, features a hybrid triple crosslink network of synthetic and natural polymers. In some embodiments, the sac-like reservoir contains an opening to a chamber of stem cells. These reservoirs can be filled with a softer bioactive gel that is activated chemically for cellular adhesion. According to some embodiments, the continuous and sinusoidal endothelium is bridged together. This device can be manufactured using, e.g., a 3D printer that forms a homogeneous matrix or an advanced (heterogeneous) digital light processing (DLP) 3D printer. In some embodiments, the use of DLP may improve construct integrity due, e.g., to tuning of gradient stiffness that better supports the vasculature geometries and the stem cell sac-like chamber so as to achieve bioprinting of a physiologically relevant human BMN model. This biomimetic device can be used as a platform for numerous applications including its use for deciphering determinants of marrow pathology (immunosuppression), development of therapeutics for cancer, infectious diseases, and bone trauma.Attorney Docket No.: 049648 / 643192

[0009] According to some embodiments, a method and associated apparatus(es) is / are provided for printing physiologically relevant BMN constructs from a bioink using a digital light processing (DLP) bioprinter. In some embodiments, the bioink comprises polyethylene glycol diacrylate (PEGDA), gelatin methacrylate (GelMA), hyaluronic acid methacrylate (HAMA), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), tartrazine, arginine-glycerine-aspartic acid (RGD), isoleucine-lysine-valine-alanine-valine (IKVAV), and Dulbecco phosphate buffer saline (dPBS) pH buffered with calcium and magnesium. In some embodiments, this bioink composition results in BMN constructs that exhibit improved vascularity / perfusability and improved stem cell growth and regulation. In some embodiments, the bioink can be formed by vortexing a solution of PEGDA, GelMA, HAMA, LAP, tartrazine, RGD, IKVAV, and dPBS at a temperature of > about 60°C. In some embodiments, DLP bioprinting is carried out layer-by-layer with an average thickness of each layer being between about 25 pm and about 100 pm. In some embodiments, an average light intensity emitted during DLP bioprinting is betw een about 10 mW / cm2and about 30 mW / cm2

[0010] According to an embodiment, a method can be carried out for forming a bioink material, the method comprising: mixing together a bioink solution comprising at least a quantity (e.g., a particular mass or a particular volume) of PEGDA, a quantity of GelMA, a quantity7of HAMA, and a quantity of tartrazine (to act as a photoabsorber) in a quantity of dPBS (a saline pH buffered solution). The PEGDA can have a molecular weight (MW) or average MW of between about 500 Da and about 10,000 Da, and / or can comprise a mixture of PEGDA having different MWs or average MWs. For example, the PEGDA can comprise a 1:1:1 ratio of PEGDA having an average MW of about 1,000 Da, PEGDA having an average MW of about 3,400 Da, and PEGDA having an average MW of about 6,000 Da. A concentration of the PEGDA in the bioink solution can be about 15 wt.%. A concentration of the GelMA in the bioink solution can be about 3 wt.%. A concentration of the HAMA in the bioink solution can be about 1 wt.%. A concentration of the tartrazine in the bioink solution can be about 1.5 mM, or about 8 wt.%. A concentration of the dPBS in the bioink solution can be about 14 wt.%.

[0011] The method may (e.g., may further) comprise mixing, with the quantities of PEGDA, GelMA, HAMA, tartrazine, and dPBS, a quantity of LAP as a photoinitiator to form the bioink solution. The method may (e.g., may further) comprise mixing with the quantities of PEGDA, GelMA, HAMA, tartrazine, and dPBS, a quantity of RGD as a peptide mimetic fibronectin to form the bioink solution. The method may (e.g., may further) comprise mixing with the quantities of PEGDA, GelMA, HAMA, tartrazine, and dPBS, a quantity of IKVAV asAttorney Docket No.: 049648 / 643192 a peptide mimetic laminin to form the bioink solution. A concentration of the LAP in the bioink solution can be about 4 mM, or about 12 wt.%. A concentration of the RGD in the bioink solution can be about 2 mM, or about 20.5 wt.%. A concentration of the IKVAV in the bioink solution can be about 5 mM, or about 26.5 wt.%.

[0012] The bioink solution comprising these components can be mixed, stirred, vortexed, or otherwise homogenized at a temperature or temperatures equal to or above about 60°C, such as at a temperature of about 75°C. The bioink, once generated, can be maintained at a temperature of above about 60°C in order to reduce gelation of the gelatin in the solution that would result in the undesirable increase in viscosity of the bioink.

[0013] According to another embodiment, a method can be carried out for forming a bioink material, the method comprising: preparing a bioink comprising PEGDA, GelMA. HAMA, LAP, tartrazine, RGD, IKVAV, and dPBS pH buffered with calcium and magnesium. A solution comprising these components can be mixed, stirred, vortexed, or otherwise homogenized at a temperature or temperatures equal to or above about 60°C, such as at a temperature of about 75 °C. The bioink, once generated, can be maintained at a temperature of above about 60°C in order to reduce gelation of the gelatin in the solution that would result in the undesirable increase in viscosity of the bioink.

[0014] According to another embodiment, a method can be carried out for 3D printing a BMN construct, the method comprising: preparing a bioink comprising PEGDA, GelMA, HAMA, LAP, tartrazine. RGD, IKVAV, and dPBS pH buffered with calcium and magnesium. The method may (e.g., may further) comprise: communicating one or more layers of the bioink into a digital light processing (DLP) printer. The method may (e.g., may further) comprise: at least partially polymerizing, crosslinking, gelling, or curing, using the DLP printer, the one or more layers of the bioink to form a three-dimensional (3D) BMN construct.

[0015] In some embodiments, the bioink comprises between about 5 wt.% and about 20 wt.% PEGDA. In some embodiments, the bioink comprises between about 1 wt.% and about 5 wt.% GelMA. In some embodiments, the bioink comprises between about 0.1 wt.% and about 2 wt.% HAMA. In some embodiments, the bioink comprises between about 5 wt.% and about 15 wt.% LAP. In some embodiments, the bioink comprises between about 5 wt.% and about 15 wt.% tartrazine. In some embodiments, the bioink comprises between about 5 wt.% and about 15 wt.% RGD. In some embodiments, the bioink comprises between about 10 wt.% and about 30 wt.% IKVAV. In some embodiments, the bioink comprises between about 10 wt.% and about 20 wt.% dPBS. In some embodiments, the bioink comprises about 15 wt.% PEGDA,Attorney Docket No.: 049648 / 643192 about 3 wt.% GelMA, about 1 wt.% HAMA, about 12 wt.% LAP, about 8 wt.% tartrazine, about 20 wt.% RGD. about 25 wt.% IKVAV, and about 15 wt.% dPBS.

[0016] In some embodiments, the bioink is bioactivated to support stem cell growth in the 3D BMN construct. In some embodiments, the at least partially polymerizing, crosslinking, gelling, or curing, using the DLP printer, the one or more layers of the bioink comprises projecting respective light emissions from one or more light emission points of the DLP printer towards respective layers of the one or more layers of the bioink. In some embodiments, a light emission duration of the respective light emissions projected tow ards respective layers of the plurality of layers of the bioactivated bioink is between about 5 sec / layer and about 20 sec / layer. In some embodiments, the projecting the respective light emissions towards respective layers of the plurality of layers of the bioactivated bioink is performed at an average temperature of between about 60°C and about 90°C. In some embodiments, an average thickness of respective layers of the one or more layers of the bioink is betw een about 25 pm and about 100 pm. In some embodiments, an average light intensity of the respective light emissions is between about 10 mW / cm2and about 30 mW / cm2In some embodiments, the bioink is prepared by vortexing a bioink solution comprising the PEGDA, the GelMA, the HAMA, the LAP, the tartrazine, the RGD, the IKVAV, and the dPBS at a temperature higher than about 75 °C.

[0017] According to another embodiment, a method can be carried out for 3D printing a BMN construct, the method comprising: providing a bioactivated bioink comprising PEGDA, GelMA, HAMA, LAP, tartrazine, RGD, IKVAV, and dPBS. The method may (e.g., may further) comprise: disposing one or more volumes of the bioactivated bioink into a printing space according to a print pathway associated with a 3D BMN construct design. The method may (e.g., may further) comprise: exposing the one or more volumes of the bioactivated bioink to emitted light to at least partially polymerize, crosslink, gel, or cure the one or more volumes of the bioactivated bioink to form a 3D BMN construct.

[0018] In some embodiments, the printing space is within a DLP printer configured to project the emitted light towards the one or more volumes of the bioactivated bioink within the printing space. In some embodiments, the bioactivated bioink comprises about 15 wt.% PEGDA, about 3 wt.% GelMA, about 1 wt.% HAMA, about 12 wt.% LAP, about 8 wt.% tartrazine, about 20 wt.% RGD, about 25 wt.% IKVAV, and about 15 wt.% dPBS. In some embodiments, the one or more volumes of the bioactivated bioink are disposed within the printing space as one or more layers associated with one or more slices of the 3D BMN construct design.Attorney Docket No.: 049648 / 643192

[0019] In some embodiments, the exposing the one or more volumes of the bioactivated bioink to the emitted light comprises projecting respective light emissions, using the DLP printer, towards respective layers of the one or more layers of the bioactivated ink. In some embodiments, a light emission duration of the respective light emissions projected towards respective layers of the one or more layers of the bioactivated bioink is between about 5 sec / layer and about 20 sec / layer. In some embodiments, the exposing the one or more layers of the bioactivated bioink to the emitted light is performed at an average temperature of between about 60°C and about 90°C. In some embodiments, an average thickness of respective layers of the one or more layers of the bioactivated bioink is between about 25 pm and about 100 pm. In some embodiments, an average light intensity of the emitted light is between about 10 mW / cm2and about 30 mW / cm2In some embodiments, the bioactivated bioink is prepared by vortexing a bioink solution comprising the PEGDA, the GelMA, the HAMA, the LAP, the tartrazine, the RGD, the IKVAV, and the dPBS at a temperature higher than about 75°C.

[0020] According to another embodiment, a method can be carried out that comprises: preparing a bioactivated bioink comprising about 15 wt.% PEGDA, about 3 wt.% GelMA, about 1 wt.% HAMA, about 12 wt.% LAP. about 8 wt.% tartrazine, about 20 wt.% RGD, about 25 wt.% IKVAV, and about 15 wt.% dPBS. The method may (e g., may further) comprise: disposing, into a printing space within a DLP printer, according to a print pathway associated with the 3D BMN construct design, the bioactivated bioink as a plurality of layers associated with a plurality of slices of the 3D BMN construct design. The method may (e.g.. may further) comprise: projecting respective light emissions, using the DLP printer, towards respective layers of the plurality of layers to at least partially polymerize, crosslink, gel, or cure the plurality of layers of the bioactivated bioink to form a 3D BMN construct.

[0021] In some embodiments, a light emission duration of the respective light emissions projected towards respective layers of the plurality of layers of the bioactivated bioink is between about 5 sec / layer and about 20 sec / layer. In some embodiments, the projecting the respective light emissions towards respective layers of the plurality of layers of the bioactivated bioink is performed at an average temperature of between about 60°C and about 90°C. In some embodiments, an average thickness of respective layers of the plurality of layers of the bioactivated bioink is between about 25 pm and about 100 pm. In some embodiments, an average light intensity of the respective light emissions is between about 10 mW / cm2and about 30 mW / cm2In some embodiments, the bioactivated bioink is prepared by vortexing a bioink solution comprising the PEGDA, the GelMA, the HAMA, the LAP, the tartrazine, the RGD, the IKVAV, and the dPBS at a temperature higher than about 75°C.Attorney Docket No.: 049648 / 643192

[0022] The above-noted aspects and features may be implemented in systems, apparatuses, methods, articles and non-transitory computer-readable media depending on the desired configuration. The subject disclosure may be implemented in and used with a number of different types of devices, such as one or more computing devices, a DLP printer, a computer-controlled DLP printer, and / or the like. An example device can comprise at least one processor and at least one memory that stores thereon instructions which, when executed by the at least one processor, cause the device to perform some or all of the elements of the above-described method, according to various embodiments. In other examples, a computer program product, such as a non-transitory computer-readable storage medium can be provided that comprises instructions stored thereon that, when executed by at least one processor of an apparatus, cause the apparatus to perform some or all elements of a method such as that descried above, according to some embodiments. In other examples, an apparatus can be provided that comprises means for carrying out a method - such means can include, e.g., a processor and a memory storing computer-executable instructions or computer codes thereon that, when executed by the processor, cause the apparatus to perform some or all of a method such as one of the methods described herein.

[0023] This summary is intended to provide a brief overview of some of the aspects and features according to the subject disclosure. Accordingly, it will be appreciated that the abovedescribed features are merely examples and should not be construed to narrow the scope of the subject disclosure in any way. Other features, aspects, and advantages of the subject disclosure will become apparent from the following detailed description, drawings and claims.Brief Summary of the Drawings

[0024] Having thus described the invention in general terms, reference will now be made to the accompanying drawings. The skilled artisan will understand that the drawings primarily are for illustrative purposes and are not intended to limit the scope of the inventive subject matter described herein. The drawings are not necessarily to scale; in some instances, various aspects of the inventive subject matter disclosed herein may be shown exaggerated or enlarged in the drawings to facilitate an understanding of different features. In the drawings, like reference characters generally refer to like features (e.g.. functionally similar and / or structurally similar elements).

[0025] FIG. 1 provides a block flow' diagram of an example process for forming a bioink and printing a bone marrow niche (BMN) construct, according to embodiments of the present disclosure.Attorney Docket No.: 049648 / 643192

[0026] FIG. 2A provides a perspective view of an example model for 3D printing a BMN construct, according to embodiments of the present disclosure.

[0027] FIG. 2B provides a rotated perspective view of the example model of FIG. 2A for 3D printing a BMN construct.

[0028] FIG. 2C provides a further rotated perspective view of the example model of FIG. 2 A for 3D printing a BMN construct.

[0029] FIG. 2D provides a top-down view of the example model of FIG. 2 A for 3D printing a BMN construct.

[0030] FIG. 3 A illustrates a model to be 3D printed using DLP of enhanced injectable bioinks, according to embodiments of the present disclosure.

[0031] FIG. 3B illustrates a 3D printed structure printed using DLP of enhanced injectable bioinks according to the model illustrated in FIG. 3 A, according to embodiments of the present disclosure.

[0032] FIG. 3C illustrates an example of reduced or low fidelity of the 3D printed structure relative to the model after 3D printing using DLP of enhanced injectable bioinks, according to embodiments of the present disclosure.

[0033] FIG. 4 is a graph illustrating print fidelity of different bioink compositions, according to some embodiments of the present disclosure.

