Micropost array apparatus and biocompatible scaffold construct

Abstract

To provide a micropost array apparatus and a biocompatible scaffold construct.SOLUTION: A biocompatible scaffold construct comprises: a biocompatible hydrogel; and at least one biomaterial microfiber strand wound to form a plurality of microfiber segments in proximity to one another and arranged in an organized configuration.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] Statement of U.S. Government Support This invention was made with government support under DARPA contract HR0011-15-90006. The U.S. Government has certain rights in this invention.

[0002] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 119,618, filed November 30, 2020, entitled "Micropost Array Apparatus and Composite Biological Scaffold", the entire disclosure of which is incorporated herein by reference.

Background Art

[0003] Skeletal tissue injury is one of the most common injuries treated in the United States. However, current treatment options often lead to dysfunction of the injured tissue and a high rate of re - injury. Therefore, many tissue engineering techniques have been developed to produce scaffold - like grafts that facilitate the regeneration of functionally native - like tissue. It is particularly important to reproduce the biochemical, morphological, and functional characteristics of the target tissue.

[0004] Three - dimensional (3D) bioprinting, an additive biomanufacturing method, is commonly performed for the fabrication of scaffolds with potential regenerative medicine applications. 3D bioprinting enables the design of cells and biomaterials and can accurately manipulate them, often into complex 3D shapes. However, bioprinting methods typically use soft hydrogels as primary structural materials, especially when dealing with biological polymers such as collagen (Mandrycky 2016). The mechanical properties of such hydrogels are often orders of magnitude lower than those of native ligaments, tendons, or other tissues. Therefore, typical bioprinting methods cannot appropriately reproduce the functional properties of skeletal or other tissues and thus cannot generate supportive scaffold structures for tissue repair.

[0005] A hybrid bioprinting method has been developed (Merceron 2015) that incorporates a thermoplastic polymer together with a hydrogel to improve the mechanical properties of the printed part. However, many printed materials still have limited mechanical strength and may have an adverse effect on injury healing and tissue regeneration.

[0006] To address these issues, many fiber-based tissue engineering approaches have been developed (Tamayol 2013) that use strong natural biomaterials such as collagen. These approaches are formed based on the established clinical use of textiles but incorporate additional means for generating cellized scaffold constructs.

[0007] Also, the use of therapeutic cells offers the potential to improve the treatment of genetic, degenerative, inflammatory, and traumatic musculoskeletal disorders (O’Keefe 2019). Unlike the case of biomaterial scaffold constructs alone, cells can improve the healing rate and overall tissue generation and functional recovery. This may be particularly true in tissues such as ligaments and tendons where passive intracellular growth may be limited in cell- and vascular-deficient environments.

[0008] Some techniques for generating biomaterial scaffold constructs utilize prefabricated fibers produced by traditional textile manufacturing processes such as weaving, knitting, and braiding. Biomaterial fibers can be produced as raw materials for these processes by wet spinning, microfluidic spinning, biospinning, interfacial complexation, and melt spinning (Tamayol 2013). Weaving can be used to generate polymer scaffold constructs with designed porosity, morphology, and shape by crossing two sets of warp or weft threads at right angles (Abrahamsson 2010). Knitting is a commonly used technique for fabricating surgical meshes, forming 3D shapes by twisting yarns or threads into a series of interconnected loops (Sahoo 2007). Braiding can form complex biomaterial structures or patterns by twisting multiple fiber strands together (Walters 2012). Additionally, relatively simple shapes such as bundles of parallel fibers tied together with sutures have been fabricated by manual assembly (Gentleman 2006).

[0009] Scaffold fabrication processes for generating biomaterial scaffold constructs that utilize fibers produced as components of the biomaterial scaffold constructs include electrospinning, wet spinning, and direct writing. These techniques utilize processes such as solvent evaporation, in-solution polymerization, or temperature-induced recrystallization to form ultrafine fiber scaffold constructs from biomaterial solutions. For example, electrospinning has been used to form random or aligned polymer fiber mats with biomimetic surface patterns aimed at tissue formation (Mauck 2009). Wet spinning can be used to fabricate scaffold constructs during fiber formation by collecting fibers on a rotating mandrel in addition to being used to form fibers as raw materials (Kaiser 2019). Direct writing can form fiber-based scaffold constructs while fully controlling porosity, fiber size, and fiber orientation (Wu 2015).

[0010] However, post-fabrication cell seeding required to generate cellular scaffolds using weaving, knitting, braiding, electrospinning, wet spinning, and direct writing can be variable by person and may strongly depend on the macroscale shape and porosity of the biomaterial scaffold. For example, small pore sizes may limit cell infiltration during seeding, particularly for scaffolds with thick or complex 3D shapes. Conversely, scaffolds with high porosity may have difficulty maintaining seeded cells evenly throughout. The dependence of cell seeding on the micro- and macroscale shape of the scaffold can limit control of overall cell distribution, particularly in the fabrication of heterologous tissues with different cell populations in the designed area.

[0011] To address the challenges associated with seeding cells onto prefabricated scaffold constructs, various fiber-based techniques have been developed to directly manipulate cells or cell-laden materials during the scaffold fabrication process (Tamayol 2013). Compared to techniques that require cell seeding, these techniques result in cellated scaffold constructs with improved consistency and control over the overall cell distribution. Polyester threads have been coated with cell-laden hydrogels and wrapped around a cylindrical mandrel to form 3D annular structures (Liberski 2011). Microfluidic systems have been used to form core-shell hydrogel fibers that surround cells that can be implanted without forming a secondary scaffold structure (Sugimoto, 2011). The micro-weaving method has been utilized to form centimeter-scale biofibers from similar cell-laden core-shell hydrogel fibers (Onoe 2011). However, the mechanical strength of cell-laden hydrogel fibers is limited, restricting the possibility of processing this using traditional textile manufacturing methods (Onoe 2011). As in typical hydrogel-based bioprinting, in the direct writing method, cell suspensions are crosslinked within a printhead to form cell-laden scaffold constructs from fiber-like extrusions. Overall, the mechanical properties of scaffold constructs formed using these hydrogel-based techniques are inferior, limiting their applicability as support scaffold constructs for treating musculoskeletal tissue injuries.

Summary of the Invention

Problems to be Solved by the Invention

[0012] It would be desirable to address one or more of the problems discussed above.

Means for Solving the Problems

[0013] In one form, the present disclosure relates to a composite scaffold construct. The composite scaffold construct can include a biocompatible hydrogel and at least one biological material strand wound to form a plurality of segments arranged in a tissue-like configuration in proximity to one another.

[0014] In another aspect, the present disclosure relates to an apparatus for creating a composite scaffold structure. The apparatus can include a first array of microposts and a second array of microposts spaced apart from the first array of microposts. The microposts can be configured to receive ultra-fine fiber strands to form a plurality of segments, and at least some of the plurality of segments are arranged in a substantially aligned configuration.

[0015] In another aspect, the present disclosure relates to a method for creating a composite scaffold structure. The method can include dispensing ultra-fine fiber strands into a biocompatible hydrogel sheath and winding the ultra-fine fiber strands around a plurality of microposts to form a plurality of segments covered with the biocompatible hydrogel and arranged in a tissue-like configuration.

[0016] Other systems, methods, features and advantages of the embodiments will be apparent to those skilled in the art upon examination of the following drawings and the following detailed description. All such additional systems, methods, features and advantages are intended to be included within this description and this summary, are within the scope of the embodiments, and are protected by the claims.

Brief Description of the Drawings

[0017] The patent or application file contains at least one color drawing. The patent or patent application publication, including the color drawing, will be provided by the Patent Office upon request and payment of the necessary fee.

[0018] The embodiments can be better understood by reference to the following drawings and description. The components in the drawings are not necessarily to scale and may be emphasized when illustrating the principles of the embodiments. Further, in the drawings, like reference numerals refer to corresponding parts throughout the various drawings.

[0019]

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DETAILED DESCRIPTION OF THE INVENTION

[0020] The novel biomanufacturing method described herein improves a method for fabricating a cell-scaffold construct for regenerative medicine. The scaffold construct is fabricated from strong and stable ultrathin fibers of clinical-grade collagen having biochemical and mechanical properties similar to native tissue. These fibers are evenly and controllably coated with cells during scaffold construct fabrication. Any cell type can be selected based on the intended use and can include stem cells, tenocytes, chondrocytes, myoblasts, osteoblasts, or numerous tissue-specific cell types. Appropriate cell culture media and substance additives must be used to facilitate the survival of the selected cell type. The scaffold construct is preferably formed to have microstructural cues to provide signals for cell alignment and a designed porosity, fiber pattern, and macroscopic dimensions suitable for the intended use. The fabrication process described herein is rapid, repeatable, scalable, and automatable. As described herein, the scaffold construct mimics the biological, morphological, and functional properties of native musculoskeletal tissue.

