Bioprinted meniscus graft and method of use thereof

Abstract

To provide synthetic tissue structures and methods for their fabrication and use, including artificial meniscus implants, comprising precisely patterned layers containing a variable synthetic tissue fiber structure dispensed from a bioprinter.SOLUTION: Provided herein are meniscus implant compositions, as well as method for making and using the same. The subject meniscus implants find use in repairing and / or replacing damaged or diseased meniscal tissue in a mammalian subject.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority as of the filing date of U.S. Provisional Patent Application No. 62 / 351,222, filed on 16 June 2016, the disclosure thereof being incorporated herein by reference in its entirety.

[0002] The present invention provides synthetic tissue structures, including artificial meniscus grafts comprising precisely patterned layers containing variable synthetic tissue fiber structures distributed from a bioprinting machine, and methods for their preparation and use. [Background technology]

[0003] The meniscus is one of the most commonly injured areas of the knee joint, with an average injury rate of 66 per 100,000 people in the United States. While complete or partial removal of the meniscus relieves acute pain, if not properly replaced, meniscus removal can lead to damage to the articular cartilage of the knee, resulting in osteoarthritis (OA). The meniscus typically exhibits poor healing potential, and none of the currently available meniscus replacement options meet the necessary load-bearing and biomechanical requirements of this unique tissue, even while successfully grafting into the surrounding tissue and providing a long-term solution for meniscus injury.

[0004] Tissue engineering techniques have long sought to create viable synthetic structures that can mimic and / or replace living organs and tissues using countless materials and methods. Historically, cells and other biological materials were seeded onto pre-formed three-dimensional scaffolds (preferably biodegradable or otherwise removable) that conferred the desired structure. See, for example, U.S. Patent No. 6,773,713. However, despite decades of development, this approach remains problematic with respect to effective cell seeding and growth, and its techniques do not work for more complex physiological structures involving more intricate spatial arrangements of different cell types.

[0005] More recently, 3D printing, a form of additive manufacturing (AM), has been applied to generate three-dimensional objects directly from digital files, where the objects are constructed layer by layer to achieve the desired three-dimensional structure. Early efforts to adapt 3D printing technology to the generation of cell constructs and tissues, known as 3D bioprinting, also followed the above convention, focusing on the initial printing of scaffold material independent of direct seeding or subsequent printing of cell material. See, for example, U.S. Patents 6,139,574; 7,051,654; 8,691,274; 9,005,972 and 9,301,925. Unfortunately, however, the polymers typically used to form scaffolds in the prior art are generally considered biocompatible, but not physiologically compatible. Thus, using this approach, which is advantageous for the mechanical stability of the required scaffold, comes at the expense of cell viability.

[0006] In particular, in meniscus graft technology, Bakarich et al. described a system in which a combination of alginate / acrylamide gel precursor solution and an expoxy-based UV-curable adhesive is used to form a printable matrix material. ACS Appl. Mater. Interfaces 6:15998-16006 (2014). The printable matrix material is used in a 3D bioprinting process, where a 2D layer of the matrix material is deposited individually, then UV light is passed over the layer for 1-5 minutes to solidify it, and then another layer is deposited on top. However, due to the non-physiological properties of the acrylamide gel and epoxy-based UV-curable matrix components, viable cells cannot be maintained within this matrix material during the bioprinting process, and the resulting scaffold still does not contribute to cell growth, differentiation, and transmission.

[0007] Alternative 3D bioprinting technologies also highlight the opposite, sacrificing mechanical structure and print fidelity for cell viability. These bioprinting systems generate synthetic tissues by depositing cellular material within a biocompatible matrix, which is then crosslinked or otherwise solidified after deposition to produce solid or semi-solid tissue structures. See, for example, U.S. Patents 9,227,339; 9,149,952; 8,931,880 and 9,315,043; U.S. Patent Publications 2012 / 0089238; 2013 / 0345794; 2013 / 0164339; and 2014 / 0287960. However, in all of these systems, the time delay between the deposition and crosslinking processes inevitably leads to a lack of control over the shape of the printed structure, as well as the cellular and matrix composition of the structure. Furthermore, cell viability is often still impaired by subsequent cross-linking or solidification events.

[0008] As one example of this problem, Markstedt et al. describe a system in which hydrogels such as collagen, hyaluronic acid, chitosan, and alginate are combined with non-physiological reinforcing fibrous materials such as nanofibrillated cellulose to be used as bioinks for 3D bioprinting. BioMacromolecules 16:1489-96 (2015). This bioink is deposited as a layer of 2D material, which is crosslinked by immersion in a divalent cation bath (CaCl2) for 10 minutes, allowing the first layer to solidify before depositing another layer on top. Although viable cells were successfully incorporated into their bioinks, cell viability analysis demonstrated a significant decrease in cell viability from approximately 95.3% before embedding to approximately 69.9% after embedding and crosslinking as a result of the crosslinking process. Furthermore, comparison with non-printed controls revealed that the decrease in cell viability was more likely attributable to the preparation and mixing of the bioink itself rather than the actual 3D printing process.

[0009] Therefore, existing 3D bioprinting technologies and materials have not been able to satisfactorily resolve the technical conflicts between structural integrity and print fidelity on the one hand, and between physiological compatibility and cell viability on the other. This invention addresses these and other unmet needs. All prior art references listed herein are incorporated in their entirety by reference. [Overview of the project]

[0010] The present invention successfully resolves the conflicting objectives in 3D bioprinting technology between structural integrity and cell viability, providing a synthetic tissue structure deposited in a solidified form that improves cell growth and / or viability characteristics and physiological function, and does not require crosslinking or other subsequent solidification steps. Aspects of the present invention include a synthetic tissue structure comprising one or more layers deposited by a bioprinter, each layer comprising synthetic tissue fibers (optional) comprising a solidified biocompatible matrix optionally containing cells and optionally containing one or more activators, wherein at least one of the matrix material, cell type, cell density, and / or amount of activator varies in at least one direction within the layer. Preferably, at least one of the layers is variable. To become It contains a single continuous synthetic tissue fiber distributed from a bioprinting machine.

[0011] In certain embodiments, a meniscus graft is provided comprising layers of synthetic tissue fibers(s) distributed from a bioprinter as a solidified biocompatible matrix optionally containing cells and optionally containing one or more activators, wherein at least one of the matrix material, cell type, cell density, and / or amount of activator varies in at least one direction within the layer. Preferably, at least one of the layers comprises continuous synthetic tissue fibers distributed from a bioprinter having a variable composition. More preferably, each of the layers comprises continuous synthetic tissue fibers having a variable composition. Even more preferably, the meniscus graft comprises a reinforcing periphery region and / or at least one anchor region as described herein.

[0012] In one embodiment, the present invention provides a synthetic tissue structure comprising a plurality of layers deposited by a bioprinting machine, wherein each layer comprises synthetic tissue fibers (may include a plurality) comprising a solidified biocompatible matrix which optionally contains cells and optionally contains one or more activators, and at least one of the layers comprises a matrix material whose type and / or amount varies in at least one direction. In some embodiments, each layer comprises a matrix material whose type and / or amount varies in at least one direction.

[0013] In another embodiment, the present invention provides a synthetic tissue structure comprising a plurality of layers, each layer comprising a plurality of synthetic tissue fibers comprising a plurality of mammalian cells distributed from a bioprinter into a solidified biocompatible matrix, and at least one of the layers comprising a cell type and / or cell density that varies in at least one direction. In some embodiments, each layer comprises a cell type and / or cell density that varies in at least one direction.

[0014] In another embodiment, the present invention provides a synthetic tissue structure comprising a plurality of layers deposited by a bioprinting machine, each layer comprising synthetic tissue fibers (may include a plurality) comprising a solidified biocompatible matrix which optionally includes cells, and at least one of the layers comprising an activator whose type and / or amount varies in at least one direction. In some embodiments, each layer comprises an activator whose type and / or amount varies in at least one direction.

[0015] In some embodiments, one or more synthetic tissue fibers are distributed in a desired pattern or configuration to form a first layer, and then one or more additional layers having the same or different patterns or configurations are distributed on top. In a given embodiment, multiple layers are stacked to form a three-dimensional structure that can be used as an artificial meniscus graft. Preferably, at least one of the layers contains a single continuous synthetic tissue fiber distributed from a bioprinter having a variable composition. More preferably, each of the layers contains a single continuous synthetic tissue fiber having a variable composition.

[0016] In some embodiments, the synthetic tissue structure may include a number of individual layers ranging from about 1 to about 250, for example, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200, 205, 210, 215, 220, 225, 230, 235, 240, or about 245 individual layers. Any suitable number of individual layers can be incorporated to generate a tissue structure having the desired dimensions.