[0034] FIGs. 5A-5J illustrate a process for bioprinting. FIGs. 5A, 5B, 5C, and 5D illustrate providing various components of the bioink including, e.g., PEGDA, GelMA. HAMA, LAP, tartrazine, etc. FIG. 5E illustrates the mixing and heating of these components. FIG. 5F illustrates the mixing or vortexing of the solution of these components. FIG. 5G illustrates the aging and heating of the solution to form the bioink. FIG. 5H illustrates a DLP bioprinting device. FIG. 5I illustrates a print head of a DLP bioprinting device. FIG. 5J illustrates the marrowPRINT construct printed.

[0035] FIGs. 6A-6H illustrate a process for forming a bioactive injectable hydrogel bioink (BoMINKA part B), according to several of the embodiments described herein. FIG. 6A illustrates the HAMA polymer that will be used for the gel formation, FIG. 6B illustrates the crosslinker dithiothreitol (DTT) that will be used to form the gel and FIG. 6C illustrates the cell media (Dubelcco MEM media) for dissolving the HAMA. FIG. 6D illustrates the RGD peptide (Fibronectin mimetic peptide) that will enhance the cell adhesion process, FIG. 6E illustrates the stem cells that will be encapsulated in the gel solution. Fig. 6F illustrates the final gel solution with cells, which then will be loaded in the stem cell chamber represented inAttorney Docket No.: 049648 / 643192 FIG. 6G. The full marrowPRINT construct with gel solution will be loaded and incubated for the gelation of second ink.

[0036] FIG. 7A illustrates a testing apparatus and FIG. 7B illustrates degradation testing results, according to several of the embodiments described herein.

[0037] FIG. 8A illustrates a mechanical testing apparatus and FIG. 8B illustrates partial operations of the mechanical testing apparatus, according to several of the embodiments described herein.

[0038] FIG. 9 illustrates example mechanical testing results, according to several of the embodiments described herein.

[0039] FIGs. 10A-10D are scanning electron microscope (SEM) images of example bioinks, according to several of the embodiments described herein.

[0040] FIG. 11 is a graph illustrating the effect of example bioink compositions on porosity of bioprinted material, according to several of the embodiments described herein.

[0041] FIGs. 12A and 12B illustrate the cellular morphology of MSCs in BoMINKA part B one day post-polymerization using, respectively, a low magnification and a high magnification, according to several of the embodiments described herein.

[0042] FIGs. 13A and 13B are graphs of, respectively, a cellular volume and a cellular sphericity index to quantity7the cell morphology' of MSCs at different timepoints, according to several of the embodiments described herein.

[0043] FIGs. 14A and 14B illustrate the cellular morphology of MSCs in BoMINKA part B four days post-polymerization using, respectively, a low magnification and a high magnification, according to several of the embodiments described herein.

[0044] FIGs. 15A-15B illustrate the cellular morphology of MSCs in BoMINKA part B one week post-polymerization using, respectively, a low magnification and a high magnification, according to several of the embodiments described herein.

[0045] FIG. 16 is an image showing the cell morphology of example cells in BoMINKA part A, according to an embodiment of the present disclosure.

[0046] FIG. 17 A is a two-dimensional (2D) representation of a computer-aided design (CAD) file used to bioprint a construct as shown in FIG. 17B. As illustrated in FIG. 17B, a 3D printed construct can be injected with one or more color dyes post-bioprinting to show various compartments and / or passages within the 3D printed construct. The blue color indicates the filled chamber for the stem cell niche. The red color represents the microchannels formed that will be endothelialized (e.g., by lining the microchannel lumen or inner surface thereof with a 3D monolayer of endothelial cells).Attorney Docket No.: 049648 / 643192

[0047] FIGs. 18A-18C illustrate, respectively, a top view, a side view, and an angled (perspective) view of a tissue engineered bone marrow microenvironment model, according to embodiments of the present disclosure.

[0048] FIGs. 19A-19B illustrate, respectively, a top view and a side view of a bioprinted construct printed based on an STL model using a LumenX bioprinter, according to embodiments of the present disclosure.

[0049] FIGs. 20A-20B illustrate, respectively, a microfluidic pump system for bioprinting tissue constructs and a subset thereof, according to embodiments of the present disclosure.

[0050] FIG. 21 provides a schematic of an example computing device configured to perform some or all aspects of 3D printing of tissues or constructs, such as BMN constructs, according to embodiments of the present disclosure.

[0051] FIG. 22 provides a schematic of an example external computing device configured to perform some or all aspects of 3D printing of tissues or constructs, such as BMN constructs, according to embodiments of the present disclosure.

[0052] FIG. 23 illustrates a process flow diagram of a method for forming bioinks for 3D printing of tissues or constructs, such as BMN constructs, according to an embodiment of the present disclosure.

[0053] FIG. 24 illustrates a process flow diagram of a method for carrying out 3D printing of tissues or constructs, such as BMN constructs, according to an embodiment of the present disclosure.

[0054] FIG. 25 illustrates a process flow diagram of a method for carrying out 3D printing of tissues or constructs, such as BMN constructs, according to an embodiment of the present disclosure.

[0055] FIG. 26 illustrates a process flow diagram of a method for carrying out 3D printing of tissues or constructs, such as BMN constructs, according to an embodiment of the present disclosure.

[0056] FIG. 27 illustrates a process flow diagram of a method for carry ing out 3D printing of tissues or constructs, such as BMN constructs, according to an embodiment of the present disclosure.

[0057] FIG. 28 illustrates a process flow diagram of a method for carrying out 3D printing of tissues or constructs, such as BMN constructs, according to an embodiment of the present disclosure.Attorney Docket No.: 049648 / 643192Detailed Description

[0058] Various embodiments of the present disclosure will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the inventions are show n. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. The term “or” is used herein in both the alternative and conjunctive sense, unless otherwise indicated. The terms “illustrative” and “exemplary” are used to be examples with no indication of qualify level. Like numbers refer to like elements throughout.

[0059] It should be understood that some, but not all embodiments are shown and described herein. Indeed, the embodiments may take many different forms, and accordingly this disclosure should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.

[0060] As used herein, the terms “about,” “substantially,” and “approximately” generally mean plus or minus 10% of the value stated, e.g., about 250 pm would include 225 pm to 275 pm, about 1,000 pm would include 900 pm to 1,100 pm. Any provided value, whether or not it is modified by terms such as “about,” “substantially,” or “approximately,” all refer to and hereby disclose associated values or ranges of values thereabout, as described above.

[0061] The following embodiments are examples. Although the specification may refer to “an”, “one”, or “some” embodiment(s) in several locations of the text, this does not necessarily mean that each reference is made to the same embodiment(s), or that a particular feature only applies to a single embodiment. Single features of different embodiments may also be combined to provide other embodiments. Further, when a particular feature, structure, or characteristic is described in connection of an embodiment, it is within the knowledge of one skilled in the art to apply such feature, structure, or characteristic in connection with other embodiments w hether or not explicitly described. It shall be understood that although the terms “first,” “second” and the like may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another.

[0062] For the purposes of the present disclosure, the phrases “at least one of A or B”, “at least one of A and B”, and “A and / or B” means (A), (B), or (A and B). For the purposes of the present disclosure, the phrase “A, B, and / or C” means (A), (B). (C), (A and B), (A and C), (B and C), or (A, B, and C).Attorney Docket No.: 049648 / 643192

[0063] As used herein, "plurality" means two or more. As used herein, a "set" of items may include one or more of such items. As used herein, whether in the subject disclosure or the claims, the terms "comprising", "including", "carrying", "having", "containing", "involving", and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of, respectively, are closed or semi-closed transitional phrases with respect to claims. Use of ordinal terms such as "first", "second", "third", etc., in the claims or the subject disclosure to modify an element does not by itself connote any priority, precedence, or order of one element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the elements. As used herein, "and / or" and "at least one of means that the listed items are alternatives, but the alternatives also include any combination of the listed items.

[0064] Bone marrow is a viscous tissue located in the center of the bone region. This microenvironment is highly heterogeneous storing a wide range of stem cells involved in tissue regeneration, hematopoiesis, and angiogenesis making it an ideal tissue for tissue engineering and pathological studies. Unfortunately, it is complicated to synthesize a model with overall similar properties. 3D Printing bone marrow microenvironment is very' complex due to the interconnection of the different niches. Microfluidic devices can be formed, according to some embodiments, using 3D Digital Light Processing (DLP) printing, using, e.g.. three separate backbones such as polyethylene glycol diacrylate (PEGDA), gelatin methacrylate (GelMA), and hyaluronic acid methacrylate (HAMA). The bioink(s) can be bioactivated with gel solution that solidifies at room temperature. To achieve that, the physical, mechanical, and cellular interactions of the substrate will be explored separately. In certain embodiments, a bioink can be provided that comprises, e.g., about 15% PEGDA, about 3% GelMA, and about 1% HAMA. In certain embodiments, the bioink can be bioactivated with collagen. In some embodiments, the resulting bioink may be printed using a DLP printer or the like to form a printed construct comprising perfusable microchannels and which is configured to allow for a diverse cellular morphology and microchannels that are durable and perfusable yet which also porous enough to support stem cell growth therein. A construct or a model made of a reservoir for softer gels combined with perfusable microchannels that also support stem cell grow th are several of the critical components of a construct or model that is physiologically relevant and usable for modeling the bone marrow niche (BMN) construct or model.Attorney Docket No.: 049648 / 643192

[0065] Systems, apparatuses, methods, and computer program products are described herein for embedded printing of thick structured meat constructs using cellular inks and a cellular yield-stress support matrix material.

[0066] Embodiments of the present invention are described below with reference to block diagrams and flowchart illustrations. Thus, it should be understood that each block of the block diagrams and flowchart illustrations may be implemented in the form of a computer program product, an entirely hardware embodiment, a combination of hardware and computer program products, and / or apparatus, systems, computing devices, computing entities, and / or the like carry ing out instructions, operations, steps, and similar words used interchangeably (e.g., the executable instructions, instructions for execution, program code, and / or the like) on a computer-readable storage medium for execution. For example, retrieval, loading, and execution of code may be performed sequentially such that one instruction is retrieved, loaded, and executed at a time. In some exemplary embodiments, retrieval, loading, and / or execution may be performed in parallel such that multiple instructions are retrieved, loaded, and / or executed together. Thus, such embodiments can produce specifically-configured machines performing the steps or operations specified in the block diagrams and flowchart illustrations. Accordingly, the block diagrams and flowchart illustrations support various combinations of embodiments for performing the specified instructions, operations, or steps.

[0067] Bone marrow is a viscous tissue located inside of the hollow of bone containing a wide range of cells which act as non-stem cells (bone, red and white blood, fat cells and more) and stem cells. The tissue contains three major niches: endosteal (stiff region where most of osteoblasts and progenitor cells are located), red marrow (vascular tissues), and yellow marrow (soft tissues containing mostly fat cells). The two main stem cells in the microenvironment are hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). HSCs can differentiate either to immune and angiogenic cells while MSCs can differentiate to cells needed for connective tissues within musculoskeletal system such as bone, cartilage, muscles, adipose, tendon, and ligaments. As such, the different niches and cell type present in this viscous tissue can be explored for regenerative medicine, disease models, and development of novel therapeutic ideas. Unfortunately, its complexity and heterogeneity make it very complicated to be synthesized in-vitro and its oversimplifying fabrication can influence the exploration of the different pathological studies.

[0068] Scaffolds may be used to mimic natural tissues for a variety of different reasons, such as for example: to promote cell adhesion and interactions, to promote transport of nutrients and growth factors allowing cellular viability and proliferations, to facilitate easier control ofAttorney Docket No.: 049648 / 643192 physical characteristics depending on the specific application, to achieve low inflammation and high cellular viability making it biocompatible, etc. Microfluidic devices are considered to be at least one possible technique to recreate bone marrow with tunable properties similar to bone marrow cavities. These devices can be used due to their abilities to control fluids and particles at microscale levels thanks to the presence of microchannels making it feasible for diverse applications such as drug screening and testing (biomarker screening), disease model diagnosis and point-of-care purposes (pathogen detection, cell counting, tissue engineering applications, etc.). However, there is an ongoing need for devices, constructs, scaffolds, approaches / techniques, methods, and compositions of matter to fabricate a complex microfluidic device with heterogeneous properties to mimic the overall bone marrow microenvironment.

[0069] Microfluidic devices can be formed either through molding, photolithography, wetitching, or 3D printing. 3D printing is the most optimal technique among the others for controlling microstructure, vascular complexity7, and tunable mechanical properties of the overall device. Methods and devices for 3D bioprinting, a type of 3D printing, are provided herein for forming biocompatible devices with high cellular viability, and mechanical and structural flexibility properties. Bioprinting can control physical and chemical properties leading to the formation of a hydrogel with extracellular matrices (ECM) abilities. Photopolymerization, a chemical crosslinking type of synthesis (polymers formed through covalent bonding), is a technique requiring the use of light (UV or blue light) on monomer in a liquid phase, also called bioink, to form a hydrogel. In some embodiments, the presence of a photoinitiator is needed since it releases free radicals upon light exposure to cause crosslinking of monomers of crosslinking materials in the bioink solution. 3D printing can comprise, e.g., the use of laser (known as laser-induced forward transfer or LIFT), selective light transmitted on the bath filled with liquid resin to form a solid adhered to a moving stage (known as stereolithography or SLA), or digital light processing (DLP). DLP works when a light source (e.g., UV or blue light) goes through a digital micromirror device (DMD), controlled by a computer array to create specific geometry. A photo absorber is added to reduce cytotoxicity, and excess heat generation, to match the absorption of the device with the bioink solution, and to improve the printability properties. Moreover, DMD is used to create geometric patterns by reflecting and focusing the light on the bioink solution. These properties may lead to a hydrogel formation with high biocompatibility7and high resolution, making it feasible for microchannel formation.Attorney Docket No.: 049648 / 643192

[0070] In some embodiments, a system for 3D printing a microfluidic device can comprise a DLP Lumen-X printer configured to print microfluidic devices that mimic the bone marrow microenvironment. Among aspects, features, or characteristics that can be mimicked in the microfluidic devices, the hard porous environment (endosteal) can be formed in certain embodiments to promote stem cell growth and differentiation, in addition to durable microchannels being formed in the microfluidic device to mimic perfusable vasculature. In some embodiments, a suitable bioink can comprise a combination of PEGDA, GelMA, and HAMA. Additionally, in some embodiments, the bioink can comprise an injectable hydrogel, a gel that acts like a liquid during extrusion / inj ection, but solidifies and acts like a gel, semisolid material, or solid material once injected. The use of an injectable hydrogel can promote, among other things, cellular response within the microfluidic device. In some embodiments, the inj ectable hydrogel can be a xeno-free hydrogel that is mechanically tunable with cell media solution and comes with a wide range of bioactivity. In some embodiments, a bioactive injectable hydrogel can be used to form portions of a physiologically relevant BMN construct, such as central marrow. Additionally, in some embodiments, an injectable hydrogel can be modified with collagen and 3D printed with a bio-ink to form a microfluidic device with clear micro-channels for the endosteal region to enhance osteogenesis.