[0021] The embodiments described herein relate to composite scaffold constructs, methods of making composite scaffold constructs, and apparatuses for making composite scaffold constructs. Such scaffold constructs include segments of aligned multiple collagen ultrathin fibers embedded in a biocompatible hydrogel. Such scaffold constructs may be suitable for use as tendon or ligament grafts or to support other surgical biomechanical repairs.

[0022] To assist in clarifying the following description of the various embodiments, various terms are defined here. Unless otherwise indicated, the following definitions apply throughout the specification (including the claims). For consistency and convenience, adjectives indicating direction are employed throughout this description corresponding to the embodiments being described.

[0023] "Biomaterials", as used throughout this detailed description and the claims, means naturally-derived proteins, glycoproteins, and glycosaminoglycan-based biopolymers and their synthetic counterparts. Possible biomaterials include those that have been used throughout the 3D bioprinting process, including, in particular, some fiber-based biomanufacturing methods. These can include, but are not limited to, collagen, elastin, fibronectin, fibrinogen, silk, synthetic polymers, proteoglycans, and hyaluronic acid (Skardal 2014 and Tamayol 2013).

[0024] With respect to collagen, the types envisioned include atelocollagen, telocollagen, gelatin, and can be sourced from collagen such as recombinant human collagen, porcine collagen, bovine collagen, jellyfish collagen, and mixtures thereof. One of ordinary skill in the art will appreciate that fibers having the tensile strength, resilience, elasticity, and toughness suitable for the particular function and use of a given implant will be produced, as discussed herein.

[0025] As used throughout this detailed description and the claims, "fiber" means a fiber, thread, or filament having a high ratio of length to diameter and is typically used as a unit. "Ultra-fine fiber" is used synonymously by virtue of the size scale of the fibers used in the preferred embodiments. "Strand" means an individual item of fiber, and fiber-based constructs are composed of a number of individual fiber strands. "Segment" means the length of a fiber with respect to a spatial location, such as a fiber segment present at a particular location in a fiber-based construct. Overall, the above terms can include fibers composed of multiple sub-fibers assembled by secondary or tertiary assembly processes such as braiding.

[0026] "Lateral", as used throughout this detailed description and the claims, means a parallel direction extending along the width of a component.

[0027] "Longitudinal", when used throughout this detailed description and in the claims, means the direction extending along the length of a component.

[0028] "Micropost", when used throughout this detailed description and in the claims, means a fixed point, protrusion, or structure around which or to which fibers can be wound or otherwise removably attached. One of ordinary skill in the art will recognize that the microposts shown in the preferred embodiments are one type of fixation structure among many possible fixation structure shapes.

[0029] "Vertical", when used throughout this detailed description and in the claims, means a direction generally orthogonal to both the lateral and longitudinal directions.

[0030] "Scaffold structure", when used throughout this detailed description and in the claims, means a 2D or 3D assembly of fibers. "Construct" and "macrostructure" are used synonymously. "Graft", when used in the preferred embodiments, means a scaffold structure intended to be implanted as a medical device.

[0031] It should be understood that each of the adjectives indicating direction can be applied to the individual components of the devices or apparatuses discussed herein. "Upward" means the vertical direction away from the ground, and "downward" means the vertical direction toward the ground. Similarly, "upper", "upper side", and other similar terms mean the part of an object that is substantially farthest from the ground in the vertical direction, and "bottom", "lower side", and other similar terms mean the part of an object that is substantially closest to the ground in the vertical direction.

[0032] In the present disclosure, "fixedly attached" means two components joined such that the components cannot be easily separated (e.g., without damaging one or both of the components). Preferred modes of fixed attachment include joining by permanent adhesives, rivets, stitches, nails, staples, welding or other thermal adhesion or other joining methods. Further, two components can be "fixedly attached" by being integrally formed, for example, in a molding process.

[0033] In the present disclosure, "removably attached" means that two components are joined such that the two components are fixed together but can be easily removed from each other. Examples of removable attachment mechanisms include hook-and-loop fasteners, friction fit connections, interference fit connections, screw connectors, camlock connectors and other such easily removable connectors of this kind.

[0034] "Strand" includes single fibers, filaments or monofilaments and regular fiber assemblies with a high length-to-diameter ratio that are typically used as a unit.

[0035] The present disclosure relates to a composite scaffold construct comprising a plurality of substantially aligned collagen ultrafine fiber segments embedded in a biocompatible hydrogel. The present disclosure further relates to methods and apparatuses for making this kind of composite scaffold construct. The disclosed systems and processes can also be applied to other types of fibers other than collagen ultrafine fibers, including other natural fibers and / or synthetic materials, and flexible polymers such as sutures, or, for example, soft steel wires and other products that those skilled in the art will recognize as suitable for use in facilitating similar scaffold constructs.

[0036] The scaffold structure is formed generally by dispensing collagen ultrafine fiber strands such that the dispensed strands are coated in a biocompatible hydrogel as a cell binder. In the present invention, the ultrafine fibers can be dispensed by pulling out the microstrands under tension or by extruding them. The collagen ultrafine fiber strands are dispensed from the central lumen of the inner conduit of the coaxial needle, and the biocompatible hydrogel precursor is dispensed from the annular lumen around the inner conduit. The hydrogel precursor is dispensed from the annular lumen by controlling the actuation of the plunger of a syringe containing the hydrogel precursor solution. In a preferred embodiment, the ultrafine fibers are dispensed from a spool. The preferred extrusion of collagen fibers is disclosed in U.S. Patent Application Publication No. 2020 / 0246505 to Francis et al., published August 6, 2020, entitled "Microfluidic Extrusion". This describes products having an ultimate tensile strength, modulus of elasticity, and elongation at break comparable to native human tendons and ligaments. The entire disclosure of U.S. Patent Application Publication No. 2020 / 0246505 is incorporated herein by reference.

[0037] Figure 1 is a schematic view of a portion of an apparatus for making a composite scaffold structure according to a preferred embodiment. As shown in Figure 1, the printing device 100 can include a 3D printing assembly including features configured to dispense collagen ultrafine fibers covered with a biocompatible hydrogel. The 3D printing assembly is provided to control the movement of the printing device 100 along the X, Y, and Z axes. In some embodiments, it will be appreciated that the printing device can have 4, 5, or 6 axes to further add degrees of freedom to the print head and receiving substrate. Further, it will be appreciated that the printing device can be configured to generate two-dimensional (2D) or three-dimensional (3D) constructs. Further, in some embodiments, the system can generate a cell scaffold structure and add a fourth dimension to the construct.

[0038] It will also be appreciated that constructs can be generated in a variety of shapes. For example, scaffolding structures having planar sheet-like shapes, prism shapes, round or cylindrical shapes, and other complex 3D shapes based on CAD models can be generated. The biomaterial strands forming such macroscopic shapes can be substantially parallel to each other, or partially aligned, or can vary the orientation and spacing of the strands in three dimensions as being substantially non-aligned. Further, some constructs can have both aligned ultra-fine fiber segments and non-aligned ultra-fine fiber segments.

[0039] As shown in FIG. 1, the printing device 100 can include a stepper motor 105 and a lead screw 110 configured to be driven by the stepper motor 105. The lead screw 110 can rotate to move the first platform 121 relative to the second platform 123 to actuate the plunger 122 of the syringe 130. A biocompatible hydrogel precursor solution 135 can be placed in the syringe. When the screw 110 rotates, the hydrogel solution 135 is pushed through the conduit 140 and into the annular conduit of the coaxial needle 125.

[0040] Simultaneously with the dispensing of the hydrogel precursor solution 135, the collagen ultra-fine fiber strands 115 can be dispensed through the inner conduit of the coaxial needle 125. The ultra-fine fiber strands can be prefabricated and used as raw materials. Thus, as shown in FIG. 1, the spool 120 can accommodate the collagen ultra-fine fiber strands 115. To effect dispensing, the free end of the collagen ultra-fine fiber strand 115 can be fixed (e.g., tied) to a frame or a micropost or other fixed structure. Then, when the print head is moved, the collagen ultra-fine fiber strand 115 is drawn from the spool 120 through the coaxial needle 125. At this time, the collagen ultra-fine fiber strand 115 is coated with the hydrogel precursor solution 135.

