Microfluidic extrusion

Abstract

Biopolymer fibers comprising collagen have excellent ultimate tensile strength, modulus, and elongation at break comparable to those of natural human tendons and ligaments. The fibers may be substantially round, oval, square, rectangular, ribbon-like, triangular, or irregular in shape. The fibers exhibit a regular longitudinal orientation, allowing for the infiltration of cell growth. Microfluidic and extrusion methods for producing the biopolymer fibers are also provided, as well as implantable biopolymer scaffolds and sutures comprising the fibers.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of concurrently pending application No. 62 / 800,317, filed on 1 February 2019, the disclosure thereof being incorporated in its entirety by reference.

[0002] Statement regarding government support The data presented herein was supported, at least in part, under DARPA Contract No. HR0011-15-9-0006. The U.S. Government has certain rights to the present invention. [Background technology]

[0003] Background of the Invention 1. Field of this disclosure This disclosure relates to a method for producing collagen fibers, a method for incorporating them into a scaffold, and an implantable biocompatible device made from such fibers. In particular, this disclosure relates to a method for extruding collagen fibers having excellent mechanical strength, biocompatibility, and immunological properties. 2. Description of Prior Art Collagen is a fibrous, insoluble protein composed of bundles of fine reticular fibrils. Collagen protein molecules bind together to form the white, shiny, inelastic fibers of tendons, ligaments, and fascia. Collagen is found in connective tissue, including skin, bones, ligaments, and cartilage.

[0004] In particular, collagen fibrils bind together to form strong connective tissues such as ligaments and tendons. Much effort has been made to manufacture collagen-containing tissues for use in the body to replace damaged collagenous body parts (especially ligaments and tendons). Damaged areas may be directly replaced with such implantable devices, or they may be used to provide a scaffold for damaged soft tissues such as tendons and ligaments, ultimately aiding in their replacement.

[0005] Such products need to function in a variety of challenging biomechanical environments that require addressing multiple functional parameters. These parameters include, for example, compatibility with biological tissues and bodily fluids, strength, flexibility, and biodegradability.

[0006] Systems and methods to address the shortcomings of the above-mentioned prior art are needed in this field. [Overview of the Initiative]

[0007] In one aspect, this disclosure relates to a biopolymer fiber containing collagen, which has the following characteristics Extreme tensile strength of approximately 1 MPa to approximately 1,700 MPa, Elastic modulus of approximately 10 MPa to approximately 20,000 MPa, Elongation at break of approximately 2 percent to 45 percent. Average fiber diameter: approximately 10 μm to 90 μm Having one or more of the following, After immersing in DPBS at room temperature for at least about 1 hour, maintain its strength. The filaments exhibit a regular longitudinal orientation structure.

[0008] In another aspect, this disclosure relates to a bundle of biopolymer fibers containing 2 to about 10,000 fibers.

[0009] In yet another aspect, the disclosure relates to an implantable biopolymer scaffold for assisting the repair of soft tissue damage, comprising biopolymer fibers or bundles thereof.

[0010] This disclosure also relates to woven sheet-like supports, patches, or orthotic devices containing biopolymer fibers.

[0011] In yet another aspect, this disclosure relates to a method for producing biopolymer fibers. The steps include: dissolving collagen in an acid solution to create a collagen solution, The step of forming a coaxial flow by simultaneously delivering a collagen solution at a first velocity through a first needle having a first diameter, and delivering a forming buffer at a second velocity through a second needle that coaxially surrounds the first needle, has a second diameter exceeding the first diameter, and forms a sheath around the collagen solution, The second flow rate of the basal buffer passed through the second needle is at least twice the first flow rate of the collagen solution passed through the first needle, step, A step of delivering collagen and formation buffer coaxially through a reaction region containing a fibril-forming bath at a time and rate sufficient to form fibers, A step of dehydrating collagen fibers at a certain extrusion rate, The process includes the step of taking the fibers onto a spool at a third speed exceeding the extrusion speed, which is sufficient to increase the molecular orientation and reduce the fiber diameter.

