Rotating frame apparatus and biocompatible scaffold construct

Abstract

To provide a rotating frame apparatus and biocompatible scaffold construct.SOLUTION: A biocompatible scaffold construct includes a plurality of collagen fiber strands, a first portion of which have been coated by a first biocompatible solution and, optionally, a second portion of which have been coated by a second biocompatible solution different than the first biocompatible solution. The coatings may include cells. Furthermore, the scaffold is constructed on rotating frame collectors.SELECTED DRAWING: Figure 7

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 the invention.

[0002] Cross-Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 119,628, filed November 30, 2020, entitled "Rotating Frame Apparatus and Composite Biological Scaffold". The entire disclosure of this U.S. Provisional Patent Application is incorporated herein by reference. This application also 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 this U.S. Provisional Patent Application 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 treatments often result in impaired function of the damaged tissue and a high rate of re-injury. Therefore, many tissue engineering techniques for manufacturing grafts such as scaffolds have been developed for the purpose of promoting the regeneration of functional native tissue. It is particularly important to reproduce the biochemical, morphological, and functional characteristics of the target tissue.

[0004] 3D bioprinting, a form of additive manufacturing, is often used to create scaffolds for regenerative medicine applications. 3D bioprinting allows for the precise manipulation of cells and biomaterials to create designed, often complex 3D shapes. However, bioprinting techniques typically utilize soft hydrogels as the main structural material [Mandrycky 2016]. The mechanical properties of such hydrogels are often orders of magnitude lower than those of native ligament and tendon tissues. Therefore, typical bioprinting techniques cannot fully replicate the functional properties of musculoskeletal tissues and cannot create load-bearing scaffolds for tissue repair.

[0005] To improve the mechanical properties of the printed parts, hybrid bioprinting techniques that incorporate thermoplastic polymers with hydrogels have been developed [Merceron 2015]. However, many of the printed synthetic materials still have limited mechanical strength and may have an adverse effect on injury healing and tissue regeneration.

[0006] To address such challenges, numerous fiber-based tissue engineering techniques using strong natural biomaterials such as collagen have been developed [Tamayol 2013]. Such techniques are based on the well-established clinical use of textiles but incorporate additional means for manufacturing cellularized scaffolds.

[0007] In addition, the use of therapeutic cells has the potential to improve the treatment of genetic, degenerative, inflammatory, and traumatic musculoskeletal diseases [O´Keefe 2019]. This may lead to improved healing rates and overall tissue regeneration and functional recovery compared to the case of using only a biomaterial scaffold. This is particularly true for ligament-like and tendon-like tissues where passive intracellular growth may be limited in a hypoxic and avascular environment.

[0008] In some techniques for manufacturing a biomaterial scaffold, premanufactured fibers produced by traditional textile manufacturing processes such as weaving, knitting, and braiding are utilized. Biomaterial fibers can be produced as raw materials for such processes by wet spinning, microfluidic spinning, biospinning, interfacial complex formation, and melt spinning [Tamayol 2013]. Using weaving, a polymer scaffold with a designed porosity, morphology, and geometry can be created by interweaving two sets of warp or weft threads at right angles [Abrahamsson 2010]. Knitting is a technique commonly used to manufacture surgical meshes, forming a 3D shape from intertwined threads or yarns within a series of interconnected loops [Sahoo 2007]. Braiding can form complex biomaterial structures or patterns by intertwining multiple fiber strands [Walters 2012]. Additionally, relatively simple shapes such as bundles of parallel fibers tied together with sutures are created by manual assembly [Gentleman 2006].

[0009] Scaffold manufacturing processes that generate a biomaterial scaffold using fibers produced as an integral part of the scaffold include electrospinning, wet spinning, and direct writing. In such techniques, processes such as solvent evaporation, in-solution polymerization, temperature-based recrystallization, etc. are utilized to form ultrafine fiber scaffolds from biomaterial solutions. For example, electrospinning is used to form randomly oriented or aligned polymer fiber mats with biomimetic surface patterns to induce tissue formation [Mauck 2009]. In addition to using wet spinning to form fibers as raw materials, it can also be utilized to create a scaffold during the fiber formation process by collecting the fibers on a rotating mandrel [Kaiser 2019]. Direct writing can form fiber-based scaffolds with excellent control over porosity, fiber size, and fiber orientation [Wu 2015].

[0010] However, the post-fabrication cell seeding process required to create cellular scaffolds using weaving, knitting, braiding, electrospinning, wet spinning, and direct writing can be subject to human variability and may strongly depend on the macroscopic scale shape and porosity of the biomaterial scaffold. For example, if the pore size is small, especially in the case of thick scaffolds or scaffolds with complex 3D shapes, cell infiltration during seeding may be limited. Conversely, in scaffolds with high porosity, it may become difficult to uniformly hold the seeded cells throughout. Since cell seeding depends on the microscale and macroscopic scale shapes of the scaffold, control of the overall cell distribution may be limited, especially when creating heterogeneous tissues containing various cell populations within the design area.

[0011] To address the challenges of seeding cells onto prefabricated scaffolds, various fiber-based techniques have been developed that directly manipulate cells or cell-containing materials during the scaffold manufacturing process [Tamayol 2013]. Compared to techniques that require cell seeding, such techniques may result in cellular scaffolds with improved consistency and control of the overall cell distribution. Polyester threads are coated with a cell-containing hydrogel and wound around a cylindrical mandrel to form a 3D tubular structure [Liberski 2011]. A microfluidic system is used to form core-shell hydrogel fibers that may be transplanted without forming a secondary scaffold structure and that contain cells [Sukimoto 2011]. A centimeter-scale biofabric is formed from core-shell hydrogel fibers containing similar cells using a micro-weaving technique [Onoe 2011]. However, since the mechanical strength of hydrogel fibers containing cells is low, the capabilities of the hydrogel fibers are limited when processed using conventional fiber manufacturing techniques [Onoe 2011]. In a direct writing technique that is almost the same as typical hydrogel-based bioprinting, a cell suspension is crosslinked within a print head to form a scaffold containing cells from a fibrous extrudate [Ghorbanian 2014]. Overall, since the mechanical properties of scaffolds formed using such hydrogel-based techniques are inferior, their applicability as load-bearing scaffolds for the treatment of musculoskeletal tissue injuries is limited.

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

SUMMARY OF THE INVENTION

[0013] In one aspect, the present disclosure is directed to a scaffold construct comprising a plurality of collagen ultrafine fiber strands fabricated on a rotating frame. Here, the strands are coated with a biocompatible solution in which cells are uniformly suspended, and the strands have properties comparable to those of living tissue.

[0014] The scaffold construct may include a first biocompatible solution, a second biocompatible solution different from the first biocompatible solution, and a plurality of collagen ultrafine fiber strands substantially aligned. A first portion of the collagen ultrafine fiber may be coated with the first biocompatible solution, and a second portion of the collagen ultrafine fiber may be coated with the second biocompatible solution.

[0015] In another aspect, the present disclosure is directed to an apparatus for creating a scaffold construct. The apparatus includes a delivery device configured to dispense collagen ultrafine fiber strands through a needle, a substantially planar frame, and a solution manifold including a first well configured to receive a first biocompatible solution, the solution manifold being disposed between the delivery device and the substantially planar frame, and a rotation device configured to rotate the substantially planar frame about an axis of rotation. The apparatus may be configured to dispense collagen ultrafine fiber strands through the needle and the solution manifold by rotation of the substantially planar frame, thereby coating the collagen ultrafine fiber strands with the first biocompatible solution within the solution manifold and winding the collagen ultrafine fiber strands around the substantially planar frame.

[0016] In another aspect, the present disclosure is directed to a method of creating a scaffold structure. The method may include attaching collagen ultra-fine fiber strands to a substantially planar frame and rotating the substantially planar frame about a rotation axis to wind the collagen ultra-fine fiber strands around the substantially planar frame. Winding the collagen ultra-fine fiber strands around the substantially planar frame causes the collagen ultra-fine fiber strands to be drawn from a delivery device, thereby pulling the collagen ultra-fine fiber strands through a needle and drawing the collagen ultra-fine fiber strands through a first well of a solution manifold filled with a first biocompatible solution.

[0017] Other systems, methods, features and advantages of the embodiments will be or become apparent to one with ordinary skill in the art upon examination of the following drawings and detailed description. 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 intended to be protected by the claims.

[0018] The following groups of clauses identify various embodiments of the disclosure. It will be understood that features of any group of clauses may be combined with and / or exchanged with one or more features of other groups of clauses.

[0019] Clause - 2 Solution Approach Clause 1 A scaffold structure, In a scaffold structure comprising a plurality of collagen ultra-fine fibers, A scaffold structure that coats a first portion of the collagen ultra-fine fibers with a first biocompatible solution and coats a second portion of the collagen ultra-fine fibers with a second biocompatible solution.

[0020] Clause 2 The scaffold structure according to clause 1, wherein the plurality of collagen microfibers are substantially parallel to each other.

[0021] Clause 3 The scaffold structure according to clause 1, wherein the plurality of collagen ultra-fine fibers include a plurality of layers of collagen ultra-fine fibers.

[0022] Clause 4 The scaffold structure according to clause 3, wherein the collagen ultra-fine fibers of adjacent layers are substantially aligned with each other.

[0023] Clause 5 The scaffold structure according to clause 1, wherein at least one of the first biocompatible solution and the second biocompatible solution contains cells suspended in a hyaluronic acid solution.

[0024] Clause 6 The scaffold structure according to clause 5, wherein the cells suspended in the hyaluronic acid solution are prepared in a cell culture medium that provides a cell-compatible environment.

[0025] Clause 7 The first portion of the collagen ultra-fine fibers coated with the first biocompatible solution is disposed in the first longitudinal portion of the scaffold, The second portion of the collagen ultra-fine fibers coated with the second biocompatible solution is discretely disposed in the second longitudinal portion of the scaffold, The scaffold structure according to clause 1, wherein the first longitudinal portion is different from the second longitudinal portion.

[0026] Clause 8 An apparatus for creating a scaffold structure, the apparatus comprising: A delivery device configured to dispense a collagen ultra-fine fiber strand through an orifice; A rotatable frame; A solution manifold including a first well configured to receive a first biocompatible solution, the solution manifold being disposed between the delivery device and the rotatable frame; A rotation device configured to rotate the rotatable frame about a rotation axis. The device is configured to draw out collagen ultrafine fiber strands through an orifice and a solution manifold by rotation of a rotatable frame, thereby coating the collagen ultrafine fiber strands with a first biocompatible solution in the solution manifold and winding the collagen ultrafine fiber strands around the rotatable frame.

[0027] Clause 9 The device according to clause 8, wherein the delivery device and the rotatable frame are configured to translate parallel to each other along a rotation axis to wind the collagen ultrafine fiber strands around different portions of the rotatable frame.

[0028] Clause 10 The device according to clause 8, wherein a first well of the solution manifold includes a first opening configured to allow passage of the collagen ultrafine fiber strands.

[0029] Clause 11 The device according to clause 10, wherein the solution manifold includes a second well configured to receive a second biocompatible solution different from the first biocompatible solution, and the second well includes a second opening configured to allow passage of the collagen ultrafine fiber strands.

[0030] Clause 12 The device according to clause 11, wherein the solution manifold includes a slot connecting the first opening and the second opening.

[0031] Clause 13 The device according to clause 12, wherein the delivery device is configured to translate the solution manifold in a direction parallel to the slot to select through which of the first well and the second well the collagen ultrafine fiber strands are drawn out.

[0032] Clause 14 A method of creating a scaffold structure, comprising: attaching collagen ultrafine fiber strands to a rotatable frame; In a method that includes rotating a rotatable frame about a rotation axis to wind a collagen ultra-fine fiber strand around the rotatable frame, a method of drawing a collagen ultra-fine fiber strand from a delivery device by winding the collagen ultra-fine fiber strand around the rotatable frame, thereby distributing the collagen ultra-fine fiber strand through an orifice and drawing the collagen ultra-fine fiber strand through a first well of a solution manifold filled with a first biocompatible solution.

[0033] Clause 15 The method according to clause 14, further comprising translating the rotatable frame and the dispensing device relative to each other along the rotation axis to wind the collagen ultra-fine fiber strand around different portions of the rotatable frame.

[0034] Clause 16 The method according to clause 14, wherein drawing the collagen ultra-fine fiber strand through the first well of the solution manifold includes drawing the collagen ultra-fine fiber strand through a first opening of the first well.

[0035] Clause 17 The method according to clause 16, further comprising selectively drawing the ultra-fine fiber strand through a first well containing a first biocompatible solution and a second well of a solution manifold containing a second biocompatible solution different from the first biocompatible solution.

[0036] Clause 18 The second well includes a second opening configured to allow the collagen ultra-fine fiber strand to pass through, the solution manifold includes a slot connecting the first opening and the second opening, The method according to clause 17, further comprising translating the solution manifold in a direction parallel to the slot to select through which of the first well and the second well the collagen ultra-fine fiber strand is drawn.

[0037] Article 19 The biocompatible solution contains one or more additives coated on the collagen ultra-fine fiber strands, The one or more additives are selected essentially from the group consisting of growth factors, antibiotics, and small molecule pharmaceuticals, according to the method described in Article 14.

[0038] Article 20 A composite scaffold comprising a plurality of elongated collagen fiber strands fabricated on a rotating frame, The strands have uniformly suspended mammalian cells, preferably have connective tissue cells including tendon cells, and are coated in a hyaluronic acid solution preferably having human and non-human mammalian cells, The strands have a cross-sectional density comparable to that of native muscle tissue of a mammal or human, composite scaffold.

[0039] Article - Single Solution Approach Article 1 A scaffold construct comprising a plurality of collagen ultra-fine fibers arranged in an organized configuration and coated with a biocompatible solution, The collagen ultra-fine fibers are arranged in a continuous loop, scaffold construct.

[0040] Article 2 The plurality of collagen ultra-fine fibers are substantially parallel to each other, the scaffold construct according to Article 1.

[0041] Article 3 The biocompatible solution contains cells suspended in a hyaluronic acid solution, the scaffold construct according to Article 1.

[0042] Article 4 The scaffold construct has mechanical properties approximating or exceeding those of human musculoskeletal tissue, the scaffold construct according to Article 1.

[0043] Article 5 The scaffold structure is the scaffold structure according to clause 4, having mechanical properties that approximate or exceed those of human tendon and ligament tissues.

[0044] Clause 6 The scaffold structure is the scaffold structure according to clause 5, having an ultimate tensile strength (UTS), tensile modulus, and breaking point strain that approximate or exceed those of the human anterior cruciate ligament (ACL), human supraspinatus tendon, and human muscle.