[0041] When drawn from a spool and distributed through a coaxial needle, a rack is provided with a plurality of microposts for organizing collagen ultrafine fibers, and collagen ultrafine fiber strands can be wound around these microposts in a tissue structure configuration.

[0042] Figure 2 is a schematic perspective view of a rack including an array of opposing microposts configured to receive collagen ultrafine fibers. As shown in Figure 2, an apparatus for creating a composite scaffold structure can include a rack 200 configured to receive collagen ultrafine fibers. In some embodiments, the rack 200 can include a first portion 205 and a second portion 210 connected by rails 215. In some embodiments, the first portion 205 and the second portion 210 can be slidable relative to each other along the rails 215. The slidable feature can facilitate the removal of the scaffold structure after it is constructed.

[0043] As shown in Figure 2, the rack 200 can further include a first array 220 of microposts. In some embodiments, the first array 220 can be disposed in a recess 225 of the first portion 205 of the rack 200. Further, the rack 200 can include a second array 230 of microposts spaced from and arranged opposite the first array 220 of microposts. For example, as shown in Figure 2, the second array 230 can be disposed in a second recess 235 of the second portion 210 of the rack 200.

[0044] The microposts are configured to receive a continuous ultrafine fiber strand that forms a plurality of adjacent ultrafine fiber segments arranged in a substantially aligned configuration.

[0045] FIG. 3 is a schematic diagram of a collagen ultra-fine fiber strand that is systematically wound between two opposing arrays of micro-posts. In some embodiments, the micro-posts of the first array 220 and the second array 230 can be arranged staggeredly. For example, as shown in FIG. 3, the first array 220 can include a first column 221 of micro-posts and a second column 222 of micro-posts that are offset from the first column 221. Similarly, the second array 230 can include a third column 231 of micro-posts and a fourth column 232 of micro-posts that are offset from the first column 221.

[0046] As shown in FIG. 3, the collagen ultra-fine fiber strand 115 can be wound back and forth in an organized configuration between the micro-posts of the first array 220 and the micro-posts of the second array 230. Further, as shown in FIG. 3, the wound strands can form a plurality of ultra-fine fiber segments that are substantially aligned. For example, the strand 115 can be wound to form a first ultra-fine fiber segment 201, a second ultra-fine fiber segment 202, a third ultra-fine fiber segment 203, a fourth ultra-fine fiber segment 204, and the like. In some embodiments, the ultra-fine fiber segments can be substantially parallel to each other as shown in FIG. 3. In another embodiment, the winding of the micro-post array and / or the ultra-fine fibers can be arranged such that the ultra-fine fiber segments are disposed at an oblique angle to each other or in other geometric configurations.

[0047] The collagen ultra-fine fiber strand and the micro-posts can be of any suitable size. Further, the spacing between the micro-posts can also vary. The following dimensions are merely illustrative, and it will be appreciated that modifications can be made to these dimensions within the scope of the disclosed concept. Also, the number of micro-posts used and the spacing between the micro-posts can be selected such that a composite scaffold structure of the desired width is obtained. Similarly, the spacing between the micro-post arrays can be selected such that a composite scaffold structure of the desired length is obtained.

[0048] As shown in FIG. 3, the first column 221 can include a first micropost 235, a second micropost 240, and a third micropost 245. The second column 222 can include a fourth micropost 250, a fifth micropost 255, a sixth micropost 260, and a seventh micropost 265. The number of microposts in each array has been reduced in FIG. 3 for clarity. Referring to FIG. 2 for an example of a rack having more microposts (specifically, 21 microposts in each array). Those skilled in the art will understand that the number of microposts can be more than the specific embodiments disclosed herein, and the number of microposts should be determined with attention to the specific product or application.

[0049] In some embodiments, the collagen ultra-fine fiber strand can be in the form of a ribbon having a width of about 50 micrometers and a thickness of about 5 micrometers. In the case of such a strand, a micropost with a diameter of about 200 micrometers can be used. For example, as shown in FIG. 3, the third micropost 245 may have a diameter of 285. In some embodiments, the diameter 285 can be about 200 micrometers.

[0050] Furthermore, the post spacing within each array can be about 1 mm. FIG. 3 shows a first distance 270 between the first micropost 235 and the fourth micropost 250, a second distance 275 between the first micropost 235 and the second micropost 240, and a third distance 280 between the first micropost 235 and the fifth micropost 255. Each of the first distance 270, the second distance 275, and the third distance 280 can be about 1 mm. Thus, all the microposts surrounding the first micropost 235 can be equally spaced from the first micropost 235, for example, at a distance of about 1 mm.

[0051] The collagen ultra-fine fiber strand can be wound around the micropost in a fluid tank that reacts with the hydrogel precursor fluid covering the ultra-fine fiber strand to solidify the hydrogel precursor into a hydrogel. That is, the ultra-fine fiber strand covered with the hydrogel precursor is dispensed into another solution tank that facilitates chemical, physical, or other reactions to convert the hydrogel precursor into a hydrogel. Thus, this reaction between the hydrogel precursor and the fluid tank can start from a cell suspension and end with a stable solid hydrogel.

[0052] In some embodiments, the fluid tank can contain a cross-linking solution such as a thrombin solution. In other embodiments, preferred hydrogel precursor / fluid tank combinations can include fibrinogen + thrombin, fibrinogen / thrombin + factor XIII, alginic acid + ionic compound, collagen + enzyme solution, silk + enzyme solution, or gelatin + enzyme solution.

[0053] In addition to reacting with the hydrogel precursor to produce a hydrogel, the fluid tank also provides a physiological hydration environment to maintain the health of the cells. Thus, the fluid tank can provide a physiological environment with appropriate temperature, pH, hydration, and biological compounds to support and maintain cell viability and health.

[0054] To provide this physiological environment, the manufacturing apparatus can also include a container configured to receive a rack of the micropost array and submerge the rack in an appropriate fluid solution. This container can be placed on a fixture that can be manually translated and rotated relative to the printing equipment.

[0055] To maintain sterility, the entire physical system can be placed in a biosafe cabinet or a filtered laminar flow hood, and all components are handled aseptically.

[0056] As described above, the collagen ultra-fine fiber strands are coaxially dispensed within a sheath of a biocompatible hydrogel precursor solution. An example of such a hydrogel precursor solution is a fibrinogen solution. When the fibrinogen solution is dispensed into the thrombin cross-linking solution bath during printing, the fibrinogen rapidly solidifies to form a stable biocompatible fibrin gel. In some embodiments, the cells are suspended within the hydrogel precursor solution. Thus, the fibrinogen is, but not necessarily, a cell suspension.

[0057] Figure 4 is a schematic view of a portion of a 3D printing apparatus and a receiving assembly. As shown in Figure 4, the fixture 400 can include a container 405 configured to receive a rack 200 submerged in a cross-linking solution.

[0058] As further shown in FIG. 4, the fixture 400 can be mounted on a stage 410 that is translatable with respect to the printing device 100 (indicated by a pair of arrows 415 in the figure). Further, the stage 410 enables the fixture 400 to be rotated with respect to the printing device 100 as indicated by arrow 420 in the figure. This mobility of the fixture 400 facilitates the setup of the manufacturing process. That is, the stage 410 is utilized to ensure that the position and orientation of the micropost array 200 are accurately known with respect to the position and direction of movement of the printing device 100. For example, the stage 410 is used to ensure that the zero point or "home position" of the printing device 100 with respect to the micropost array 200 is accurately known. Thus, during use, the 3D printing assembly moves the printing device 100 to the "home position", and then the stage 410 translates so that the coaxial needle 125 is accurately positioned between the microposts within the micropost array 200 prior to printing. Further, the stage 410 is also used to ensure that the lateral and longitudinal movements of the printing device 100 are accurately aligned with the lateral and longitudinal directions of the microarray 200. For example, the 3D printing assembly first moves the printing device 100 purely in the longitudinal direction and then purely in the lateral direction. After each movement of the printing device 100, the stage 410 is rotated so that the coaxial needle 125 is accurately positioned between the microposts within the micropost array 200 prior to printing. Once the ultra-fine fiber strands are fixed to the microposts, when the printing device 100 then moves, the collagen ultra-fine fiber strands are drawn through the needle from the spool 120.