[0017] In some embodiments, one or more individual fibers and / or layers are organized to generate one or more zones within the tissue structure, each zone having one or more desired properties (e.g., one or more mechanical and / or biological properties). As used herein, the term “region” refers to a portion of the tissue structure defined in the xy plane (e.g., a region or portion of an individual layer where each layer of the tissue structure defines the xy plane), while the term “zone” refers to a portion of the tissue structure defined in the z direction and comprising at least two adjacent regions from separate xy planes or layers (e.g., a “macrolayer” comprising a plurality of individual “microlayers”).

[0018] The zones according to embodiments of the present invention may have any desired three-dimensional shape and may occupy any desired portion of the synthetic tissue structure. For example, in some embodiments, the zone may extend over the entire length, width, or height of the synthetic tissue structure. In some embodiments, the zone may extend over only a portion of the length, width, or height of the synthetic tissue structure. In some embodiments, the synthetic tissue structure includes several different zones organized along the length, width, height, or a combination thereof of the synthetic tissue structure. In one preferred embodiment, the synthetic tissue structure includes three different zones organized along the height of the synthetic tissue structure, so that a path through the synthetic tissue structure from bottom to top passes through all three zones.

[0019] In some embodiments, a zone may include a number of layers ranging from about 2 to about 250, for example, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200, 205, 210, 215, 220, 225, 230, 235, 240, or about 245 individual layers. In some embodiments, individual layers within a zone are organized in a manner that imparts one or more mechanical and / or biological properties to the zone. For example, in some embodiments, individual layers within a zone include one or more reinforcing materials that impart increased mechanical strength to the zone. In some embodiments, individual layers within a zone include one or more materials that impart desired cell growth properties to the zone. In some embodiments, individual layers within a zone, or multiple individual compartments of a fibrous structure passing through a zone, may be alternating in a manner that imparts desired properties to the zone. For example, in some embodiments, individual layers or regions within a zone are alternating such that odd-numbered layers contain one or more reinforcing materials that impart desired mechanical properties to the zone, and even-numbered layers contain one or more materials that impart desired biological properties to the zone (e.g., softer materials that contribute to cell migration, growth, viability, etc.). In some embodiments, the zone comprises several adjacent individual layers (e.g., about 2, 3, 4, 5, 6, 7, 8, 9 or about 10 or more adjacent layers) containing one or more reinforcing materials that impart increased mechanical strength to the zone, and the adjacent layers are alternating with other several adjacent individual layers (e.g., about 2, 3, 4, 5, 6, 7, 8, 9 or about 10 or more adjacent layers) containing one or more materials that impart desired biological properties to the zone (e.g., softer materials that contribute to cell migration, growth, viability, etc.).

[0020] In one embodiment, the artificial meniscus graft comprises at least one base zone, at least one internal zone, and at least one surface zone, at least one of which comprises a layer comprising synthetic tissue fibers (may include a solidified biocompatible matrix) wherein the type and / or quantity of the matrix material varies between the center and the periphery of the layer. In some embodiments, one or more matrix materials around or near the periphery of the layer comprise a reinforcing matrix material.

[0021] Embodiments of the present invention also include a meniscus graft comprising one or more anchor regions. As used herein, the term “anchor region” refers to a region comprising one or more reinforcing matrix materials. A meniscus graft according to embodiments of the present invention may comprise any preferred number of anchor regions, e.g., 1 to 12, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In some embodiments, the meniscus graft does not comprise any anchor regions.

[0022] In another embodiment, an artificial meniscus graft is provided, comprising at least one base zone, at least one internal zone, and at least one surface zone, wherein at least one zone comprises a layer comprising at least one synthetic tissue fiber comprising a plurality of mammalian cells distributed from a bioprinter into a solidified biocompatible matrix, and at least one layer comprising a cell density varying in at least one direction. In some embodiments, each of the layers comprises a cell density varying in at least one direction. In some embodiments, the cell density is 0 to about 100 × 10⁻⁶ 6 This is within the range of cells / mL.

[0023] In another aspect, an artificial meniscus graft according to an embodiment of the present invention includes at least one basal zone, at least one internal zone, and at least one surface zone, and at least one layer in one of the zones includes synthetic tissue fiber(s) comprising a solidified biocompatible matrix and at least one active agent, and the at least one active agent varies in type and / or amount between the center and the periphery of the layer.

[0024] In some embodiments, the biocompatible matrix around the layer may include at least one active soluble agent that is released from the matrix over time to promote ingrowth of host vascular cells and ingrowth of chondrocytes. Such bioactive agents include, but are not limited to, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), insulin-like growth factor-1 (IGF-1), bone morphogenetic factor, hepatocyte scatter factor, urokinase plasminogen activator, transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), or any combination thereof.

[0025] In some embodiments, the biocompatible matrix around the layer may include at least one insoluble factor for promoting ingrowth of cells. Non-limiting examples of such insoluble factors include hyaluronic acid or sulfated hyaluronic acid, fibronectin, fibrin, and collagen I. Additional bioactive factors may be incorporated into the matrix disposed inside the artificial meniscus graft to promote collagen deposition by the chondrocytes contained therein. Non-limiting examples of such additional bioactive factors include insulin, connective tissue-derived growth factor (CTGF), or a combination thereof.

[0026] In some embodiments, the surrounding portion or region contains at least one active agent. In some embodiments, the entire surrounding of the layer contains at least one active agent. In some embodiments, the surrounding contains a plurality of active agents. In some embodiments, the entire surrounding of the layer contains an active agent that reduces the host's inflammatory response by inclusion of one or more steroid compounds contained in one or more microparticles, for example, to ensure sustained release over a long period of time.

[0027] In some embodiments, the artificial meniscus implant has an arcuate shape having a front end portion, a rear end portion, an intermediate portion therebetween that defines a curved path between the front end portion and the rear end portion, an inner side, and an outer side. In some embodiments, the cell density increases in a radial pattern from the inner side towards the outer side. In some embodiments, the concentration of the reinforcing matrix material increases in a radial pattern from the inner side towards the outer side. In some embodiments, the amount of the active agent increases in a radial pattern from the inner side towards the outer side.

[0028] In some embodiments, the basal zone includes one layer containing randomly oriented synthetic tissue fibers; the internal zone includes one or more layers containing circumferentially oriented synthetic tissue fibers and radially oriented synthetic tissue fibers; the surface zone includes one or more layers containing randomly oriented synthetic tissue fibers. In some embodiments, the circumferentially oriented synthetic tissue fibers have a first diameter and the radially oriented synthetic tissue fibers have a second different diameter. In some embodiments, the circumferentially oriented synthetic tissue fibers and the radially oriented synthetic tissue fibers have the same diameter. In some embodiments, the synthetic tissue fibers have a diameter in the range of about 20 μm to about 500 μm.

[0029] In some embodiments, circumferentially oriented synthetic tissue fibers(s) comprise a first solidified biocompatible matrix, and radially oriented synthetic tissue fibers(s) comprise a second, different solidified biocompatible matrix. In some embodiments, circumferentially oriented and radially oriented synthetic tissue fibers(s) comprise the same solidified biocompatible matrix.

[0030] In some embodiments, the internal zone includes a layer containing synthetic tissue fibers(or more) configured to promote the deposition of collagen fibers aligned along the longitudinal direction of the synthetic tissue fibers(or more). In some embodiments, the internal zone includes a layer containing circumferentially oriented synthetic tissue fibers(or more) configured to promote the deposition of collagen fibers aligned along the longitudinal direction of the circumferentially oriented synthetic tissue fibers(or more). In some embodiments, the internal zone includes a layer containing radially oriented synthetic tissue fibers(or more) configured to promote the deposition of collagen fibers aligned along the longitudinal direction of the radially oriented synthetic tissue fibers(or more).

[0031] The solidified biocompatible matrix may contain any of the wide variety of natural or synthetic polymers that support the viability of viable cells, including, for example, alginate, laminin, fibrin, hyaluronic acid, poly(ethylene) glycol-based gels, gelatin, chitosan, agarose, or combinations thereof. In a preferred embodiment, the solidified biocompatible matrix contains alginate or other suitable biocompatible polymers that can solidify instantaneously while being dispensed from the printhead. In a more preferred embodiment, the solidified biocompatible matrix contains a homogeneous composition of alginate across the radial cross-section of each synthetic tissue fiber.

[0032] In particularly preferred embodiments, the solidified biocompatible matrix is ​​physiologically compatible, i.e., contributes to cell growth, differentiation, and transmission. In some such embodiments, the physiologically compatible matrix comprises alginates combined with one or more of the following: collagen, fibronectin, thrombospongin, glycosaminoglycans (GAGs), deoxyribonucleic acid (DNA), adhesion glycoproteins, elastin, and combinations thereof. In certain embodiments, the collagen is selected from the group consisting of collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, or collagen XVIII. In certain embodiments, the GAG ​​is selected from the group consisting of hyaluronic acid, chondroitin-6-sulfate, dermatan sulfate, chondroitin-4-sulfate, or keratin sulfate.