[0045] Clause 7 An apparatus for creating a scaffold structure, the apparatus comprising: A delivery device configured to dispense collagen ultrafine fiber strands through an orifice; A rotatable frame; A strand coating system configured to coat the collagen ultrafine fiber strands with a biocompatible solution during dispensing of the strands, the solution strand coating system being disposed between the delivery device and the rotatable frame; A rotation device configured to rotate the rotatable frame about a rotation axis, in an apparatus comprising: The apparatus is configured such that rotation of the rotatable frame draws the collagen ultrafine fiber strands through the orifice, thereby coating the collagen ultrafine fiber strands with the biocompatible solution and winding the collagen ultrafine fiber strands around the rotatable frame.

[0046] Clause 8 The apparatus according to clause 7, wherein the delivery device and the substantially planar frame are configured to translate relative to each other along the rotation axis to wind the collagen ultrafine fiber strands around different portions of the substantially planar frame.

[0047] Clause 9 The rotatable frame comprises at least two parallel beams around which the collagen ultra-fine fiber strands can be wound, thereby forming a scaffold structure configured as a continuous loop, the apparatus according to clause 7.

[0048] Clause 10 The rotatable frame is spring-biased to maintain the continuous loop of the scaffold structure under tension, the apparatus according to clause 9.

[0049] Clause 11 The rotatable frame includes removable end pieces that fix the parallel beams to each other, and by removing the removable end pieces, the continuous loop scaffold structure can be slipped off the parallel beams, the apparatus according to clause 9.

[0050] Clause 12 The rotatable frame is configured to be immersed in the cell culture solution, the apparatus according to clause 7.

[0051] Clause 13 The delivery device is configured to simultaneously dispense a plurality of strands of collagen ultra-fine fibers, the apparatus according to clause 7.

[0052] Clause 14 A method of creating a scaffold structure, comprising: attaching collagen ultra-fine fiber strands to a rotatable frame; and rotating the rotatable frame about a rotation axis to wind the collagen ultra-fine fiber strands around the rotatable frame. A method of drawing collagen ultra-fine fiber strands from a delivery device by winding the collagen ultra-fine fiber strands around a rotatable frame, thereby dispensing the collagen ultra-fine fiber strands through an orifice and drawing the collagen ultra-fine fiber strands through a biocompatible solution.

[0053] Clause 15 The method according to clause 14, further comprising moving a substantially planar frame and a dispensing device parallel to each other along a rotation axis to wind collagen ultra-fine fiber strands around different parts of the rotatable frame.

[0054] Clause 16 The method according to clause 14, wherein the rotatable frame includes at least two parallel beams around which collagen ultra-fine fiber strands can be wound, thereby forming a scaffold structure configured as a continuous loop.

[0055] Clause 17 The method according to clause 16, further comprising removing removable end pieces that fix the parallel beams to each other and sliding the scaffold structure off the parallel beams.

[0056] Clause 18 The method according to clause 14, further comprising immersing the rotatable frame wound with collagen ultra-fine fibers in a cell culture solution.

[0057] Clause 19 The method according to clause 14, wherein a plurality of collagen ultra-fine fibers are simultaneously drawn out by the rotation of the rotatable frame.

[0058] Clause 20 The biocompatible solution includes one or more additives coated on the collagen ultra-fine fiber strands, The method according to clause 14, wherein the one or more additives are essentially selected from the group consisting of growth factors, antibiotics, and small molecule pharmaceuticals.

[0059] Clause - In-liquid Rotating Frame Technique Clause 1 A scaffold structure comprising a plurality of collagen ultra-fine fibers arranged in an organized configuration and coated with a biocompatible hydrogel, The scaffold structure, wherein the collagen ultra-fine fibers are arranged in a continuous loop.

[0060] Clause 2 The biocompatible hydrogel is the scaffold construct according to item 1, which contains cells.

[0061] Item 3 The biocompatible hydrogel is the scaffold construct according to item 1, which contains a fibrin gel.

[0062] Item 4 The scaffold construct is the scaffold construct according to item 1, which has mechanical properties approximating or exceeding those of human skeletal tissues.

[0063] Item 5 The scaffold construct is the scaffold construct according to item 4, which has mechanical properties approximating or exceeding those of human tendon and ligament tissues.

[0064] Item 6 The scaffold construct is the scaffold construct according to item 5, which has an ultimate tensile strength (UTS), tensile elastic modulus, and breaking point strain approximating or exceeding those of human anterior cruciate ligament (ACL), human supraspinatus tendon, and human muscle.

[0065] Item 7 An apparatus for creating a scaffold construct, the apparatus comprising: A delivery device configured to distribute collagen ultra-fine fiber strands through an orifice; A rotatable frame; A strand coating system configured to coat the collagen ultra-fine fiber strands with a first biocompatible solution during distribution of the strands, the solution strand coating system being disposed between the delivery device and the rotatable frame; A rotation device configured to rotate the rotatable frame about a rotation axis. The device is configured to draw out collagen ultra-fine fiber strands through an orifice by rotation of a rotatable frame, thereby coating the collagen ultra-fine fiber strands with a first biocompatible solution and winding the collagen ultra-fine fiber strands around the rotatable frame. The device further includes a tank configured to contain a second biocompatible solution into which the rotatable frame can be immersed while winding the collagen ultra-fine fiber strands around the rotatable frame.

[0066] Clause 8 The device according to clause 7, wherein the delivery device and the rotatable frame are configured to translate parallel to each other along the axis of rotation in order to wind collagen ultra-fine fiber strands around different portions of the rotatable frame.

[0067] Clause 9 The device according to clause 7, wherein the rotatable frame includes at least two parallel beams around which the collagen ultra-fine fiber strands can be wound, thereby forming a scaffold structure configured as a continuous loop.

[0068] Clause 10 The device according to clause 9, wherein the rotatable frame is spring-biased to maintain a continuous loop of the scaffold structure under tension.

[0069] Clause 11 The device according to clause 9, wherein the rotatable frame includes removable end pieces that fix the parallel beams to each other, and by removing the removable end pieces, the continuous loop scaffold structure can be slipped off the parallel beams.

[0070] Clause 12 The device according to clause 7, wherein the rotatable frame is configured to be immersed in a cell culture solution.

[0071] Clause 13 The device according to clause 7, wherein the delivery device is configured to simultaneously dispense a plurality of strands of collagen ultra-fine fibers.

[0072] Clause 14 A method of creating a scaffold structure, attaching a collagen ultra-fine fiber strand to a rotatable frame, and rotating the rotatable frame about a rotation axis to wind the collagen ultra-fine fiber strand around the rotatable frame, in a method comprising: By winding the collagen ultra-fine fiber strand around the rotatable frame, the collagen ultra-fine fiber strand is drawn out from a delivery device, thereby distributing the collagen ultra-fine fiber strand through an orifice and drawing the collagen ultra-fine fiber strand through a first biocompatible solution, wherein the rotatable frame is immersed in a second biocompatible solution while winding the collagen ultra-fine fiber strand around the rotatable frame, The first biocompatible solution is mixed with the second biocompatible solution to form a biocompatible hydrogel, the method.

[0073] Clause 15 The method according to clause 14, further comprising moving the substantially planar frame and the dispensing device parallel to each other along the rotation axis to wind the collagen ultra-fine fiber strand around different portions of the rotatable frame.

[0074] Clause 16 The method according to clause 14, wherein the rotatable frame includes at least two parallel beams around which the collagen ultra-fine fiber strand can be wound, thereby forming a scaffold structure configured as a continuous loop.

[0075] Clause 17 The first biocompatible solution contains thrombin and the second biocompatible solution contains fibrinogen, so that a fibrin gel is formed when the strand coated with the first biocompatible solution is immersed in the second biocompatible solution, the method according to clause 14.

[0076] Clause 18 The method according to clause 14, wherein the first biocompatible solution is a cell suspension.

[0077] Clause 19 The method according to clause 14, wherein a plurality of collagen ultrafine fibers are simultaneously drawn out by the rotation of a rotatable frame.

[0078] Clause 20 At least one of the first biocompatible solution and the second biocompatible solution contains one or more additives coated on a collagen ultrafine fiber strand. The method according to clause 14, wherein the one or more additives are essentially selected from the group consisting of growth factors, antibiotics, and small molecule pharmaceuticals.

[0079] Clause - Treatment method for volumetric muscle loss Clause 1 A method for treating volumetric muscle loss (VML), comprising applying a scaffold construct formed of a plurality of collagen ultrafine fibers arranged in an organized sheet-like configuration and coated with a biocompatible solution into a VML wound site.

[0080] Clause 2 The method according to clause 1, wherein the scaffold construct comprises a plurality of sheet-like layers of collagen ultrafine fibers.

[0081] Clause 3 The method according to clause 1, wherein the biocompatible solution is hyaluronic acid.

[0082] Clause 4 The method according to clause 1, wherein the biocompatible solution is a cell suspension.

[0083] Clause 5 The method according to clause 4, wherein the cell suspension contains muscle progenitor cells (MPC).

[0084] Clause 6 The method according to clause 5, wherein the cell suspension contains about 4,000,000 cells / mL.

[0085] Clause 7 The scaffold construct has mechanical properties that substantially approximate or exceed those of the human tendon, the method according to clause 1.

[0086] Clause 8 The scaffold construct has an average ultimate tensile strength (UTS), tensile modulus, and breaking point strain that substantially approximate or exceed those of the human tendon, the method according to clause 7.

[0087] Clause 9 A method for treating volumetric muscle loss (VML), In a method that includes attaching a scaffold construct formed from a plurality of collagen ultrafine fibers arranged in an organized sheet-like configuration within the VML wound site, The scaffold construct includes a plurality of sheet-like layers of collagen ultrafine fibers, the method.

[0088] Clause 10 The scaffold construct is coated with a biocompatible solution, the method according to clause 9.

[0089] Clause 11 The biocompatible solution is hyaluronic acid, the method according to clause 10.

[0090] Clause 12 The biocompatible solution is a cell suspension, the method according to clause 10.

[0091] Clause 13 The cell suspension includes muscle progenitor cells (MPC), the method according to clause 12.

[0092] Clause 14 The cell suspension includes about 4,000,000 cells / mL, the method according to clause 12.

[0093] Clause 15 The scaffold construct has mechanical properties that substantially approximate or exceed those of the human tendon, the method according to clause 9.

[0094] Article 16 The scaffold structure has an average ultimate tensile strength (UTS), tensile elastic modulus, and breaking point strain that substantially approximate or exceed those of the average ultimate tensile strength (UTS), tensile elastic modulus, and breaking point strain of the human tendon, and is the method according to Article 15.

Brief Description of the Drawings

[0095] The patent or application file includes at least one drawing created in color. A copy of this patent or patent application publication that includes color drawings will be provided by the Patent Office upon request and payment of the required fees.

[0096] The embodiments can be better understood by referring to the following drawings and description. The components in the drawings are not necessarily to scale; instead, emphasis is placed on explaining the principles of the embodiments. Further, in the drawings, like reference numerals designate corresponding parts throughout different figures.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27

Figure 28

Figure 29

Figure 30

Figure 31

Figure 32

Figure 33

Figure 34

Figure 35

Figure 36

Figure 37

Figure 38

Figure 39

Figure 40

Figure 41

Figure 42

Figure 43

Figure 44

Figure 45

Figure 46

Figure 47

Figure 48

Figure 49

Figure 50

Figure 51

Figure 52

Figure 53

DETAILED DESCRIPTION OF THE INVENTION

[0097] The present invention relates to the formation of biocompatible scaffold constructs using high - strength collagen fibers. In particular, the present disclosure relates to the use of Rotational Frame Fabrication (RFF) to produce a cellularized scaffold composed of high - density collagen ultra - fine fibers for treating ligament injury, tendon injury, and volumetric muscle loss (VML) injury. The collagen ultra - fine fibers are controllably seeded with cells and formed into viable 3D grafts having a designed porosity, fiber pattern, and macroscopic dimensions in an automated, scalable biomanufacturing process.

[0098] The following description in this specification relates to tests conducted and documented in "Assembled Cell-Decorated Collagen (AC-DC) bioprinted implants mimic musculoskeletal tissue properties and promote functional recovery" by 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, which was published on July 2, 2021 and the preprint is available from bioRxiv at https: / / doi.org / 10.1101 / 2021.06.22.449431. The entire disclosure of this published document is incorporated herein by reference.

[0099] Collagen, as a natural polymer, is preferred as it provides inherent biocompatibility and bioactivity, as well as cues of a microstructure that mimics natural tissues to promote cell attachment, alignment, and proliferation. As described herein, the grafts provide excellent strength and stability and closely match the mechanical properties of native tendon and ligament tissues immediately after fabrication and after 4 weeks of in vitro culture. Furthermore, such grafts promote native tissue regeneration and functional recovery in vivo using a validated VML injury model.

[0100] The novel biomanufacturing method described herein provides an improved method for manufacturing cell-laden scaffolds for regenerative medicine applications. The implantable scaffolds are manufactured from strong and stable ultra-fine fibers of clinical-grade collagen with biochemical and mechanical properties suitable for medical use.

[0101] This fiber can be uniformly and controllably coated with cells during the manufacture of the scaffold, and selectively coated with multiple types of cells to create a scaffold with designed non-uniformity. The scaffold is formed using a microstructure cue that indicates cell alignment, as well as a designed porosity, fiber pattern, and macroscopic dimensions. The manufacturing process is rapid, reproducible, and automated. The resulting scaffold mimics the biological, morphological, and functional properties of natural ligament and tendon tissues.

[0102] The embodiments described herein relate to scaffold constructs, methods for creating scaffolds, and apparatuses for creating scaffolds. Such scaffolds include a plurality of aligned collagen nanofibers coated with a biocompatible solution. Such scaffolds may be suitable for use as tendon or ligament grafts or other biomechanical surgical repairs.

[0103] To aid and clarify the following description of the various embodiments, various terms are defined herein. Unless otherwise specified, the following definitions apply throughout this specification (including the claims). For consistency and convenience, adjectives representing directions corresponding to the illustrated embodiments are employed throughout this detailed description.

[0104] The term "biocompatible solution" as used throughout this detailed description and in the claims refers to a liquid solution, preferably a cell culture medium, that maintains the viability of the cells applied to coat the fibers by the method of the present invention and supports their growth and function.

[0105] The term "collagen" includes expected types such as atelocollagen, telocollagen, and gelatin derived from collagen, and may be collagen from various sources such as recombinant human collagen, porcine collagen, bovine collagen, jellyfish collagen, and mixtures thereof. One of ordinary skill in the art will understand that nanofibers having tensile strength, elasticity, resilience, and toughness suitable for the particular function and use of a given implant being considered herein will be manufactured.