[0059] In some embodiments, the coaxial needle can include a flexible extension on the outer cannula to protect the collagen microfiber strands from damage. During distribution of the strands, the strands are drawn from the tip of the inner cannula of the coaxial needle at an angle of approximately 90 degrees relative to the central axis of the needle. Thus, a flexible extension can be included at the end of the outer cannula of the coaxial needle to protect the strands from damage that may occur by drawing the strands through the relatively sharp end of the needle. The flexible extension bends relative to the central axis of the needle, so the strands emerge from the flexible extension at an angle of less than 90 degrees. Further, the tip of the flexible extension may be made considerably softer than the metal needle tip. Both the reduced angle and the soft extension tip can contribute to reducing the potential for damage to the strands when they are withdrawn from the needle.

[0060] FIG. 5 is an enlarged cross-sectional schematic view of the tip of a coaxial needle with a flexible extension fitted to the outer cannula. As shown in FIG. 5, the coaxial needle 125 may have an outer cannula 500 and an inner cannula 505. The outer cannula 500 and the inner cannula 505 can be formed of a relatively hard material such as stainless steel or rigid plastic. An annular outer lumen 510 is formed between the outer cannula 500 and the inner cannula 505. The annular outer lumen 510 is configured to dispense a hydrogel precursor sheath around the collagen microstrands 515 as they are drawn through the inner cannula 505.

[0061] To protect the strand from damage as it is drawn through the tip 520 of the inner cannula 505, a flexible tip 525 (formed of, for example, rubber, silicone, flexible plastic, etc.) can be fitted to and extend from the tip 530 of the outer cannula 500. Thus, as shown in FIG. 5, the tip 535 of the flexible extension 525 can be bent in the direction in which the strand 515 is withdrawn from the needle 125.

[0062] FIG. 6 is a schematic diagram of a printhead path for winding collagen ultra-fine fiber strands around a micropost. As shown in FIG. 6, the collagen ultra-fine fiber strand 115 can be wound around the micropost 600 by a printhead moving along the dotted line path 605. That is, the coaxial needle tip can move along the route along the dotted line path 605 shown in FIG. 6. It can be seen that the path 605 reverses and overlaps in some areas. Further, the path 605 is generally equally spaced from all the microposts 600. For example, in the embodiment shown in FIG. 6, the microposts 600 are spaced approximately 1 mm (i.e., 1000 micrometers) apart from each other. Thus, the path 605 maintains a distance of at least 0.5 mm from each micropost 600. Also, since multiple strands can be wound around the micropost, multiple layers of ultra-fine fibers can be formed. In some embodiments, by continuously winding the same strand around the micropost 600, a layered ultra-fine fiber of a determined thickness can be formed in different vertical planes. Generally, the thickness of each layer can be approximately equal to the diameter of the fibers forming the layer. The orientation of the fibers (parallel, orthogonal, or oblique) can vary within each layer or between layers. Those skilled in the art will appreciate that the orientation of the fibers can be selected and controlled throughout the structure fabricated to adjust the stability and mechanical properties in various directions.

[0063] FIG. 7 is an enlarged schematic view of a portion of a composite scaffold structure formed using the materials and methods described herein. As shown in FIG. 7, the scaffold structure can include collagen ultra-fine fiber strands arranged in a plurality of tissue-like ultra-fine fiber segments 700. As shown in FIG. 7, the ultra-fine fiber segments 700 can be substantially aligned with each other. In some embodiments, the ultra-fine fiber segments 700 can be substantially parallel to each other. In some embodiments, non-aligned / non-parallel ultra-fine fiber segments can be used. Further, multiple layers of ultra-fine fibers 700 are stacked on top of each other.

[0064] The ultra-fine fiber segment 700 can include a loop 705 that serves as a starting point when it is wound around the micropost during generation. Further, FIG. 7 shows a plurality of cells 710 disposed in a cell biocompatible hydrogel in which the ultra-fine fiber segment 700 is disposed. In some embodiments, the hydrogel can be cross-linked, as part of the manufacturing process, for example, by a cross-linking solution in which a scaffold construct is generated.

[0065] FIG. 7 shows an approximate scale and shows that the ultra-fine fiber segments are approximately 200 micrometers apart from each other and that the loop 705 has a diameter of approximately 200 micrometers. The loop is created by a micropost having a diameter of about 200 micrometers. In some embodiments, the segments can have different spacings therebetween, i.e., spacings greater than or less than 200 micrometers. It will be understood by those skilled in the art that the fiber spacing depends on the fiber diameter and the dimensions of the micropost array and can be selected based on the desired total amount of fiber and the macroscopic dimensions of the construct being generated.

[0066] FIG. 8 is a flowchart showing the steps of a method of making a composite scaffold construct according to a preferred embodiment. As shown in FIG. 8, the method includes distributing collagen ultra-fine fiber strands within a biocompatible hydrogel precursor sheath (step 800). Further, in step 805, the method includes winding the collagen ultra-fine fiber strands around a plurality of microposts to form a plurality of substantially aligned collagen ultra-fine fiber segments embedded within the biocompatible hydrogel. As described above, by winding the collagen ultra-fine fiber strands around the plurality of microposts, a plurality of substantially parallel ultra-fine fiber segments can be formed. Steps 800 and 805 can be repeated to form multiple layers of ultra-fine fibers.

[0067] Furthermore, in step 810, the method can include immersing the wound strand in a crosslinking solution. For example, as described above, the winding of the collagen ultra-fine fiber strand around the micro-post can be carried out in a container including a crosslinking solution bath. That is, the immersion is carried out simultaneously with the winding of the strand around the micro-post.

[0068] Furthermore, since the scaffold construct is a biological tissue, it must be maintained under appropriate conditions. After printing is completed, the scaffold construct can be removed from the micro-post array and placed in a cell culture container such as a dish or multi-well plate. For example, in step 815, the method further includes maintaining the ultra-fine fiber and hydrogel scaffold construct under typical cell culture conditions. In some cases, the cell culture conditions are maintained for a period of 7 days or more without significant degradation of the strength or ultra-fine fiber structure.

[0069] The following is a further description of the materials and methods used to generate the scaffold constructs discussed herein.

[0070] The novel additive manufacturing method was developed to generate a cellularized composite scaffold construct consisting of a biocompatible hydrogel reinforced by highly aligned strong collagen nanofibers. Specifically, in a preferred embodiment, a robotic print head winds collagen nanofibers between and around an array of microposts while extruding or dispensing a cell-laden hydrogel precursor. The array of microposts is placed within a crosslinking solution bath. The crosslinking solution bath serves to solidify the cell-laden hydrogel when extruded or dispensed around the simultaneously withdrawn collagen fibers. As a result, a composite three-dimensional (3D) structure is formed consisting of layers of parallel fibers surrounded by a cell-laden hydrogel that maintains the macroscopic shape and orientation of the fibers and has a designed pattern and dimensions. Structures on the millimeter or centimeter scale are printed with a pattern and dimensions designed from collagen fibers of approximately 50 μm width and approximately 5 μm thickness (ribbon shape) using microposts having a diameter of approximately 200 μm and a post spacing within the array of approximately 1 mm.

[0071] To enable this technique, a custom extrusion print head (Figure 1) was designed and attached to a commercially available 3D printer, Folger Tech FT-5 R2. The print head uses a lead screw driven by a planetary gear stepper motor to mechanically press a disposable syringe to extrude a cell suspension with a resolution of less than microliters. The extruded cell suspension passes through the outer needle of a coaxial needle assembly during printing. A spool of collagen nanofibers is loaded into the print head and dispensed through the inner needle of the coaxial needle assembly. At the exit of the needle assembly, the collagen fibers are evenly coated by the extruded cell suspension. The amount of cell suspension extruded per millimeter of fiber withdrawn is a user-set process parameter and provides a means to control the cell density obtained throughout the scaffold construct. In a preferred embodiment, the cell density varies between 0 and 10 million cells / mL depending on the printing parameters and the desired number of cells in the resulting scaffold construct.

[0072] A custom-made micropost array (Figure 2), consisting of two halves each with two parallel rows of steel microposts, was designed as the receiving substrate. The microposts have a diameter of about 200 μm, a spacing between the posts of each half of about 1 mm, and the distance between the two halves of the post array assembly is adjustable to facilitate the fabrication of structures with various lengths.