[0033] As outlined above, anchor regions can be generated by incorporating harder synthetic materials, including but not limited to polycaprolactone (PCL), poly(lactic acid-coglycolic acid) (PLGA), polyurethane (PU), or any combination thereof, into specific zones (i.e., suture points) of the graft. In some embodiments, anchor regions may contain a bi-network hydrogel produced by combining at least two different hydrogel materials, examples of which include, but are not limited to, alginate, gelatin methacryloyl (GelMA), methacryloyl polyethylene glycol (PEGMA), gellan gum, agarose, polyacrylamide, or any combination thereof. High-strength fibers can also be generated from high concentrations of biological polymers, including, but are not limited to, collagen, chitosan, silk fibroin, or any combination thereof, which can be incorporated into one or more anchor regions. In some embodiments, the anchor region and / or reinforced periphery of the graft comprises one or more layers of high-strength material, alternately deposited in the z-direction, and one or more layers of softer matrix material containing, for example, hydrogel material, which contributes to the survival and inward growth of the cells as described above. In this way, the softer, cell-compatible hydrogel material provides one or more desired biological functions, and the harder material provides one or more desired mechanical functions, thereby creating a hybrid structure with appropriate mechanical and biological functions.

[0034] In some embodiments, mammalian cells are selected from the group consisting of fibroblasts, chondrocytes, fibrochondrocytes, early human meniscus-derived chondrocytes, stem cells, myeloid cells, embryonic stem cells, mesenchymal stem cells, myeloid-derived mesenchymal stem cells, induced pluripotent stem cells, differentiated stem cells, tissue-derived cells, microvascular endothelial cells, and combinations thereof. In preferred embodiments, the cell viability within the synthetic viable tissue structure is in the range of about 70% to a maximum of about 100%, for example, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, about 99.5%, or about 99.9%, compared to the cell viability before printing.

[0035] In some embodiments, the meniscus graft further includes a cell-free sheath positioned below the basal zone. In some embodiments, the meniscus graft further includes a cell-free sheath positioned above the surface zone. In some embodiments, the meniscus graft further includes a first cell-free sheath positioned below the basal zone and a second cell-free sheath positioned above the surface zone.

[0036] In some embodiments, the meniscus graft further comprises at least one activator. In some embodiments, the at least one activator is selected from the group consisting of TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-4, BMP-6, BMP-12, BMP-13, basic fibroblast growth factor, fibroblast growth factor-1, fibroblast growth factor-2, platelet-derived growth factor-AA, platelet-derived growth factor-BB, platelet-rich plasma, IGF-I, IGF-II, GDF-5, GDF-6, GDF-8, GDF-10, vascular endothelial cell-derived growth factor, pleiotrophin, endothelin, nicotinamide, glucagon-like peptide-I, glucagon-like peptide-II, parathyroid hormone, tenascin-C, tropoelastin, thrombin-derived peptide, laminin, biological peptides containing a cell-binding domain and biological peptides containing a heparin-binding domain, therapeutic agents, and any combination thereof.

[0037] In a preferred embodiment, the bioprinter distributes a solidified biocompatible matrix containing multiple mammalian cells through a single orifice. In a particularly preferred embodiment, the single orifice is contained within a printhead as described and claimed in WO2014 / 197999, the disclosure thereof is incorporated herein by reference in its entirety. [Brief explanation of the drawing]

[0038] [Figure 1] This is a schematic diagram of the synthetic tissue fiber deposition process for each layer. [Figure 2]This is a schematic diagram of the knee joint, illustrating the lateral and medial menisci. (Adapted from *The knee meniscus: structure-function, pathophysiology, current repair techniques, and prospects for regeneration*. Biomaterials. 2011 October;32(30):7411-7431.doi:10.1016 / j.biomaterials.2011.06.037, Eleftherios A. Makris, MD1, Pasha Hadidi, BS1, and Kyriacos A. Athanasiou, Ph.D., PE1). [Figure 3] This is a schematic diagram of the meniscus, illustrating both a top view and a cross-sectional view. The lateral (red-red), central (white-red), and medial (white-white) regions are shown. (Adapted from *The knee meniscus: structure-function, pathophysiology, current repair techniques, and prospects for regeneration*. Biomaterials. 2011 October;32(30):7411-7431.doi:10.1016 / j.biomaterials.2011.06.037, Eleftherios A. Makris, MD1, Pasha Hadidi, BS1, and Kyriacos A. Athanasiou, Ph.D., PE1). [Figure 4] Panel A is a schematic diagram of the meniscus, illustrating a cross-section. The circumferential and radial alignment of collagen fibers imparts biomechanical properties to the meniscus. Panel B illustrates the surface zone, layered zone, and internal (deep) zone. Collagen fibers in the surface and layered zone, near the meniscus surface, are randomly oriented. Deeper fibers within the meniscus are oriented in both the circumferential and radial directions. [Figure 5]This is a force diagram illustrating the components of the axial load force F on various parts of the meniscus. The axial load force (F) perpendicular to the meniscus surface and the horizontal force (fr) are generated by compressing the femur (Ff). F is counteracted by the tibial uplift force (Ft), while fr leads to radial meniscal extrusion, which is counteracted by tensile forces from the anterior and posterior inserting ligaments. Consequently, tensile hoop stress is generated along the circumferential direction during axial compression, which is resisted by circumferentially oriented collagen fibers. (The knee meniscus:structure-function,pathophysiology,current repair techniques,and prospects for regeneration.Biomaterials.2011 October;32(30):7411-7431.doi:10.1016 / j.biomaterials.2011.06.037,Eleftherios A.Makris,MD1,Pasha Hadidi,BS1,and Kyriacos (Adapted from A. Athanasiou, Ph.D., PE1). [Figure 6] Images are provided of two cell-free 3D meniscus-like structures with pre-programmed zone-specific scaffold content and harmonized patterning of printed synthetic tissue fiber structures. Scale bar = 1 cm. [Figure 7] A series of microscopic images illustrating the alignment of native collagen fibers in smaller diameter fibers. Orientation of polymerized collagen fibers in microfluidic channels of different diameters, including 30 μm (panel a), 100 μm (panel b), 400 μm (panel c), and no channel (panel d) (Lee et al., 2006). [Figure 8] This panel shows a series of images illustrating on-the-fly adjustment of the diameter of alginate fibers printed using a 3D bioprinting system, and a graph comparing the average fiber diameters. Panels A, B, and C illustrate three different diameters of alginate fibers produced by printing at three different pressure settings in the 3D bioprinting system. Quantification of the widths of multiple fibers demonstrates consistency in the average diameter at each pressure setting (graph, right). [Figure 9] This is a diagram of the patterning of synthetic tissue fibers in various layers of a 3D bioprinted meniscus. The extracellular matrix (ECM), e.g., the synthetic tissue fiber structure of a specific diameter loaded with collagen, is patterned using techniques that reproduce the fine patterning of collagen and the zonal structure of the meniscus. The basal and surface zones contain randomly oriented fibers printed with larger diameter fibers. The internal zones contain circumferentially and radially aligned collagen aligned within patterned fibers of smaller diameter. [Figure 10] This panel shows data from two-photon imaging of collagen fibers in artificial 3D tissue. Panel A: Formaldehyde-fixed H&E-stained portion of a 3D co-culture of early human airway epithelial cells and fibroblasts after 90 days of culture on an electrospun gelatin (ESG) scaffold. Panel B: Two-photon imaging of an unstained portion demonstrating the deposition of fibrous collagen (purple) oriented parallel to the surface of the ESG scaffold, in the same direction as the fibroblasts that deposit collagen. Panel C: Emission spectrum of unstained tissue demonstrates that non-centrosymmetric collagen fibers generate a specific second harmonic signal (SHG) (Wadsworth et al., 2014). [Figure 11] This is a diagram of meniscus tissue with zone-specific cell and ECM content. "Red-red bioink" and "white-white bioink" are used to generate tissue with zone structure. The desired cell type (e.g., MSC-derived chondrocytes or early meniscus-derived cells) is seeded into red-red and white-white zones at appropriate physiological densities. The specific ECM content of the scaffold is modified according to the tissue zone. The "white-red" zone in the central zone of the tissue contains a mixture of red-red and white-white bioink and cells. This bioprinting system facilitates control over both cells (cell type and cell density) and ECM content in any given zone of a meniscus graft. [Modes for carrying out the invention]

[0039] Aspects of the present invention include a synthetic tissue structure comprising one or more layers deposited by a bioprinter, each layer comprising synthetic tissue fibers (may include) comprising a solidified biocompatible matrix optionally containing cells and optionally containing one or more activators, wherein at least one of the matrix material, cell type, cell density, and / or amount of activator varies in at least one direction within the layer. Preferably, at least one of the layers comprises a single continuous synthetic tissue fiber dispensed from a bioprinter having a variable composition. As used herein, the term “solidified” refers to a solid or semi-solid state of a material that maintains its shape fidelity and structural integrity upon deposition. As used herein, the term “shape fidelity” means the ability of a material to maintain its three-dimensional shape. In some embodiments, the solidified material has the ability to maintain its three-dimensional shape for about 30 seconds or longer, e.g., about 1, 10, or 30 minutes or longer, e.g., about 1, 10, 24, or 48 hours or longer. As used herein, the term “structural integrity” means the ability of materials to hold together under loads including their own weight while resisting breakage or bending.