[0106] As used throughout this detailed description and in the claims, the term "fiber" refers to fibers, yarns, or filaments having a high ratio of length to diameter and typically used as a group. The term "ultra-fine fiber" is used synonymously in accordance with the scale of the size of the fibers used in the preferred embodiments. The term "strand" refers to an individual item of fiber, although fiber-based constructs may be composed of many individual fiber strands. Throughout, the above terms may include fibers composed of multiple secondary fibers assembled by secondary or tertiary assembly processes such as braiding.

[0107] As used throughout this detailed description and in the claims, the term "lateral" refers to the left-right direction extending along the width of a component.

[0108] As used throughout this detailed description and in the claims, the term "longitudinal" refers to the direction extending along the length of a component.

[0109] It will be understood that each of the adjectives representing such directions can be applied to the individual components of the device or apparatus under consideration. The term "upward" refers to the vertical direction away from the ground, and the term "downward" refers to the vertical direction toward the ground. Similarly, the terms "topmost", "upper", and other similar terms refer to the part of an object that is substantially farthest from the ground in the vertical direction, and the terms "bottom", "lower", and other similar terms refer to the part of an object that is substantially closest to the ground in the vertical direction.

[0110] For purposes of this disclosure, the term "fixedly attached" shall refer to two components joined such that the components cannot be readily separated (e.g., without destroying one or both of the components). Exemplary modes of fixed attachment may include joining by permanent adhesives, rivets, stitches, nails, staples, welding or other thermal bonding, or other joining techniques. Further, two components may be "fixedly attached" by being integrally formed in a molding process, for example.

[0111] As used throughout this detailed description and in the claims, the term "scaffold" refers to a two-dimensional or three-dimensional assembly of fibers. The terms "construct" and "macrostructure" are used synonymously. The terms "scaffold construct", "graft", and "implant" refer to scaffolds intended for implantation as medical devices in preferred embodiments.

[0112] As used throughout this detailed description and in the claims, the term "vertical" refers to a direction generally orthogonal to both the lateral and longitudinal directions.

[0113] Preferred embodiments of the present disclosure are directed to scaffold constructs coated with a biocompatible solution and comprising a plurality of substantially aligned collagen ultrafine fibers. One of ordinary skill in the art will understand that the ultrafine fibers in the construct may be substantially parallel to each other, partially aligned, or not substantially aligned, in a state where the orientation and spacing of the ultrafine fibers vary three-dimensionally. Further, some constructs may include both aligned and non-aligned ultrafine fibers. For example, in some embodiments, some of the ultrafine fibers may be oriented at an oblique angle to other ultrafine fibers in the construct. Additionally, not only may scaffold constructs produced by the disclosed methods be manufactured in a closed-loop configuration, but the completed scaffold constructs may have other complex three-dimensional shapes in addition to planar sheet configurations, prismatic configurations, circular configurations, or cylindrical configurations. In some cases, the shape of the scaffold construct may be based on a CAD model.

[0114] In a preferred embodiment, to print a specific cell scaffold, the biocompatible solution contains cells suspended in a hyaluronic acid solution prepared in Dulbecco's Modified Eagle Medium (DMEM). Hyaluronic acid is a major component of the extracellular matrix of connective tissue and is utilized herein as a "cell adhesive" that promotes the adhesion of cells to collagen nanofibers. An exemplary hyaluronic acid concentration may be 5 mg / mL. Any cell type may be selected based on the intended use, and any cell type may include stem cells, tendon cells, chondrocytes, myoblasts, osteoblasts, or a number of tissue-specific cell types. To promote the survival of the selected cell type, appropriate cell culture media and material additives need to be utilized.

[0115] The present disclosure is directed to methods and apparatuses for creating such scaffolds. It should be noted that the disclosed systems and processes are applicable to other types of fibers other than collagen nanofibers, including other natural fibers and / or synthetic materials or combinations thereof that have sufficient mechanical properties (such as strength, flexibility, etc.) for operation by the disclosed processes. Further, it should be noted that in some embodiments, a plurality of collagen nanofibers need not necessarily be aligned with each other.

[0116] The scaffold is generally formed by dispensing collagen nanofiber strands in such a way that the dispensed strands are coated within the biocompatible solution. For the purposes of the present invention, the nanofibers may be dispensed by pulling out the nanofiber strands under tension or by extruding the nanofiber strands. In some embodiments, the collagen nanofiber strands may be dispensed from the central lumen of the inner conduit of a coaxial needle with the biocompatible solution being dispensed from an annular lumen around the inner conduit. The biocompatible solution may be dispensed from the annular lumen via controlled actuation of a plunger of a syringe containing the biocompatible solution.

[0117] In a preferred embodiment, the ultra-fine fibers are supplied from a spool. Exemplary extrusion of collagen fibers is disclosed in U.S. Patent Application Publication No. 2020 / 0246505 to Francis et al., published on August 6, 2020, entitled "Microfluidic Extrusion". This U.S. Patent Application Publication describes products having an ultimate tensile strength, elastic modulus, and breaking point strain comparable to those of native human tendons and ligaments. The entire disclosure of U.S. Patent Application Publication No. 2020 / 0246505 is incorporated herein by reference as Appendix A.

[0118] 2-solution method FIG. 1 is a schematic view of a part of an apparatus for creating a scaffold structure according to an exemplary embodiment. As shown in FIG. 1, the printing device 100 may include a 3D printing assembly. The 3D printing assembly further comprises a function configured to dispense collagen ultra-fine fibers covered with a biocompatible solution. The 3D printing assembly is provided to control the movement of the printing device 100 along the X, Y, and Z axes. As shown in FIG. 1, the printing device 100 may include a stepping motor 105 and a lead screw 110 configured to be driven by the stepping motor 105. The lead screw 110 may rotate to move the first platform 112 relative to the second platform 113 in order to operate the plunger 114 of the syringe 115, which may contain the biocompatible solution 152. When the screw 110 rotates, the biocompatible solution 152 may be pushed through the conduit 116 into the annular conduit of the coaxial needle 125.

[0119] Simultaneously with the dispensing of the biocompatible solution 152, the collagen ultra-fine fiber strand 150 may be drawn through the inner conduit of the coaxial needle 125. As shown in FIG. 1, the spool 120 may contain the collagen ultra-fine fiber strand 150. To achieve dispensing, the free end of the collagen ultra-fine fiber strand 150 may be fixed (e.g., tied) to the frame. Next, as the frame rotates, the collagen ultra-fine fiber strand 150 is drawn from the spool 120 and passes through the coaxial needle 125. As it moves, the collagen ultra-fine fiber strand 150 is coated with the biocompatible solution 152.

[0120] A frame 135 may be provided to organize the collagen ultra-fine fibers as the collagen ultra-fine fiber is drawn from the spool and supplied through the coaxial needle. The frame 135 may rotate to wind the collagen ultra-fine fiber strand 150 around the frame 135, thereby forming a scaffold 155. The device 100 may include a rotating device configured to rotate the frame 135. For example, as shown in FIG. 1, the rotating device may include one or more motors such as a first motor 130 and a second motor 132. As shown in FIG. 1, the frame holding device may be rotatable about the rotation axis 137 as indicated by the arrow 140.

[0121] Furthermore, to facilitate the attachment of the frame 135 to the frame holding device, one or both of the motors may be movable, for example, as indicated by arrow 145. In some embodiments, one or both of such motors may be manually movable or movable via an electric mechanism. Additionally, the frame holding device may be movable to determine which portion of the frame 135 forms the scaffold. As the strand 150 is wound around the frame 135, the device may translate the frame 135 as indicated by arrow 147, or translate the printing device 100 along the axis of rotation, so that subsequent windings of the strand 150 are formed side by side to provide width to the scaffold 155. Further, a plurality of such windings may be formed to provide thickness to the scaffold 155.

[0122] The frame may have any suitable shape. In some embodiments, the frame may have a substantially planar configuration. In some embodiments, the frame may include protrusions or extensions to facilitate gripping the frame without contacting the scaffold constructed on the frame. In some embodiments, the frame may include features to facilitate attachment to a rotating device. In some embodiments, the frame may include a plurality of zones configured such that, for example, a plurality of scaffolds can be constructed on the same frame. In some embodiments, the frame may include a mechanism such as a fastener that can slide over the frame after winding to tie the strand to the frame and prevent the strand from unraveling. In some embodiments, the frame may include a spring-loading mechanism configured to maintain a predetermined amount of tension on the scaffold.

[0123] Figure 2 is a schematic perspective view of a substantially planar frame with a collagen ultra-fine fiber strand wound around the frame. As shown in Figure 2, the frame 135 may include a first crossbar 200 and a second crossbar 205 configured to function as a winding surface around which the scaffold is to be wound. Further, the frame 135 may include a first side portion 210 and a second side portion 215 connecting the first crossbar 200 to the second crossbar 205. For this reason, as shown in Figure 2, in some embodiments, the frame may include a central opening 217. The central opening 217 may prevent the scaffold 155 from being joined to the frame 135. Further, the central opening 217 may limit the influence of cell migration from the scaffold 155 to the frame 135. Further, the central opening 217 may potentially improve the diffusion of nutrients from the surrounding cell culture medium to the scaffold 155.

[0124] In addition, as shown in Figure 2, the frame 135 may further include a plurality of protrusions 220 extending outwardly in the same plane as the entire frame. The protrusions 220 may be provided as a location for gripping / handling the frame 135 without damaging the scaffold 155. In some embodiments, as shown in Figure 2, at least a portion of the protrusions 220 may be raised with respect to the plane of the entire frame. Such raised portions may facilitate attachment to a rotating device and may also maintain the distance between the scaffold and the surrounding of its solid surface, for example, the bottom of a microwell plate during cell culture.

[0125] Figure 3 is a schematic cross-sectional view of the needle and the rotating frame with a collagen ultra-fine fiber strand wound around the frame along line 3-3 of Figure 2. As shown in Figure 3, the frame 135 may rotate about an axis 137, as indicated by arrow 300. Due to this rotation, the strand 150 is drawn through the needle 125 and the strand 150 is wrapped or wound around the frame 135 to form the scaffold 155.

[0126] Other frame configurations and functions are also possible. For example, FIG. 4 is a schematic view of a part of another embodiment of a frame around which collagen ultra-fine fiber strands are wound. As shown in FIG. 4, the frame 400 may have a first cross member 405, a second cross member 410, a first end 415, and a second end 420. The frame 400 may be attached to a rotating device including a first frame fixture 425 and a second frame fixture 430. By winding around the frame 400 as described above, a scaffold 435 may be formed. In addition, as shown in FIG. 4, in some embodiments, the ends of the frame 400 may include an extension 440 configured to facilitate gripping / handling of the frame 400 without damaging the scaffold 435.

[0127] FIG. 5 is a schematic view of another embodiment of a frame around which collagen ultra-fine fiber strands are wound in three parts. As shown in FIG. 5, the frame 500 may be configured to form a plurality of scaffolds on the same frame. For example, the frame 500 may include a first area 505 configured to receive a first scaffold 510, a second area 515 configured to receive a second scaffold 520, and a third area 525 configured to receive a third scaffold 530. It will be understood that the frame may be configured to receive fewer or more scaffolds in order to optimize the manufacture / formation of the scaffolds.

[0128] In addition, as shown in FIG. 5, in some embodiments, the frame 500 may include a first clip 535 and a second clip 540 configured to slide on the frame 500 after the scaffold is formed to prevent the scaffold from unraveling.

[0129] FIG. 6 is a schematic view of another embodiment of a frame around which collagen ultra-fine fiber strands are wound. As shown in FIG. 6, the frame 600 may include a peripheral portion 605 configured to receive a winding of collagen ultra-fine fiber strands to form a scaffold 610. To substantially maintain a predetermined tension on the scaffold 610, the frame 600 may be spring-loaded, for example, by a spring mechanism 615.

[0130] In some embodiments, it may be desirable to form different portions of scaffolds with different compositions. For example, in some cases, it may be desirable to form the end portions of the scaffolds of ligaments or tendons adjacent to bone with a different composition than other portions of the ligament or tendon. This reproduces the different structures / compositions of natural ligaments and tendons. To manufacture a scaffold having different compositions in different portions, an apparatus that selectively distributes collagen ultra-fine fiber strands through different biocompatible solutions may be utilized.

[0131] FIG. 7 is a schematic view of an apparatus for creating a scaffold construct according to another exemplary embodiment, the apparatus being configured to selectively distribute collagen ultra-fine fiber strands through different biocompatible solutions. As shown in FIG. 7, the apparatus 700 may include two separate columns for distributing two different biocompatible solutions, respectively. The first column may include a first stepping motor 705 and a first lead screw 710 configured to be driven by the stepping motor 705. The first lead screw 710 may rotate to move the first platform 712 relative to the second platform 713 in order to operate the plunger of a first injector 760 that may contain a biocompatible solution. As the screw 710 rotates, the biocompatible solution may be distributed to the solution manifold 727.

[0132] Simultaneously with the distribution of the biocompatible solution, the collagen ultra-fine fiber strand may be drawn through the needle 725. As shown in FIG. 7, the spool 720 may contain the collagen ultra-fine fiber strand and distribute the strand through the needle 725. In some embodiments, a plurality of spools may distribute a plurality of strands simultaneously. For example, as shown in FIG. 8, in some embodiments, one or more additional spools may be disposed on both sides of the spool 720.

[0133] To achieve the distribution, the free end of the collagen ultra-fine fiber strand 750 may be fixed (e.g., tied) to the frame. Then, as the frame rotates, the collagen ultra-fine fiber strand 750 is wound around the frame and thus drawn from the spool 720 through the needle 725.

[0134] A frame 735 may be provided to organize the collagen ultra-fine fibers as they are drawn from the spool and supplied through the coaxial needle. The frame 735 may rotate to wind the collagen ultra-fine fiber strand 750 around the frame 735, thereby forming a scaffold 755. The apparatus 700 may include a rotating device configured to rotate the frame 735. For example, as shown in FIG. 7, the rotating device may include one or more motors such as a first motor 730 and a second motor 732. As shown in FIG. 7, the frame holding device may be rotatable about the rotation axis 737 as indicated by the arrow 740.

[0135] Further, to facilitate attachment of the frame 735 to the frame holding device, one or both of the motors may be movable, for example, as indicated by arrow 745. In some embodiments, one or both of such motors may be manually movable or movable via an electric mechanism. Additionally, the frame holding device may be movable to determine which portion of the frame 735 forms a scaffold. As the strand 750 is wrapped around the frame 735, the device may translate the frame 735 as indicated by arrow 747 or translate the device 700 along the axis of rotation, such that subsequent wraps of the strand 750 are formed side by side to give the scaffold 755 a width. Further, a plurality of layers of such wraps may be formed to give the scaffold 755 a thickness.