[0071] FIG. 1 provides, according to one or more embodiments of the present disclosure, an exemplary process 100 of forming a physiologically relevant BMN construct / model from a bioink 101. While various of the embodiments described herein refer to models and constructs of the bone marrow niche specifically, the same and / or similar processes, protocols, materials, bioinks, and / or the like can be used to print / form other tissues or models. For example, one or more other stem cell niches can be modeled using a 3D construct modeling, e.g., an endosteal niche, a hematopoietic stem cell niche, a hair follicle stem cell niche, an intestinal stem cell niche, a neural stem cell niche, a cancel stem cell niche, a germline stem cell niche, spermatogonial stem cell niche, and / or the like.

[0072] As illustrated in FIG. 1 according to some embodiments, a first bioink material 102 can be provided that is or comprises, e g., Polyethylene Glycol Diacrylate (PEGDA). A second bioink material 103 can be provided that is or comprises, e.g.. Gelatin Methacrylate (GelMA). A third bioink material 104 can be provided that is or comprises, e.g.. Hyaluronic Acid Methacry late (HAMA). The first bioink material 102, the second bioink material 103, and the third bioink material 104 can be combined to form a Bioink Part A 101. The Bioink Part A 101 can be printed using digital light processing 105 to form a BMN Construct Part A 106.Attorney Docket No.: 049648 / 643192

[0073] In some embodiments, the Bioink Part A 101 can, optionally, further comprise other optional components, such as, e.g., arginyl-glycyl-aspartic acid (RGD) peptides, LAP, tartrazine, IKVAV, etc., illustrated in FIG. 1 as RGD / LAP / Tartrazine / IKVAV 107. In some embodiments, the further components comprise only a RGD, only LAP, only tartrazine, only IKVAV, a combination of some of these, and / or a combination of all of these. In some embodiments, one or more of the optional other components (e.g., RGD / LAP / Tartrazine / IKVAV 107) can be added to an existing mixture of one or more of the first bioink material 102, the second bioink material 103, and / or the third bioink material 104 with a fluid, such as dPBS before mixing / formation thereof to form the Bioink Part A 101.

[0074] In other embodiments, one or more of the optional other components (e.g., RGD / LAP / Tartrazine / IKVAV 107) can be added to the Bioink Part A 101 after mixing one or more of the first bioink material 102, the second bioink material 103, and / or the third bioink material 104 with a fluid, such as dPBS to form the Bioink Part A 101. In some embodiments, the Bioink Part A 101 can comprise components identified in Table 1. In some embodiments, the Bioink Part A 101 can be the only bioink used during printing of an initial construct structure, such as the BMN Construct Part A 106, and one or more other bioinks, such as Bioink Part B loaded with RGD 109 can be added to the BMN Construct Part A 106 after bioprinting thereof. In other embodiments, the Bioink Part A 101 can be the only bioink used to print a finalized article, model or construct, such as a BMN Construct 110.Table 1. Example composition of a Bioink Part A (e.g., BoMINKA). Components Form Stock Concentration Amount needed for Concentration 5mL (total volume) PEGDA 3 solids N / A 1,000 Da: 5% 1,000 Da: 250 mg (5 mg / 100 pL)3,400 Da: 5% 3,400 Da: 250 mg (5 mg / 100 pL)6,000 Da: 5% 6,000 Da: 250 mg (5 mg / 100 pL)GelMA Solid N / A 3 % (3 mg / 100 pL) 150 mg HAMA Solid N / A 1 % (1 mg / 100 pL) 50 mgLAP Solid 10 mM 4 mM 2000 pL Tartrazine Solid 10 mM 1.5 mM 750 pLRGD Solid 40 mM 2 mM 250 pLIKVAV Solid 20 mM 5 mM 1250 pLDPBS Liquid N / A Add to 5mLAttorney Docket No.: 049648 / 643192

[0075] Bioprinting of the BMN construct part A 106 can be based on, e.g., a computer-aided design (CAD) model 108. A DLP printer or the like can, based on the CAD model 108, cause communication of one or more volumes of the Bioink Part A 101 into a print space using, e.g., a printhead, a printing tip, or the like. The DLP printer or a system / apparatus comprising the DLP printer can then, based on the CAD model 108 and / or a print pathway, tool pathway, printing instructions, and / or the like associated with the CAD model 108, cause movement of the printhead, or a printing platform or support surface, or the like through the print space as the plurality of volumes of the Bioink Part A 101 are communicated therethrough and into the print space of the DLP printer or apparatus comprising the DLP printer. Polymerization of the Bioink Part A 101 can be carried out using, e.g., DLP 105. In some embodiments, DLP 105 can be carried out (e.g.. concurrently with or following extrusion / communication of Bioink Part A 101 into the print space) by emitting light towards the Bioink Part A 101, such as towards each layer of the Bioink Part A 101, e.g., using a polymerize-while-printing approach or a print-then-polymerize approach. In some embodiments, a Bioink Part B loaded with RGD 109 can, optionally, be provided that further comprises HAMA mixed with RGD / DTT in Dulbecco’s Modified Eagle’s Medium (DMEM). The Bioink Part B loaded with RGD 109 can, optionally, be added to stem cell chambers, incubated for about one hour then complete media will be added to the top of hydrogel. In some embodiments, the approach 100 can continue with the Bioink Part B loaded with RGD 109 being communicated into one or more regions (e.g.. Stem Cell Chambers) within the BMN Construct Part B 106. such as to cause stem cell adhesion, proliferation and differentiation in the chambers in the construct thereby forming a finished model / construct, i.e., the BMN construct 110.

[0076] In some embodiments, the CAD model 108 can include a 3D model of the BMN construct 106 that is abstracted or subdivided into slices (e.g., to form a sliced 3D model), such as a plurality of stacked layers, a plurality of flat layers, a plurality of portions or regions of layers, etc. In some embodiments, the CAD model 108 can also comprise or be modified to comprise a print pathway, a tool path, printing instructions, and / or the like. The CAD model 108 can then be loaded into or communicated to, e.g., a DLP printer, a sy stem / device comprising a DLP printer, and / or the like.

[0077] Referring now' to FIGs. 2A-2D, different BMN models can be formed as inputs / printing instructions for 3D printing (e.g., DLP printing) of example BMN constructs printed from bioinks, bioink gels, bioink hydrogels, and / or the like. Example BMN constructs can be used to study, e.g., endosteal, yellow marrow, and / or red marrow stem cell growth. A 3D printed microfluidic construct is illustrated that includes vasculature and stem cellAttorney Docket No.: 049648 / 643192 growth / differentiation regions. As shown in FIG. 2A, a particular BMN construct / model can comprise, e.g., a stem cell niche maturation chamber and a perfusable vasculature comprising one or more channels / vessels formed through or within a bioprinted BMN construct formed as a tunable bioprinted scaffold / matrix for stem cell niches. The perfusable vasculature can comprise a perfusion inlet and a perfusion outlet. The perfusable vasculature can be or comprise a sinusoidal vasculature network. One or more multi-vascular bridges can be formed between different portions of a sinusoidal vasculature network. In accordance with the same embodiment illustrated in FIG. 2A, FIGs. 2B-2D illustrate the same BMN model as shown in FIG. 2A, but from, respectively, a 45° rotated view, a 75° rotated view, and a top-down view.

[0078] An example 3D printing model / CAD model comprises vascular tissue that mimics condition of human vascular tissue through microchannel(s) in the BMN. In some embodiments, the tissue may go through a support part (endosteal), and a reservoir (e.g., mimicking a central portion also called yellow marrow). In some embodiments, hybrid hydrogels can be formed by combining natural modified with methacrylates (e.g., GelMA and HAMA) to facilitate covalent linking, with synthetic polymers (e.g., PEGDA). According to some embodiments, natural polymers can be chosen since they contain natural molecules found in extracellular matrix (ECM) material, which may be important for mimi eking / studying cellular interaction, proliferation, and differentiation. Additionally or alternatively, synthetic polymer(s) may be used since they are physically and chemically tunable. For example, PEGDA can be the synthetic polymer, or one of the synthetic polymers, used because, e.g., it is biocompatible, biostable, and a strong polymer when polymerized / crosslinked that can form a hydrogel in the presence of a photoinitiator and when exposed to a light source. In some embodiments, PEGDA may be used for forming constructs of different applications or materials, such as bone, bone tissue, cartilage, etc., which makes it suitable for use in bone marrow formation. Unfortunately, polymers such as PEGDA can lack the bioactivity of natural tissues.

[0079] As such, in some embodiments, GelMA can be added as a natural polymer in the bioink due to its high biocompatibility and bioactivity, e.g.. in connection to signaling needed for musculoskeletal tissues such as bone and cartilage. In some embodiments, other / another natural polymer, such as HAMA, can be used also or instead since, e.g., hyaluronic acid is biocompatible, has properties that make it suitable for many orthopedic applications and bone or bone tissue engineering applications, can make it mechanically tunable, has suitable printing properties, and can enhance cellular adhesion. In some embodiments, a bioink can comprise a 3D printing hydrogel comprising PEGDA, GelMA, and HAMA.Attorney Docket No.: 049648 / 643192

[0080] In some embodiments, compositional characteristics such as the molecular weight makeup of PEGDA and process parameters such as printing time were varied to improv e / optimize formation of a micro-fluidic device with sufficient / improved physical / mechanical properties. The improved physical / mechanical strength / durability of at least certain portions of the construct, such as the vasculature, in certain embodiments, improved the perfusability of the 3D printed construct in terms of the vascular network as a microfluidic device. Printing intensity was chosen based at least on the parameters of the bioink. In some embodiments, bioprinting was carried out using a low resolution (e.g., high granularity ) to increase the quality of printing and a high bum-in layer to increase adhesion of the gels to the printing bed. In some embodiments, various molecular weights or molecular weight combinations of PEGDA were used. In certain embodiments, the use of higher molecular weight PEGDA can increase crosslinking of the hydrogel and result in stiffer gels.

[0081] In some embodiments, during different portions of the bioprinting process, such as during printing of different portions or features of the BMN construct, different bioink gel solutions, different exposure times / intensities, PEGDA molecular weights, and / or other aspects / combinations of printing material / process parameters can be used to differentiate between the different features or portions. In some embodiments, the intensity and bum-in layer may be, e.g., 20 mW / cm2, for a number of layers, such as four initial layers. In some embodiments, a printing rate of, e.g., 10 sec / layer can be used, while in other embodiments, printing can be carried out at a faster rate (e.g.. 20 sec / layer) or a slower rate to achieve a varied print quality / resolution. Printed gels in some embodiments, can have a measured diameter (Dm) between 2 / 3 and % of the Dewith 6,000 Da having 40% of the proposed one.

[0082] Referring now to FIGs. 3A-3C, illustrated are a 3D model design and printed 3D model illustrating printability properties that vary based on, e.g., the printing parameters. As illustrated, a printing rate of about 10 sec / layer, the ratio was very low (<0.6), and the grids were very slushy, and, in some embodiments, this may worsen for higher molecular weight, which may be due to increased presence of macromolecules but insufficient time to crosslink them. The ratio of the PEGDA mixture was the same as 1,000 Da and 3,400 Da. This could be because 1,000 Da and 3,400 Da were crosslinked more than 6,000 Da due to the lower presence of macromer of 1,000 Da and 3,400 Da.

[0083] Referring now to FIG. 4, for 15 sec printing parameters, all groups were very close to a ratio of 1. As a result, in some embodiments, 15 sec / layer was used as the average or target print rate. In some embodiments, the PEGDA mixture experienced an interconnection between all chosen molecular weights but print fidelity was a little lower than 6,000 Da (-0.75). For 20Attorney Docket No.: 049648 / 643192 sec printing, inconclusive results were observed. 1,000 Da’s printing ratios were not significant implying that the printing ratio peaked after 15 sec printing parameters. The printing ratio decreased since 3,400 Da had more macromer than 1,000 Da but not as much as photocrosslink present in the bioink solution. For the 6,000 Da PEGDA, the printing ratio increased significantly, which may be because the 6,000 Da PEGDA has more macromer than photocrosslink. The mixture’s ratio decreased but was considered high (>0.8). As such, other than when using the 3,400 Da PEGDA, FIG. 4 illustrates that a 20 sec / layer printing rate was the most optimized printing setting among all the groups illustrated in FIG. 4.

[0084] FIGs. 5A-5I illustrate a process 200 for bioprinting. In FIGs. 5A-5D, materials are provided for forming a bioink. In some embodiments, the bioink was formed using at least the components shown in Table 1. In other embodiments, the bioink was formed using at least the components shown in Table 4. In some embodiments, the bioink comprises 15% of PEGDA (advanced Biometrix) made of different molecular weights groups (1,000 Da, 3,400 Da, 6,000 Da, and an equal mixture of the three molecular weights, called a mixture group), 3% GelMA (50% degree of freedom, Advanced Biometrix), 1% HAMA (Advanced Biometrix) containing 4 mM lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, Advanced Biometrix) and 1.25 mM tartrazine (Sigma Aldrich) all disposed, dispersed, or dissolved in dPBS. In some embodiments, once all the components were added, the bioink was left overnight at a minimum temperature of 37°C then was covered with aluminum foil then mixed for a minimum of one hour before was heated again to a temperature of 37°C.

[0085] In some embodiments, the procedure for the formation of bioinks and 3D printing gels includes formation of macromers for bioink formation including PEGDA (see, e.g., FIG. 5A), GelMA (see, e g., FIG. 5B), HAMA (see. e g., FIG. 5C), and in FIG. 5D, LAP photoinitial (i.e., in the small tube) and tartrazine (i.e., in the big bottle). In FIG. 5E. all items illustrated in FIGs. 5A-5D were mixed in dPBS and dissolved using the heat process. In FIG. 5F, the mixture was headed overnight. In FIG. 5G, the gels were vortexed and became clear. In FIG. 5H, the gels were injected into a DLP printer, such as the printer Lumen-X. In FIG. 51, a print head is used to print the bioink during the printing process.