[0040] In some embodiments, the solidified composition has an elastic modulus greater than about 15, 20, or 25 kilopascals (kPa), more preferably greater than about 30, 40, 50, 60, 70, 80, or 90 kPa, and even more preferably greater than about 100, 110, 120, or 130 kPa. The preferred range of elastic moduli includes from about 15, 25, or 50 Pa to about 80, 100, 120, or 140 kPa.

[0041] An additional aspect of the present invention is an artificial meniscus graft for use in repairing and / or replacing damaged or affected meniscus tissue in a mammalian subject, comprising synthetic tissue fibers(s) distributed from a bioprinter as a solidified biocompatible matrix optionally containing cells and optionally containing one or more activators, wherein at least one of the matrix material, cell type, cell density, and / or type and / or amount of activators varies in at least one direction within the fibers.

[0042] As shown in Figure 1, a solidified biocompatible matrix, optionally containing multiple mammalian cells, is dispensed from a bioprinter that forms one or more synthetic tissue fibers on the deposition surface, ultimately creating layers. Thus, after the dispensing of the already solidified matrix from the printhead, no subsequent crosslinking or other solidification steps are required. Therefore, a second layer can be rapidly deposited on top of the first layer while maintaining the structural integrity of the first layer, and this process can be continued to deposit multiple layers one after another until a three-dimensional shape with the desired shape is obtained.

[0043] The solidified biocompatible matrix may, advantageously, comprise alginate or any other suitable biocompatible polymer that can solidify instantaneously while being dispensed from the printhead. In a preferred embodiment, the alginate-based matrix is ​​printed and simultaneously crosslinked at the time of printing by contacting it with a divalent cation crosslinking solution (e.g., CaCl2 solution) before or during dispensing from the printhead. In a particularly preferred embodiment, the alginate-based biocompatible matrix further comprises one or more physiological materials, as described in more detail herein. In a further preferred embodiment, the solidified biocompatible matrix comprises a homogeneous composition of alginate across the radial cross-section of each synthetic tissue fiber.

[0044] In some embodiments, the synthetic tissue fiber structure comprises a plurality of individual compartments (organized along the length of the synthetic tissue fiber) produced by sequentially depositing different matrix materials (e.g., natural and / or synthetic polymers), different cell types, different cell concentrations, and / or different types and / or amounts of activators within each compartment of the same continuous synthetic tissue fiber structure. For example, in some embodiments, the synthetic tissue fiber structure comprises a first compartment containing a first matrix material and a second compartment containing a second matrix material. In some embodiments, the synthetic tissue fiber structure comprises a first compartment containing a first cell type and a second compartment containing a second cell type. In some embodiments, the synthetic tissue fiber structure comprises a first compartment containing a first cell concentration and a second compartment containing a second cell concentration. In some embodiments, the synthetic tissue fiber structure comprises a first compartment containing a first activator and a second compartment containing a second activator. To achieve desired biomechanical properties and / or biological activity, any combination of matrix material, cell type, cell concentration, and / or type and / or amount of activator can be used in different compartments of the synthetic tissue fiber structure.

[0045] The synthetic tissue fiber structures according to embodiments of the present invention may include controlled patterning of different matrix materials (e.g., natural and / or synthetic polymers) and crosslinking techniques for generating a desired cross-sectional profile within a given compartment. For example, in some embodiments, the synthetic tissue fiber structure includes compartments having solid, tubular, or porous cross-sectional profiles. Non-limiting examples of cross-sectional profiles that can be generated in the synthetic tissue fiber structures according to embodiments of the present invention include those described in Jun, Yesl, et al. “Microfluidic spinning of micro- and nano-scale fibers for tissue engineering.” Lab on a Chip 14.13(2014):2145-2160, the disclosure of which is incorporated herein by reference in its entirety.

[0046] In some embodiments, the resulting synthetic tissue fibers are patterned using a software tool to form layers optionally containing multiple mammalian cells and / or multiple biocompatible matrix materials. In certain embodiments, the multiple layers are deposited sequentially to produce a multilayer meniscal graft containing multiple zones. In some embodiments, the meniscal graft includes at least one base zone, at least one internal zone, and at least one surface zone, the internal zone including at least one layer containing at least one circumferentially oriented synthetic tissue fiber and at least one radially oriented synthetic tissue fiber. Preferably, at least one of the layers contains a single continuous synthetic tissue fiber dispensed from a bioprinter having a variable composition.

[0047] One advantage of this meniscus graft is that the matrix composition, cell type, cell density, and the type and / or concentration of activators can be controlled at any point in any part of any layer of the graft, thereby facilitating the production of a meniscus graft that more closely resembles the natural structure of meniscus tissue and possesses desired biomechanical properties, including but not limited to reinforcing anchor regions around the graft, circumferential and radially oriented fibrous structures within the meniscus graft, and specific cell types and cell densities within specific regions and / or zones of the graft.

[0048] Another advantage of the present invention may be the selective addition of one or more activators (described in more detail herein) to different compartments of synthetic tissue fibers, thereby enabling precise localization of the activator within one or more layers of the meniscal graft, which includes, but is not limited to, increasing the appropriate concentration of the activator around the cell-free graft to promote intrinsic cell growth. The meniscal graft is described in more detail below.

[0049] Meniscal anatomy: The meniscus is a pair of crescent-shaped fibrocartilages consisting of both medial and lateral portions, located between the corresponding femoral condyle and the tibial plateau (Figure 2). Anterior and posterior insertive ligaments firmly connect the meniscus, and they securely fix it to the tibial plateau. The meniscus is generally wedge-shaped, with the lateral meniscus being approximately 32.4–35.7 mm long and 26.6–29.3 mm wide, while the medial meniscus is approximately 40.5–45.5 mm long and 27 mm wide. Each is a complex, glossy white tissue composed of cells, specialized extracellular matrix (ECM) material, and zone-specific innervation and angiogenesis. The meniscus is well angiogenic at birth, but over time, the blood vessels retract laterally until (in humans) approximately 10–30% of the surrounding meniscus is angiogenic by age 10. Therefore, the adult human meniscus has two distinct zones: an outer vascular / nerve zone (red-red zone) and an inner, completely avascular / nerve-free zone (white-white zone). These areas are separated by a narrow central (red-white) zone that contains features of both the outer (red-red) and inner (white-white) zones (Figure 3). Importantly, the self-healing capacity of each area is directly related to blood supply and leaves the inner white-white zone vulnerable to trauma and degenerative lesions.

[0050] Meniscus cells and biochemical compositions The meniscus is a highly hydrated tissue containing approximately 72% water, with the remaining 28% consisting mainly of extracellular matrix (ECM) and cells. Collagen makes up the majority of the ECM (75%), followed by glycosaminoglycans (GAGs, 17%), DNA (2%), adhesion glycoproteins (<1%), and elastin (<1%). These ratios vary depending on the zone, age, and condition of the tissue. The cellular components of the meniscus are zone-specific and include both fibrochondrocytes and chondrocyte-like cells.

[0051] The composition of the meniscus differs in each zone. In the outer red-red zone, the cells are morphologically more fibroblast-like, undergoing many processes. The ECM in this zone is primarily fibrous collagen type I (80%). The inner white-white zone has an ECM that closely resembles hyaline cartilage, with more collagen-II (42%), a reduced percentage of collagen-I (28%), and a higher GAG concentration. The cells in this zone are referred to as fibrochondrocytes, or chondrocyte-like cells. The surface layer of the meniscus has another distinguishable cell type with potential stem cell-like properties. Zone-specific ECM components of the meniscus are produced by cells present in the tissue; therefore, phenotypic markers of meniscal cells may include ECM protein expression or gene expression, such as COL1A1 (collagen-1), COL2A2 (collagen-2), VCAN (versican), ACAN (agrecan), CSPG4 (chondroitin-6-sulfate), Sox9, and Col10a (collagen-10a). Cell density also varies within each zone, as do cell types specific to each meniscal zone. Vascular (red-red) and avascular (white-red, white-white) zones each have a cell density of 12,820 cells / mm³. 3 and 27,199 cells / mm³ 3 The average cell density is high, and it has more fibrochondrocytes than fibroblast-like cells (Cengiz et al., 2015). The meniscus is highly heterogeneous and exhibits zone-specific changes in cellular phenotype and ECM composition.