[0136] The solution manifold 727 may contain a first biocompatible solution, and the collagen ultra-fine fiber strand 750 may be drawn through an opening in the manifold 727. The opening may extend vertically through a portion of the manifold 727 that includes a well containing the biocompatible solution. Thus, when the strand 750 is drawn through the manifold 727, the strand 750 becomes coated with the biocompatible solution.

[0137] A delivery device configured to dispense the collagen ultra-fine fiber strand 750 through the needle 725 may include a second columnar portion configured to dispense a second biocompatible solution. For example, the device 700 may include a second stepping motor 706 and a second lead screw 711 configured to be driven by the second stepping motor 706. The second lead screw 711 may rotate to move a third platform 714 relative to a fourth platform 715 to actuate a plunger of a second injector 765 that may contain the second biocompatible solution. When the second screw 711 rotates, the second biocompatible solution may be dispensed into the solution manifold 727.

[0138] The manifold 727 may comprise a second well configured to contain a second biocompatible solution. The apparatus may be configured to select which well the strand passes through, and thus at any given time select through which of the two biocompatible solutions the strand 750 will be drawn. This is accomplished by translating the solution manifold. Figures 8 through 12 illustrate this process in greater detail.

[0139] Figure 8 is a schematic diagram of a portion of the apparatus shown in Figure 7. As shown in Figure 8, different biocompatible solutions may be delivered separately to the solution manifold 727. For example, a first biocompatible solution may be delivered from a first injector 760 through a first conduit 770 to a first side of the manifold 727. A second biocompatible solution may be delivered from a second injector 765 through a second conduit 775 to a second side of the manifold 727.

[0140] Figure 9 is a schematic diagram of a solution manifold according to one embodiment. As shown in Figure 9, the solution manifold 727 may comprise a first well 900 configured to receive a first biocompatible solution and a second well 905 configured to receive a second biocompatible solution. The first well 900 of the solution manifold 727 may comprise a first opening 910 configured to allow a collagen ultra-fine fiber strand to pass therethrough. Further, the second well 905 may comprise a second opening 915 configured to allow a collagen ultra-fine fiber strand to pass therethrough. Further, the manifold 727 may comprise a slot 920 connecting the first opening 910 and the second opening 915. As shown in Figure 9, the slot 920 may be relatively wide with respect to the openings of the wells. In other embodiments, the slot 920 may be relatively narrow.

[0141] FIG. 9 also shows a rail 925 capable of receiving the manifold 727 by an operating mechanism. The manifold 727 may be translated in a parallel direction in opposite directions to deliver the strand through a biocompatible solution in one well or the other well during dispensing. For this reason, the dispensing device is configured to translate the solution manifold in a direction parallel to the slot to select through which of the first well and the second well the collagen ultra-fine fiber strand is drawn.

[0142] FIG. 10 is a schematic view of an apparatus for creating a scaffold structure with the solution manifold translated to the left. As shown in FIG. 10, the solution manifold 727 may be translated to the left as indicated by arrow 1005 so that the strand is delivered through the right side portion of the manifold 727. A thick cable 1000 and a motor (not shown) may be used to operate / translate the manifold 727 back and forth.

[0143] FIG. 11 is a schematic view of an apparatus for creating a scaffold structure with the solution manifold translated to the right. As shown in FIG. 11, the solution manifold 727 may be translated to the right as indicated by arrow 1010 so that the strand is delivered through the left side portion of the manifold 727.

[0144] FIG. 12 is a schematic view of a substantially planar frame with a multi-solution scaffold wound around the frame. As shown in FIG. 12, a scaffold 1205 may be formed on the frame 735. Further, as shown in FIG. 12, the first end 1210 of the scaffold 1205 may be different from the second end 1215 of the scaffold 1210. Such a difference may occur by using different solutions. For example, one solution may be cellular and the other may be acellular. In another example, one solution may contain bone cells and the other solution may contain tendon cells or ligament cells.

[0145] Note that other elements may be suspended in the biocompatible solution and thus incorporated with the collagen strands to form a scaffold. That is, one or more additives may be included in the biocompatible solution such that they are coated on the collagen strands. For example, in some embodiments, the biocompatible solution may include growth factors, antibiotics, small molecule pharmaceuticals, and the like.

[0146] To manufacture such a multi-solution scaffold, a first portion of the collagen ultrafine fibers coated with a first biocompatible solution may be disposed in a first longitudinal portion of the scaffold, and a second portion of the collagen ultrafine fibers coated with a second biocompatible solution may be disposed in a second longitudinal portion of the scaffold. Here, the first longitudinal portion is different from the second longitudinal portion.

[0147] In the example shown in FIG. 12, the first longitudinal portion is the first end 1210, and the second longitudinal portion is the second end 1215. In other embodiments, scaffold portions having different compositions may be arranged laterally rather than from end to end. Further, in some embodiments, the scaffold may have three or more different portions having different compositions.

[0148] FIG. 13 is a flowchart showing steps of a method for creating a scaffold construct according to an exemplary embodiment. As shown in FIG. 13, the method may include distributing collagen ultrafine fiber strands through a needle (step 1300). Further, in step 1305, the method may include winding the collagen ultrafine fiber strands around a frame. Further, as reflected in step 1310, the solution manifold may be translated along the frame rotation axis to select between two solutions within the solution manifold. These three steps may be performed sequentially or simultaneously within a loop.

[0149] Furthermore, since the scaffold may be implanted to support living tissue, it needs to be maintained under appropriate conditions. For example, in step 1315, the method further includes a step of maintaining the scaffold for more than two days under cell culture conditions. In some cases, the cell culture conditions may be maintained for up to seven days without significant reduction in strength or ultra-fine fiber tissue.

[0150] The following is an additional description of the materials and methods used to manufacture the scaffolds discussed herein, as well as a description of sample scaffolds manufactured using the rotational frame technology discussed herein.

[0151] A new additive manufacturing technique has been developed for producing cellularized scaffolds composed of highly dense and highly aligned strong collagen ultra-fine fibers. Specifically, cells are uniformly coated on continuous collagen ultra-fine fibers having a width of about 50 μm and a thickness of about 5 μm (ribbon-like), and the ultra-fine fibers are wound three-dimensionally (3D) around a rigid frame to form a scaffold. In some embodiments, collagen ultra-fine fibers having a substantially round / circular cross-section may be utilized.

[0152] When mimicking the tissue of the ultrastructure of natural ligaments and tendons, the fibers coated with these cells are wound parallel to their own neighbors and their own upper ends to form a rectangular macrostructure of a designed width, length, and thickness. The scaffold is cultured on such a frame to maintain the tension and alignment of the wound fibers before removal. Such scaffolds are intended to promote the regeneration and recovery of function in musculoskeletal tissue injuries.

[0153] To implement this technique, a custom extrusion printhead (Figure 1) was designed and attached to a Folger Tech FT-5 R2 commercial 3D printer. The printhead mechanically compresses a disposable injector using a lead screw driven by a planetary gear stepper motor to dispense cell suspensions with a resolution of less than 1 microliter. The dispensed cell suspension passes through the outer needle of the coaxial needle assembly during printing. Up to three spools of collagen ultrafine fibers are loaded onto the printhead, and each spool is fed through the inner needle of the coaxial needle assembly to simultaneously dispense three strands of fibers. At the exit of the needle assembly, the collagen fibers are uniformly coated by the dispensed cell suspension. The amount of cell suspension dispensed per millimeter of extended fiber is a process parameter determined by the user and provides a means of controlling the resulting cell density and the total number of cells across the scaffold. In a preferred embodiment, the cell density can vary in the range of 0 to 10 million cells / mL depending on the printing parameters and the desired number of cells within the resulting scaffold.

[0154] A custom receiving assembly was designed to hold a small disposable 3D printing frame between two stepper motors (Figure 1). Prior to printing, the fiber is drawn through the coaxial needle and attached to the first fixation point on the frame. When the frame is rotated by the motor, the fiber is drawn through the coaxial needle under tension and coated by the dispensed cell suspension (Figure 3). By adjusting the rotation of the frame and the linear movement (feed rate) of the printhead along the width of the frame, a cellularized scaffold of highly aligned collagen ultrafine fibers at high density is produced (Figure 2). The stepper motor driving the frame rotation is attached to a manual linear stage, allowing for easy attachment and removal of the frame between successive prints.

[0155] In a preferred embodiment, to print the cellular scaffold, cells are suspended in a hyaluronic acid solution prepared in Dulbecco's Modified Eagle Medium (DMEM). Further, the control of the dispensing amount is improved, and since the viscosity is high, the influence of sedimentation of cells during printing is limited. DMEM provides the nutrients and cell-compatible environment necessary to maintain the health of cells during printing.

[0156] The Folger Tech FT-5 R2 hardware and firmware were modified to facilitate the printing method. The commercially available FDM print head was removed and replaced with a custom extrusion print head. The Z-axis control of the printer was reused as a new R rotation axis of a custom rotary frame assembly attached to the build plate of the printer. Off-the-shelf parts of the frame assembly including the print head and the frame were 3D printed in-house from PLA using a MakerBot Replicator+. All the stepping motors and drive pulleys were replaced to improve the resolution on the X-axis, Y-axis, and the new R-axis. To accommodate such hardware changes, the firmware of the printer was also modified.

[0157] The cellular collagen ultra-fine fiber scaffold can also be formed on a frame of any size and shape, such as a rigid rectangular frame for culturing in a microwell plate, or directly between two sutures to facilitate future transplantation. As the rigid object rotates, it is possible to wrap the fibers in any number of shapes and various directions, increasing the complexity achievable, for example, by multi-axis manipulation of the "frame" by a robot. A scaffold having a complex 3D geometry, including planar sheet-like geometries, prismatic geometries, circular or cylindrical geometries, may be manufactured based on a CAD model.

[0158] Custom Python code was developed to receive user input of the designed scaffold shape and printing parameters and output the corresponding G-code file. This control system will be described in more detail below.

[0159] To maintain sterility, the entire physical system may be located inside a biosafety cabinet or a filtered laminar flow hood, and all components may be handled aseptically.

[0160] Cellular collagen ultrafine fiber scaffolds (Figs. 2 and 4) having various geometric shapes were rapidly and reproducibly fabricated on 3D printed PLA frames. Multiple samples were printed on a single frame with retaining ends to prevent fiber unraveling (Fig. 5). Additionally, scaffolds may be printed between two lengths of suture thread and held under tension during culture using a dedicated attachment assembly (Fig. 6). The PLA frame was found to maintain the fiber alignment and macrostructure of the printed scaffolds and the stability during culture over several weeks. The printed collagen scaffolds were lifted from the surface of the well plate during culture, and the frame was designed with "legs" to prevent cell migration from the scaffold to the plate. During bioreactor testing, etc., the frame was designed with "ears" as attachment points for improved handling with tweezers and loading into fixtures. As a benchmark, scaffolds with a width of 2 mm, a length of 10 mm, and a thickness of 0.5 mm can be printed within 20 minutes at an elongation rate of the collagen ultrafine fibers from 1 to 1000 mm / min, and even up to a maximum of 10,000 mm / min or more. In some embodiments, the elongation rate may be about 20 mm / min (1.2 m / hour). In other embodiments, the elongation rate may exceed 300 mm / min. In some cases, for example, it will be understood that the frame can be rotated at any suitable speed to wind the collagen ultrafine fibers. In yet other embodiments, the frame may rotate at any speed between 1 and 240 rpm.

[0161] Figure 14 is a transmitted light microscope image of a printed scaffold showing uniform parallel fibers. Figure 15 is an image of Figure 14 shown at twice the magnification of Figure 14. Figure 16 is a fluorescence microscope image showing the elongation of cells in addition to a uniform distribution of cells throughout. Figure 17 is an image of Figure 16 shown at twice the magnification of Figure 16.

[0162] By light microscopy, after 3 days of culture, a highly dense and highly aligned parallel fiber scaffold is shown with good dimensional fidelity (Figs. 14 and 15). Fluorescent imaging of human tendon cells labeled with the Cell Tracker Red CMPTX fluorescent probe after 11 days of culture shows cells uniformly distributed throughout the printed scaffold and longitudinal cell elongation (Figs. 16 and 17). The fluorescence of the cells is overlaid with the autofluorescence of the fibers at 495 nm.

[0163] Similar to the method developed for single-solution rotary frame printing, a new layer manufacturing technique was developed to produce a cellularized scaffold composed of highly dense, highly aligned, strong collagen ultra-fine fibers coated with multiple cell types in clearly defined different regions. The ability to control the location of multiple cell types or printed solutions within a single printed scaffold aims to enable and promote the repair of damage and the regeneration of damaged tissue at the interfaces of different tissue types such as the myotendinous junction (muscle-tendon interface) and the attachment site (tendon / ligament and bone interface).

[0164] To implement this method, a dual-solution print head (Fig. 7) was designed and fabricated to interact with a Folger Tech FT-5 R2 commercial 3D printer. Two independent planetary gear stepping motors mechanically compress disposable syringes individually to dispense cell suspensions with a resolution of less than 1 microliter. Instead of passing the cell suspension through a coaxial needle as in the single-solution rotary frame method, it is dispensed at both ends of an electric slide manifold. At each end of the manifold, the dispensed cell suspension accumulates in a small coating container. During printing, the automatic manifold slides back and forth to bring each container of cell suspension into contact with or out of contact with the collagen fibers being printed. As the fibers pass through each container, they are uniformly coated with the respective cell suspension. The amount of cell suspension dispensed per millimeter of extended fiber is a process parameter determined by the user and provides a means to control the cell density obtained throughout the scaffold.

[0165] Load a maximum of three collagen fiber spools onto the print head and attach them to an adjustable slip clutch that can control the tension of the fibers during printing. The fibers are fed through the center of the slide manifold. Here, the fibers do not come into contact with any of the coating containers. For example, to print an area of fibers coated with solution A, the electric manifold slides until the container of dispensed solution A is in the path of the fibers. Next, the rotating frame rotates to draw the fibers through the container and coat them with solution A. Next, to print an area of fibers coated with solution B, the electric manifold slides in the opposite direction until the container of dispensed solution B is in the path of the fibers, and the fibers are drawn out and coated again. By adjusting the coating of the fibers with two solutions and the rotation of the rotating frame, scaffolds can be created that contain two different regions of different solutions or cell types in the case of different cell suspensions. This technique can be conveniently extended to print scaffolds containing any number of solutions and cell types in different regions using a manifold that can position the fibers within multiple coating containers.