[0012] In another aspect, this disclosure relates to a method for producing biopolymer fibers. The steps include: dissolving collagen in an acid solution to create a collagen solution, A step of delivering a collagen solution to a forming buffer at a first velocity through a first needle having a first diameter, A step of delivering collagen and forming buffer through a reaction region containing a fibril-forming bath at a time and rate sufficient to form fibers, A step of dehydrating collagen fibers at a certain extrusion rate, The process includes the step of taking the fibers onto a spool at a speed of about twice to about ten times the extrusion speed, which is sufficient to increase the molecular orientation and reduce the diameter of the fibers.

[0013] In yet another aspect, this disclosure relates to a method for producing biopolymer fibers, The steps include: dissolving clinical-grade collagen in an acid solution to prepare a collagen solution, A step of sending a collagen solution at a first volumetric flow rate through a first needle to produce a first velocity, and simultaneously sending a forming buffer at a second velocity through a tube that coaxially surrounds the first needle to form a sheath around the collagen solution to form a coaxial flow, A step in which the velocity of the base buffer is from about 2 times to about 20 times the first velocity of the collagen solution through the first needle, A step of sending the collagen and the forming buffer flowing coaxially through a reaction region including a fibril formation bath at a time and velocity sufficient to form fibers, A step of dehydrating the collagen fibers at a certain extrusion rate, A step of taking out the fibers at a third velocity exceeding the extrusion rate, which is sufficient to enhance the orientation of the molecules and reduce the diameter of the fibers, and relates to a method including these steps.

[0014] In an additional aspect, the present disclosure relates to a method for manufacturing biopolymer fibers, A step of dissolving clinical-grade collagen in an acid solution to prepare a collagen solution, A step of extruding the solution through a nozzle into a guide that sends the extruded solution into a bath of a forming buffer, A step of dehydrating the fibers formed in the forming buffer bath, A step of recovering the fibers, and relates to a method including these steps.

[0015] In a further additional aspect, the present disclosure relates to a method for manufacturing biopolymer fibers, A step of dissolving clinical-grade collagen in an acid solution to prepare a collagen solution, A step of sending the collagen solution into a forming buffer at a first velocity through a first needle having a first diameter, A step of sending the collagen and the forming buffer through a reaction region including a fibril formation bath at a time and velocity sufficient to form fibers, A step of dehydrating the collagen fibers at a certain extrusion rate, A step of taking out the fibers at a third velocity exceeding the extrusion rate, which is sufficient to enhance the orientation of the molecules and reduce the diameter of the fibers, and relates to a method including these steps. The present invention relates to a method comprising one or more steps of taking the fibers onto a spool at a speed of about twice to about twelve times the extrusion speed, which is sufficient to increase the molecular orientation and reduce the diameter of the fibers.

[0016] This disclosure also includes aspects of providing an implantable biomolecular scaffold containing biomolecular fibers to assist in the repair of soft tissue damage, and a method for assisting in the repair of soft tissue damage, including the implantation of the biomolecular scaffold.

[0017] Upon closer examination of the following drawings and detailed description, other systems, methods, features, and advantages of the present invention will be apparent or will become apparent to those skilled in the art. All such additional systems, methods, features, and advantages are contained herein and in this abstract, are within the scope of the present invention, and are intended to be protected by the following claims.

[0018] The present invention can be better understood by referring to the following drawings and description. The components in the drawings are not necessarily to scale, and instead, emphasis is placed on illustrating the principles of the present invention. Furthermore, in the drawings, similar reference numbers indicate corresponding parts in different drawings. [Brief explanation of the drawing]