[0052] Table 1 shows the heterogeneous distribution of cell types and biochemical scaffold content in the knee meniscus. The red-red zone is characterized by fibroblast-like cells and collagen-I dominant extracellular matrix (ECM) with trace amounts of collagen-II. The white-red and white-white zones contain fibrochondrocytes and a collagen-II-rich matrix, as well as a higher proportion of glycosaminoglycans (GAGs). [Table 1]

[0053] The patterning of collagen fibers imparts the biomechanical properties of the meniscus. The microanatomical shape of the meniscus is closely related to its biomechanical properties. The hydration properties of the meniscus (approximately 72% water) provide resistance to compressive stress because water is incompressible, but the meniscus also possesses considerable tensile strength, which is imparted through the regular arrangement of collagen fibers with a diameter of 10 μm throughout the tissue (Figure 4) (Baker et al., 2007). The surface and layered zones of the meniscus are composed of randomly oriented collagen fibers, while the deeper fibers within the meniscus are oriented circumferentially and radially. Under normal use, forces several times the body weight are generated within the knee, and the meniscus transmits 50-100% of this load through a high-density network of circumferentially aligned collagen fibers (Figure 4). This regular structure results in very high tensile properties (50-300 MPa) in the fiber direction (Baker et al., 2007). When the knee is subjected to axial load, tensile hoop stress is generated circumferentially, and this stress attempts to push the meniscus out of the knee joint (Figure 5). However, the tensile strength of the circumferentially aligned collagen fibers and the rigid bonds in the anterior and posterior insertive ligaments help prevent meniscal extrusion, significantly reducing stress and protecting the tibial cartilage. In contrast, if the anterior or posterior insertive ligaments or the surrounding circumferential collagen fibers rupture, the load transfer mechanism changes, which damages the tibial cartilage. Compressive strength has been measured in fresh-frozen human menisci, with axial and radial compressive coefficients at 12% strain being 83.4 kPa and 76.1 kPa, respectively, and tensile modulus being several orders of magnitude larger (Chia & Hull, 2008).

[0054] The goal of tissue engineering is to generate structures that replicate the function of the original tissue. In the case of the meniscus, the challenge is to generate viable tissue that can survive long-term grafting in the knee joint while also possessing the biomechanical strength necessary to withstand the considerable compressive forces encountered in daily life. The meniscus is a remarkably complex tissue with specific structures on the mm, μm, and nm scales, all of which contribute to the tissue's biomechanical function. To date, meniscal engineering has been somewhat limited by the fabrication tools available to researchers, such as molding hydrogels using castings or seeding cells onto pre-fabricated scaffolds. These approaches cannot generate the microscale structures necessary to replicate function. In contrast, the meniscal grafts described herein can achieve point-to-point control over matrix material(s), cell type, cell density, and activator composition, facilitating the creation of grafts that more closely resemble the original structural characteristics of the meniscus.

[0055] The meniscus is a heterogeneous tissue with cells and ECM components distributed in specific zones. Zone specificity is essential for conferring regenerative and biomechanical functions. This artificial meniscus graft utilizes the precise placement of different matrix materials, cell types, cell densities, and activator compositions in accurate regions and / or zones of 3D tissue, thereby enabling the reproduction of the red-red, white-red, and white-white zone structure of the meniscus (Figure 6).

[0056] It has been demonstrated that the cell density within the human meniscus changes in a zone-specific manner (approximately 13 × 10 in the red-red zone). 6 Cells / ml, 28 × 10 in white-white and white-red zones 6 Cells / ml (Cengiz et al., 2015). Cell density plays a crucial role in maintaining proper cellular phenotype, ECM organization, and corresponding tissue biomechanics. In some embodiments, the meniscal graft has a density of approximately 0 to approximately 100 × 10⁻⁶ cells / ml. 6The cell density ranges from cells / mL or higher. Thus, in some embodiments, the meniscus graft may have a cell density that varies from one location to another within the graft. For example, in a given embodiment, the meniscus graft includes layers having a cell density that varies in at least one direction. In other embodiments, the graft is acellular and designed for inward growth of endogenous cells.

[0057] Collagen provides tensile strength to most tissues, and multiple collagen fibrils with a diameter of approximately 100 nm combine to form strong coiled-coil fibers with a diameter of approximately 10 μm. The biomechanical function of the meniscus is conferred through the alignment of collagen fibers in the circumferential and radial directions (Figure 4). In some embodiments, the meniscal graft contains patterned collagen fibrils produced by adjusting the diameter of the synthetic tissue fiber structure used to generate the graft.

[0058] Previous studies have shown that microfluidic channels of different diameters can guide the polymerization of collagen fibrils, forming fibers oriented along the length of the channel, but only at channel diameters of 100 μm or less (Lee et al., 2006) (Figure 7). Early endothelial cells grown within these oriented matrices have been shown to align in the direction of the collagen fibers. In another study, Martinez et al. demonstrated that 500 μm channels in a cellulose-bead scaffold can direct the alignment of collagen and cells (Martinez et al., 2012). In some embodiments, the meniscal graft contains synthetic tissue fibrous structures having diameters ranging from approximately 20 μm to approximately 500 μm, for example, approximately 50 μm, 75 μm, 100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 225 μm, 250 μm, 275 μm, 300 μm, 325 μm, 350 μm, 375 μm, 400 μm, 425 μm, 450 μm, or approximately 475 μm (Figure 7). By adjusting the fiber diameter, the orientation of collagen fibers within the meniscal graft can be controlled. Therefore, by patterning the synthetic tissue fiber structure and the collagen fibers within it in this way, it is possible to produce a meniscus graft with a physiologically precise arrangement of circumferentially and radially aligned collagen fibers, which is essential for imparting the necessary biomechanical properties to the meniscus graft (Figure 8).

[0059] The meniscus is an inherently heterogeneous structure with zones of varying compositions and structures. This meniscal graft includes, but is not limited to, a complex biological structure with its own material composition and structure, including fiber diameter, ECM composition, cellular composition, and cell density. The ability to control these and other aspects of the synthetic tissue fiber structure used to generate this meniscal graft allows for the construction of the zone structure found in intrinsic meniscal tissue.

[0060] In a given embodiment, the meniscus graft is produced using an automated control system that modulates one or more properties of the synthetic tissue fibers to achieve material switching at any point, for example, within individual fiber structures, between separate fiber structures, within or across layers, within or across zones, and essentially across the entire structure. As a result, point-to-point control of the meniscus graft composition is achieved. Furthermore, important parameters such as fiber diameter and layer thickness can also be adjusted as desired. This level of automated control is essential for accurately reproducing the heterogeneous composition and morphology found in the original knee meniscus.

[0061] These synthetic tissue fibers support the viable growth of a wide variety of human cells. The synthetic tissue fiber structures can be finely tuned to include, for example, different ECM proteins, GAGs, and growth factors to optimize the matrix for specific cell types. Computer-controlled deposition of the synthetic tissue fiber structures allows for precise placement of cells and matrix materials at specific locations, generating physiologically relevant heterogeneous meniscal grafts.

[0062] In certain embodiments, the mechanical properties of the meniscus graft are controlled by adjusting the collagen patterning and / or by adjusting one or more properties of the matrix material (e.g., alginate, collagen) used to generate the synthetic tissue fiber structure. For example, in some embodiments, one or more anchor regions as described above are disposed around the graft to facilitate attachment and / or fixation, e.g., via suturing. The anchor region can be generated by incorporation of a stiffer synthetic material including, but not limited to, a higher strength material such as polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), polyurethane (PU), or any combination thereof. Anchor regions according to embodiments of the present invention can contain a double network hydrogel generated by combining at least two different hydrogel materials including, but not limited to, alginate, gelatin methacrylate (GelMA), methacryloyl polyethylene glycol (PEGMA), gellan gum, agarose, polyacrylamide, or any combination thereof. Also, high strength fibers can be generated from high concentration biological polymers including, but not limited to, collagen, chitosan, silk fibroin, or any combination thereof, and these can be incorporated into one or more anchor regions.