[0166] The rotating frame receiving substrate is the same as that described for the single-solution rotating frame printing technique above. To print the cellular scaffold, cells are suspended in a hyaluronic acid solution prepared in Dulbecco's Modified Eagle Medium (DMEM). Hyaluronic acid is an important component of the extracellular matrix and is utilized herein as a "cell adhesive" that promotes the adhesion of cells to the collagen ultrafine fibers. Additionally, control of the dispensed volume is improved, and the effect of cell sedimentation during printing is limited due to the high viscosity. DMEM provides the nutrients and cell-compatible environment necessary to maintain the health of the cells during printing.

[0167] The modified Folger Tech FT-5 R2 considered above is also used for multi-solution printing with the single-solution print head described above replaced by a multi-solution print head (Figure 7). Further, the Y-axis drive is changed to control the electric sliding motion of the slide manifold on the print head. That is, the motion in the Y direction is no longer automated on the print head. This does not affect the function because there is no required motion in the Y direction when using the rotary frame printing method. To accommodate such hardware changes, the printer's firmware was also changed.

[0168] The custom Python code described above was developed to accept user input of the designed scaffold shape and printing parameters and output the corresponding G-code file. User input includes the number of scaffolds per frame, the distance between scaffolds, the width of the scaffold, the height of the scaffold, the number of fiber layers, the dispensing amount of cell suspension per millimeter of extended fiber, the feed distance between parallel fibers, the Z-direction standoff distance from the frame to the print head during printing, the Y-direction standoff distance from the frame to the print head during printing, a value specifying whether to print a double cell type in separate regions or a single cell type, the length of region A, the length of region B, the length of the frame, and the rotational speed of the frame. The Python code calculates and outputs a G-code file containing all the parameters and motion / extrusion commands for executing the designed print. This file is sent to the printer to manufacture the designed scaffold. Repetier-Host is used as a user interface for executing such commands, as well as manual homing, motion, and extrusion commands.

[0169] A scaffold with two separate regions was fabricated rapidly and reproducibly using the multi-solution rotary frame printing method (Figure 12). Here, a collagen fiber scaffold printed with different regions of hyaluronic acid stained with two different colors of food coloring for visualization is shown.

[0170] Figure 18 is a fluorescence microscope image of a scaffold printed with rat muscle progenitor cells (MPCs), showing live cells, dead cells, and collagen fiber autofluorescence at 405 nm. Figure 19 is a fluorescence microscope image of the scaffold shown in Figure 18 after 7 days of culture. After 7 days, the cells had proliferated, as indicated by the more prominent presence of green in Figure 19. Figure 20 is a graph showing the results of alamarBlue analysis indicating the metabolic activity of scaffolds printed with human tendon cells after 1, 3, and 7 days of culture.

[0171] As a first characterization step, scaffolds were fabricated using a multi-solution rotary frame print head, but only one solution was used. That is, the slide manifold with the coating container was used to coat the fibers during printing, but remained stationary during printing and did not switch to coating with another solution. To evaluate cell viability and distribution, rat muscle progenitor cells (MPCs) (from the laboratory of Dr. George Christ) suspended at 4E6 cells / mL were printed onto muscle-like scaffolds. Adherent cells were labeled with the fluorescent lipophilic tracer DiD according to a standard protocol before being collected for printing. Scaffolds 5 mm wide, 10 mm long, and 0.25 mm thick were printed onto a PLA frame. Immediately after printing, dead cell nuclei were labeled with the fluorescent ethidium homodimer-1 according to a standard protocol. The scaffolds were imaged with an inverted fluorescence microscope (Axiovert, Zeiss). Fluorescence images showing live cells, dead cells, and autofluorescence of collagen fibers at 405 nm were overlaid (Figure 18). The cells could be confirmed uniformly across the scaffold at a reasonable ratio of live to dead cells. Labeling and imaging were repeated after 7 days of culture (Figure 19). The cells could be confirmed across the scaffold with increased density due to significant proliferation and an increased ratio of live to dead cells.

[0172] In addition, tendon-like scaffolds were printed using human tendon cells or tendon cells (ZenBio) suspended at 1E6 cells / mL, and the cell metabolic activity over time was evaluated using alamarBlue analysis. The scaffolds were cultured in a 10% alamarBlue solution in tendon cell growth medium for 4 hours, and fluorescence was measured according to the standard protocol. It was observed that the metabolic activity of the cellularized scaffolds during culture (n = 7) increased over 7 days of culture (Figure 20). This indicates an increase in cell health, activity, and proliferation.

[0173] Figure 21 is a fluorescence image of a dual-solution scaffold printed with MPC and tendon cells in different regions, and the image shows a high-density region of MPC (left) and a low-density region of tendon cells (right). Figure 22 is a graph showing the results of alamarBlue analysis indicating the metabolic activity of scaffolds printed with MPC and tendon cells after 1 day, 3 days, and 7 days of culture.

[0174] To verify multi-solution rotational frame printing, scaffolds were fabricated with low-density human tendon cells and high-density muscle progenitor cells in different regions. Specifically, the final tendon cell printing solution was composed of 1.5E6 human tendon cells / mL (ZenBio, NC), and the human muscle progenitor cell (MPC) printing solution was composed of 4E6 human muscle progenitor cells / mL (obtained from the laboratory of Dr. George Christ, UVA, VA). The printed samples were 9 mm in total length, with a 4-mm tendon cell region at one end and a 4-mm MPC region at the opposite end. For visualization, both cell types were labeled with the CellTracker Red CMPTX fluorescent probe. Fluorescent images taken immediately after printing showed different regions with high cell density (left side) and low cell density (right side) (cell fluorescence shown in white), and there was a clearly defined boundary interface between them (Figure 21). This demonstrates the ability to fabricate scaffolds using different materials, in this case cell suspensions, in the designed regions. Perhaps this could be extended to various patterns, such as finger-like patterns at the boundary interfaces as present in natural tissues, overlapping each other. Such dual-solution scaffolds with tendon cell and MPC regions were also used to evaluate the metabolic activity of the cells over time using alamarBlue analysis. The scaffolds were cultured in a 10% alamarBlue solution in muscle progenitor cell growth medium for 4 hours, and fluorescence was measured according to the standard protocol. It was observed that the metabolic activity of the cellularized scaffolds during culture (n = 4) increased over 7 days of culture (Figure 22). This indicates an increase in the overall cell health, activity, and proliferation across these two cell types during co-culture.

[0175] A scaffold printed with human mesenchymal stem cells (hMSCs) and a scaffold without the printed cells were cultured for 1 day and 28 days under typical cell culture conditions to evaluate the mechanical properties of the scaffolds. Figure 23 is a graph showing the peak loads of the cell-free scaffold and the cell scaffold printed using the rotational frame method after 1 day and 28 days of culture. Figure 24 is a graph showing the ultimate tensile strength (UTS) of the cell-free scaffold and the cell scaffold printed using the rotational frame method after 1 day and 28 days of culture. Figure 25 is a graph showing the tangent modulus of the cell-free scaffold and the cell scaffold printed using the rotational frame method after 1 day and 28 days of culture. Figure 26 is a graph showing the breaking point strain of the cell-free scaffold and the cell scaffold printed using the rotational frame method after 1 day and 28 days of culture.

[0176] Scaffolds with and without printed cells were created and the mechanical properties of the collagen fiber scaffolds printed using the rotational frame method and the influence of the cells were characterized. Immediately before the test, the samples were removed from the culture and excess medium was removed using lint-free wiping fibers. The cross-section of the samples was measured using calipers. Each end was fastened to the gripping part of a uniaxial tensile testing machine (MTS Systems Corporation, Eden Prairie, MN) equipped with a 100 N load cell. The samples were pulled to failure at a gripping part movement speed of 0.5 mm / second, and the load and movement data were recorded. The ultimate tensile strength (UTS) was determined using the recorded maximum load, and the tangent modulus was determined by the linear region of the stress-strain curve. The peak load, UTS, tangent modulus, and breaking point strain of the same cell-free scaffold and cell scaffold tested for failure after 1 day and 28 days of culture are shown in Figures 23, 24, 25, and 26, respectively.

[0177] In some embodiments, the scaffold construct may be formed using a single biocompatible solution. That is, the scaffold construct may include a plurality of collagen ultrafine fibers arranged in an organized configuration and coated with a biocompatible solution such as a hyaluronic acid solution. In some embodiments, the biocompatible solution may include cells suspended in the biocompatible solution. In some embodiments, the biocompatible solution may include one or more additives that will be coated onto the collagen ultrafine fiber strands. The one or more additives may include, for example, growth factors, antibiotics, small molecule pharmaceuticals, or any other suitable additives.

[0178] In some embodiments, the collagen ultrafine fibers may be arranged in a continuous loop, for example, so as to be wound around a rotatable frame by the above method. In some embodiments, the plurality of collagen ultrafine fibers may be substantially parallel to each other. In other embodiments, at least some of the collagen ultrafine fibers may be arranged at an oblique angle with respect to other collagen ultrafine fibers in the scaffold construct.

[0179] Such a single-solution scaffold construct may have mechanical properties that approximate or exceed those of human skeletal tissue. In some embodiments, the scaffold construct may have mechanical properties that exceed those of human tendon and ligament tissue. For example, in some embodiments, the scaffold construct may have an ultimate tensile strength (UTS), tensile modulus, and break point strain that approximate or exceed the average ultimate tensile strength (UTS), tensile modulus, and break point strain of human anterior cruciate ligament (ACL), human supraspinatus tendon, and human muscle.

[0180] Single-solution approach The scaffold structure formed from a single solution may be formed on substantially the same apparatus as described above. The apparatus may comprise a delivery device configured to dispense collagen ultra-fine fiber strands through an orifice. The apparatus may comprise a rotatable frame. Further, the apparatus may include a strand coating system configured to coat the collagen ultra-fine fiber strands with a biocompatible solution during dispensing of the strands, and the solution strand coating system is disposed between the delivery device and the rotatable frame. Further, the apparatus may comprise a rotation device configured to rotate the rotatable frame about an axis of rotation.

[0181] The apparatus may be configured to draw the collagen ultra-fine fiber strands through the orifice by rotation of the rotatable frame, thereby coating the collagen ultra-fine fiber strands with the biocompatible solution and winding the collagen ultra-fine fiber strands around the rotatable frame. The apparatus may be further configured such that the delivery device and the substantially planar frame translate relative to each other along the axis of rotation to wind the collagen ultra-fine fiber strands around different portions of the substantially planar frame.

[0182] The rotatable frame may have any suitable configuration. In some cases, the rotatable frame may comprise at least two parallel beams around which the collagen ultrafine fiber strands can be wound, resulting in a scaffold structure configured as a continuous loop. In some embodiments, the rotatable frame may be spring-biased to maintain the continuous loop of the scaffold structure under tension. In some embodiments, the rotatable frame may comprise removable end pieces that fix the parallel beams relative to each other. Here, by removing the removable end pieces, the continuous loop scaffold structure can be slid off the parallel beams. In some embodiments, the rotatable frame may be configured to be immersed in a cell culture fluid. In some embodiments, the frame may be formed of a material such that cells in a biocompatible solution are less likely to move towards the frame material than towards the collagen material of the scaffold. For example, in some embodiments, the rotatable frame may be formed of a material such as stainless steel. Cells are more likely to move towards the collagen strands than towards the stainless steel beams of the rotatable frame. In some embodiments, the frame may be formed of a sterilizable material.

[0183] In some embodiments, the delivery device may be configured to simultaneously dispense a plurality of strands of collagen ultrafine fibers. The plurality of strands may be dispensed through the same needle / orifice. For example, in some cases, three strands of collagen ultrafine fiber may be simultaneously dispensed and wound around the rotatable frame. It will be appreciated that any practical number of strands of collagen ultrafine fibers can be simultaneously dispensed. Simultaneously withdrawing a plurality of strands of collagen ultrafine fibers provides several advantages including increased cell attachment, increased size and strength of the scaffold structure, rapid construction of the structure, increased surface area that can be coated with the biocompatible solution (and cells), and space between the collagen ultrafine fibers to which the cells may bind.

[0184] A method of creating a scaffold construct using a single biocompatible solution may be substantially the same as the above process for creating a dual-solution scaffold construct, except that instead of alternately replacing the solution through which the strands pass, the strands may be continuously dispensed through a single solution. The method of creating a scaffold construct may include attaching collagen ultrafine fiber strands to a rotatable frame and rotating the rotatable frame about a rotation axis to wind the collagen ultrafine fiber strands around the rotatable frame. Winding the collagen ultrafine fiber strands around the rotatable frame causes the collagen ultrafine fiber strands to be drawn from a delivery device, whereby the collagen ultrafine fiber strands are dispensed through an orifice and the collagen ultrafine fiber strands are drawn through the biocompatible solution. Thereby, a scaffold construct configured as a continuous loop may be formed.

[0185] This method may further include translating a substantially planar frame and a dispensing device parallel to each other along a rotation axis to wind the collagen ultrafine fiber strands around different portions of the rotatable frame. Optionally, this method may include simultaneously drawing a plurality of collagen ultrafine fibers by rotation of the rotatable frame.

[0186] In some embodiments, the rotatable frame may comprise at least two parallel beams around which the collagen ultrafine fiber strands may be wound. Optionally, the rotatable frame may comprise two end pieces that hold the parallel beams a predetermined distance apart. At least one of the end pieces may be removable. In such embodiments, the method may include removing at least one of the removable end pieces and sliding the scaffold construct off the parallel beams.

[0187] When using a cell solution, this method may include immersing the rotatable frame with the wound collagen ultrafine fibers in a cell culture medium. The scaffold construct on the frame may be left in the cell culture medium for a predetermined time.

[0188] Immersion (hydrogel) method It will be appreciated that the rotary frame printing method can be used to form a scaffold construct comprising a biocompatible hydrogel. Such a construct may be formed by dispensing collagen ultrafine fibers through a first biocompatible solution and immersing the rotary frame in a second biocompatible solution. The second biocompatible solution, when combined with the first biocompatible solution, produces a hydrogel and thereby encapsulates the winding of a plurality of collagen ultrafine fibers around the rotatable frame in the biocompatible hydrogel.

[0189] Exemplary procedures include dispensing collagen ultrafine fibers through a fibrinogen solution (which may be a cell solution) and winding the ultrafine fibers onto a rotary frame immersed in a thrombin solution. Fibrinogen and thrombin, when combined, form a fibrin hydrogel material that encapsulates or otherwise coats the scaffold construct. Fibrin is a natural protein formed during blood clotting and is widely used in FDA-approved medical procedures and is promising as a tissue engineering material due to its biocompatibility, bioactivity, and mechanical properties.