[0086] Referring now to FIGs. 6A-6E, the process 200 is illustrated according to another embodiment, to form a bioactive system using a composition of 70% bioink and 30% Vitro-Gel Collagen. As illustrated in FIG. 6A, the process 200 can include forming or providing a bioink. A VitroGel Collagen can be diluted (e g., 1:3) and mixed with a-MEM media. An injectable gel solution can then be mixed with the bioink. The composite mixture can then beAttorney Docket No.: 049648 / 643192 3D printed and then washed thoroughly. The same overall testing can be done (e.g., printability', degradation, and porosity) on constructs printed using this embodiment of the process 200.

[0087] In some embodiments, a cell-laden bioactive system is thereby created using two-step techniques. First, the cell-laden injectable hydrogel may be formed following the same steps described above. Then, the equivalent amount of bioink was added to the injectable hydrogel and 3D printed (FIG. 6D) with the same parameters described above. After the synthesis, the cells were fixed and stained, then imaged (FIG. 6E) using a confocal microscope.

[0088] Bioinks were 3D printed using the Lumen X in a grid configuration, e.g., as shown in FIGs. 3A and 3B. The gel pattern was used as a platform to optimize the lumen formation related to the microfluidic devices. The fixed parameters were intensity (20 mW / cm2) and burn-in layer (e.g., four times, or 4x). The exposure time (e.g., 10 sec, 15 sec, and 20 sec per layer) and molecular weight of PEGDA were varied as shown in Tables 2-4.Table 2. Example properties of bioinks formed using 1,000 Da PEGDA, 3,400 Da PEGDA,6,000 Da PEGDA, or a 1: 1: 1 mixture thereofComponent 1000 Da 3400 Da 6000 Da Mixture1PEGDA 15% 15%J 15% 15% GelMA 3% 3% 3% 3% HAMA 1% 1% 1% 1% LAP 4 mM 4 mM 4 mM 4 mM Tartrazine 1.25 mM 1.25 mM 1.25 mM 1.25 mM dPBS lx2lx2lx2lx21The mixture was formed by adding 5% of 1000 Da, 5% of 3400 Da, and 5% 6000 Da2The lOx dPBS was diluted by 1 / 10 with pure waterTable 3. Example bioprinting parametersParame- Description Characteristics Intensity Intensity of blue light of 3D Printer 20 mW / cm2ExposureTime exposed for each layer 10-20 sec timeLayer Thickness 50 μm Burn-in, Increases exposure for early layers 4 xLayerAttorney Docket No.: 049648 / 643192 Table 4. Exam ole composition of bioink materialConcentr Composition Properties Description-ation Synthetic Biostable, biocompatible, strong Polymer malleable, and a bioinert polymer. Forms Polyethylene Molecular rigid hydrogels that can supports small- Glycol Diacrylate Weight (1000 hollowed network geometries (to later 15% (PEGDA) Da, 3400 Da, become endothelialized) and a large- 6000 Da): hollowed chamber (to later become a1:1:1 large stem cell maturation chamber).Biofunctional polymer containing a wide GelatinNatural range of proteins; Denatured collagen;Methacrylate 3%Polymer the presence of methacrylate is used as(GelMA)catalyst for polymerization.Biofunctional glycosaminoglycanprotein; highly abundant in boneHyaluronic Acid Hydratedmarrow; enhances cell proliferation, Methacrylate Natural 1%migration and differentiation; the(HAMA) Polymerpresence of methacrylate is used ascatalyst for polymerization.At the exposure of a light source emitting Lithium a 405 nm wavelength, it initiates free Phenyl(2,4,6- radical chain polymerization with4 mM Trimethylbenzoyl) Photoinitiator methacrylate, diacrylate, and thiol group;(IL 77%) Phosphinate it is used in DLP for bioprinting due to(LAP) low cytotoxicity of the chemical andlight source.It limits the light scattering beyond agiven area; It maintains bioprinting 1.5 mM Tartrazine Photoabsorberresolution; it stops over-excitation of (8.02%) LAP in already formed layers.It enhances cell attachment to thehydrogel; it is found in majorArginine-Glycine- Peptideextracellular matrices (collagen, 2 mM Aspartic Acid mimeticfibronectin, laminin); it ensures firm (20.50%) (RGD) Fibronectinattachment while promoting cell growthand proliferation.Isoleucine-lysine- PeptideIt functions in cell adhesion; it promotes 5 mM valine- alaninemimeticangiogenesis of endothelial cells. (26.44%) valine (IKVAV) LamininDPBS is the solvent used for dissolving Dulbeccoand mixing all reagents needed to form Phosphate Buffer A saline pHthe bioink solution; the presence of traceSaline (DPBS) + buffered 14.27% elements (i.e., calcium, magnesium) incalcium and solutionDPBS also helps control the swelling ofmagnesium.the formed hvdrogel.Attorney Docket No.: 049648 / 643192

[0089] In some embodiments, the intensity' of DLP light emission can be chosen based on, e.g., an exposure time per layer and the presence or concentration of tartrazine as a photoabsorber in the bioink. In some embodiments, fine layers (e.g., 50 μm) can be used to increase the resolution of the prints. In some embodiments, one or more initial layers of the bioink disposed within a printing space of the DLP printer can represent one or more bum-in layers. In some embodiments, the bum-in layers can be exposed to relatively increased emitted light exposure, such as by increasing the light exposure duration, increasing the light exposure intensity, or both. In certain embodiments, a bum-in layer light exposure can be between about 200% and about 600% of a non-bum-in layer light exposure. For example, if a print has a nonbum-in layer emission duration of about 15 sec / layer, the bum-in layer emission duration can be between about 30 sec / layer and about 60 sec / layer assuming no material change in emission intensity between the bum-in layers and the non-bum-in layers. In some embodiments, the bum-in layers may provide for improved adherence of the gel / hydrogel construct to the print head and / or print substrate.

[0090] In some embodiments, other non-bum-in exposure durations such as about 10 sec / layer and about 20 sec / layer, or greater than about 20 sec / layer can be used for nonbum-in layers. To test for patentability, a test grid of 3D printed gel discs having a diameter of about 600 pm was printed. After printing, the 3D gels discs were washed thoroughly and then imaged using a bright-field microscope EVOS FL Auto and analyzed using FIJI software. The percent (%) printability was calculated as:An% printability = ——where Dm is the diameter measure using the microscope (pm) and De is the expected diameter put using 3D model design (pm). Some results from this analysis can be found, e.g., in FIG. 7A and FIG. 7B.

[0091] The hydrolysis of the gels was investigated because degradation may occur due to hydrolysis of the end group of acrylate ester of PEGDA over time. GelMA and HAMA can be impacted by hydrolysis only at high pH or high temperature. Even though GelMA and HAMA were present in the bioink solution at a standard condition (neutral pH and room temperature for the most part), there was a high amount of PEGDA (15%). As previously described, four groups of six different discs were studied for the impact of hydrolysis on degradation. The gel discs 300 such as those shown in FIG. 7A were printed with the parameters discussed previously (e.g., intensity: 20 mW / cm2, exposure time: 20 sec / layer, and burn-in layer fabricated at 400% exposure duration) and tested for degradability purposes.Attorney Docket No.: 049648 / 643192

[0092] FIG. 7A illustrates an array of gel discs 300 3D printed from different example bioinks. The gel discs 300 were 3D printed and merged in dPBS. The rate of degradation of the gel discs 300 is illustrated in FIG. 7B. Six disks having an average diameter of about 8 mm an average height of about 1.5 mm were printed for a variety of different bioink compositions. 3D printing was carried out at a light intensity of about 20 mW / cm2, for a light exposure duration of about 20 sec / layer, using a 400% light exposure duration for the bum-in layer. The gel discs 300 were washed thoroughly and then weighed right after. The six gel discs 300 were then stored in six individual wells filled with dPBS at about 4°C. The gel discs 300 were weighed again after a variety of aging durations (e.g., one day, four days, seven days, and 14 days). For each timestamp in FIG. 7B, the gel discs 300 were air-dried for an hour, then their mass was measured, and then the gel discs 300 were placed back into dPBS in their individual wells and stored again at about 4°C until the next weigh time.

[0093] From FIG. 7B, it is shown that the 3D printed gel discs 300 were susceptible to degradation but at a different rate depending on, e.g., composition and molecular weight of PEGDA. For example, for gel discs 300 printed from bioinks comprising 1,000 Da PEGDA and the mixture of dilferent molecular weights of PEGDA, the initial weight was lower in comparison to other groups. On the other hand, apart from gel discs 300 printed from a bioink comprising 3,400 Da PEGDA, most of the other gel discs 300 had more than about 35% mass reduction after 14 days of aging in dPBS. As such, for at least some example bioinks disclosed herein, the average initial weight of the gel discs 300 increased when increasing the molecular weight of the PEGDA in the bioink while keeping the mass of the gel discs 300 equivalent and while maintaining an equivalent wt.% of PEGDAin the bioinks. One exception is that the mass of the gel discs 300 formed from a bioink comprising the mixture of different molecular weights of PEGDA. which was slightly lower as compared to gel discs 300 formed from bioinks comprising 3,400 Da and 6,000 Da PEGDA. In some examples, increasing molecular weight of PEGDA in the bioink slowed down the hydrolysis rate. For example, after 14 days of aging, it was found that gel discs 300 printed from bioinks comprising 3,400 Da PEGDA were significantly higher than gel discs 300 printed from bioinks comprising 1,000 Da PEGDA and gel discs 300 printed from bioinks comprising the mixture of dilferent molecular weight of PEGDA. Therefore, generally, increases in molecular weight appear to increase the amount of macromer in the gel solution, which can increase the weight of the gel discs 300, making it harder for the acrylate ester to get detached from PEGDA. Without wishing to be bound by any particular theory, this may be important for cellular response because MSCs may help remodel the tissues and adapt to substrates. Additionally, the molecular weight of PEGDA in the bioinkAttorney Docket No.: 049648 / 643192 was considered an important aspect of the degradation of the gel discs 300. This physical aspect made the synthesized gel feasible for the formation of the BMN constructs at least because of the relative importance of acrylate ester detachment from PEGDA to promoting tissue remodeling and substrate adaptation.

[0094] Referring now to FIGs. 8 A and 8B, another aspect of the testing of the gel discs 300 was testing mechanical properties of the gel discs 300. In order to test mechanical properties, four groups of six gel discs 300 were 3D printed. The gel discs 300 had a diameter of about 8 mm and a height of about 2 mm. The gel discs 300 were washed thoroughly and left overnight at about 4°C to swell. The diameter and the height of the gel discs 300 were measured before conducting the mechanical testing using a compression mechanical tester 400. A continuous force of about 100 N and a strain rate of about 10% / min up to a maximum of about 30% of an initial height of each gel disc 300 was applied using the compression mechanical tester 400.The stress was calculated by dividing the compressed force by the cross-section area and the strain was calculated by dividing the compressed height by initial height. A graph of stressstrain curve was formed using Excel™ and Young’s Modulus was calculated as the slope of stress vs strain between 10% and 20% strain.

[0095] The Young’s Modulus of the hydrogels was tested using the gel discs 300 to explore the impact of, e.g., different molecular weights of PEGDA, on the mechanical properties. For the four groups of six gel discs 300 tested, the Young's Modulus testing results are provided in FIG. 9. The purpose of the Young’s Modulus testing was to explore the impact of molecular weight of PEGDA in the bioink on the resulting stiffness of the gel discs 300. The gel discs 300 were put on top of a plate as shown in FIG. 8A, and then compressed to 30% of their initial height as shown in FIG. 4B. It was found that the Young’s Modulus of all groups was very high (the lowest being almost 300 kPa). Additionally, the Young’s Modulus for gel discs 300 formed from bioinks comprising 1,000 Da PEGDA, 3,400 Da PEGDA, and the mixture of different molecular weight PEGDA were higher than the Young’s Modulus for gel discs 300 formed from bioinks comprising 6,000 Da PEGDA, with a change of 20-30%. On the other hand, the Young’s Modulus of gel discs 300 formed from bioink comprising the mixture of different molecular weight PEGDA was not significantly different from the Young’s Modulus of gel discs 300 formed from bioinks comprising 1,000 Da PEGDA and 6,000 Da PEGDA but was significantly different from the Young’s Modulus of gel discs 300 formed from bioinks comprising 3,400 Da PEGDA. This may mean that the stiffness of gel discs 300 peaked, at least locally if not globally, when using bioinks comprising PEGDA having a molecular weight or average molecular weight of about 3,400 Da, as shown in FIG. 9.Attorney Docket No.: 049648 / 643192

[0096] All the groups had a Young’s Modulus as low as 280 kPa. It was found that the endosteum region of the bone marrow was very stiff (e.g., more than about 35 kPa). As such, the stiffness of the formed gel discs 300 was within the target range. The results showed that there was an increase of Young’s Modulus when increasing the molecular weight of PEGDA in bioinks from 1,000 Da to 3,400 Da (-25%), but a decrease of 37% occurred when the molecular weight of PEGDA in the bioink increased from 3,400 Da to 6,000 Da. Moreover, the Young’s Modulus of gel discs 300 formed from bioink comprising the mixture of different molecular weight PEGDA was much lower in comparison to gel discs 300 formed from many other groups or composition of bioink except, e.g., bioink comprising 6,000 Da PEGDA. Without wishing to be bound by any particular theory, this may be due to the presence of more macromer than photocrosslink in the bioink comprising 6,000 Da PEGDA.

[0097] The porosity of the gels was another physical characteristic analyzed. One purpose for testing porosity was to anticipate the impact of the molecular weight of PEGDA in the bioink on the porous regions of the resulting BMN construct. Here again four groups of six gel discs 300 were 3D printed and tested for porosity. The gel discs 300 were formed, washed thoroughly, and left at 4°C overnight, and then tested using a scanning electron microscope (SEM) the following day. Referring now to FIGs. 10A-10D, the SEM images of the gel discs 300 for each group are shown. The gels had very' low pore zones. FIG. 11 is a graph that illustrates the results of data analysis of the porosity testing. The pore zones / regions in gel discs 300 formed from bioink comprising 1.000 Da PEGDA and 3,400 Da PEGDA were not statistically different each from the other, even though it was thought that the higher molecular weight of PEGDA would lead to higher crosslinking. However, when the molecular weight of PEGDA in the bioink increased to 6,000 Da, there was a 10% drop in the pore zone / porosity due to an increase of macromer in the gel discs 300. For gel discs 300 formed from bioinks comprising the mixture of different molecular weight PEGDA, the porosity dropped by about 50%, which may be due to the presence of more macromer in the gel discs 300.