[0063] A meniscus graft according to an embodiment of the present invention can include from 0 to about 12 anchor regions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 anchor regions. An anchor region according to an embodiment of the present invention can range in size from about 5 mm 2 to about 40 mm 2 , such as about 6, 7, 8, 9, or 10 mm 2 , or about 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38 mm 2 .

[0064] Anchor regions according to embodiments of the present invention may be generated by incorporating a harder synthetic material into the suture point, such as polycaprolactone (PCL), poly(lactic acid-coglycolic acid) (PLGA), polyurethane (PU), or any combination thereof. Anchor regions according to embodiments of the present invention may optionally contain a double-network hydrogel generated by combining at least two different hydrogel materials, including but not limited to alginate, gelatin methacryloyl (GelMA), methacryloyl polyethylene glycol (PEGMA), gellan gum, agarose, polyacrylamide, or any combination thereof. High-strength fibers may also be generated from high concentrations of biological polymers, including but not limited to collagen, chitosan, silk fibroin, or any combination thereof. In some embodiments, one or more of these biological polymers may be incorporated into one or more anchor regions. In some embodiments, the entire perimeter of the layer of the artificial meniscus graft includes a reinforcing matrix material. In some embodiments, the perimeter includes a plurality of reinforcing anchor regions, each containing one or more reinforcing matrix materials.

[0065] In some embodiments, high-strength fibers may be incorporated (e.g., patterned) into one or more reinforced periphery regions of the meniscus graft to increase strength along the periphery of the graft. In some embodiments, high-strength fibers are incorporated around the entire periphery of the graft. Within the anchor region and / or reinforced periphery of the meniscus graft, layers of high-strength material may be alternating with layers of softer material optimized for cell survival and inward growth. Increased strength within the anchor region and / or reinforced periphery may be imparted by increasing the concentration of fibrous material, increasing the packing density of printed fibers, increasing the diameter of printed fibers, or any combination thereof. In some embodiments, the anchor region may be colored, for example, by incorporating a non-toxic dye into the printable anchor material to act as a visual guide during surgery, thereby informing the surgeon of the location of the reinforced region of the meniscus graft adapted to the placement of sutures.

[0066] In the human meniscus, the correct orientation and alignment of collagen fibers are crucial for conferring appropriate biomechanical properties to the tissue. As previously discussed, the orientation of native collagen fibers and subsequent cell alignment can be directed by restricting the crosslinking process to channels with a small diameter or fibers less than approximately 100 μm (Lee et al., 2006) (Onoe et al., 2006). In a given embodiment, the meniscal graft includes a layer in which one or more synthetic tissue fiber structures are configured to promote the deposition of collagen fibers aligned with the longitudinal direction of the synthetic tissue fibers. Thus, in a given embodiment, the synthetic tissue fibers are deposited in a radial and / or circumferential orientation and are configured to promote the deposition of collagen fibers aligned with the radial and / or circumferential orientation of the synthetic tissue fibers. In this way, the circumferential and / or radial orientation of collagen fibers can be achieved.

[0067] In some embodiments, the diameter of the synthetic tissue fibers is adjusted so that the collagen fibers are properly aligned, for example, the surface and periphery of the meniscus contain randomly oriented (e.g., irregular) collagen fibers, while the internal region(s) contain circumferentially and radially aligned fibers. Figure 9 shows non-limiting examples of synthetic tissue fiber orientation in each of the multiple layers within the meniscus graft.

[0068] Meniscus injury and surgical repair options Meniscus injuries are very common in the knee joint. Meniscus disorders are typically classified by different age groups. Meniscus injuries in younger human patients (<40 years) are usually caused by trauma or congenital meniscal disease, while those in older human patients (>40 years) tend to be associated with degenerative tears. Meniscus injuries can simply be classified clinically into perimeniscal lesions and avascular meniscal lesions. Numerous surgical techniques have been developed to repair meniscal tears in the vascular (red-red) zone with high overall success rates (63-91%) in younger patients with stable knees. Injuries and tears in the avascular (white-white) zone of the meniscus are often associated with poor post-repair prognosis, and consequently, several different treatment strategies have been attempted with varying outcomes. Most notably, these include the use of pseudo-meniscal synovial tissue, perforation of the peripheral meniscus with meniscal tear suturing, creation of vascular access channels, and the use of mesenchymal stem cells and / or growth factors. Since none of the above techniques are commonly employed, the primary strategy for orthopedic surgeons is to perform partial meniscectomy in cases of irreparable or degenerative meniscal injury, even if this treatment strategy does not prevent the development of knee OA. Partial meniscectomy can lead to OA by reducing the contact area between the femoral condyle and the tibial platform. Altering the load-bearing properties of articular cartilage can lead to progressive degeneration of the meniscus and articular cartilage through a vicious cycle of injury, inflammation, and further tissue degeneration.

[0069] Artificial meniscal graft: As outlined above, aspects of the present invention include an artificial meniscus graft comprising at least one base zone, at least one internal zone, and at least one surface zone, each of which comprises a layer comprising at least one synthetic tissue fiber distributed from a bioprinter as a solidified biocompatible matrix optionally containing cells and optionally containing one or more activators, as described herein. In some embodiments, one or more of the matrix material, cell types, cell density, and / or types and / or amounts of activators may vary across at least one direction of a given layer. For example, in some embodiments, a layer of the meniscus graft may have a cell density that is lower along a first side and increases (linearly or nonlinearly) across the layer toward the opposite side. In a given embodiment, the cell density in a given layer may vary in two directions. For example, in some embodiments, the cell density in a given layer may increase (linearly or nonlinearly) in both the x and y directions across the layer. In a given embodiment, the cell density may be 0 to 100 × 10¹⁶ per mL 6 Changes can occur at the individual cell level or even at a higher level.

[0070] In some embodiments, at least one layer of the artificial meniscus graft may contain at least one circumferentially and / or radially oriented synthetic tissue fiber. The circumferentially and / or radially oriented fibers may have the same or different diameters, the same or different matrix materials, the same or different cell types, and the same or different cell densities. In certain embodiments, the diameter of the synthetic tissue fiber may vary from 20 μm to 500 μm.

[0071] In a given embodiment, the synthetic tissue fibers are configured to promote the deposition of collagen fibers aligned along the longitudinal direction of the synthetic tissue fibers. In a given embodiment, the synthetic tissue fibers are configured to promote the deposition of randomly oriented collagen fibers. As provided in Figure 10, the collagen fibers in the 3D fabricated tissue take on an orientation that depends on one or more features of the scaffold material used to generate the 3D tissue. Similarly, embodiments of the present meniscus graft can be modified to control the orientation of collagen fibers within the graft material.

[0072] In a given embodiment, as provided in Figure 11, the meniscus graft is constructed using sequential layering, as described above, so that the meniscus graft includes internal, central, and lateral zones. In a given embodiment, the cell types, cell density, and / or matrix material present in any given zone can be controlled, thereby producing a meniscus graft that resembles the intrinsic structure and biomechanical properties of natural meniscus tissue.

[0073] Biocompatible matrix materials: The solidified biocompatible matrix may contain any of the wide variety of natural or synthetic polymers that support the viability of viable cells, including, for example, alginate, laminin, fibrin, hyaluronic acid, poly(ethylene) glycol-based gels, gelatin, chitosan, agarose, or combinations thereof. In a preferred embodiment, the solidified biocompatible matrix contains alginate or other suitable biocompatible polymers that can solidify instantaneously while being dispensed from the printhead. In a more preferred embodiment, the solidified biocompatible matrix contains a homogeneous composition of alginate across the radial cross-section of each synthetic tissue fiber.

[0074] In a particularly preferred embodiment, the solidified biocompatible matrix is ​​physiologically compatible, i.e., contributes to cell growth, differentiation, and transmission. "Physiological matrix material" means the biological material found in intrinsic mammalian tissues. Non-limiting examples of such physiological matrix materials include fibronectin, thrombospongin, glycosaminoglycans (GAGs) (e.g., hyaluronic acid, chondroitin-6-sulfate, dermatan sulfate, chondroitin-4-sulfate, or keratin sulfate), deoxyribonucleic acid (DNA), adhesion glycoproteins, and collagen (e.g., collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, or collagen XVIII).

[0075] Mammalian cell types: Non-limiting examples of mammalian cell types that may be used in this meniscus graft include fibroblasts, chondrocytes, meniscal fibrochondrocytes, stem cells, bone marrow stromal (stem) cells, embryonic stem cells, mesenchymal stem cells, induced pluripotent stem cells, differentiated stem cells, tissue-derived cells, smooth muscle cells, skeletal muscle cells, epithelial cells, endothelial cells, myoblasts, chondrocytes, osteoblasts, osteoclasts, and any combination thereof.