[0190] On the other hand, in the non-immersion procedure, the first biocompatible solution is a hyaluronic acid solution (which may contain cells such as hMSCs in some cases), while in the immersion procedure, the first biocompatible solution may instead contain cells suspended in a 40 mg / mL bovine-derived fibrinogen solution (1 to 10 million cells / mL). As before, the rotary frame bioprinting process may be used to coat the collagen ultrafine fibers while controlling with this cell suspension. At this time, the fibers are wound around their own sides and on top to form a 3D graft with cells distributed throughout.

[0191] In some embodiments, instead of performing scaffold formation in air, the dipping method may utilize a bath of 7 U / mL bovine-derived thrombin solution prepared in hMSC medium in which the graft remains immersed during printing. When the collagen fibers coated with the dispensed fibrinogen cell suspension contact the bath, thrombin rapidly initiates the coagulation of fibrinogen and starts the formation of a stable fibrin gel. This process binds the cells to the fibers and significantly reduces the number of cells that detach from the printed scaffold construct. It is necessary to select solutions that solidify (gelate) within seconds upon mixing.

[0192] The thrombin bath may additionally be warmed to 37 °C to maintain scaffold hydration and limit cell stress during printing. Once printing is complete, the bath descends to provide access to a temporary frame for transfer to a culture plate on which a rotatable frame can be placed.

[0193] A 14-day test was conducted using alamarBlue analysis of cell metabolic activity to evaluate cell health and proliferation with fibrin grafts. Since the cell state can change, cell metabolic activity does not directly indicate cell number, but alamarBlue has been used to estimate the total number of cells within the graft by generating a standard curve. Known numbers of cells were seeded into well plates using serial dilution, allowed to attach for 3 hours, and evaluated using a standard alamarBlue protocol. Overall, an exponential increase in cell number was observed, indicating excellent cell compatibility of implants fabricated with fibrin gels using the dipping-rotation frame technique.

[0194] The immersion rotation frame technique may be applied to various cell-compatible reagent pairs that form hydrogels upon contact with each other, such as, for example, a collagen solution printed in a neutralizing buffer or sodium alginate printed in an ionic crosslinking solution. In other embodiments, exemplary hydrogel precursor / fluid bath combinations may include fibrinogen + thrombin, fibrinogen / thrombin + factor XIII, alginate + ionic compound, collagen + enzyme solution, silk + enzyme solution, or gelatin + enzyme solution. Other reagent pairs will be known to those skilled in the art.

[0195] Furthermore, the first biocompatible solution may include one or more additives that are to be coated onto the collagen ultrafine fiber strands. The one or more additives may include, for example, growth factors, antibiotics, or small molecule pharmaceuticals.

[0196] The scaffold construct formed using the biocompatible hydrogel may have mechanical properties that approximate or exceed those of human musculoskeletal tissue. For example, the scaffold construct may have mechanical properties that approximate or exceed those of human tendon and ligament tissue. Notably, the scaffold construct may have an ultimate tensile strength (UTS), tensile modulus, and break point strain that approximate or exceed those of the human anterior cruciate ligament (ACL), human supraspinatus tendon, and human muscle.

[0197] Figure 27 is a schematic front view of an apparatus for creating a scaffold construct on a rotatable frame while immersed in a solution. Such a scaffold construct may include a plurality of collagen ultrafine fibers arranged in an organized configuration and coated with a biocompatible hydrogel. The collagen ultrafine fibers may be arranged in a continuous loop. Additionally, other shapes may be formed using the immersion rotation frame. For example, at least a portion of the winding on the rotation frame may be arranged at an angle oblique to the direction of the winding.

[0198] As shown in FIG. 27, an apparatus 2700 for creating a scaffolding structure may have substantially the same mechanical structure as the above-described apparatus. For example, apparatus 2700 may include a delivery device configured to dispense collagen ultra-fine fiber strands. The delivery device may include a spool 2705 configured to dispense one or more collagen ultra-fine fiber strands. In some embodiments, spool 2705 may include multiple spools (see FIG. 28). Thus, as discussed above, spool 2705 may be configured to dispense two or more ultra-fine fiber strands, such as three strands.

[0199] The apparatus may further include a strand coating system configured to coat the collagen ultra-fine fiber strands with a first biocompatible solution during dispensing of the strands, the solution strand coating system being disposed between the delivery device and a rotatable frame. As shown in FIG. 27, the strand coating system may include an injector 2720 containing a first biocompatible solution 2725 and a needle or nozzle 2710 having an orifice 2715 through which the collagen ultra-fine fiber strands may pass and be drawn out. It will be appreciated that in some embodiments, the orifice may be provided on a different type of structure other than a needle or nozzle. Injector 2720 may be configured to dispense the first biocompatible solution 2725 in a manner that coats the collagen ultra-fine fiber strands as the first biocompatible solution 2725 is dispensed through orifice 2715.

[0200] Device 2700 may be configured to draw a collagen ultra-fine fiber strand through orifice 2715 by rotation of a rotatable frame, thereby coating the collagen ultra-fine fiber strand with a first biocompatible solution 2725 and winding the collagen ultra-fine fiber strand around the rotatable frame. For this purpose, device 2700 may include a rotation device configured to rotate the rotatable frame about a rotation axis. For example, as further shown in FIG. 27, device 2700 may include a rotation system 2740 configured to rotate the rotatable frame. Rotation system 2740 may include a rotation motor 2745. Rotation motor 2745 may be any suitable type of rotation motor and may have any of the characteristics of the motors discussed above. For example, in some embodiments, rotation motor 2745 may be a stepping motor.

[0201] Device 2700 may further include a tank configured to contain a second biocompatible solution in which the rotatable frame can be immersed while winding the collagen ultra-fine fiber strand around the rotatable frame. For example, as shown in FIG. 27, device 2700 may include a tank 2730 containing a second biocompatible solution 2735. As shown in the following figures, tank 2730 may be configured to immerse the rotatable frame therein, rotate it, and wind the collagen ultra-fine fiber strand around the rotatable frame.

[0202] Furthermore, device 2700 may include a rotatable frame support structure 2750 configured to hold the rotatable frame that will be rotated by rotation motor 2745. Additionally, device 2700 may include a rotatable frame rearrangement system 2755 configured to relocate the attachment position of the rotatable frame downward relative to rotation motor 2745. This enables the rotatable frame to be placed within tank 2730 while rotating it.

[0203] The rotatable frame may have any suitable configuration capable of winding the collagen ultra-fine fiber strands. For example, in some embodiments, the rotatable frame may comprise at least two substantially parallel beams spaced apart. Winding the collagen ultra-fine fiber strands around such parallel beams produces a scaffold structure configured as a continuous loop. Across various geometries of rotatable frames, it has been observed that a rigid frame maintains the alignment of the fibers during cultivation over several weeks and the macrostructure of the printed scaffold structure.

[0204] Figure 28 is a schematic front perspective view of the apparatus of Figure 27. As shown in Figure 28, the rotatable frame 2760 may comprise at least two parallel beams capable of winding the collagen ultra-fine fiber strands, and thus may form a scaffold structure configured in a continuous loop (see also Figures 34 to 36). For example, as shown in Figure 28, the rotatable frame 2760 may comprise a first beam 2765 and a second beam 2770. In some embodiments, the first beam 2765 and the second beam 2770 may be substantially parallel to each other when the rotatable frame 2760 is assembled. In some embodiments, the rotatable frame 2760 may additionally comprise a first end piece 2775 and a second end piece 2780 that fix the first beam 2765 and the second beam 2770 to each other. In some embodiments, at least one of such end pieces may be removable so that the continuous loop scaffold structure can slide off the beam.

[0205] The rotatable frame may have any of various suitable configurations and / or characteristics, including the configurations and / or characteristics of the designs shown in Figures 2 to 6 and 12 and discussed above. In some embodiments, the rotatable frame may be spring-biased to maintain a continuous loop of the scaffold structure under tension. See, for example, Figure 6.

[0206] After wrapping the collagen ultra-fine fiber strands around the rotatable frame, the entire frame may be immersed in the cell culture medium. For this reason, in some embodiments, the rotatable frame may be configured to be immersed in the cell culture medium without deterioration.

[0207] In addition, as shown in FIG. 28, the apparatus 2700 may include an additional rotating assembly configured to facilitate underwater printing. For example, the rotatable frame repositioning system (2755, shown in FIG. 27) may include a gear system 2800. The gear system 2800 may transmit the rotation of the rotating motor to a lower location, such that the rotatable frame rotates at a location offset from the axis of the motor output shaft.

[0208] The method of creating a scaffold structure may include, as discussed above, attaching the collagen ultra-fine fiber strands to a rotatable frame and rotating the rotatable frame about a rotation axis to wind the collagen ultra-fine fiber strands around the rotatable frame. In addition, as described above, by winding the collagen ultra-fine fiber strands around the rotatable frame, the collagen ultra-fine fiber strands are drawn out from the delivery device, thereby distributing the collagen ultra-fine fiber strands through the orifice and drawing the collagen ultra-fine fiber strands through the first biocompatible solution. The rotatable frame is immersed in the second biocompatible solution while winding the collagen ultra-fine fiber strands around the rotatable frame, and the first biocompatible solution and the second biocompatible solution are combined to form a biocompatible hydrogel.

[0209] FIG. 29 is a schematic front perspective view of the apparatus of FIG. 27 with the rotatable frame immersed. As shown in FIG. 29, to immerse the rotating frame 2760, the position of the tank 2730 may be raised using the support block 2900.

[0210] Figure 30 is a schematic front perspective view of the gear system 2800 of the apparatus 2700. As shown in Figure 30, the gear system 2800 may include a first gear 3000 attached to the output shaft of a rotating motor. The gear system 2800 may also include a second gear 3005 driven by the first gear 3000 and a third gear 3010 driven by the second gear 3005. The third gear 3010 may be attached to the rotatable frame support structure 2750 (see Figure 27).

[0211] Figure 31 is a schematic enlarged front view of the gear system of the apparatus of Figure 27. It can be clearly seen that the rotatable frame 2760 is attached to the apparatus.

[0212] Figure 32 is a schematic front view of the apparatus of Figure 27 with the rotatable frame immersed. As shown in Figure 32, the tank 2730 may be lifted and a support block 2900 may be placed beneath it. In some embodiments, the tank 2730 may be manually movable. In other embodiments, the tank 2730 may be raised and lowered vertically using a mechanical lifting system. Separately, the rotatable frame 2760 may be lowered into the tank 2730. Figure 33 is a schematic front perspective view of the rotatable frame 2760 attached to the apparatus 2700 and immersed in the tank 2730.

[0213] Figure 34 is a schematic rear perspective view of the rotatable frame 2760. As shown in Figure 34, the rotatable frame 2760 may be composed of a first beam 2765 and a second beam 2770 that can be fixed to each other by a first end piece 2775 and a second end piece 2780. The first end piece 2775 and the second end piece 2780 may be configured to be attachable to a rotating mechanism. Further, although it is repetitive, one or both of the end pieces may be removable from the beam to allow the scaffolding structure (3405) to slide off the beam. In some embodiments, the end pieces of the frame may be formed from a flexible material such as rubber, so that the tension on the scaffolding structure is reduced and the beams may be compressed together to allow the scaffolding structure to be removed from the rotatable frame.

[0214] Furthermore, the first end piece 2775 and / or the second end piece 2780 may include a hole 3400. After the collagen ultra-fine fiber strand is wound, it may be cut from the spool, leaving an unknotted end. The unknotted end of the strand may be tied through the hole 3400 so that the scaffold structure does not come undone. This can be seen in FIG. 35 where the unknotted end 3430 is tied through the hole 3400. In some cases, the hole 3400 may be used to secure the tip of the collagen ultra-fine fiber to the rotatable frame before winding is performed.

[0215] As described with respect to the embodiments discussed above, the device may be configured such that the delivery device and the rotatable frame translate parallel to each other along the axis of rotation to wind the collagen ultra-fine fiber strand around different portions of the rotatable frame. FIG. 35 is a schematic front view of the rotatable frame of FIG. 34 with the collagen ultra-fine fiber wound in three separate regions. By moving the frame laterally with respect to the orifice (or vice versa), the collagen ultra-fine fiber may be collected in different regions of the rotatable frame.

[0216] In particular, the first scaffold structure 3410 may be formed in the first area of the rotatable frame 2760. The second scaffold structure 3415 may be formed in the second area of the rotatable frame 2760. Furthermore, the third scaffold structure 3420 may be formed in the third area of the rotatable frame 2760. The number of scaffold structures that can be formed on the rotatable frame may depend on the overall size of the frame, the number of strands being drawn / wound simultaneously, the thickness of the collagen ultra-fine fiber strand, and the overall size of the scaffold structure. FIG. 36 is a schematic front view of the rotatable frame 2760 having three regions of wound collagen ultra-fine fiber and suture 3425 joining the ends of the regions to form three separate scaffold structures.

[0217] Cell alignment, distribution, and mechanical properties In the following section, aspects of cell alignment, cell distribution, and mechanical properties of scaffold constructs formed using the methods disclosed herein will be described. The manufacturing methods for each of the evaluated samples are described below. However, since most of the strength is provided by the collagen nanofibers common to all types of scaffold constructs of the disclosure, it will be understood that the mechanical properties of the scaffold constructs formed by each of the different manufacturing methods described herein are expected to be substantially the same.

[0218] Examination revealed excellent fidelity and cellularity of the implant. The cellularized collagen nanofiber scaffold construct was formed using a single solution technique (i.e., using collagen nanofiber strands dispensed through a cellularized hyaluronic acid solution). After the winding operation, the scaffold construct was fixed into individual bundles using suture as described above.

[0219] hMSCs were printed on the reference implant, and alamarBlue analysis was used to evaluate the cell metabolic activity 1 day, 4 days, and 7 days after culturing. It was observed that the metabolic activity of the cellularized implant increased 5-fold during the 7-day culture period, indicating an increase in cell health, activity, and proliferation, as shown by fluorescence.

[0220] The cell viability of the entire printed implant was qualitatively and quantitatively evaluated by fluorescence imaging of hMSCs. Figure 37 is an image of three collagen nanofibers dispensed through a cell hyaluronic acid solution. Qualitatively, generally, a high viability is indicated by live cells (green) significantly outnumbering dead cells (red). Furthermore, Figure 37 shows cells 3700 placed directly on the surface of the collagen nanofibers. This is one of the advantages of the single solution approach, namely that cells tend to be attracted to the strands, in contrast to the two-solution approach where cells are slightly separated from the surface of the strands and remain floating or suspended in the gel to form a hydrogel. On the other hand, the hydrogel becomes harder, so there is a possibility that the distribution of cells on the scaffold construct will remain more uniform.