[0073] The two halves are attached to a bath while maintaining a designed distance between them. Prior to printing, the bath is filled with a cross-linking solution (e.g., thrombin solution) and the post array is submerged in it. During printing, the robotic print head moves between the posts in a designed pattern. The collagen fibers are pulled out under tension by the posts to maintain the pattern followed by the print head. An example pattern to form a single layer of fibers is shown in Figure 3. The instrument is controlled to incrementally adjust the vertical distance of the print head from the micropost array so that the pattern can be repeated as it is printed to form the 3D structure.

[0074] The reservoir and post array assembly is preferably mounted on manually adjustable linear and rotational stages. These stages are used for initial alignment of the print head and post positions and to precisely align the orientation of the post array with the printer's directional axis. After printing, the construct is removed from the micropost array by lifting the construct vertically.

[0075] To prevent breakage of the fiber at the tip of the coaxial needle where a sharp angle is formed during printing, a thin-walled semi-flexible tube is preferably fitted to the outer surface of the outer needle of the coaxial assembly such that about 1 mm of the tube extends beyond the end of the needle. This allows the fiber to be pulled from the needle during printing without bending at a sharp angle at the rough edge of the steel needle. The semi-flexible tube bends slightly to provide a softer surface and more gradual bends while maintaining printing accuracy.

[0076] In a preferred embodiment for printing a cell scaffold construct, the cells are suspended in a fibrinogen solution prepared in Dulbecco's Modified Eagle Medium (DMEM) and printed onto collagen fibers wound around micro posts that are immersed in a thrombin solution also prepared in DMEM. Fibrinogen and thrombin are, respectively, a preferred pair of a clinically relevant biocompatible hydrogel precursor and a crosslinking solution. In this example, the fibrinogen cell suspension rapidly solidifies when extruded into the thrombin crosslinking solution bath during printing to form a stable biocompatible fibrin gel. Fibrin gels have been widely used in FDA-approved medical treatments. Those skilled in the art will appreciate that the mechanical and bioactive properties of fibrin can be adjusted by the user-set concentrations of fibrinogen and thrombin.

[0077] The micropost array printing method described herein can be implemented using various pairs of cell-compatible reagents that form hydrogels when in contact with each other, such as a collagen solution printed in a neutralizing buffer or sodium alginate printed in an ionic crosslinking solution. Other pairs of reagents will be known to those skilled in the art. In some embodiments, hyaluronic acid can be utilized as a cell adhesive that facilitates cell attachment to collagen fibers without using a crosslinking reagent. Other types of bioadhesives or biomaterials, such as Pluronic® F127, gelatin, collagen, alginic acid, silk, etc. can also be used. Those skilled in the art will appreciate that such bioadhesives and biomaterials have appropriate adhesive and sticky properties to enable easy cell attachment to collagen fibers.

[0078] The hardware and firmware of the Folger Tech FT-5 R2 have been improved to facilitate the printing method of the present invention. The commercially available FDM print head has been removed and replaced with a custom extruder print head (Figure 4). Off-the-shelf components for the print head and micro-post array assembly were 3D printed in-house or machined from PLA. All stepper motors and drive pulleys were replaced to improve the resolution in the X, Y, and Z axes. The printer firmware was improved to accommodate these hardware changes.

[0079] A custom Python code was developed to accept user input regarding the design scaffold structure shape and print parameters and output the corresponding g-code file. G-code is a common numerical control programming language typically used to control automated computer-aided manufacturing processes, including 3D printing. User input to the Python code includes the number of X-direction posts for winding the fibers (which determines the width of the print sample), the diameter of the posts, the distance between the rows of the innermost posts (which determines the length of the print sample), the number of fiber layers (which determines the thickness of the printed sample), the vertical distance between fiber layers (layer thickness), the X-direction distance between posts, the collagen fiber width, the extruded filament diameter (which determines the amount of extruded cell suspension), the print head movement speed in the case of linear and circular segments, and the graphical output options for the resulting fiber pattern. The Python code calculates and outputs a g-code file containing all the parameters and motion / extrusion commands for executing the designed print. The parameters and commands are sent to the printer to generate the designed scaffold structure. Repetier-Host is used as the user interface to execute these commands as well as the manual homing, motion, and extrusion commands. The Python code also outputs a scaled graphical representation of the resulting fiber pattern and the print head motion path (example shown in Figure 6). This aids in construct design and troubleshooting.

[0080] Shown below is a description of a preferred scaffold construct printed using the micro - post array technique discussed herein and tested for strength and other properties.

[0081] Figure 9 is a macroscopic image of a printed scaffold construct submerged in a medium within a microwell plate. Figure 10 is a macroscopic image of a printed scaffold construct held with forceps. Figure 11 is a transmitted - light microscope image of a cell - free printed scaffold construct containing a pattern of parallel collagen ultrathin fiber segments. Figure 12 is a transmitted - light microscope image of a cell - printed scaffold construct containing a pattern of parallel collagen ultrathin fiber segments.

[0082] Composite hydrogel and collagen ultrathin fiber scaffold constructs were fabricated using the micro - post array printing method as described herein. Macroscopic views show that the printed structures maintained their designed shape when stored in the medium (Figure 9) and when handled by forceps after removal from the micro - post array (Figure 10). Transmitted - light microscope views of the printed structures immediately after printing, without cells (Figure 11) and including tendon cells harvested from a rat's tail (Figure 12), showed fiber segments that were parallel and evenly spaced. In the case of the cellularized scaffold construct, the cells were evenly distributed between the parallel collagen ultrathin fiber segments and suspended in the fibrin gel.

[0083] Figure 13 is a microscopic image of a scaffold construct pattern formed by winding collagen fibers around a micro - post array after 2 days under standard cell - culture conditions. Figure 14 is a magnification of the microscopic image of Figure 13 by a factor of 2, visually showing the elongated cell morphology indicative of interaction with the hydrogel environment.

[0084] A unique pattern formed by winding fibers around alternating microposts within the array was maintained after 2 days under typical cell culture conditions (Figure 13). The cell morphology appears to become more elongated as the culture exceeds 2 days compared to the substantially spherical morphology of the cells seen immediately after printing (Figure 12), showing the interaction (elongation, adhesion, migration) of cell substances with the bioactive fibrin environment. The printed scaffold construct was found to maintain the original fiber pattern for several weeks of culture. Notably, in the case of the cellularized scaffold construct, it has been observed that the fibrin gel contracts and more tightly surrounds the collagen fiber segments during the extended culture period. This is thought to be due to the cell forces present when culturing tendon cells in a soft, compressible substrate.

[0085] Figure 15A is a fluorescence microscopy image of the scaffold construct after 7 days of culture, showing the cytoskeletal protein vimentin, cell nuclei using DAPI, and the autofluorescence of collagen fibers at 495 nm. Figure 15B is a composite image from 12 fields of view of the printed cell construct after 7 days of culture.

[0086] Figure 16 is a graph showing the results of an alamarBlue assessment of the metabolic activity of scaffold constructs printed with human tendon cells after 7 and 14 days of culture.

[0087] Fluorescent imaging of scaffolds printed with human tendon cells or tendon cells (ZenBio) after 7 days of bioreactor culture under a certain tension shows that cells are distributed relatively evenly overall when visualized by labeling the cytoskeletal protein vimentin (Figure 15). Parallel collagen nanofibers maintaining a distinct pattern formed from the micro-post array can be seen due to the autofluorescence of the fibers at 495 nm. Cells mainly disappear from the left and right ends of the scaffold construct where the fibrin gel moves by a compression clamp that compresses the ends of the scaffold construct for culture in the bioreactor. Furthermore, the scaffold construct was printed with human tendon cells to evaluate the time-dependent cell metabolic activity using alamarBlue assessment while culturing under a certain tension. Samples were taken out of the bioreactor chamber after 7 and 14 days of culture. At each time point, the scaffold constructs were incubated in a 10% alamarBlue solution of tendon cell growth medium for 4 hours, and fluorescence was measured according to the standard protocol. It was found that the metabolic activity of the cellularized scaffold constructs in culture (n = 4) increased from 7 to 14 days of culture (Figure 16), indicating an increase in cell health, activity, and proliferation.