[0098] In order to conduct the SEM testing and porosity analysis, gel discs 300 for each group were 3D printed using parameters as described previously herein. The porosity of each gel disc 300 was tested using SEM. The gel discs 300 were adhered to a metal hook, then were inserted inside of the SEM machine using a high vacuum pump. A distance between the detector and the gel discs 300 was set to 10 mm. A spot of 3 and power of 10 kV was used to analyze the gel discs 300. The SEM images in FIGs. 10A-10D were then taken, and the pore zone size / porosity was analyzed using FIJI.Attorney Docket No.: 049648 / 643192

[0099] While the porosity of gel discs 300 formed from bioinks comprising 1,000 Da PEGDA and 3,400 Da PEGDA were not significantly different, it was found that the higher the molecular weight of PEGDA, the lower the pore size of the gels. For the lesser molecular weight, this could be because the initial increase of the crosslink amount of PEGDA did not impact the porosity of the gels as more drops were observed at higher molecular weight. It was also found that the mixture significantly lowered the porosity. The pore zone was lower than 8 pm diameter in total which is proportional to a stiffer scaffold.

[0100] Referring now to FIGs. 12A-12D, another aspect of this disclosure, according to some embodiments, is to illustrate and explain how modification of injectable hydrogel bio inks, gel with lower Young’s Modulus and coupled with RGD peptide, with different peptides, may lead to different MSC morphological spreading overtime. For example, different chemical signaling of the gels could lead to heterogeneity of the 3D-printed microfluidic device, which may be important because 3D printing bioactive microfluidic devices may require appropriate chemical signaling, especially since there are three niches with different biophysical and biochemical characteristics in the BMN being modeled. While implementing growth factors or proteins was contemplated, the use of injectable hydrogels (e.g., MeHA injectable hydrogel) was determined to be preferable. These hydrogels were characterized by, e g., their ability to be chemically modified with peptides, and their low mechanical properties which may make it feasible to model central marrow niches. As such, in certain examples, MeHA coupled with RGD hydrogel led to MSCs spreading at or after about four days post-polymerization.

[0101] In some embodiments, these gels were mixed with MSCs at a cell density of about 500,000 cells / mL, and cellular morphology was investigated as a platform for exploring the overall properties of the gels after one day, four days, and seven days of encapsulation.

[0102] As shown in FIGs. 12A-12B, respectively, cells were successfully encapsulated in MeHA hydrogel. As shown in FIG. 13, results showed that cellular volume increased over time and that the circularity spherical index decreased over time.

[0103] MSCs in VGCOL proliferated the most after about four days, as shown in FIG. 14 and FIG. 15. Therefore this example environment for stem cell growth is shown to be compatible for the cells.

[0104] FIGs. 14A-14B illustrate cellular morphology according to certain embodiments at a low magnification versus a high magnification. In these examples, the cells exhibited more spread as compared to those illustrated FIGs. 12A-12B, which is confirmed by the data presented in the graphs of FIGs. 13A-13B.Attorney Docket No.: 049648 / 643192

[0105] Further, cellular morphology after seven days was imaged and is illustrated in FIGs. 15A-15B, which illustrate the difference when using, respectively, a low magnification versus a high magnification. As shown, the cells elongated and spread, which is confirmed by the data presented in the graphs of FIGs. 13A-13B.

[0106] MSCs were encapsulated in the 3D printed gel formed from BoMINKA part A to explore the cellular behavior, such as shown in FIG. 16. For the single system, the bioink solution was added directly to the cell pellets, resuspended then 3D printed using the previous printing parameters. The cellular morphologies were shown to be spread and linear and were confluent. Additionally, there were a mixture of rounded and spread cells (see, e.g., FIG. 16).

[0107] The microfluidic device was printed following the CAD model shown in FIG. 17B and the lumen was shown after the thorough washing (see. e.g., FIG. 17A). Therefore, the lumen formation was not impacted even when the different hydrogel synthesis was introduced. The dye was injected into the middle chamber and the microchannels, as illustrated in FIG. 17 A.

[0108] What follows are select non-limiting examples provided for the purpose of illustrating the processes, methods, apparatuses, and systems described herein.

[0109] For MeHA hydrogel, 3 wt% MeHA was mixed with DMEM, 10 mM Dithiothreitol and 2 mM RGD (cells pellets, labeled with CellTracker Red CMPTX, w ere resuspended in the gel solution). The gel solution covered each bottom glass of a 35 mm dish and were incubated for one hour. Then, growth media and Fetal Bovine Serum (FBS) was added on top of the gel solution and then changed once every two days for a total period of seven days. The gels were then imaged with a Leica confocal microscope and the cellular volume was measured using FIJI, an image processing tool, to analyze the images of the gels taken using the Leica confocal microscope. Based at least on the cellular volume measurements, a circular spherical index (CSI) was calculated as follows:1 27T3 X (6 X F)3CSI = - - - —Awhere / stands for cellular volume and A stands for the surface area of the cells.

[0110] Provided below in Table 5 is an example composition of an injectable MeHA injectable hydrogel bioink and properties of the same. Among other differences between this and other hydrogels and hydrogel bioinks described is the use of dithiothreitol (DTT), in addition to HAMA and RGD in a DMEM media.Attorney Docket No.: 049648 / 643192 Table 5. Example composition of an injectable MeHA hydrogel bioink material Components Form ConcentrationHAMA Solid 3% (3 mg / 100 pL)DTT Solid 10 mM (15.43%)RGD Solid 2 mM (205%)DMEM Media 64.07%

[0111] In some examples, different PEGDA molecular weights, along with GelMA and HAMA, were investigated in a function of bone marrow cavities. The bioink solution was 3D printed at different exposure times to optimize the printability. The degradation, porosity, and stiffness of the gels were investigated. Certain bioactive gels were also explored. It was found that when the molecular weight of PEGDA was equally mixed, the gels had a slow rate of degradation, low porosity, and stiffness closer to the endosteal region. The addition of the bioactive injectable hydrogel increased the cellular proliferation and the physical properties of the system. Moreover, for a longer period, a solid robust microfluidic device was formed with a microchannel. Consequently, the bioactive system was proven to be ideal to mimic the properties of the marrow niche found in the bone marrow. The interface interaction between the two different types of hydrogels was considered as the foundation of the formation of artificial bone marrow.

[0112] Referring now to FIGs. 18A-18C, a design for a tissue construct model is illustrated from, respectively, a top view, a side view, and an angled (perspective) view, for a tissue engineered bone marrow microenvironment model, with a stem cell niche, specialized vasculature, and physiological fluid flow perfusion.. The tissue construct was modeled as a construct polygon design using Blender 5.0 software. The construct polygon design was dimensioned and configured to represent a tissue engineered bone marrow microenvironment construct comprising a sinusoidal vascular compartment, a continuous vascular compartment, and a stem cell niche maturation chamber.

[0113] The Blender 5.0 model from FIGs. 18A-18C were converted into *. STL format for 3D bioprinting with a LumenX bioprinter (CelLink). Referring now to FIGs. 19A and 19B, respectively, a top view and a side view of such an STL format model is illustrated.

[0114] Conversion into STL file format for 3D printing allows for representation of the 3D model's surface geometry to be used in bioprinters. The use of one or more BoMINKA bioink(s) may permit or achieve various mechanical properties needed for the bioengineered tissue construct to have sufficient stability and favorable microenvironment as required for recapitulating the bone marrow stem cell niche. Shown with the top view- and side view of theAttorney Docket No.: 049648 / 643192 bioprinted construct in FIGs. 19A and 19B is a ruler for dimensionality. Note, two different color dyes were used to show the presence of open lumens for the vascular compartments and the stem cell maturation chamber.

[0115] Referring now to FIGs. 20A and 20B, illustrated therein are, respectively, an image of a microfluidic pump system and a subset thereof comprising the microfluidic configuration with the perfusable bioprinted construct therein. The microfluidic pump illustrated in FIG. 20A is dimensioned and configured for bioprinting of perfusable tissue constructs. The microfluidic pump illustrated in FIG. 20A comprises at least a syringe, tubing, and ports. The microfluidic pump illustrated in FIG. 20A can be configured to prepare the marrowPRINT construct for continuous perfusion. As shown in FIG. 20B, a unique multi-vascular bridge allows for the seeding and culturing of two distinct populations of endothelial cells.

[0116] Some or all of the elements, steps, or components of the approaches described herein can be carried out by a computing device or an apparatus comprising a processor and memory. Examples of such computing devices and apparatuses are described in more detail below.

[0117] Embodiments of the present invention may be implemented in various ways, including as computer program products that comprise articles of manufacture. Such computer program products may include one or more software components including, for example, software objects, methods, data structures, or the like. A software component may be coded in any of a variety of programming languages. An illustrative programming language may be a lower-level programming language such as an assembly language associated w ith a particular hardware architecture and / or operating system platform. A software component comprising assembly language instructions may require conversion into executable machine code by an assembler prior to execution by the hardware architecture and / or platform. Another example programming language may be a higher-level programming language that may be portable across multiple architectures. A software component comprising higher-level programming language instructions may require conversion to an intermediate representation by an interpreter or a compiler prior to execution.

[0118] Other examples of programming languages include, but are not limited to, a macro language, a shell or command language, a job control language, a script language, a database query or search language, and / or a report writing language. In one or more example embodiments, a software component comprising instructions in one of the foregoing examples of programming languages may be executed directly by an operating system or other software component without having to be first transformed into another form. A software componentAttorney Docket No.: 049648 / 643192 may be stored as a file or other data storage construct. Software components of a similar type or functionally related may be stored together such as, for example, in a particular directory, folder, or library. Software components may be static (e.g., pre-established or fixed) or dynamic (e.g., created or modified at the time of execution).

[0119] A computer program product may include a non-transitory computer-readable storage medium storing applications, programs, program modules, scripts, source code, program code, object code, byte code, compiled code, interpreted code, machine code, executable instructions, and / or the like (also referred to herein as executable instructions, instructions for execution, computer program products, program code, and / or similar terms used herein interchangeably). Such non-transitory computer-readable storage media include all computer-readable media (including volatile and non-volatile media).

[0120] In one embodiment, a non-volatile computer-readable storage medium may include a floppy disk, flexible disk, hard disk, solid-state storage (SSS) (e.g., a solid-state drive (SSD), solid state card (SSC), solid state module (SSM), enterprise flash drive, magnetic tape, or any other non-transitory magnetic medium, and / or the like. A non-volatile computer-readable storage medium may also include a punch card, paper tape, optical mark sheet (or any other physical medium with patterns of holes or other optically recognizable indicia), compact disc read only memory (CD-ROM), compact disc-rewritable (CD-RW), digital versatile disc (DVD), Blu-ray disc (BD), any other non-transitory optical medium, and / or the like. Such a non-volatile computer-readable storage medium may also include read-only memory (ROM), programmable read-only memory (PROM), erasable programmable read-only memory (EPROM), electrically erasable programmable read-only memory (EEPROM), flash memory (e.g., Serial, NAND, NOR, and / or the like), multimedia memory cards (MMC), secure digital (SD) memory’ cards, SmartMedia cards, CompactFlash (CF) cards, Memory Sticks, and / or the like. Further, a non-volatile computer-readable storage medium may also include conductive-bridging random access memory (CBRAM), phase-change random access memory (PRAM), ferroelectric random- access memory (FeRAM), non-volatile randomaccess memory (NVRAM), magnetoresistive random-access memory (MRAM), resistive random-access memory (RRAM), Silicon-Oxide-Nitride-Oxide-Silicon memory (SONOS), floating junction gate random access memory (FJG RAM), Millipede memory, racetrack memory, and / or the like.

[0121] In one embodiment, a volatile computer-readable storage medium may include random access memory (RAM), dynamic random access memory (DRAM), static random access memory (SRAM), fast page mode dynamic random access memory (FPM DRAM),Attorney Docket No.: 049648 / 643192 extended data-out dynamic random access memory (EDO DRAM), synchronous dynamic random access memory (SDRAM), double data rate synchronous dynamic random access memory (DDR SDRAM), double data rate type two synchronous dynamic random access memory (DDR2 SDRAM), double data rate type three synchronous dynamic random access memory (DDR3 SDRAM), Rambus dynamic random access memory (RDRAM), Twin Transistor RAM (TTRAM), Thyristor RAM (T-RAM), Zero-capacitor (Z-RAM), Rambus inline memory module (RIMM), dual in-line memory module (DIMM), single in-line memory module (SIMM), video random access memory' (VRAM), cache memory (including various levels), flash memory, register memory, and / or the like. It will be appreciated that where embodiments are described to use a computer-readable storage medium, other types of computer-readable storage media may be substituted for or used in addition to the computer-readable storage media described above.

[0122] As should be appreciated, various embodiments of the present invention may also be implemented as methods, apparatus, systems, computing devices, computing entities, and / or the like. As such, embodiments of the present invention may take the form of an apparatus, system, computing device, computing entity, and / or the like executing instructions stored on a computer-readable storage medium to perform certain steps or operations. Thus, embodiments of the present invention may also take the form of an entirely hardware embodiment, an entirely computer program product embodiment, and / or an embodiment that comprises combination of computer program products and hardware performing certain steps or operations.

[0123] FIG. 21 provides a schematic of such a computing device 500 according to one embodiment of the present invention. In general, the terms computing device, computing entity, computer, entity, device, system, and / or similar words used herein interchangeably may refer to, for example, one or more computers, computing entities, desktops, mobile phones, tablets, phablets, notebooks, laptops, distributed systems, kiosks, input terminals, servers or server networks, blades, gateways, switches, processing devices, processing entities, set-top boxes, relays, routers, network access points, base stations, the like, and / or any combination of devices or entities adapted to perform the functions, operations, and / or processes described herein. Such functions, operations, and / or processes may include, for example, transmitting, receiving, operating on, processing, displaying, storing, determining, creating / generating, monitoring, evaluating, comparing, and / or similar terms used herein interchangeably. In one embodiment, these functions, operations, and / or processes can be performed on data, content, information, and / or similar terms used herein interchangeably.Attorney Docket No.: 049648 / 643192

[0124] As shown in FIG. 21, in one embodiment, the computing device 500 may include or be in communication with one or more processing elements 502 (also referred to as processors, processing circuitry, and / or similar terms used herein interchangeably) that communicate with other elements within the computing device 500 via a bus, for example. As will be understood, the processing element 502 may be embodied in a number of different ways. For example, the processing element 502 may be embodied as one or more complex programmable logic devices (CPLDs), microprocessors, multi-core processors, coprocessing entities, application-specific instruction-set processors (ASIPs), microcontrollers, and / or controllers. Further, the processing element 502 may be embodied as one or more other processing devices or circuitry. The term circuitry may refer to an entirely hardware embodiment or a combination of hardware and computer program products. Thus, the processing element 502 may be embodied as integrated circuits, application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), programmable logic arrays (PLAs), hardware accelerators, other circuitry, and / or the like. As will therefore be understood, the processing element 502 may be configured for a particular use or configured to execute instructions stored in volatile or non-volatile media or otherwise accessible to the processing element 502. As such, whether configured by hardware or computer program products, or by a combination thereof, the processing element 502 may be capable of performing steps or operations according to embodiments of the present invention when configured accordingly.