[0221] Quantitatively, ImageJ was used together with established cell counting techniques to compare the number of live and dead cells in the entire implant immediately after printing. In representative implants printed with typical process parameters, hMSCs were found to be 93.2 ± 1.7% viable immediately after printing, and the cell viability consistently exceeded 90% across different implant shapes and printing conditions.

[0222] Furthermore, fluorescence imaging of cells and fiber autofluorescence showed implants 1 day after printing with a uniform initial distribution of hMSCs throughout, and implants 26 days after culture with dense, high-density cellularization. Printed cells were found to adhere to collagen fibers within 24 hours and grow along the collagen fibers, continuing to proliferate until they reached a confluent state at a rate dependent on cell type, initial cell printing density, and culture conditions.

[0223] In long-term culture, the overall appearance of the implant changes from almost translucent with a visible fibrous surface texture to an opaque white-yellowish color with a smooth surface texture. This indicates a significant accumulation of the deposited extracellular matrix (ECM). Additionally, high-density intracellular growth was observed when cells bridged the gaps between adjacent fibers.

[0224] Over time, the cells elongate along the longitudinal length of the collagen superfine fibers. The substrate orientation has been shown to affect function, the potential for cell remodeling [Foolen 2018], and the alignment of the extracellular matrix produced by the cells [Wang 2003]. Therefore, ImageJ's orientation analysis was used to quantify the substrate orientation across the entire assembled cell-decorated collagen (AC-DC) implant using fluorescence imaging and image processing techniques. A representative composite image of a typical 2×2 mm field of view of an implant printed with MPCs after 14 days of culture is shown in Figure 38.

[0225] This alignment is quantified and shown in FIGS. 39 and 40. In particular, FIG. 39 is a graph showing the directional analysis of only the fiber component of the scaffold structure shown in FIG. 38. In the analysis of only the fiber component, a narrow frequency distribution is shown indicating fibers that are fairly parallel with almost all of the directional features within ±10° of the peak direction.

[0226] FIG. 40 is a graph showing the directional analysis of only the cell component of the scaffold structure shown in FIG. 38. In the analysis of only the cell component, a frequency distribution is shown with almost all components within ±20° of the peak and having an orientation of the peak that is essentially the same as the fiber direction, indicating that there is a fairly high degree of cell alignment parallel to the fibers. When this analysis was performed on the entire implant printed with both hMSCs and MPCs, it was concluded that the implant consistently showed highly aligned parallel collagen fibers and significant cell elongation in the fiber direction with a high degree of directionality.

[0227] Furthermore, a uniform cell distribution was achieved. The applicant has developed a method for quantifying the cell distribution across the entire scaffold structure by adapting means for analyzing the distribution of particles within the field of view [Han2014, Ober2015]. Such a method provides a quantitative means for verifying the AC-DC process control and the reproducibility of the uniform placement of cells throughout the implant. The results shown herein are representative and demonstrate the functionality of the cell distribution analysis method.

[0228] FIG. 41 is an image of the scaffold structure shown in FIG. 38 after processing (e.g., fluorescent labeling of the MPC cell plasma membrane) for cell distribution analysis with the cells shown in white. Note that the cells are distributed relatively uniformly across the structure along the structure. This uniform distribution is quantified and shown in FIGS. 42 and 43.

[0229] The printed implant image (e.g., the image shown in FIG. 41) was processed according to our protocol. The relative cell confluence along the horizontal and vertical directions of the image, determined by the number of white pixels indicating cell material compared to black pixels indicating cell-free spaces, was calculated and plotted. FIG. 42 is a graph showing the relative cell confluence along the horizontal axis of the scaffold construct shown in FIG. 41. FIG. 43 is a graph showing the relative cell confluence along the vertical axis of the scaffold construct shown in FIG. 41.

[0230] Plots of relative cell confluence provide a means to easily visualize the cell distribution across the entire printed implant. Peaks, valleys, and distortions indicate changes in the number and arrangement of cells across the field of view. Linear regression analysis can be further used as a simple method to evaluate cell confluence. In the case of a completely uniform cell distribution analyzed in 100 small containers, a horizontal line with a y-intercept of 0.01 is obtained as a result of linear regression analysis. Thus, the relative cell confluence of each small container would be 1 / 100 of the total number of cells. When measured across both the horizontal and vertical directions from a representative field of view of an AC-DC implant printed with MPC, the results of linear regression are nearly horizontal lines (FIGS. 42 and 43), indicating an essentially uniform distribution of cells across the entire print.

[0231] As shown in FIGS. 42 and 43, the applicant further implemented an additional method to quantify the cell distribution using a uniformity measure U based on Shannon entropy [Han1014, Ober2015]. Briefly, the range of the uniformity measure U is from 0 to 1, with a score of 0 for a completely non-uniform distribution where cells are present in exactly half of the field of view, and a score of 1 for a completely uniform distribution where cells are present exactly evenly throughout. Similar to the calculation and plotting of relative cell confluence, the uniformity was calculated across the horizontal direction (U in FIG. 42 trans ) and vertical direction (U in FIG. 43 long ) and determined using the same imaging and image processing techniques. For example, in the case of a representative AC-DC implant with MPC, cell uniformity analysis showed that, as shown in FIG. 42, U trans=0.87 is obtained, and as shown in FIG. 43, U long =0.95 is obtained.

[0232] The scaffold constructs formed using the processes described herein exhibit mechanical properties that approximate or exceed those of human skeletal tissue. MPCs were used for seeding in biomechanical tests because they are difficult to isolate and culture, well characterized, well adapted to culture, and widely used in various laboratories. The applicant evaluated the mechanical properties of AC-DC implants printed with hMSCs or AC-DC implants not printed with hMSCs at 1 day and 28 days in static culture to evaluate the load-bearing capacity, stability, and effect of in vitro cell remodeling. It was found that the custom 2-pin attachment method for tensile testing yielded significantly more consistent results compared to implant attachment with standard compression grips, which often leads to implant damage, slippage, or staggered breakage of individual fibers within the implant.

[0233] FIG. 44 is a graph showing the stress-strain curves of cell-free and cell-containing scaffold constructs. As shown, the representative stress-strain curves for each test group shown in FIG. 44 exhibit a distinct "toe" region with a gradually increasing slope, followed by a linear region of maximum slope, and finally a sharp decrease in stress indicating failure, which is clearly defined.

[0234] The cross-sectional area (Figure 45), ultimate tensile strength (UTS) (Figure 46), tangent modulus (Figure 47), and break point strain (Figure 48) were also measured in the same way. In the plots showing UTS (Figure 46) and tangent modulus (Figure 47), horizontal lines indicating the average UTS and tensile modulus of human ACL (a) [Chandrashekar 2006], the strongest part of the human supraspinatus tendon (b) [Itoi 1995], and a typical collagen gel used in tissue engineering (c) [Achilli 2010] are drawn. As shown in Figures 46 to 48, the acellular implants and cell implants generated using AC-DC bioprinting almost match or exceed the important mechanical properties of typical natural human tendons immediately after printing and continue to maintain that state even after 28 days of culture.

[0235] Notably, the UTS and modulus of the collagen nanofiber implants are several orders of magnitude greater than the strength and stiffness of collagen gels typical of biomanufacturing techniques, with a typical UTS of about 20 kPa and a tensile modulus of about 200 kPa [Achilli 2010]. Such values are characteristic of the collagen gel itself, regardless of the type of cells included or the intended maturation of the 3D-printed construct. Both the acellular implants and cell implants after 1 day and 28 days of culture underwent strains exceeding 20% before breaking (Figure 48). Thus, the AC-DC implants provide sufficient elasticity to withstand typical strain values in vivo, such as the 13.2% peak strain of the ACL during normal walking [Taylor 2013]. Although biomechanical testing of muscle is very difficult to accurately evaluate due to its anisotropy, viscoelasticity, hyperelasticity, and highly non-linear elastic behavior and has thus been little studied in this field, in general, the tensile properties of the AC-DC implants significantly exceed those of muscle. These tensile properties can be a UTS of at least 0.44 Mpa, a modulus of about 2.0 Mpa, and a break point strain exceeding 40% along the fiber direction.

[0236] Treatment of volumetric muscle loss Injuries to musculoskeletal tissues, 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 nanofiber implant for promoting the repair and regeneration of the function of such musculoskeletal soft tissues. Clinically relevant cells are controllably positioned along clinically relevant high-strength collagen fibers to biomanufacture a musculoskeletal tissue analog for restoring the form and function of damaged tissue.

[0237] Human mesenchymal stem cells (hMSCs) or rat muscle progenitor cells (MPCs) are bioprinted to create artificial implants that may be valuable for a variety of applications, including tendon or muscle regeneration. Mesenchymal stem cells offer excellent potential for enhancing the repair and regeneration of musculoskeletal tissues 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 shown significant therapeutic effects in promoting functional recovery of volumetric muscle loss injuries in validated animal models [Mintz (2020), Passipieri (2019)].

[0238] The present invention is based in part on the discovery that glutaraldehyde-crosslinked collagen fibers having high tensile strength can be used as filaments for bioprinting, and that such fibers can reproduce the structural, cellular, and mechanical similarities of natural tissues in an automated scalable manufacturing process. This was previously an ambitious and unfulfilled task [Murphy (2014), Murphy (2020)].

[0239] Results using implants manufactured using an aggregate cell-decorated collagen (“AC-DC”) bioprinting process showed that the cell orientation and distribution throughout the implant mimicked the cellular properties of native skeletal tissue. The bioprinted implants according to the invention approximate and can be tuned to exceed the strength and stiffness of human skeletal tissue. Furthermore, the implants far exceeded the properties of common collagen hydrogels.

[0240] In addition, the regenerability of such implants was evaluated in vivo in a murine VML model. A significant-sized muscle injury was created and repaired in the hindlimb, and the potential for torque generation in the limb was measured over 12 weeks. Both acellular and cellular implants were found to promote functional recovery compared to the unrepaired group, with AC-DC implants containing therapeutic muscle progenitor cells promoting the highest degree of recovery.

[0241] Histological analysis of explanted muscle cross-sections and automated image processing revealed an increase in total muscle fiber number, median muscle fiber size, and increased cellularization of the injury repaired with the cellularized implant. Such tests raise the great potential of advanced bioprinting methods to generate tissue analogs with the potential to repair many challenging skeletal injuries and with near-native biological and biomechanical properties.

[0242] Example: Functional recovery in the VML model An in vivo skeletal muscle repair trial was conducted over 12 weeks in a validated murine VML model. Details of such a trial are available in “Assembled Cell-Decorated Collagen (AC-DC) bioprinted implants mimic musculoskeletal tissue properties and promote functional recovery” by 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, published July 2, 2021, and a preprint is available via bioRxiv at https: / / doi.org / 10.1101 / 2021.06.22.449431. The entire disclosure of this document is incorporated herein by reference.

[0243] 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 directly evaluated against an untreated control group, a cell-free implant group that received repair with an AC-DC implant without cell components, and a cell implant group that received repair with an AC-DC implant printed with murine MPCs. The creation of the defect, the initial placement of the implant, the placement of sutures for implant attachment, and the replacement of the fascia are shown in FIGS. 49A through 49D, respectively. Specifically, FIG. 49A shows the creation of a VML injury that is approximately 1 cm × 0.7 cm × 0.5 cm in size and weighs at least 20% of the total weight of the TA. FIG. 49B shows a cell-free AC-DC implant inserted into the injury site. This implant is sutured to the injury site in FIG. 49C, and the arrows indicate the attachment points. FIG. 49D shows the fascia sutured over the injury site to further secure the implant in place.

[0244] All animals recovered after surgery, and no signs of infection or death were observed. Across the experimental groups, the body weights of the animals increased similarly over 12 weeks (Figure 49E). This shows the body weights of the animals before injury and at 4, 8, and 12 weeks after injury, corresponding to the time points of the functional tests. As shown in Figure 49F, the defect weights measured at the time of surgery were not statistically different. Figure 49F shows the weights of the defects that occurred in the "no repair", "acellular implant", and "cell implant" (NR, AI, and CI, respectively) experimental groups (p = 0.8, no significant difference). In Figures 49E to 49I, all data are based on n = 7 per group at each time point (*p < 0.05 indicates significance).

[0245] Functional tests were performed in vivo before defect creation and at 4, 8, and 12 weeks after repair to evaluate the postoperative muscle recovery. Briefly, the hindlimbs of the rats were attached to an electric treadmill and electrically stimulated to measure the maximum isometric torque generation [Mintz (2020), Passipieri (2019), Corona (2014)]. The average value was expressed as torque normalized to the body weight of the animal (N-mm / kg body weight) at each time point to control for the increase in torque generation due to the growth of the animal. As shown in Figure 49G, the baseline torque generation ability before defect creation did not change statistically among the treatment groups (p = 0.9, no significant difference). The torque generation after repair was expressed as raw torque (Figure 49H), and the percentage of the baseline torque generation is shown in Figure 49I. The percentage of the measured torque to the baseline torque is shown at 4, 8, and 12 weeks after repair. Such numbers indicate that the functional recovery is promoted by implant implantation. Although all methods show a similar trend, there are slight differences in statistical significance.

[0246] Most notably, a significant improvement in torque generation capacity was observed over 12 weeks for the injuries repaired using cell-based implants containing therapeutic MPCs. Four weeks later, the native torque generation was significantly lower in both the acellular implant group and the cell implant group than in the no repair group, and the percentage of the reference torque was significantly lower in the cell implant group. This initial decrease in torque generation capacity may be due to the initial wound healing process or may be related to the initial tensile properties of the implant. However, no differences were observed between the treatment groups by 8 weeks after repair.

[0247] At 12 weeks after repair, in contrast to the findings at 4 weeks, the native torque generation was found to be significantly higher in the cell implant group compared to the no repair group, and the percentage of the reference torque was significantly higher in both the acellular implant group and the cell implant group, revealing an important trend in the functional recovery of VML injury between the treatment groups. Furthermore, in animals that did not receive repair, a significant decline in function was observed over 12 weeks. In contrast, in animals repaired with acellular implants, the torque generation remained almost consistent, indicating that the presence of the cell-free collagen fiber implant reduced the functional decline associated with no repair.

[0248] Notably, resection of the synergistic muscle during defect creation removed approximately 20% of the torque generation in the anterior compartment [Mintz(2020)]. Thus, the normalized torque will be limited to a maximum of 85 N-mm / kg across all treatment groups (average 106 N-mm / kg at the reference value). The average functional recovery of the cell-based implant group at 12 weeks was 76% of the theoretical maximum recovery after synergistic muscle resection, compared to 67% in the acellular group and 57% in the no repair group. Furthermore, in 3 out of 7 animals repaired with cell implants, a functional recovery exceeding 87% was observed, and 1 animal recovered to almost the theoretical maximum recovery compared to the pre-injury level (99%).