[0088] Composite scaffold constructs were printed with and without cells using the micro-post array method, and their mechanical properties were evaluated. Figure 17 is a graph showing the peak loads of acellular and cell composite collagen nanofiber scaffold constructs printed using the micro-post array method after 7 days of bioreactor culture under a constant tension. Figure 18 is a graph showing the ultimate tensile strength (UTS) of acellular and cell composite collagen nanofiber scaffold constructs printed using the micro-post array method after 7 days of bioreactor culture under a constant tension. Figure 19 is a graph showing the Young's modulus of acellular and cell composite collagen nanofiber scaffold constructs printed using the micro-post array method after 7 days of bioreactor culture under a constant tension.

[0089] Figure 20 is a graph showing the cross-sectional area of cell-free and cell-composite collagen ultra-fine fiber scaffold constructs printed using the micropost array method after 7 days of cultivation in a constant tension bioreactor.

[0090] Figure 21 is a graph showing the breaking strain of cell-free and cell-composite collagen ultra-fine fiber scaffold constructs printed using the micropost array method after 7 days of cultivation in a constant tension bioreactor.

[0091] As a pilot study, two cell-free scaffold constructs were fabricated, and two scaffold constructs with rat tail tendon cells were fabricated. For both the cell-free and cell-containing scaffold constructs, fibrinogen was prepared to 40 mg / mL in DMEM, and the thrombin bath solution was prepared to 0.2265 mg / mL in DMEM. In the case of the cell-containing scaffold constructs, the cells were suspended to 750,000 cells / mL in the fibrinogen solution.

[0092] Both scaffold constructs were cultured at a constant tension in a bioreactor for 7 days. Immediately before the test, the samples were removed from the culture, and the excess medium was removed by wiping without leaving lint. The cross-sectional area of the samples was measured using calipers. Each end was clamped into the grips of a uniaxial tension testing machine (MTS Systems Corporation (Eden Prairie, Minnesota)) with a 100 N load cell. The samples were pulled until they broke at a grip displacement of 0.5 mm / sec, and the load and displacement data were recorded. The Young's modulus (E) was measured in the linear region of the stress-strain curve, and the ultimate tensile strength (UTS) was determined using the maximum recorded load. The peak load, UTS, and Young's modulus for the cell-free and cell-containing scaffold constructs that underwent the breaking test are shown in FIGS. 17, 18, and 19, respectively. From this pilot study, no significant differences were seen between the mechanical properties of the cell-free and cell-containing scaffold constructs.

[0093] Treatment for muscle mass reduction Skeletal tissue injuries, including volumetric muscle loss (VML), are common and often lead to permanent disability and deformity. One aspect of the present invention relates to the preparation and use of a cellularized collagen ultra-fine fiber implant to facilitate functional recovery and regeneration of such skeletal soft tissue. For a collagen ultra-fine fiber scaffold construct similar to the above, clinically relevant cells are controllably positioned along clinically relevant high-strength collagen fibers to biomanufacture a skeletal tissue analog of the damaged tissue with respect to the form and function of recovery. Therefore, the scaffold construction method described herein can be utilized to form a scaffold for use in the treatment of volumetric muscle loss (VML). Shown below are details regarding the description of the VML treatment procedure and testing of such procedures using a scaffold similar to that described above.

[0094] Human mesenchymal stem cells (hMSCs) or rat muscle progenitor cells (MPCs) are bioprinted to generate artificial implants that can be valuable for a variety of utilities, including tendon or muscle regeneration. Mesenchymal stem cells confer excellent potential for enhancing skeletal tissue repair and regeneration due to their immune evasion properties (Ankrum 2014, Zhang 2015), therapeutic effects (Zhang 2015, Jang 2015, Lee 2017), multi-lineage differentiation potential (Pittenger 1999), and availability as a commercially available clinically relevant cell type. Similarly, MPCs have demonstrated prominent therapeutic effects in facilitating functional recovery of volumetric muscle loss in valid animal models (Mintz 2020, Passipieri 2019).

[0095] The present invention is based in part on the discovery that glyoxal cross-linked collagen fibers having high tensile strength can be used as filaments for bioprinting to generate structural, cellular, and mechanical analogs of native tissue in an automated and scalable fabrication process (which was previously a daunting and unrealistic challenge) (Murphy 2014, Murphy 2020).

[0096] The results using implants generated using assembled cell-decorated collagen (AC-DC) showed that the cell orientation and distribution throughout the implant mimicked the cell characteristics of native skeletal tissue. The bio-printed implants according to the present invention approximate and can be adjusted to exceed the strength and stiffness of human skeletal tissue. Further, this implant far exceeded the properties of a general collagen hydrogel.

[0097] The regenerative potential of such implants was also evaluated in vivo in a rodent VML model. A critically sized muscle injury of the hindlimb was created and repaired, and leg torque regenerative potential was measured over 12 weeks. It was found that both acellular and cellular implants promoted functional recovery compared to the non-repaired group, and that the AC-DC implant containing therapeutic muscle progenitor cells promoted the highest degree of recovery.

[0098] Histological analysis and automated image processing of the in vitro transplanted muscle cross-sections revealed an increase in the total number of muscle fibers, the median muscle fiber size, and the cellularization of the injury repaired with the cellularized implant. These studies have the potential to repair a number of difficult skeletal injuries and lead to great promise for advanced bioprinting methods to generate tissue analogs with properties close to native biological and biomechanical properties.

[0099] Example: Functional Recovery in the VML Model In vivo skeletal muscle repair studies were conducted over 12 weeks in a valid rodent VML model using implants similar to those described above. Details of this study are available in K. W. Christensen, J. Turner, K. Coughenour, Y. Maghdouri-White, A. A. Bulysheva, O. Sergeant, M. Rariden, A. Randazzo, A. J. Sheean, G. J. Christ, M. P. Francis, “Assembled Cell-Decorated Collagen (AC-DC) bioprinted implants mimic musculoskeletal tissue properties and promote functional recovery”, published on July 2, 2021, and can be preprinted via bioRxiv at https: / / doi.org / 10.1101 / 2021.06.22.449431. The entire disclosure of this publication is incorporated herein by reference.

[0100] At least 20% of the total muscle weight was removed from the tibialis anterior (TA) muscle of the left hindlimb of Lewis rats (Mintz 2020, Corona 2014). Three repair methods were evaluated head-to-head: an untreated control group, a cell-free implant group receiving repair with an acellular component AC-DC implant, and a cell implant group receiving repair with an AC-DC implant printed with rodent MPCs. Defect generation, initial placement of the implant, suture placement for implant attachment, and fascia replacement are shown in FIGS. 22A-D, respectively. Specifically, FIG. 22A shows the generation of a VML injury with dimensions of approximately 1 cm x 0.7 cm x 0.5 cm and a weight of at least 20% of the total TA weight. FIG. 22B shows the acellular AC-DC implant inserted into the injury site, and in FIG. 22C, the implant is sutured into the injury site, with the arrow indicating the attachment point. FIG. 22D shows the fascia sutured over the injury site to further secure the implant in place.

[0101] All animals recovered after surgery, and there were no signs of infection or death. Across the experimental groups, the body weight of the animals increased similarly over 12 weeks (Figure 22E). This shows the body weights of the animals before surgery, 4 weeks, 8 weeks, and 12 weeks after surgery corresponding to the time points of the functional tests. As shown in Figure 22F, the defect weights measured at the time of surgery showed no statistical difference and represent the defect weights generated for the "no repair", "acellular implant", and "cell implant" (NR, AI, and CI, respectively) experimental groups (p = 0.8, no significant difference). In Figures 22E - I, all data are based on n = 7 per group per time point (*p < 0.05 indicates significance).

[0102] To evaluate muscle recovery after surgery, functional tests were performed in vivo at 4 weeks, 8 weeks, and 12 weeks after defect generation and repair, and before defect generation. Briefly, the rat hindlimb was attached to a motorized footplate, and electrical stimulation was applied to measure maximum isometric torque generation (Mintz 2020, Passipieri 2019, Corona 2014). The mean values are represented as torque normalized by the body weight of the animal at each time point to control for the increase in torque generation due to animal growth (N - mm / kg of body weight). As shown in Figure 22G, the baseline torque generation function before defect generation did not change statistically between treatment groups (p = 0.9, no significant difference). The torque generation after repair is represented as the raw torque (Figure 22H), and the percentage of the baseline torque generation is shown in Figure 22I. The measured torque and the percentage of the baseline torque are shown for 4 weeks, 8 weeks, and 12 weeks after repair. These numbers indicate that functional recovery is facilitated by implant implantation. Both methods show a similar trend with only a small statistically significant variation.