[0125] In one embodiment, the computing device 500 may include or be in communication with non-volatile media (also referred to as non-volatile storage, memory, memory storage, memory- circuitry and / or similar terms used herein interchangeably). In one embodiment, the non-volatile storage or memory may include one or more non-volatile storage or memory media 503. including but not limited to hard disks, ROM, PROM, EPROM, EEPROM, flash memory, MMCs, SD memory cards, Memory Sticks, CBRAM, PRAM, FeRAM, NVRAM, MRAM, RRAM, SONOS, FJG RAM, Millipede memory, racetrack memory, and / or the like. As will be recognized, the non-volatile storage or memory media may store databases, database instances, database management systems, data, applications, programs, program modules, scripts, source code, object code, byte code, compiled code, interpreted code, machine code, executable instructions, and / or the like. The term database, database instance, database management system, and / or similar terms used herein interchangeably may refer to a collection of records or data that is stored in a computer-readable storage medium using one or more database models, such as a hierarchical database model, network model, relational model,Attorney Docket No.: 049648 / 643192 entity-relationship model, object model, document model, semantic model, graph model, and / or the like.

[0126] In one embodiment, the computing device 500 may include or be in communication with volatile media (also referred to as volatile storage, memory, memory storage, memory circuitry and / or similar terms used herein interchangeably). In one embodiment, the volatile storage or memory’ may also include one or more volatile storage or memory media 504, including but not limited to RAM, DRAM, SRAM, FPM DRAM, EDO DRAM, SDRAM, DDR SDRAM, DDR2 SDRAM, DDR3 SDRAM, RDRAM, TTRAM, T-RAM, Z-RAM, RIMM, DIMM, SIMM, VRAM, cache memory, register memory, and / or the like. As will be recognized, the volatile storage or memory media may be used to store at least portions of the databases, database instances, database management systems, data, applications, programs, program modules, scripts, source code, object code, byte code, compiled code, interpreted code, machine code, executable instructions, and / or the like being executed by, for example, the processing element 502. Thus, the databases, database instances, database management systems, data, applications, programs, program modules, scripts, source code, object code, byte code, compiled code, interpreted code, machine code, executable instructions, and / or the like may be used to control certain aspects of the operation of the computing device 500 with the assistance of the processing element 502 and operating system.

[0127] In some embodiments, the computing device 500 may also include one or more network interfaces, such as a transceiver 508 for communicating with various computing entities, such as by communicating data, content, information, and / or similar terms used herein interchangeably that can be transmitted, received, operated on, processed, displayed, stored, and / or the like. Such communication may be executed using a wired data transmission protocol, such as fiber distributed data interface (FDDI), digital subscriber line (DSL), Ethernet, asynchronous transfer mode (ATM), frame relay, data over cable service interface specification (DOCSIS), or any other wired transmission protocol. Similarly, the computing device 500 may be configured to communicate via wireless external communication networks using any of a variety of protocols, such as general packet radio service (GPRS), Universal Mobile Telecommunications System (UMTS), Code Division Multiple Access 2000 (CDMA2000), CDMA2000 IX (IxRTT), Wideband Code Division Multiple Access (WCDMA), Global System for Mobile Communications (GSM), Enhanced Data rates for GSM Evolution (EDGE), Time Division-Synchronous Code Division Multiple Access (TD-SCDMA), Long Term Evolution (LTE), Evolved Universal Terrestrial Radio Access Network (E-UTRAN), Evolution-Data Optimized (EVDO), High Speed PacketAttorney Docket No.: 049648 / 643192 Access (HSPA), High-Speed Downlink Packet Access (HSDPA), IEEE 802.11 (Wi-Fi), Wi-Fi Direct, 802.16 (WiMAX), ultra-wideband (UWB), infrared (IR) protocols, near field communication (NFC) protocols, Wibree, Bluetooth protocols, wireless universal serial bus (USB) protocols, and / or any other wireless protocol.

[0128] Although not shown, the computing device 500 may include or be in communication with one or more input elements, such as a keyboard input, a mouse input, a touch screen / display input, motion input, movement input, audio input, pointing device input, joystick input, keypad input, and / or the like. The computing device 500 may also include or be in communication with one or more output elements (not shown), such as audio output, video output, screen / display output, motion output, movement output, and / or the like.

[0129] FIG. 22 provides an illustrative schematic representative of an external computing device 600 that can be used in conjunction with embodiments of the present invention. In general, the terms device, system, computing entity, entity, and / or similar words used herein interchangeably may refer to, for example, one or more computers, computing entities, desktops, mobile phones, tablets, phablets, notebooks, laptops, distributed systems, kiosks, input terminals, servers or server networks, blades, gateways, switches, processing devices, processing entities, set-top boxes, relays, routers, network access points, base stations, the like, and / or any combination of devices or entities adapted to perform the functions, operations, and / or processes described herein. External computing entities 600 can be operated by various parties. As shown in FIG. 22, the external computing device 600 can include an antenna 608, a transmitter 609 (e g., radio), a receiver 610 (e.g., radio), and a processing element 602 (e.g., CPLDs, microprocessors, multi-core processors, coprocessing entities, ASIPs, microcontrollers, and / or controllers) that provides signals to and receives signals from the transmitter 609 and receiver 610, correspondingly.

[0130] The signals provided to and received from the transmitter 609 and the receiver 610, correspondingly, may include signaling information / data in accordance with air interface standards of applicable wireless systems. In this regard, the external computing device 600 may be capable of operating with one or more air interface standards, communication protocols, modulation types, and access types. More particularly, the external computing device 600 may operate in accordance with any of a number of wireless communication standards and protocols, such as those described above with regard to the computing device 500. In a particular embodiment, the external computing device 600 may operate in accordance with multiple wireless communication standards and protocols, such as UMTS, CDMA2000, IxRTT, WCDMA, GSM, EDGE, TD-SCDMA, LTE, E-UTRAN, EVDO, HSPA, HSDPA, Wi-Attorney Docket No.: 049648 / 643192 Fi, Wi-Fi Direct, WiMAX, UWB, IR, NFC, Bluetooth, USB, and / or the like. Similarly, the external computing device 600 may operate in accordance with multiple wired communication standards and protocols, such as those described above with regard to the computing device 600 via a network interface 606.

[0131] Via these communication standards and protocols, the external computing device 600 can communicate with various other entities using concepts such as Unstructured Supplementary Service Data (USSD), Short Message Service (SMS), Multimedia Messaging Service (MMS), Dual-Tone Multi -Frequency Signaling (DTMF), and / or Subscriber Identity Module Dialer (SIM dialer). The external computing device 600 can also download changes, add-ons, and updates, for instance, to its firmware, software (e.g., including executable instructions, applications, program modules), and operating system.

[0132] According to one embodiment, the external computing device 600 may include location determining aspects, devices, modules, functionalities, and / or similar words used herein interchangeably. For example, the external computing device 600 may include outdoor positioning aspects, such as a location module adapted to acquire, for example, latitude, longitude, altitude, geocode, course, direction, heading, speed, universal time (UTC), date, and / or various other information / data. In one embodiment, the location module can acquire data, sometimes known as ephemeris data, by identifying the number of satellites in view and the relative positions of those satellites (e.g.. using global positioning systems (GPS)). The satellites may be a variety of different satellites, including Low Earth Orbit (LEO) satellite systems, Department of Defense (DOD) satellite systems, the European Union Galileo positioning systems, the Chinese Compass navigation systems, Indian Regional Navigational satellite systems, and / or the like. This data can be collected using a variety of coordinate systems, such as the Decimal Degrees (DD); Degrees, Minutes, Seconds (DMS); Universal Transverse Mercator (UTM); Universal Polar Stereographic (UPS) coordinate systems; and / or the like. Alternatively, the location information / data can be determined by triangulating a position of the external computing entity 600 in connection with a variety of other systems, including cellular towers, Wi-Fi access points, and / or the like. Similarly, the external computing device 600 may include indoor positioning aspects, such as a location module adapted to acquire, for example, latitude, longitude, altitude, geocode, course, direction, heading, speed, time, date, and / or various other information / data. Some of the indoor systems may use various position or location technologies including RFID tags, indoor beacons or transmitters, Wi-Fi access points, cellular towers, nearby computing devices (e.g., smartphones, laptops) and / or the like. For instance, such technologies may include theAttorney Docket No.: 049648 / 643192 iBeacons, Gimbal proximity beacons, Bluetooth Low Energy (BLE) transmitters, NFC transmitters, and / or the like. These indoor positioning aspects can be used in a variety of settings to determine the location of someone or something to within inches or centimeters.

[0133] The external computing device 600 may also comprise a user interface (that can include a display 607 coupled to the processing element 602) and / or a user input interface (coupled to the processing element 602). For example, the user interface may be a user application, browser, user interface, and / or similar words used herein interchangeably executing on and / or accessible via the external computing device 600 to interact with and / or cause display of information / data from the computing device 600, as described herein. The user input interface can comprise any of a number of devices or interfaces allowing the external computing device 600 to receive data, such as a keypad 611 (hard or soft), a touch display, voice / speech or motion interfaces, or other input device. In embodiments including a keypad 611, the keypad 611 can include (or cause display of) the conventional numeric (0-9) and related keys (#, *), and other keys used for operating the external computing device 600 and may include a full set of alphabetic keys or set of keys that may be activated to provide a full set of alphanumeric keys. In addition to providing input, the user input interface can be used, for example, to activate or deactivate certain functions, such as screen savers and / or sleep modes.

[0134] The external computing device 600 can also include volatile storage or memory 604 and / or non-volatile storage or memory 603, which can be embedded and / or may be removable. For example, the non-volatile memory may be ROM, PROM, EPROM, EEPROM, flash memory’, MMCs, SD memory cards, Memory Sticks, CBRAM, PRAM, FeRAM, NVRAM, MRAM, RRAM, SONOS, FJG RAM, Millipede memory. racetrack memory, and / or the like. The volatile memory’ may be RAM, DRAM, SRAM, FPM DRAM, EDO DRAM, SDRAM, DDR SDRAM, DDR2 SDRAM, DDR3 SDRAM, RDRAM, TTRAM, T-RAM, Z-RAM, RIMM, DIMM, SIMM, VRAM, cache memory’, register memory’, and / or the like. The volatile and non-volatile storage or memory’ can store databases, database instances, database management systems, data, applications, programs, program modules, scripts, source code, object code, byte code, compiled code, interpreted code, machine code, executable instructions, and / or the like to implement the functions of the external computing device 600. As indicated, this may include a user application that is resident on the entity or accessible through a browser or other user interface for communicating with the computing entity 500 and / or various other computing entities.Attorney Docket No.: 049648 / 643192

[0135] In another embodiment, the external computing device 600 may include one or more components or functionality that are the same or similar to those of the computing device 500, as described in greater detail above. As will be recognized, these architectures and descriptions are provided for exemplary purposes only and are not limiting to the various embodiments.

[0136] In some embodiments, the computing device 500 can comprise the external computing device 600, the computing device 500 suitable to carry out movement of the various components of the external computing device 600, flow rates or deposition / dispersal volumes, or the like. In some embodiments, the computing device 500 or a component thereof can be configured to be in communication with the external computing device 600, which can be configured to provide instructions for printing, a design file for a printed article, printing pathway directions, printhead movement instructions, support surface / build surface movement instructions, or the like to the computing device 500, which is configured to carry out printing.

[0137] Referring now to FIG. 23, a method 700 for preparing bioinks is illustrated. The method 700 can comprise: preparing a bioink comprising polyethylene glycol diacrylate (PEGDA). gelatin methacrylate (GelMA), hyaluronic acid methacrylate (HAMA), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), tartrazine, arginine-glycerine-aspartic acid (RGD), isoleucine-lysine-valine-alanine-valine (IKVAV), and Dulbecco phosphate buffer saline (dPBS) pH buffered with calcium and magnesium, at 701.

[0138] Some or all of the method 700 can be carried out according to a process (e.g., 100), by an apparatus, or by a computing device, such as 200, 500, or 600. For example, an apparatus can comprise at least one processor and at least one memory storing instructions thereon that, when executed by the at least one processor, cause the apparatus to perform some or all of the method 700. Additionally, a computer program product can be provided that comprises a non-transitory computer-readable storage medium storing instructions thereon that, when executed by a processor, cause a machine or apparatus to perform some or all of the method 700.

[0139] Referring now to FIG. 24, a method 800 for preparing bioinks is illustrated. The method 800 can comprise: preparing a bioink comprising polyethylene glycol diacrylate (PEGDA). gelatin methacrylate (GelMA), hyaluronic acid methacrylate (HAMA), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), tartrazine, arginine-glycerine-aspartic acid (RGD), isoleucine-lysine-valine-alanine-valine (IKVAV), and Dulbecco phosphate buffer saline (dPBS) pH buffered with calcium and magnesium, at 801. In some embodiments, the method 800 may (e.g., may further) comprise: communicating one or more layers of the bioink into a digital light processing (DLP) printer, at 802. In some embodiments,Attorney Docket No.: 049648 / 643192 the method 800 may (e.g., may further) comprise: at least partially polymerizing, crosslinking, gelling, or curing, using the DLP printer, the one or more layers of the bioink to form a three-dimensional (3D) construct, at 803.

[0140] Some or all of the method 800 can be carried out according to a process (e.g., 100), by an apparatus, or by a computing device, such as 200, 500, or 600. For example, an apparatus can comprise at least one processor and at least one memory storing instructions thereon that, when executed by the at least one processor, cause the apparatus to perform some or all of the method 800. Additionally, a computer program product can be provided that comprises a non-transitory computer-readable storage medium storing instructions thereon that, when executed by a processor, cause a machine or apparatus to perform some or all of the method 800.

[0141] Referring now to FIG. 25, a method 900 for preparing bioinks is illustrated. The method 900 can comprise: preparing a bioink comprising about 15 wt.% PEGDA, about 3 wt.% GelMA, about 1 wt.% HAMA, about 12 wt.% LAP, about 8 wt.% tartrazine, about 20 wt.% RGD, about 25 wt.% IKVAV, and about 15 wt.% dPBS, at 901. In some embodiments, the method 900 may (e.g., may further) comprise: disposing, into a printing space within a DLP printer, according to a print pathway associated with the 3D BMN construct design, the bioink as a plurality of layers associated with a plurality of slices of the 3D BMN construct design, at 902. In some embodiments, the method 900 may (e.g., may further) comprise: projecting respective light emissions, using the DLP printer, towards respective layers of the plurality of layers to at least partially polymerize, crosslink, gel. or cure the plurality of layers of the bioink to form a 3D BMN construct, at 903. Injectable hydrogel comprising about 3% HAMA, about 20.50% RGD, about 15.4% DTT, about 61.1% dMEM w as communicated into the chamber of the construct then was incubated for one hour and a complete media w as communicated into the top of the hydrogel, at 904.