[0249] After evaluating in vivo functional recovery at 12 weeks, TA muscles isolated for morphological and histological examination were harvested. The overall morphology of the muscles repaired by the acellular AC-DC implant and the cellular AC-DC implant appeared more similar to the control muscle than the unrepaired group with a convex indentation at the injury site. Additionally, more fascia was identified in the repaired groups. The distinction between the implant and the surrounding tissue was not clear, showing ingrowth of the tissue inside the collagen fiber implant or absorption of the implant. The isolated muscles were excised in the abdominal cross-section and processed for H&E staining. Representative images of each experimental group are shown in FIGS. 50A to 50D.

[0250] In FIGS. 50A to 50D, representative H&E images of the tibialis anterior (TA) muscle for the (A) uninjured control group, (B) unrepaired group, (C) acellular implant group, and (D) cellular implant experimental group at 12 weeks are shown. The black dashed line indicates the approximate area of lesion occurrence. The green dashed ellipse identifies the location of the AC-DC implant.

[0251] In FIGS. 50E to 50F, enlarged views of the location of the (E) acellular implant and (F) cellular implant, including the enlarged window views, show intracellular ingrowth and myofiber formation at the location of the cellular implant (yellow dashed ellipse). The scale bars in FIG. 50 are all 1 mm unless otherwise noted.

[0252] Similar to the macroscopic examination, the non-repaired group showed a distinct depression at the injury site, indicating a lack of tissue regeneration (Figure 50B). In contrast, in animals repaired with acellular implants and cellular implants, the tissue was more substantial and the cross-section was uniform, similar to the uninjured control group, resulting in an improved cosmetic effect. The remaining collagen fibers from the implant appeared as a dark pink, somewhat circular cross-section with a diameter of approximately 100 μm within the injury site. Inward growth of cells was visible within and around the implant (Figures 50E and 50F). The fiber cross-sections were more apparent in the acellular implant group than in the cellular implant group, probably indicating an increased rate of fiber absorption in the cellularized implant. For injuries repaired with the cell AC-DC implant, the presence of new muscle fibers at the implant site was noted (Figure 50F).

[0253] As the magnification of the images from the H&E sections increased, new muscle fibers and angiogenesis within the implant area in Figure 51 became more apparent, along with the deposition of new collagen qualitatively shown by Masson trichrome staining around the implant area in Figure 52. In Figure 51, prominent new muscle fibers are labeled "MF". These muscle fibers were found to be growing within the AC-DC implant as defined by the implant collagen fibers (labeled with "*"). In addition, prominent nearby blood vessels are shown in the implant area, indicated by the black arrows. Nerve bundles are indicated by "N". In Figure 52, it can be seen that collagen is present within and around the implant area and the defect area in the sections of each group.

[0254] Additional sections from the TA muscle belly were processed for analysis using SMASH, a semi - automated muscle fiber analysis software as shown in FIGS. 53A - 53K. In FIGS. 53A - 53D, representative laminin - stained sections of the TA muscle are shown for (A) the sham control group, (B) the no - repair group, (C) the acellular implant group, and (D) the cell implant experimental group. The dashed ellipse indicates the approximate area of injury. In FIGS. 53E - 53H, color - coded outputs from the software that identifies individual muscle fibers within the sections corresponding to FIGS. 53A - 53D respectively are shown. FIG. 53I shows the total fiber number, FIG. 53J shows the median fiber cross - sectional area (FCSA), and FIG. 53K shows the product of the fiber number and FCSA for the sham control (Ctrl) group, no - repair (NR) group, acellular implant (AI) group, and cell implant (CI) experimental group. The scale bar in all of FIGS. 53 is 1 mm. The data presented are based on n = 7 per group at each time point, and *p<0.05 indicates significance.

[0255] Referring to FIG. 53, laminin and fluorophore 488 staining identify the outlines of muscle fibers throughout the sections (FIGS. 53A - 53D), and SMASH analysis enables the discrimination of individual fibers as seen when the coloring is applied (FIGS. 53E - 53H). In the analysis of the total number of fibers, no significant difference occurs among the sham control group, no - repair group, acellular implant group, and cell implant group (FIG. 53I). However, the median fiber cross - sectional area (FCSA) of muscle sections repaired with acellular AC - DC implants and cell AC - DC implants was significantly larger than that of the no - repair group and not significantly different from that of the sham control group (FIG. 53J). The cell - seeded implant group and the control group show the largest differences from the no - repair group with p - values of 0.0007 and 0.0002 respectively.

[0256] Multiplying the total number of fibers by the median fiber cross - sectional area gives the total muscle fiber cross - sectional area (FIG. 53K). Repeating, this product shows no significant difference between the sham control group and the injury after 12 weeks of survival repaired with acellular implants and cell implants, supporting that the AC - DC implant promoted an increase in the total muscle fiber area.

[0257] In summary, a method of treating volumetric muscle loss (VML) includes applying a scaffold construct formed from a plurality of collagen ultrafine fibers arranged in an organized sheet-like configuration and coated with a biocompatible solution into the VML wound site. Optionally, applying the scaffold construct into the VML wound site may 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 may be sutured within the wound site. Further optionally, the method may further include replacing the fascia on the scaffold construct after applying the scaffold construct into the VML wound site.

[0258] It should also be noted that, optionally, a scaffold construct implanted within a given VML wound site may comprise a plurality of sheet-like layers of collagen ultrafine fibers. The number of layers utilized may vary depending on the depth of the wound site. Similarly, the overall size of the scaffold construct used may vary depending on the size of the wound site.

[0259] Optionally, the biocompatible solution coating the collagen ultrafine fibers may be hyaluronic acid. Optionally, the biocompatible solution may be a cell suspension. In such a case, the cell suspension may include muscle progenitor cells (MPCs). The cell suspension may include about 4,000,000 cells / mL. Alternatively, other concentrations may be used to form the scaffold construct.

[0260] The scaffold construct implanted to treat VML may have mechanical properties that substantially approximate or exceed those of the human tendon. Notably, the scaffold construct may have an average ultimate tensile strength (UTS), tensile modulus, and break point strain that substantially approximate or exceed those of the human tendon.

[0261] Preferred biocompatible scaffold construct In a preferred embodiment of the scaffold construct according to the present invention, the length dimension and the width dimension would independently range from about 1 mm to 10 mm depending on the size and shape of the damaged area where repair is intended. Preferably, the length and width of the scaffold construct to be used in VML would independently be about 2 cm to 9 cm, 3 cm to 8 cm or 4 cm to 7 cm. In an alternative embodiment, it may be standardized in a structure of 4 cm (width) × 10 cm (length), more preferably 6 cm × 10 cm, 8 cm × 10 cm and 10 cm × 10 cm.

[0262] The thickness of the scaffold construct according to the present invention may be limited by the ability of the recipient host's surrounding tissue to form blood vessels in the implant so that the cells adhered to the construct continue to survive. For this reason, in a preferred scaffold construct, the depth (thickness) would be 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 manufactured by forming about 2 to 8 layers of distributed and coated fibers. This reflects about 2 to 4 passes of the print head along the frame, since each pass of the print head wraps 360 degrees around the frame to form two layers.

[0263] For transplantation purposes, a physician may use multiple scaffold constructs for VML repair. Such constructs may be stacked or arranged sequentially along the area where repair is intended.

[0264] The print head may be configured to generate a scaffold construct capable of adjusting the spacing between fibers as discussed above. For example, the spacing between fibers would preferably range from an average of about 0 mm (i.e., the fibers are directly adjacent) to about 1 mm. In a preferred embodiment, the average spacing between fibers is about 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1,000 microns.

[0265] The cell suspension formulation according to the present invention will have a density sufficient to effectively coat the dispensed fibers of the scaffold construct. Preferred cell densities are in the range of about 0 to about 10 million cells / mL, as discussed above. Preferred cell suspension densities are about 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 a preferred embodiment, it is preferred that about 0.1 to 10 microliters of cell suspension is extruded per millimeter of the elongate fiber.

[0266] For a cell population adhering to a scaffold construct, the preferred number of cells on the construct will be in the range of about 100,000 to about 1 million cells per implant, or more than 1 million cells per implant. Preferred ranges are 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.

[0267] In the preferred hydrogels as described above, it is necessary to start solidification within seconds after contacting the crosslinking solution and rapidly stabilize.

[0268] Although various embodiments have been described, this description is not intended to be limiting but is exemplary, and it will be apparent to those skilled in the art that many more embodiments and implementation examples are possible within the scope of the embodiments. Many possible combinations of features are shown in the accompanying figures and are considered in this detailed description, but many other combinations of the disclosed features are possible. Unless otherwise restricted, any feature of any embodiment may be used in combination with any other feature or element of any other embodiment, or used in place of any other feature or element. Thus, it will be understood that any of the features shown and / or considered in this disclosure may be implemented together in any suitable combination. The features, parameters, characteristics, and measurement criteria related to the various embodiments discussed above apply to other embodiments disclosed herein unless the contrary is explicitly shown or they are non-practical. For this reason, the embodiments should not be restricted except when considering the appended claims and their equivalents. Additionally, various modifications and changes may be made within the scope of the appended claims.

[0269] References

Prior Art Documents

Non-Patent Documents

[0270]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Non-Patent Document 19

Non-Patent Document 20

Non-Patent Document 21

Non-Patent Document 22

Non-Patent Document 23

Non-Patent Document 24

Non-Patent Document 25

Non-Patent Document 26

Non-Patent Document 27

Non-Patent Document 28

Non-Patent Document 29

Non-Patent Document 30

Non-Patent Document 31

Non-Patent Document 32

Claims

1. A scaffold structure comprising a plurality of collagen ultra-fine fibers, wherein a first portion of the collagen ultra-fine fibers is coated with a first biocompatible solution and a second portion of the collagen ultra-fine fibers is coated with a second biocompatible solution.

2. An apparatus for creating a scaffold structure, the apparatus comprising: a delivery device configured to dispense a collagen ultra-fine fiber strand through an orifice; a rotatable frame; a solution manifold including a first well configured to receive a first biocompatible solution, the solution manifold being disposed between the delivery device and the rotatable frame; a rotation device configured to rotate the rotatable frame about a rotation axis, wherein the apparatus is configured to draw the collagen ultra-fine fiber strand through the orifice and the solution manifold by rotation of the rotatable frame, thereby coating the collagen ultra-fine fiber strand with the first biocompatible solution in the solution manifold and winding the collagen ultra-fine fiber strand around the rotatable frame.

3. A method for creating a scaffold structure, the method comprising: attaching a collagen ultra-fine fiber strand to a rotatable frame; rotating the rotatable frame about a rotation axis to wind the collagen ultra-fine fiber strand around the rotatable frame, wherein winding the collagen ultra-fine fiber strand around the rotatable frame draws the collagen ultra-fine fiber strand from a delivery device, thereby dispensing the collagen ultra-fine fiber strand through an orifice and drawing the collagen ultra-fine fiber strand through a first well of a solution manifold filled with a first biocompatible solution.

4. A scaffold structure comprising a plurality of collagen ultra-fine fibers arranged in an organized configuration and coated with a biocompatible solution, wherein the collagen ultra-fine fibers are arranged in a continuous loop.

5. An apparatus for creating a scaffold structure, the apparatus comprising: a delivery device configured to dispense a collagen ultra-fine fiber strand through an orifice; a rotatable frame; A strand coating system configured to coat the collagen ultra-fine fiber strand with a biocompatible solution during distribution of the strand, wherein the solution strand coating system is disposed between the delivery device and the rotatable frame, the strand coating system; A rotation device configured to rotate the rotatable frame about a rotation axis, and; The apparatus is configured to draw out the collagen ultra-fine fiber strand through the orifice by rotation of the rotatable frame, thereby coating the collagen ultra-fine fiber strand with the biocompatible solution and winding the collagen ultra-fine fiber strand around the rotatable frame. apparatus. **Claim 6** A method of creating a scaffold structure, comprising: Attaching a collagen ultra-fine fiber strand to a rotatable frame; Rotating the rotatable frame about a rotation axis to wind the collagen ultra-fine fiber strand around the rotatable frame, and; By winding the collagen ultra-fine fiber strand around the rotatable frame, the collagen ultra-fine fiber strand is drawn out from a delivery device, thereby distributing the collagen ultra-fine fiber strand through an orifice and drawing the collagen ultra-fine fiber strand through a biocompatible solution. method. **Claim 7** A scaffold structure comprising a plurality of collagen ultra-fine fibers arranged in an organized configuration and coated with a biocompatible hydrogel, The scaffold structure, wherein the collagen ultra-fine fibers are arranged in a continuous loop. **Claim 8** An apparatus for creating a scaffold structure, the apparatus comprising: A delivery device configured to distribute a collagen ultra-fine fiber strand through an orifice; A rotatable frame; A strand coating system configured to coat the collagen ultra-fine fiber strand with a first biocompatible solution during distribution of the strand, wherein the solution strand coating system is disposed between the delivery device and the rotatable frame, the strand coating system; A rotation device configured to rotate the rotatable frame about a rotation axis, and; The device is configured to draw out the collagen ultrafine fiber strand through the orifice by the rotation of the rotatable frame, thereby coating the collagen ultrafine fiber strand with the first biocompatible solution and winding the collagen ultrafine fiber strand around the rotatable frame. The device further comprises a tank configured to contain a second biocompatible solution in which the rotatable frame can be immersed while winding the collagen ultrafine fiber strand around the rotatable frame. **Claim 9** A method of creating a scaffold structure, comprising: attaching a collagen ultrafine fiber strand to a rotatable frame; and rotating the rotatable frame about a rotation axis to wind the collagen ultrafine fiber strand around the rotatable frame. By winding the collagen ultrafine fiber strand around the rotatable frame, the collagen ultrafine fiber strand is drawn out from a delivery device, thereby distributing the collagen ultrafine fiber strand through an orifice and drawing the collagen ultrafine fiber strand through a first biocompatible solution. While winding the collagen ultrafine fiber strand around the rotatable frame, the rotatable frame is immersed in a second biocompatible solution. The method, wherein the first biocompatible solution is combined with the second biocompatible solution to form a biocompatible hydrogel. **Claim 10** A method of treating volumetric muscle loss (VML), comprising: applying a scaffold construct formed from a plurality of collagen ultrafine fibers arranged in an organized sheet-like configuration and coated with a biocompatible solution within a VML wound site. **Claim 11** A method of treating volumetric muscle loss (VML), comprising: applying a scaffold construct formed from a plurality of collagen ultrafine fibers arranged in an organized sheet-like configuration within a VML wound site, wherein the scaffold construct comprises a plurality of sheet-like layers of the collagen ultrafine fibers.

Images