[0103] Most notably, a marked improvement in torque generation ability was observed over 12 weeks for injuries repaired with cell-based MPC-containing implants. At 4 weeks, native torque generation was significantly lower in the acellular and cell implant groups compared to no repair, and the percentage of baseline torque was significantly lower in the cell implant group. This initial decrease in torque generation ability is thought to be due to the early wound healing process or related to the initial tensile properties of the implant. However, no differences between treatment groups were observed by 8 weeks post-surgery.

[0104] At 12 weeks post-repair, in contrast to 4 weeks, native torque generation was found to be significantly higher in the cell implant group compared to the no repair group, and the percentage of baseline torque was significantly higher in both the acellular and cell implant groups, revealing the main trend in functional recovery of VML injury among treatment groups. Furthermore, a marked decline in function became evident over 12 weeks for the non-repaired animals. In contrast, for animals repaired with acellular implants, torque generation was generally consistently maintained, indicating that the presence of cell-free collagen fiber implants reduced the functional deterioration associated with non-repair.

[0105] Notably, detachment of the synergistic muscle during deficit creation removes 20% of torque generation in the anterior compartment (Mintz 2020). Thus, normalized torque will be limited to 85 N-mm / kg across treatment groups (mean 106 N-mm / kg at the baseline). The average functional recovery of the cell-based implant group at 12 weeks was 76% of the maximum theoretical recovery after synergistic muscle detachment, compared to 67% in the acellular group and 57% in the non-repair group. Furthermore, 3 out of 7 animals receiving repair with cell implants were observed to have a functional recovery higher than 87%, and 1 animal recovered to nearly the maximum theoretical recovery (99%) compared to the pre-injury level.

[0106] After evaluation of the in vivo functional recovery at week 12, the isolated TA muscles were harvested for morphological and histological examinations. The gross morphology of those repaired by acellular and cellular AC-DC implants appeared more similar to the control muscle than the unrepaired group that showed a convex notch at the injury site. More fascia was also noted in the repair groups. The difference between the implant and the surrounding tissue was not obvious, showing tissue ingrowth or absorption around the collagen fiber implant. The muscles were separated across the muscle bellies and processed with H&E staining. Representative images for each experimental group are shown in FIGS. 23A to D.

[0107] In FIGS. 23A to D, representative H&E images of the tibialis anterior (TA) muscle are shown for the experimental groups of (A) uninjured control, (B) unrepaired, (C) acellular implant, and (D) cellular implant, at 12 weeks later. The black dashed line indicates the approximate area of defect generation. The green dashed ellipse indicates the AC-DC implant position.

[0108] In FIGS. 23E to F, enlarged views of the (E) acellular implant and (F) cellular implant positions are shown, together with enlarged windows showing intracellular growth and muscle fiber formation at the cellular implant position (yellow dashed ellipse). All scale bars in FIG. 23 are 1 mm unless otherwise noted.

[0109] Similar to the macroscopic examination, the non-repaired group presented a distinct depression at the injury site, indicating insufficient tissue regeneration (Figure 23B). In contrast, animals repaired with acellular and cellular implants showed greater tissue filling and an even cross-section similar to that of the non-injured control, thus demonstrating an aesthetic improvement. The remaining collagen fibers from the implant appeared within the injury site as somewhat round, dark pink cross-sections with a diameter of approximately 100 μm. Intracellular growth was visible within and around the implant (Figures 23E and 23F). The fiber cross-sections were more evident in the acellular implant group than in the cellular implant group, which probably indicates an increased rate of fiber absorption in the case of the cellularized implant. In the case of injuries repaired with the cellular AC-DC implant, the presence of new muscle fibers at the implant site was noted (Figure 23F).

[0110] Images of more magnified H&E sections further revealed new muscle fibers and angiogenesis within the implant area of Figure 24, together with new collagen deposition, as quantitatively shown by Masson's trichrome staining around the implant area of Figure 25. In Figure 24, it was found that prominent new muscle fibers labeled "MF" grew within the AC-DC implant, distinguishable from the implant collagen fibers (labeled "*"). In the implant area, adjacent blood vessels were also prominent, indicated by black arrows. Nerve bundles are denoted by "N". In Figure 25, sections of each group showed the presence of collagen inside and around the implant area and the defect area.

[0111] The additional sections of the TA muscle belly were processed for analysis using SMASH semi - automated muscle fiber analysis software as shown in FIGS. 26A - K. In FIGS. 26A - D, representative laminin - stained sections of the TA muscle are shown by dashed ellipses indicating the approximate area of injury for the experimental groups of (A) non - injured control, (B) no repair, (C) acellular implant, and (D) cellular implant. In FIGS. 26E - H, the colored output from the software identifies individual muscle fibers within the sections corresponding to FIGS. 26A - D respectively. FIG. 26I shows the total fiber count, FIG. 26J shows the median fiber cross - sectional area (FCSA), and FIG. 26K shows the product of the fiber number and FCSA for the experimental groups of non - injured control (Ctrl), no repair (NR), acellular implant (AI), and cellular implant (CI). All scale bars in FIG. 26 are 1 mm. The data presented are based on n = 7 per group per time point, and *p < 0.05 indicates significance.

[0112] Referring to FIG. 26, laminin and phalloidin 488 staining identifies the outlines of muscle fibers throughout the section (FIGS. 26A - D), and SMASH analysis adds coloration to distinguish individual fibers when viewed (FIGS. 26E - H). Analysis of the total fiber count does not result in a significant difference among the groups of non - injured control, no repair, acellular implant, and cellular implant (FIG. 26I). However, the median fiber cross - sectional area (FCSA) in muscle sections repaired with acellular and cellular AC - DC implants is significantly larger than that of the no - repair group and not significantly different from that of the non - injured control (FIG. 26J). The cellularized implant and control groups have p - values of 0.0007 and 0.0002 respectively, showing the greatest difference from the no - repair group.

[0113] Multiplying the total fiber number by the median fiber cross - sectional area gives the total muscle fiber cross - sectional area (FIG. 26K). Again, this product shows no significant difference between the non - injured control and the injury repaired with acellular and cellular implants after 12 weeks of survival, supporting the ability of AC - DC implants to facilitate an increase in total muscle fiber area.

[0114] Briefly, a method of treating volumetric muscle loss (VML) can include securing a scaffold construct formed of a plurality of collagen ultrafine fibers arranged in an organized sheet-like configuration and coated with a biocompatible solution within the VML wound site. In some instances, securing the scaffold construct within the VML wound can include suturing two or more portions of the scaffold construct to muscle tissue within the VML wound site. For example, the four corners of the scaffold construct can be sutured within the wound site. Additionally, in some instances, the method can further include replacing the fascia overlying the scaffold construct after securing the scaffold construct within the VML wound site.

[0115] In some instances, a scaffold construct implanted within a given VML wound site can include a plurality of sheet-like layers of collagen ultrafine fibers. The number of layers used can vary depending on the depth of the wound site. Similarly, the overall size of the scaffold construct used can vary depending on the size of the wound site.

[0116] In some instances, the biocompatible solution coating the collagen ultrafine fibers can be hyaluronic acid. In some instances, the biocompatible solution is a cell suspension. In such instances, the cell suspension can include muscle progenitor cells (MPCs). The cell suspension can include about 4,000,000 cells / mL. Other concentrations can be used instead to form the scaffold construct composition.

[0117] The scaffold construct implanted to treat VML can have mechanical properties that substantially approximate or exceed those of a human tendon. The scaffold construct can have an average ultimate tensile strength (UTS), tensile modulus, and elongation at break that substantially approximate or exceed those of a human tendon.

[0118] Preferred Scaffold Construct The preferred embodiments of the scaffold structures according to the present invention have individual length and width dimensions in the range of about 1 mm to 10 mm, depending on the size and shape of the damaged area scheduled for repair. Preferably, the length and width of the scaffold structures used for VML are individually about 2 cm to 9 cm, 3 cm to 8 cm or 4 cm to 7 cm. Another embodiment can be standardized to a configuration of 4 cm (width) × 10 cm (length), more preferably 6 cm × 10 cm, 8 cm × 10 cm and 10 cm × 10 cm.