[0142] Some or all of the method 900 can be carried out according to a process (e.g., 100), by an apparatus, or by a computing device, such as 200, 500, or 600. For example, an apparatus can comprise at least one processor and at least one memory storing instructions thereon that, when executed by the at least one processor, cause the apparatus to perform some or all of the method 900. Additionally, a computer program product can be provided that comprises a non-transitory computer-readable storage medium storing instructions thereon that, w hen executed by a processor, cause a machine or apparatus to perform some or all of the method 900.

[0143] Referring now to FIG. 26, a method 1000 for preparing bioinks is illustrated. The method 1000 can comprise: communicating a mass of GelMA into a volume of dPBS, at 1001.In some embodiments, the method 1000 may (e.g., may further) comprise: communicating aAttorney Docket No.: 049648 / 643192 mass of PEGDAinto the mixture of GelMA and dPBS, the mass of PEGDA comprising equal mass amounts of 1,000 Da PEGDA, 3,400 Da PEGDA, and 6,000 Da PEGDA, at 1002. In some embodiments, the method 1000 may (e.g., may further) comprise: communicating a mass of HAMA into the mixture of GelMA, PEGDA, and dPBS, at 1003. In some embodiments, the method 1000 may (e.g., may further) comprise: communicating a mass of LAP into the mixture of GelMA, PEGDA, HAMA, and dPBS, at 1004. In some embodiments, the method 1000 may (e.g., may further) comprise: communicating a mass of tartrazine into the mixture of GelMA, PEGDA, HAMA, LAP, and dPBS, at 1005. In some embodiments, the method 1000 may (e.g., may further) comprise: communicating a mass of RGD peptides and a mass of IKVAV into the mixture of GelMA, PEGDA, HAMA, LAP, tartrazine, and dPBS to form a bioink, at 1006.

[0144] Some or all of the method 1000 can be carried out according to a process (e.g., 100), by an apparatus, or by a computing device, such as 200, 500, or 600. For example, an apparatus can comprise at least one processor and at least one memory storing instructions thereon that, when executed by the at least one processor, cause the apparatus to perform some or all of the method 1000. Additionally, a computer program product can be provided that comprises a non-transitory computer-readable storage medium storing instructions thereon that, when executed by a processor, cause a machine or apparatus to perform some or all of the method 1000.

[0145] Referring now to FIG. 27, a method 1100 for preparing bioinks is illustrated. The method 1100 can comprise: communicating a mass of GelMA into a volume of dPBS, at 1101.In some embodiments, the method 1100 may (e.g., may further) comprise: communicating a mass of PEGDA into the mixture of GelMA and dPBS, the mass of PEGDA comprising equal mass amounts of 1,000 Da PEGDA, 3,400 Da PEGDA, and 6,000 Da PEGDA, at 1102. In some embodiments, the method 1100 may (e.g., may further) comprise: communicating a mass of HAMA into the mixture of GelMA, PEGDA, and dPBS, at 1103. In some embodiments, the method 1100 may (e.g., may further) comprise: communicating a mass of LAP into the mixture of GelMA, PEGDA, HAMA, and dPBS, at 1104. In some embodiments, the method 1100 may (e.g., may further) comprise: communicating a mass of tartrazine into the mixture of GelMA, PEGDA, HAMA, LAP, and dPBS, at 1105. In some embodiments, the method 1100 may (e.g., may further) comprise: communicating a mass of RGD peptides into the mixture of GelMA, PEGDA, HAMA, LAP, tartrazine, and dPBS, at 1106. In some embodiments, the method 1100 may (e.g., may further) comprise: communicating a mass of IKVAV into the mixture of GelMA, PEGDA, HAMA, LAP, tartrazine, and dPBS to form a bioink, at 1107. In some embodiments, the method 1100 may (e.g., may further) comprise: maintaining the bioink at a temperature of greater than or equal to about 75°C, at 1108.Attorney Docket No.: 049648 / 643192

[0146] Some or all of the method 1100 can be carried out according to a process (e.g., 100), by an apparatus, or by a computing device, such as 200, 500, or 600. For example, an apparatus can comprise at least one processor and at least one memory storing instructions thereon that, when executed by the at least one processor, cause the apparatus to perform some or all of the method 1100. Additionally, a computer program product can be provided that comprises a non-transitory computer-readable storage medium storing instructions thereon that, when executed by a processor, cause a machine or apparatus to perform some or all of the method 1100.

[0147] Referring now to FIG. 28, a method 1200 for preparing bioinks is illustrated. The method 1200 can comprise: preparing a hydrogel bioink comprising about 15 wt.% PEGDA, about 3 wt.% GelMA, about 1 wt.% HAMA, about 12 wt.% LAP, about 8 wt.% tartrazine, about 20 wt.% RGD, about 25 wt.% IK. VAV, and about 15 wt.% dPBS. at 1201. In some embodiments, the method 1200 may (e.g., may further) comprise: maintaining the bioink hydrogel at an average temperature of greater than or equal to about 75°C, at 1202. In some embodiments, the method 1200 may (e.g., may further) comprise: communicating one or more layers of the bioink into a digital light processing (DLP) printer according to a printing pathway, the panting pathway being associated with a three-dimensional model of a BMN construct, at 1203. In some embodiments, the method 1200 may (e.g., may further) comprise: emitting light, using the DLP printer, towards the one or more layers of the hydrogel bioink to at least partially polymerize, crosslink, gel, or cure the hydrogel bioink, thereby forming the BMN construct, wherein an average light intensity of the light emitted towards the hydrogel bioink is between about 10 mW / cm2and about 30 mW / cm2and an average emission duration of the light emitted towards the one or more layers of the hydrogel bioink is between about 5 sec / layer and about 20 sec / layer, at 1204. Injectable hydrogel comprising about 3% HAMA, about 20.50% RGD, about 15.4% DTT, about 61.1% dMEM was communicated into the chamber of the construct then was incubated for one hour and a complete media was communicated into the top of the hydrogel, at 1205.

[0148] Some or all of the method 1200 can be carried out according to a process (e.g., 100), by an apparatus, or by a computing device, such as 200, 500, or 600. For example, an apparatus can comprise at least one processor and at least one memory storing instructions thereon that, when executed by the at least one processor, cause the apparatus to perform some or all of the method 1200. Additionally, a computer program product can be provided that comprises a non-transitory computer-readable storage medium storing instructions thereon that, when executed by a processor, cause a machine or apparatus to perform some or all of the method 1200.Attorney Docket No.: 049648 / 643192

[0149] Described herein are systems, apparatuses, methods, and computer program products for developing a humanized, or physiologically relevant MPS of a BMN. The development of a humanized MPS of the BMN as described herein provides for, among other things, a BMN construct / model that exhibits improved physiological relevance with regard to the human BMN, improved stem cell growth and regulation therein, and improved perfusability. The BMN construct / model described herein further provides for improved physiological, medical, and pharmaceutical research, making the described BMN construct / model a groundbreaking translational tool. Current BMN models lack the structure, heterogeneity and cellular composition to be sufficiently physiologically relevant with regard to the human BMN. Moreover, animal models for the study of the bone marrow are limited given the significant differences between BMNs and differences in stem cell regulation between humans and other species.

[0150] Described herein are systems, apparatuses, methods, and computer program products for developing a humanized, or physiologically relevant MPS of a BMN. The tissue engineered MPS device bioprinted construct described herein incorporates key aspects of the human bone marrow, such as relevant vascular geometries and composition (human continuous and sinusoidal forming endothelial cells), human hematopoietic cells, bioprinted 3D gel matrices that support cell polarization and differentiation, and perfusion of vascular spaces. Using tissue engineering techniques and material science, a 3D bioprinted device was developed that contains various (e.g., three) different compartments (e.g., a porous gel matrix, a sac-like reservoir, and interconnected vascular compartment) that mimics the characteristics of the human BMN. According to some embodiments, this device, called MarrowPrint, features a hybrid triple crosslink network of synthetic and natural polymers. In some embodiments, the sac-like reservoir contains an opening to a chamber of stem cells. These reservoirs can be filled with a softer bioactive gel that is activated chemically for cellular adhesion. According to some embodiments, the continuous and sinusoidal endothelium is bridged together. This device can be manufactured using, e.g., a 3D printer that forms a homogeneous matrix or an advanced (e.g., heterogeneous) digital light processing (DLP) 3D printer. In some embodiments, the use of DLP may improve construct integrity due, e.g., to tuning of gradient stiffness that better supports the vasculature geometries and the stem cell sac-like chamber so as to achieve bioprinting of a physiologically relevant human BMN model. This biomimetic device can be used as a platform for numerous applications including its use for deciphering determinants of marrow pathology (immunosuppression), development of therapeutics for cancer, infectious diseases, and bone trauma.Attorney Docket No.: 049648 / 643192

[0151] According to an embodiment, a method can be carried out for forming a bioink material, the method comprising: preparing a bioink comprising polyethylene glycol diacrylate (PEGDA), gelatin methacrylate (GelMA), hyaluronic acid methacrylate (HAMA), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), tartrazine, arginine-glycerine-aspartic acid (RGD), isoleucine-lysine-valine-alanine-vahne (IKVAV), and Dulbecco phosphate buffer saline (dPBS) pH buffered with calcium and magnesium.

[0152] According to another embodiment, a method can be carried out for 3D printing a BMN construct, the method comprising: preparing a bioink comprising PEGDA, GelMA, HAMA, LAP, tartrazine, RGD, IKVAV, and dPBS pH buffered with calcium and magnesium. The method may (e.g., may further) comprise: communicating one or more layers of the bioink into a digital light processing (DLP) printer. The method may (e.g., may further) comprise: at least partially polymerizing, crosslinking, gelling, or curing, using the DLP printer, the one or more layers of the bioink to form a 3D BMN construct.

[0153] In some embodiments, the bioink comprises between about 5 wt.% and about 20 wt.% PEGDA. In some embodiments, the bioink comprises between about 1 wt.% and about 5 wt.% GelMA. In some embodiments, the bioink comprises between about 0.1 wt.% and about 2 wt.% HAMA. In some embodiments, the bioink comprises between about 5 wt.% and about 15 wt.% LAP. In some embodiments, the bioink comprises between about 5 wt.% and about 15 wt.% tartrazine. In some embodiments, the bioink comprises between about 5 wt.% and about 15 wt.% RGD. In some embodiments, the bioink comprises between about 10 wt.% and about 30 wt.% IKVAV. In some embodiments, the bioink comprises between about 10 wt.% and about 20 wt.% dPBS. In some embodiments, the bioink comprises about 15 wt.% PEGDA, about 3 wt.% GelMA, about 1 wt.% HAMA, about 12 wt.% LAP, about 8 wt.% tartrazine, about 20 wt.% RGD. about 25 wt.% IKVAV, and about 15 wt.% dPBS.

[0154] In some embodiments, the bioink is bioactivated to support stem cell growth in the 3D BMN construct. In some embodiments, the at least partially polymerizing, crosslinking, gelling, or curing, using the DLP printer, the one or more layers of the bioink comprises projecting respective light emissions from one or more light emission points of the DLP printer towards respective layers of the one or more layers of the bioink. In some embodiments, a light emission duration of the respective light emissions projected tow ards respective layers of the plurality of layers of the bioactivated bioink is between about 5 sec / layer and about 20 sec / layer. In some embodiments, the projecting the respective light emissions towards respective layers of the plurality of layers of the bioactivated bioink is performed at an average temperature of between about 60°C and about 90°C. In some embodiments, an average thickness of respectiveAttorney Docket No.: 049648 / 643192 layers of the one or more layers of the bioink is between about 25 pm and about 100 pm. In some embodiments, an average light intensity of the respective light emissions is between about 10 mW / cm2and about 30 mW / cm2. In some embodiments, the bioink is prepared by vortexing a bioink solution comprising the PEGDA, the GelMA, the HAMA, the LAP, the tartrazine, the RGD, the IKVAV, and the dPBS at a temperature higher than about 75°C.

[0155] According to an embodiment, a method can be carried out for 3D printing a BMN construct, the method comprising: providing a bioactivated bioink comprising PEGDA, GelMA, HAMA, LAP, tartrazine, RGD, IKVAV, and dPBS. The method may (e.g., may further) comprise: disposing one or more volumes of the bioactivated bioink into a printing space according to a print pathway associated with a 3D BMN construct design. The method may (e.g., may further) comprise: exposing the one or more volumes of the bioactivated bioink to emitted light to at least partially polymerize, crosslink, gel, or cure the one or more volumes of the bioactivated bioink to form a 3D BMN construct.

[0156] In some embodiments, the printing space is within a DLP printer configured to project the emitted light towards the one or more volumes of the bioactivated bioink within the printing space. In some embodiments, the bioactivated bioink comprises about 15 wt.% PEGDA, about 3 wt.% GelMA, about 1 wt.% HAMA, about 12 wt.% LAP, about 8 wt.% tartrazine, about 20 wt.% RGD, about 25 wt.% IKVAV, and about 15 wt.% dPBS. In some embodiments, the one or more volumes of the bioactivated bioink are disposed within the printing space as one or more layers associated with one or more slices of the 3D BMN construct design.

[0157] In some embodiments, the exposing the one or more volumes of the bioactivated bioink to the emitted light comprises projecting respective light emissions, using the DLP printer, towards respective layers of the one or more layers of the bioactivated ink. In some embodiments, a light emission duration of the respective light emissions projected towards respective layers of the one or more layers of the bioactivated bioink is between about 5 sec / layer and about 20 sec / layer. In some embodiments, the exposing the one or more layers of the bioactivated bioink to the emitted light is performed at an average temperature of between about 60°C and about 90°C. In some embodiments, an average thickness of respective layers of the one or more layers of the bioactivated bioink is between about 25 pm and about 100 pm. In some embodiments, an average light intensity of the emitted light is between about 10 mW / cm2and about 30 mW / cm2In some embodiments, the bioactivated bioink is prepared by vortexing a bioink solution comprising the PEGDA. the GelMA, the HAMA, the LAP, the tartrazine, the RGD, the IKVAV, and the dPBS at a temperature higher than about 75°C.Attorney Docket No.: 049648 / 643192

[0158] According to another embodiment, a method can be carried out that comprises: preparing a bioactivated bioink comprising about 15 wt.% PEGDA, about 3 wt.% GelMA, about 1 wt.% HAMA, about 12 wt.% LAP, about 8 wt.% tartrazine, about 20 wt.% RGD, about 25 wt.% IKVAV, and about 15 wt.% dPBS. The method may (e.g., may further) comprise: disposing, into a printing space within a DLP printer, according to a print pathway associated with the 3D BMN construct design, the bioactivated bioink as a plurality of layers associated with a plurality of slices of the 3D BMN construct design. The method may (e.g., may further) comprise: projecting respective light emissions, using the DLP printer, towards respective layers of the plurality of layers to at least partially polymerize, crosslink, gel, or cure the plurality of layers of the bioactivated bioink to form a 3D BMN construct.