[0076] Cells can be obtained from a donor (allogeneic) or recipient (autologous). Cells may also be from an established cell culture system or may be cells that have undergone genetic technology and / or manipulation to achieve a desired genotype of phenotype. In some embodiments, tissue pieces that can provide a number of different cell types within the same structure may also be used. In one preferred embodiment, the meniscus graft contains patient-specific bone marrow-derived mesenchymal stem cells. In one preferred embodiment, the meniscus graft contains early meniscal chondrocytes. In one preferred embodiment, the meniscus graft contains microvascular endothelial cells. In one preferred embodiment, the meniscus graft contains patient-specific induced pluripotent stem cell-derived chondrocytes.

[0077] In some embodiments, cells can be obtained from a suitable human or animal donor, or from the subject to which the cells are transplanted. Mammalian species include, but are not limited to, humans, monkeys, dogs, cattle, horses, pigs, sheep, goats, cats, mice, rabbits, and rats. In one embodiment, the cells are human cells. In other embodiments, the cells may originate from animals such as dogs, cats, horses, monkeys, or any other mammal.

[0078] Without being bound by any particular theory, the number of cells seeded does not limit the final tissue produced (e.g., meniscus), but the optimal cell density can improve one or more properties of the meniscus graft.

[0079] Cells can be present anywhere within the meniscus graft, for example, within the basal zone, the internal zone, and / or the surface zone. In some embodiments, different types of cells may be spatially arranged in predetermined zones of the meniscus graft to mimic the original meniscal fibrocartilage structure. For example, in some embodiments, one or more fibroblasts may be arranged in a first region and / or individual layers of the meniscus graft. In some embodiments, one or more chondrocytes may be arranged in a first region and / or individual layers of the meniscus graft.

[0080] Certain types of cells having a specific density can be placed in any desired zone of the meniscus graft. In some embodiments, one or more stem cells, for example, bone marrow stem cells, can be placed in at least a portion of the meniscus graft and / or its individual layers. In some embodiments, at least a portion of the stem cells can be differentiated into a chondroplastic phenotype. Those skilled in the art can easily differentiate stem cells into a desired phenotype (e.g., chondroplastic phenotype) by, for example, exposing the cells to art-recognized cell differentiation factors and / or commercially available differentiation media.

[0081] Appropriate growth conditions for mammalian cells are well known in the art (Freshney, RI (2000) Culture of Animal Cells, a Manual of Basic Technique. Hoboken NJ, John Wiley & Sons; Lanza et al. Principles of Tissue Engineering, Academic Press; 2nd edition May 15, 2000; and Lanza & Atala, Methods of Tissue Engineering, Academic Press; 1st edition October 2001). Cell culture media generally contain essential nutrients and optionally additional elements such as growth factors, salts, minerals, and vitamins, which may be selected depending on the type of cell being cultured. Specific components may be selected to enhance cell growth, differentiation, secretion of specific proteins, etc. Generally, standard growth media include Dulbecco's Modified Eagle Medium Low Glucose (DMEM) supplemented with 10-20% fetal bovine serum (FBS) or calf serum, containing 110 mg / L of pirubate and glutamine, and 100 U / ml of penicillin, as is appropriate for various other standard media well known to those skilled in the art. Growth conditions vary depending on the type of mammalian cells used and the desired tissue.

[0082] Additional sources of human cells include, but are not limited to, bone marrow-derived mesenchymal stem cells (MSCs) and early human meniscus-derived chondrocytes. MSCs are attractive for regenerative medicine purposes because they can be isolated from patients and readily expanded for use as autologous tissue replacement. Unlike donor allogeneic grafts, recipient-derived MSC autografts have zero risk of interhuman disease transmission or immune-mediated tissue rejection. An additional advantage of MSCs is that culture protocols for differentiating MSCs into fibrochondrocyte-like cells are clearly defined. Chemically defined media have been shown to induce chondrogenic phenotypes in cultured MSCs and promote the deposition of fibrochondrocyte-like extracellular matrix (ECM) by MFCs in pellet cultures over 10 weeks (Mauck 2006 & Brendon 2007).

[0083] Dedifferentiation of chondrocytes under 2D cell culture conditions has prompted investigations into the effects of more complex physiological culture conditions. Oxygen has a fundamental impact on cell behavior, and cells in the avascular zone of the meniscus are hypoxic due to insufficient oxygenated blood supply. Several studies have investigated the effects of hypoxic growth conditions on chondrocyte phenotype; bovine articular chondrocytes grown in hypoxic (5% O2) cultures were shown to reexpress significantly more collagen-II at the protein level compared to the same cells grown under normal oxygen (21% O2) conditions (Domm et al., 2002). The meniscus is subjected to habitual compressive stress, and mechanical stimulation is thought to be necessary to induce the appropriate chondrocyte phenotype. Ultrasound stimulation at a frequency of 1 MHz has been shown to increase ECM deposition by chondrocytes in 2D and 3D cultures, but the effect is transient, lasting only 28 days (Hsu et al., 2006). Upton et al. isolated cells from the medial and lateral zones of the meniscus and grew them as a monolayer on the soft membrane. Exposure to 5% biaxial strain has been shown to increase NO and total protein expression in both cell populations (Upton et al., 2006). For optimal meniscal biomechanical performance, hypoxic culture and mechanical strain can be used to maximize phenotypic differentiation of MSC-derived chondrocytes or early meniscal cells in 3D culture.

[0084] Activating agent: In some embodiments, the meniscal graft according to embodiments of the present invention may contain at least one activator. Non-limiting examples of such activators include TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-4, BMP-6, BMP-12, BMP-13, basic fibroblast growth factor, fibroblast growth factor-1, fibroblast growth factor-2, platelet-derived growth factor-AA, platelet-derived growth factor-BB, platelet-rich plasma, IGF-I, IGF-II, GDF-5, GDF-6, GDF-8, GDF-10, vascular endothelial cell-derived growth factor, pleiotrophin, endothelin, nicotinamide, glucagon-like peptide-I, glucagon-like peptide-II, parathyroid hormone, tenascin-C, tropoelastin, thrombin-derived peptide, laminin, biological peptides containing cell-binding domains and biological peptides containing heparin-binding domains, therapeutic agents, and any combination thereof.

[0085] As used herein, the term “therapeutic agent” refers to any chemical part that is a biologically, physiologically, or pharmacologically active substance acting locally or systemically on a subject. Non-limited examples of therapeutic agents, also referred to as “drugs,” are listed in well-known references such as the Merck Index, Physician's Desk Reference, and The Pharmacological Basis of Therapeutics, which include, but are not limited to, pharmaceuticals; vitamins; mineral supplements; substances used to treat, prevent, diagnose, cure or alleviate disease or illness; substances that affect the structure or function of the body; or prodrugs that become biologically active or more active after being placed in a physiological environment. In some embodiments, one or more therapeutic agents may be used during implantation into the subject, which can be released from the meniscus graft described herein into adjacent tissue or fluids. Examples of therapeutic agents include, but are not limited to, antibiotics, anesthetics, any therapeutic agent that promotes meniscus regeneration or tissue healing, or reduces pain, infection, or inflammation, or any combination thereof.

[0086] Additional activators may include, but are not limited to, proteins, peptides, nucleic acid analogs, nucleotides, oligonucleotides, nucleic acids (DNA, RNA, siRNA), peptide nucleic acids, aptamers, antibodies or fragments or parts thereof, antigens or epitopes, hormones, hormone antagonists, growth factors or recombinant growth factors and their fragments and variants, cytokines, enzymes, antibiotics or antimicrobial compounds, anti-inflammatory agents, antifungal agents, antiviral agents, toxins, prodrugs, small molecules, drugs (e.g., drugs, dyes, amino acids, vitamins, antioxidants) or any combination thereof.

[0087] Non-limiting examples of antibiotics suitable for inclusion in the meniscus graft of the present invention include aminoglycosides (e.g., neomycin), ansamycin, carbasephalosporins, carbapenems, cephalosporins (e.g., cefazolin, cefaclor, cefditoren, ceftoviprole), glycopeptides (e.g., vancomycin), macrolides (e.g., erythromycin, azithromycin), monobactams, penicillins (e.g., amoxicillin, ampicillin, cloxacillin, dicloxacillin, flucloxacillin), polypeptides (e.g., bacitracin, polymyxin B), and quinolones (e.g., ciprofloxacin, enoxacin). This includes (e.g., gatifloxacin, ofloxacin), sulfonamides (e.g., sulfasalazine, trimethoprim, trimethoprim-sulfamethoxazole (cotrimoxazole)), tetracyclines (e.g., doxycycline, minocycline, tetracycline, etc.), chloramphenicol, lincomycin, clindamycin, ethambutol, mupirocin, metronidazole, pyrazinamide, thianphenicol, rifampicin, thianphenicol, dapsone, clofazimine, quinupristin, metronidazole, linezolid, isoniazid, fosfomycin, fusidic acid, or any combination thereof.