[0119] The thickness of the scaffold structure according to the present invention may be limited by the ability of the surrounding tissue of the receiving site through which blood vessels are passed through the implant so that the cells attached to the construct remain viable. Thus, the preferred scaffold structures have a depth (thickness) of about 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm and 5 mm. In a preferred construct, the implant is produced by forming about 2 to 8 layers of distributed and coated fibers.

[0120] For implantation, a plurality of scaffold structures can be used by the surgeon in the repair of VML. These constructs can be sequentially stacked or arranged along the area where the repair is scheduled.

[0121] The print head can be configured to produce a scaffold structure and the spacing between the fibers can be adjusted as described above. For example, the spacing between the fibers ranges on average from about 0 (i.e., the fibers are directly adjacent) to about 1 mm. In a preferred embodiment, the average spacing between the fibers is about 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 microns.

[0122] The cell suspension according to the present invention is formulated at a density sufficient to effectively coat the dispensed fibers of the scaffold construct. Preferred cell densities range from 0 to 10 million cells / mL as described above. Preferred cell suspension densities are approximately 100,000, 200,000, 400,000, 600,000, 800,000, 1 million, 1.5 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, and 10 million cells / mL. In the case of a preferred embodiment, approximately 0.1 to 10 microliters of cell suspension are extruded per millimeter of the drawn fiber.

[0123] In the case of a cell population adhering to the scaffold construct, the preferred number of cells on the construct is from about 100,000 to 1 million cells per implant or more than 1 million cells per implant. Preferred ranges are from about 200,000 to 900,000, 300,000 to 800,000, 400,000 to 700,000, and 500,000 to 600,000 cells per implant.

[0124] As described above, the preferred hydrogel must rapidly stabilize and begin to solidify within seconds of contact with the crosslinking solution.

[0125] Although various embodiments of the present invention have been described, the description is illustrative rather than limiting, and it will be apparent to those skilled in the art that many further embodiments and implementations within the scope of the present invention are possible. Many possible combinations of features are shown in the accompanying drawings and discussed in this detailed description, but many other combinations of the disclosed features and methods are within the scope of the ordinary skill level in this field. Any feature of any embodiment can be combined with or used in place of any other feature or element of any other embodiment, unless particularly restricted. Therefore, it should be understood that any of the features illustrated and / or discussed in this disclosure can be implemented in suitable combinations. Therefore, the embodiments should not be limited except in consideration of the claims and their equivalents. Also, various improvements and changes can be made by those skilled in the art as desired, and these also remain within the scope of the claims.

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Claims

1. A scaffold structure, comprising: a biocompatible hydrogel; and at least one ultra-fine fiber strand of a biomaterial wound to form a plurality of ultra-fine fiber segments arranged in an organized configuration and adjacent to each other. The scaffold structure.

2. The scaffold structure according to claim 1, wherein in the organized configuration, at least some of the plurality of ultra-fine fiber segments are aligned substantially parallel to each other.

3. The scaffold structure according to claim 1, wherein in the organized configuration, at least some of the plurality of ultra-fine fiber segments are arranged obliquely to each other.

4. The scaffold structure according to claim 1, wherein the plurality of ultra-fine fiber segments includes a plurality of layers of ultra-fine fibers stacked to form a three-dimensional structure.

5. The scaffold structure according to claim 1, wherein the biomaterial is selected from the group consisting of collagen, elastin, hyaluronic acid, fibrinogen, fibrin, fibronectin, silk, alginic acid, and Pluronic®.

6. The scaffold structure according to claim 5, wherein the biomaterial is collagen.

7. The scaffold structure according to claim 6, wherein the biocompatible hydrogel further includes cells distributed therein.

8. The scaffold structure according to claim 1, wherein the biocompatible hydrogel is crosslinked.

9. An apparatus for making a scaffold structure, the apparatus comprising: a first array of microposts; and a second array of microposts disposed spaced apart from the first array of microposts. The microposts are configured to receive an ultra-fine fiber strand to form a plurality of segments, and at least some of the plurality of segments are arranged in a substantially aligned configuration. The apparatus.

10. The apparatus according to claim 9, wherein the microposts are arranged such that the ultra-fine fiber strands received by the apparatus form a plurality of segments, and at least some of them are aligned substantially parallel to each other.

11. The apparatus according to claim 9, wherein the microposts are configured to receive the ultra-fine fiber strand to form a plurality of ultra-fine fiber segments, and at least some of the ultra-fine fibers are arranged in a plurality of stacked layers to form a three-dimensional structure.

12. The apparatus according to claim 9, wherein the ultra-fine fiber is a biomaterial selected from the group consisting of collagen, elastin, hyaluronic acid, fibrinogen, fibrin, fibronectin, silk, alginic acid, and Pluronic (registered trademark).

13. The apparatus according to claim 9, wherein the micropost is disposed in a container configured to receive a fluid reservoir capable of forming the scaffold structure therein.

14. Furthermore, it includes a 3D printing device configured to distribute ultra-fine fiber strands, The apparatus according to claim 9, wherein the container and the micropost are translatable with respect to the 3D printing device.

15. Furthermore, it includes a 3D printing device configured to distribute ultra-fine fiber strands, The apparatus according to claim 9, wherein the container and the micropost are rotatable with respect to the 3D printing device.

16. Furthermore, it includes a 3D printing device configured to distribute ultra-fine fiber strands, The apparatus according to claim 9, wherein the 3D printing device includes a coaxial needle configured to distribute the ultra-fine fiber strands into a hydrogel sheath.

17. The coaxial needle is an inner conduit and an outer conduit, the inner conduit defines an inner lumen configured to distribute the ultra-fine fiber strands, an annular outer lumen is defined between the outer conduit and the inner conduit, and the annular outer lumen is configured to distribute the hydrogel sheath, the inner conduit and the outer conduit, and a flexible extension tube extending from the tip of the outer conduit, The apparatus according to claim 16, comprising.

18. A method of creating a scaffold structure, comprising: distributing ultra-fine fiber strands into a biocompatible hydrogel sheath; and wrapping the ultra-fine fiber strands around a plurality of microposts to form a plurality of segments covered by the biocompatible hydrogel and arranged in an organized configuration. The method comprising.

19. The method according to claim 18, wherein wrapping the ultra-fine fiber strands to form the organized configuration of the segments includes arranging at least some of the segments substantially in alignment with each other.

20. The method according to claim 19, wherein winding the ultra-fine fiber strands around a plurality of the micro-posts forms a plurality of substantially parallel segments. **Claim 21** The method according to claim 19, wherein arranging at least some of the segments of the ultra-fine fibers substantially aligned with each other includes arranging at least some of the plurality of ultra-fine fibers aligned on the same plane with each other. **Claim 22** The method according to claim 19, wherein arranging at least some of the segments of the ultra-fine fibers substantially aligned with each other includes arranging at least some of the ultra-fine fibers in a plurality of layers stacked to form a three-dimensional structure. **Claim 23** The method according to claim 18, wherein the ultra-fine fibers are a biomaterial selected from the group consisting of collagen, elastin, hyaluronic acid, fibrinogen, fibrin, fibronectin, silk, alginic acid, and Pluronic (registered trademark). **Claim 24** The method according to claim 23, wherein the biomaterial is collagen. **Claim 25** The method according to claim 24, wherein winding the ultra-fine fiber strands of the collagen around the micro-posts is carried out in a container containing a bath of a cross-linking solution. **Claim 26** The method according to claim 25, wherein the cross-linking solution is a thrombin solution. **Claim 27** The method according to claim 24, wherein winding the ultra-fine fiber strands of the collagen around the micro-posts is carried out in a container containing a bath of a cross-linking solution. **Claim 28** The method according to claim 27, wherein the cross-linking solution contains factor XIII. **Claim 29** The method according to claim 27, further including translating and rotating the container containing the bath of the cross-linking solution and the micro-posts to reposition them in a known orientation with respect to a 3D printing device. **Claim 30** The biocompatible hydrogel is a cell hydrogel, The method according to claim 18, further including maintaining the ultra-fine fibers and the hydrogel scaffold construct under cell culture conditions for one day or several days. **Claim 31** The method according to claim 18, wherein winding the ultra-fine fiber strands of the collagen around a plurality of micro-posts includes winding a plurality of layers of collagen ultra-fine fiber strands around the plurality of micro-posts to form a three-dimensional structure. **Claim 32** A method of treating volumetric muscle loss (VML) comprising anchoring the scaffold construct according to claim 1 within the VML wound site.

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