[0159] In some embodiments, a light emission duration of the respective light emissions projected towards respective layers of the plurality of layers of the bioactivated bioink is between about 5 sec / layer and about 20 sec / layer, greater than about 20 sec / layer, less than about 5 sec / layer, and / or the like, inclusive of all values and ranges therebetween, such as values of about 30 sec / layer, about 40 sec / layer, about 50 sec / layer, etc.

[0160] In some embodiments, the projecting the respective light emissions towards respective layers of the plurality of layers of the bioactivated bioink is performed at an average temperature of between about 60°C and about 90°C. In some embodiments, an average thickness of respective layers of the plurality of layers of the bioactivated bioink is between about 25 pm and about 100 pm. In some embodiments, an average light intensity of the respective light emissions is between about 10 mW / cm2and about 30 mW / cm2In some embodiments, the bioactivated bioink is prepared by vortexing a bioink solution comprising the PEGDA, the GelMA, the HAMA, the LAP, the tartrazine, the RGD, the IKVAV, and the dPBS at a temperature higher than about 75°C.

[0161] The above-noted aspects and features may be implemented in systems, apparatuses, methods, articles and non-transitory computer-readable media depending on the desired configuration. The subject disclosure may be implemented in and used with a number of different types of devices, such as one or more computing devices, a DLP printer, a computer-controlled DLP printer, and / or the like. An example device can comprise at least one processor and at least one memory that stores thereon instructions which, when executed by the at least one processor, cause the device to perform some or all of the elements of the above-described method, according to various embodiments. In other examples, a computer program product, such as a non-transitory computer-readable storage medium can be provided that comprises instructions stored thereon that, when executed by at least one processor of an apparatus, causeAttorney Docket No.: 049648 / 643192 the apparatus to perform some or all elements of a method such as that descried above, according to some embodiments. In other examples, an apparatus can be provided that comprises means for carrying out a method - such means can include, e.g., a processor and a memory storing computer-executable instructions or computer codes thereon that, when executed by the processor, cause the apparatus to perform some or all of a method such as one of the methods described herein.

[0162] In some embodiments, one or more of the operations, steps, elements, or processes described herein may be modified or further amplified as described below. Moreover, in some embodiments, additional optional operations may also be included. It should be appreciated that each of the modifications, optional additions, and / or amplifications described herein may be included with the operations previously described herein, either alone or in combination, with any others from among the features described herein.

[0163] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of teachings presented in the foregoing descriptions and the associated drawings. Although the figures only show certain components of the apparatus and systems described herein, it is understood that various other components may be used in conjunction with the system. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Moreover, the steps in the method described above may not necessarily occur in the order depicted in the accompanying diagrams, and in some cases one or more of the steps depicted may occur substantially simultaneously, or additional steps may be involved. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Specific equipment and materials described in the examples are for illustration only and not for purposes of limitation. For instance, any and all articles, portions of articles, structures, bulk materials, and / or the like, having any form factor, scale, dimensions, aesthetic attributes, material properties, internal structures, and / or mechanical properties, which are formed according to any of the disclosed methods, approaches, processes, or variations thereof, using any devices, equipment, apparatuses, systems, or variations thereof, using any of the build material, printing mixture, ink, yield-stress support material, or other material compositions described herein or variations thereof, are all contemplated and covered by the present disclosure. None of the examples provided are intended to, nor should they, limit in any way the scope of the present disclosure.

Claims

Attorney Docket No.: 049648 / 643192ClaimsWhat is claimed is:

1. A method comprising:preparing a bioink comprising polyethylene glycol diacrylate (PEGDA), gelatin methacrylate (GelMA), hyaluronic acid methacrylate (HAMA), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), tartrazine, arginine-glycerine-aspartic acid (RGD), isoleucine-lysine-valine-alanine-valine (IKVAV), and Dulbecco’s Phosphate-Buffered Saline (dPBS) pH buffered with calcium and magnesium;communicating one or more layers of the bioink into a digital light processing (DLP) printer; andat least partially polymerizing, using the DLP printer, the one or more layers of the bioink to form a three-dimensional (3D) construct.

2. The method of claim 1, wherein the bioink comprises between about 5 wt.% and about 20 wt.% PEGDA.

3. The method of any prior claim, wherein the bioink comprises between about 1 wt.% and about 5 wt.% GelMA.

4. The method of any prior claim, wherein the bioink comprises between about 0.1 wt.% and about 2 wt.% HAMA.

5. The method of any prior claim, wherein the bioink comprises between about 5 wt.% and about 15 wt.% LAP.

6. The method of any prior claim, wherein the bioink comprises between about 5 wt.% and about 15 wt.% tartrazine.

7. The method of any prior claim, wherein the bioink comprises between about 5 wt.% and about 15 wt.% RGD.

8. The method of any prior claim, wherein the bioink comprises between about 10 wt.% and about 30 wt.% IKVAV.Attorney Docket No.: 049648 / 6431929. The method of any prior claim, wherein the bioink comprises between about 10 wt.% and about 20 wt.% dPBS.

10. The method of any prior claim, wherein the bioink comprises about 15 wt.% PEGDA, about 3 wt.% GelMA, about 1 wt.% HAMA, about 12 wt.% LAP, about 8 wt.% tartrazine, about 20 wt.% RGD, about 25 wt.% IKVAV, and about 15 wt.% dPBS.

11. The method of any prior claim, wherein the bioink is bioactivated to support stem cell growth in the 3D construct.

12. The method of any prior claim, wherein the at least partially polymerizing, using the DLP printer, the one or more layers of the bioink comprises projecting respective light emissions from one or more light emission points of the DLP printer towards respective layers of the one or more layers of the bioink.

13. The method of claim 12, wherein a light emission duration of the respective light emissions projected towards respective layers of the plurality of layers of the bioactivated bioink is between about 5 sec / layer and about 20 sec / layer.

14. The method of any one of claims 12-13, wherein the projecting the respective light emissions towards respective layers of the plurality of layers of the bioactivated bioink is performed at an average temperature of between about 60°C and about 90°C.

15. The method of any one of claims 1-14, wherein an average thickness of respective layers of the one or more layers of the bioink is between about 25 pm and about 100 pm.

16. The method of any one of claims 12-15, wherein an average light intensity of the respective light emissions is between about 10 mW / cm2and about 30 mW / cm217. The method of any prior claim, wherein the bioink is prepared by vortexing a bioink solution comprising the PEGDA, the GelMA, the HAMA, the LAP, the tartrazine, the RGD, the IKVAV. and the dPBS at a temperature higher than about 75°C.Attorney Docket No.: 049648 / 643192 18. The method of any prior claim, wherein the 3D construct is a 3D bone marrow niche (BMN) construct.

19. The method of claim 18, wherein the 3D BMN construct comprise at least perfusable vasculature and one or more stem cell growth regions.

20. The method of any prior claim, wherein the bioink comprises a hydrogel.

21. An apparatus comprising at least one processor and at least one memory storing instructions thereon that, when executed by the at least one processor, cause the apparatus to perform a method according to any prior claim.

22. An apparatus comprising means for performing a method according to any one of claims 1-20.

23. Anon-transitory computer-readable storage medium comprising instructions stored thereon that, when executed by at least one processor of an apparatus, cause the apparatus to perform a method according to any one of claims 1-20.

24. A method comprising:preparing a bioink comprising about 15 wt.% PEGDA, about 3 wt.% GelMA, about 1 wt.% HAMA, about 12 wt.% LAP, about 8 wt.% tartrazine, about 20 wt.% RGD, about 25 wt.% IKVAV, and about 15 wt.% dPBS;disposing, into a printing space within a DLP printer, according to a print pathway associated with a 3D construct design, the bioink as a plurality of layers associated with a plurality of slices of the 3D construct design; andprojecting respective light emissions, using the DLP printer, towards respective layers of the plurality of layers to at least partially polymerize the plurality of layers of the bioink to form a 3D construct.

25. The method of claim 24, wherein a light emission duration of the respective light emissions projected towards respective layers of the plurality of layers of the bioink is between about 5 sec / layer and about 20 sec / layer.Attorney Docket No.: 049648 / 643192 26. The method of any one of claims 24-25, wherein the projecting the respective light emissions towards respective layers of the plurality of layers of the bioink is performed at an average temperature of between about 60°C and about 90°C.

27. The method of any one of claims 24-26, wherein an average thickness of respective layers of the plurality' of layers of the bioactivated bioink is between about 25 pm and about 100 pm.

28. The method of any one of claims 24-27, wherein an average light intensity' of the respective light emissions is between about 10 mW / cm2and about 30 mW / cm229. The method of any one of claims 24-28, wherein the bioactivated bioink is prepared by vortexing a bioink solution comprising the PEGDA, the GelMA, the HAMA, the LAP, the tartrazine, the RGD, the IKVAV, and the dPBS at a temperature higher than about 75°C.

30. The method of any one of claims 24-29, wherein the 3D construct is a 3D BMN construct.

31. The method of claim 30, wherein the 3D BMN construct comprise at least perfusable vasculature and one or more stem cell growth regions.

32. An apparatus comprising at least one processor and at least one memory' storing instructions thereon that, when executed by the at least one processor, cause the apparatus to perform a method according to any one of claims 24-31.

33. An apparatus comprising means for performing a method according to any one of claims 24-31.

34. Anon-transitory computer-readable storage medium comprising instructions stored thereon that, when executed by at least one processor of an apparatus, cause the apparatus to perform a method according to any one of claims 24-31.

35. A method comprising:heating a mass of GelMA to a temperature of between about 40°C and about 60°C;Attorney Docket No.: 049648 / 643192 communicating the mass of GelMA into a volume of dPBS;communicating a mass of PEGDA into the mixture of GelMA and dPBS, the mass of PEGDA comprising equal mass amounts of 1,000 Da PEGDA, 3,400 Da PEGDA, and 6,000 Da PEGDA;vortexing the mixture of GelMA, PEGDA, and dPBS at a second temperature of about 60°C and a vortex rate of about 600 rotations per minute (RPM) to fully or substantially fully dissolve the PEGDA and / or GelMA within the dPBS;communicating a mass of HAMA into the mixture of GelMA, PEGDA, and dPBS; sonicating the mixture of GelMA, PEGDA, HAMA, and dPBS for about 5 minutes; vortexing the mixture of GelMA, PEGDA, HAMA, and dPBS at the second temperature and the vortex rate of about 600 RPM to fully or substantially fully dissolve the HAMA in the dPBS;communicating a mass of LAP into the mixture of GelMA, PEGDA, HAMA, and dPBS;vortexing the mixture of GelMA, PEGDA, HAMA, LAP, and dPBS at the second temperature and the vortex rate of about 600 RPM to fully or substantially fully dissolve the LAP in the dPBS;communicating a mass of tartrazine into the mixture of GelMA, PEGDA, HAMA, LAP, and dPBS;maintaining the mixture of GelMA, PEGDA, HAMA. LAP. tartrazine, and dPBS at the second temperature; andcommunicating a mass of RGD peptides and a mass of IKVAV into the mixture of GelMA, PEGDA, HAMA, LAP, tartrazine, and dPBS to form the bioink,wherein a composition of the bioink comprises about 5 mg / 100 pL of the 1,000 Da PEGDA, about 5 mg / 100 pL of the 3,400 Da PEGDA, about 5 mg / 100 pL of the 6,000 Da PEGDA, about 3 mg / 100 pL of the GelMA, about 1 mg / 100 pL of the HAMA, about 4 mM of the LAP, about 1.5 mM of the tartrazine, about 2 mM of the RGD peptides, and about 5 mM of the IKVAV.

36. The method of claim 35, further comprising:disposing one or more volumes of the bioink into a printing space according to a print pathway associated with a 3D construct design; andexposing the one or more volumes of the bioink to emitted light to at least partially polymerize the one or more volumes of the bioink to form a 3D construct.Attorney Docket No.: 049648 / 64319237. The method of claim 36, wherein the printing space is within a DLP printer configured to project the emitted light towards the one or more volumes of the bioink within the printing space.

38. The method of any one of claims 36-37, wherein the bioink comprises 15 wt.% PEGDA. 3 wt.% GelMA, 1 wt.% HAMA. 12 wt.% LAP, 8 wt.% tartrazine. 20 wt.% RGD.25 wt.% IKVAV, and 15 wt.% dPBS.

39. The method of any one of claims 37-38, wherein the one or more volumes of the bioink are disposed within the printing space as one or more layers associated with one or more slices of the 3D construct design.

40. The method of claim 39, wherein the exposing the one or more volumes of the bioink to the emitted light comprises projecting respective light emissions, using the DLP printer, towards respective layers of the one or more layers of the ink.

41. The method of claim 40, wherein a light emission duration of the respective light emissions projected towards respective layers of the one or more layers of the bioink is between about 5 sec / layer and about 20 sec / layer.

42. The method of any one of claims 39-41, wherein the exposing the one or more layers of the bioink to the emitted light is performed at an average temperature of between about 60°C and about 90°C.

43. The method of any one of claims 39-42, wherein an average thickness of respective layers of the one or more layers of the bioink is between about 25 pm and about 100 pm.

44. The method of any one of claims 36-43, wherein an average light intensity of the emitted light is between about 10 mW / cm2and about 30 mW / cm2.

45. The method of any one of claims 36-44, further comprising:exposing a first subset of layers of the bioink to emitted light having an average light intensity of between about 30 mW / cm2and about 120 mW / cm2: andAttorney Docket No.: 049648 / 643192 exposing a second subset of layers of the bioink to emitted light having an average light intensity of between about 10 mW / cm2and about 30 mW / cm246. The method of any one of claims 36-45, wherein the 3D construct is a 3D BMN construct.

47. The method of claim 46, wherein the 3D BMN construct comprise at least perfusable vasculature and one or more stem cell growth regions.

48. An apparatus comprising at least one processor and at least one memory' storing instructions thereon that, when executed by the at least one processor, cause the apparatus to perform a method according to any one of claims 35-47.

49. An apparatus comprising means for performing a method according to any one of claims 35-47.

50. Anon-transitory computer-readable storage medium comprising instructions stored thereon that, when executed by at least one processor of an apparatus, cause the apparatus to perform a method according to any one of claims 35-47.

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