[0088] Non-exclusive examples of antibodies include absiximab, adalimumab, alemtuzumab, basiliximab, bevacizumab, cetuximab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, ibritumomab tiuxetan, infliximab, muromonab-CD3, natalizumab, ofatumumab, omalizumab, palivizumab, panitumumab, ranibizumab, rituximab, tositumomab, trastuzumab, altumomab penteteate, alsitumomab, atlizumab, vectumomab, belimumab, besilesomab, bisilomab, canakinumab, and capromab pendeti. This includes katumakisomab, denosumab, edrecolomab, efungumab, erzumakisomab, etalacizumab, fanolesomab, fontrizumab, gemtuzumab ozogamicin, golimumab, igobomab, imusilomab, rabetuzumab, mepolizumab, motabizumab, nimotuzumab, nofetumomab merpentan, olegobomab, pemtumomab, pertuzumab, loberizumab, luprizumab, thresomab, takatuzumab tetraxetan, tefivazumab, tocilizumab, ustekinumab, bicilizumab, botumumab, zaltumumab, zanorimumab, or any combination thereof.

[0089] Non-limiting examples of enzymes suitable for use with meniscal grafts as described herein include peroxidases, lipases, amyloses, organophosphate dehydrogenases, ligases, restriction endonucleases, ribonucleases, DNA polymerases, glucose oxidases, and laccases.

[0090] Additional, non-limiting examples of activators suitable for use with this meniscus graft include: cell growth media, e.g., Dulbecco's Modified Eagle Medium, fetal bovine serum, non-essential amino acids, and antibiotics; growth and morphogenesis factors, e.g., fibroblast growth factor, transforming growth factor, vascular endothelial growth factor, epidermal growth factor, platelet-derived growth factor, insulin-like growth factor), bone morphogenesis growth factor, bone morphogenesis-like proteins, transforming growth factors, nerve growth factor, and related proteins (growth factors are known in the art, e.g., Rosen & Thies, CELLULAR & MOLECULAR BASIS BONE FORMATION & REPAIR (RGLanes) See Co., Austin, TeX, 1995; anti-angiogenic proteins, e.g., endostatins, and other naturally occurring or genetically modified proteins; polysaccharides, glycoproteins, or lipoproteins; anti-infective agents, e.g., antibiotics and antivirals, chemotherapeutic agents (i.e., anticancer agents), anti-rejection agents, analgesics and combinations of analgesics, anti-inflammatory agents, steroids, or any combination thereof.

[0091] Methods for repairing meniscus defects: Aspects of the present invention include methods for repairing and / or replacing at least a portion of the meniscus in a subject. To achieve meniscus repair or regeneration, any of the meniscal grafts described herein can be implanted in a subject in need. Thus, methods for repairing meniscal defects or promoting meniscal regeneration in a subject are also provided herein. In one embodiment, the method includes implanting a meniscal graft described herein into a defective site requiring meniscal repair or regeneration.

[0092] The term "subject" includes, but is not limited to, non-human primates such as humans, chimpanzees, and other apes and monkey species; domestic animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; and laboratory animals such as rodents such as mice, rats, and guinea pigs. The term does not indicate a specific age or sex. Therefore, it includes adult and newborn subjects, as well as fetuses, regardless of whether they are male or female. In one embodiment, the subject is a mammal. In one embodiment, the subject is a human subject.

[0093] In some embodiments, the method may include fixing a meniscus graft or its anchor regions to the defective site and / or fixing one or more anchor regions of the meniscus graft to at least one anatomical structure within the subject. In some embodiments, the method may further include removing at least a portion of the defective meniscus from the subject.

[0094] In some embodiments, the method may further include administering at least one of the activators described herein systemically and / or topically (e.g., to a meniscus graft site).

[0095] All patents and patent publications referenced herein are incorporated herein by reference in their entirety.

[0096] Those skilled in the art will likely recognize the aforementioned modifications and improvements. Not all such modifications and improvements are included herein for the sake of brevity and readability, but it should be understood that they are appropriately within the scope of the appended claims.

Claims

1. A synthetic tissue structure comprising multiple layers deposited by a bioprinting machine, wherein each layer comprises one or more synthetic tissue fibers containing a solidified biocompatible matrix, the type of matrix material changes in at least one direction within one or more synthetic tissue fibers within at least one layer, and the synthetic tissue structure further comprises one or more anchor regions.

2. The synthetic tissue structure according to claim 1, further comprising cells.

3. The synthetic tissue structure according to claim 1, further comprising an activator.

4. The synthetic structure according to claim 1, wherein each of the layers comprises a matrix material whose type changes in at least one direction.

5. The synthetic tissue structure according to claim 2, wherein at least one of the layers comprises a cell type and / or cell density that changes in at least one direction within one or more synthetic tissue fibers.

6. The synthetic tissue structure according to claim 5, wherein each of the layers comprises a cell type and / or cell density that changes in at least one direction within one or more synthetic tissue fibers.

7. The synthetic tissue structure according to claim 3, wherein at least one of the layers contains one or more activators whose type and / or amount changes in at least one direction within one or more synthetic tissue fibers.

8. The synthetic tissue structure according to claim 7, wherein each of the layers contains one or more activators whose type and / or amount changes in at least one direction within one or more synthetic tissue fibers.

9. The synthetic tissue structure according to claim 1, further comprising one or more reinforced surrounding regions.

10. The synthetic tissue structure according to claim 1, wherein the periphery of the synthetic tissue structure includes one or more anchor regions, and the one or more anchor regions include one or more reinforcing matrix materials.

11. The synthetic tissue structure according to claim 1, wherein the entire perimeter of the layer of the synthetic tissue structure comprises a reinforcing matrix material.

12. The synthetic tissue structure according to claim 1, wherein the matrix material comprises alginate, laminin, fibrin, hyaluronic acid, poly(ethylene) glycol-based gel, gelatin, chitosan, agarose, or a combination thereof.

13. The synthetic tissue structure according to claim 12, wherein the matrix material comprises alginate.

14. The synthetic tissue structure according to claim 1, wherein the solidified biocompatible matrix is ​​physiologically compatible.

15. The synthetic tissue structure according to claim 14, wherein the solidified biocompatible matrix comprises one or more of the following: collagen, fibronectin, thrombospongin, glycosaminoglycans (GAGs), deoxyribonucleic acid (DNA), adhesive glycoproteins, elastin, and combinations thereof.

16. The synthetic tissue structure according to claim 15, wherein the collagen is collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, or collagen XVIII.

17. The synthetic tissue structure according to claim 15, wherein the GAG ​​is hyaluronic acid, chondroitin-6-sulfate, dermatan sulfate, chondroitin-4-sulfate, or keratin sulfate.

18. The synthetic tissue structure according to claim 2, wherein the cells are mammalian cells, and the mammalian cells are selected from the group consisting of fibroblasts, chondrocytes, fibrochondrocytes, early human meniscus-derived chondrocytes, stem cells, bone marrow cells, embryonic stem cells, mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, induced pluripotent stem cells, differentiated stem cells, tissue-derived cells, microvascular endothelial cells, and combinations thereof.

19. The synthetic tissue structure according to claim 1, wherein at least one of the layers comprises a single continuous synthetic tissue fiber distributed from a bioprinting machine.

20. The synthetic tissue structure according to claim 19, wherein a single continuous synthetic tissue fiber distributed from the bioprinting machine has a variable composition.

21. The synthetic tissue structure according to claim 1, wherein one or more anchor regions comprise one or more double-network hydrogels produced by combining polycaprolactone (PCL), poly(lactic acid-coglycolic acid) (PLGA), polyurethane (PU), any combination thereof, or at least two different hydrogel materials.

22. The synthetic tissue structure according to claim 1, wherein one or more anchor regions contain a non-toxic dye.

23. The synthetic tissue structure according to claim 1, wherein one or more anchor regions include one or more layers of high-strength material alternately laminated with one or more layers of softer matrix material, and the softer matrix material includes a material that contributes to cell growth and viability.

24. The synthetic tissue structure according to claim 23, wherein the layer of one or more high-strength materials comprises one or more double-network hydrogels produced by combining polycaprolactone (PCL), poly(lactic acid-coglycolic acid) (PLGA), polyurethane (PU), any combination thereof, or at least two different hydrogel materials.

25. The synthetic tissue structure according to claim 24, wherein the at least two different hydrogel materials include alginate, gelatin methacryloyl (GelMA), methacryloyl polyethylene glycol (PEGMA), gellan gum, agarose, polyacrylamide, or any combination thereof.

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