[0019] [Figure 1] Figure 1 is a schematic diagram of an embodiment of the method disclosed herein. [Figure 2] Figure 2 is a schematic diagram showing the first step in the formation of a collagen solution in one embodiment of the present disclosure. [Figure 3] Figure 3 is a schematic diagram of the use of a degassing apparatus in collagen production in one embodiment of the method disclosed herein. [Figure 4] Figure 4 is a schematic diagram of a centrifuge suitable for use in embodiments of the methods disclosed herein. [Figure 5] Figure 5 is a schematic diagram illustrating the use of a centrifuge in one embodiment of the present disclosure. [Figure 6]Figure 6 is a schematic diagram of an embodiment of a coaxial needle used to form collagen fibers in one embodiment. [Figure 7] Figure 7 is a schematic diagram of a collagen fiber reaction region including a fibril-forming bath in one embodiment of the present disclosure. [Figure 8] Figure 8 shows a schematic embodiment of a dehydration bath in one embodiment of the present disclosure. [Figure 9] Figure 9 is a schematic diagram of an embodiment of a device for separating collagen fibers from a dehydration bath. [Figure 10] Figure 10 is a schematic diagram of a fiber recovery system in one embodiment of the present disclosure. [Figure 11] Figure 11 is a schematic diagram of a fiber recovery spool in one embodiment of the present disclosure. [Figure 12] Figure 12 is a schematic diagram of the termination process according to an embodiment of the present disclosure. [Figure 13] Figure 13 is a schematic diagram of a method according to one embodiment of the present disclosure. [Figure 14] Figure 14 is a schematic diagram of a method according to one embodiment of the present disclosure. [Figure 15] Figure 15 is a schematic diagram of one embodiment of equipment suitable for use in manufacturing the products of this disclosure. [Figure 16] Figure 16 shows additional details of the equipment shown in Figure 15. [Figure 17] Figure 17 is a table summarizing the compositions used in this disclosure. [Figure 18A] Figure 18A is a table summarizing the conditions used in the embodiment. [Figure 18B] Figure 18B is a table summarizing the conditions used in the embodiment. [Figure 18C] Figure 18C is a table summarizing the conditions used in the embodiment. [Figure 19] Figure 19 is a graph summarizing the mechanical properties related to this disclosure. [Figure 20] Figure 20 is a graph summarizing other mechanical properties related to this disclosure. [Figure 21]Figure 21 is a graph summarizing yet another mechanical property related to this disclosure. [Figure 22] Figure 22 is a magnified image of a composition manufactured in accordance with this disclosure. [Figure 23] Figure 23 is a graph summarizing the width of the embodiments of this disclosure. [Figure 24] Figure 24 is a graph summarizing the thickness of the embodiments of this disclosure. [Figure 25] Figure 25 is a graph summarizing the mechanical properties of the embodiments of this disclosure. [Figure 26] Figure 26 is a graph summarizing another mechanical property of an embodiment of the present disclosure. [Figure 27] Figure 27 is a graph summarizing the relationships of the mechanical properties of the embodiments of this disclosure. [Figure 28] Figure 28 is a graph summarizing the relationships of other mechanical properties in embodiments of this disclosure. [Figure 29] Figure 29 is a graph summarizing the characteristics of the embodiments of this disclosure. [Figure 30] Figure 30 is a color image of an embodiment of the present disclosure. [Figure 31] Figure 31 is a graph summarizing the characteristics of the embodiments of this disclosure. [Figure 32] Figure 32 is a graph summarizing another characteristic of the embodiment of the present disclosure. [Figure 33] Figure 33 is a graph summarizing yet another characteristic of the embodiment of the present disclosure. [Figure 34] Figure 34 is an enlarged color image of one embodiment of the present disclosure. [Figure 35] Figure 35 is a magnified color image of a microfiber product. [Figure 36] Figure 36 is a magnified color image of a control microfiber product. [Figure 37] Figure 37 is an image of a feature of one embodiment of the present disclosure. [Figure 38] Figure 38 is an image of a feature of one embodiment of the present disclosure. [Figure 39]Figure 39 is a graph summarizing the features of the embodiments of this disclosure. [Figure 40] Figure 40 is a graph summarizing how the size of one embodiment of the present disclosure changes over time. [Figure 41] Figure 41 is a graph summarizing how the mechanical properties of one embodiment of the present disclosure change over time. [Figure 42] Figure 42 is a graph summarizing how another mechanical property of one embodiment of the present disclosure changes over time. [Figure 43] Figure 43 is a graph summarizing how yet another characteristic of one embodiment of the present disclosure changes over time. [Figure 44] Figure 44 is a graph summarizing how yet another characteristic of one embodiment of the present disclosure changes over time. [Figure 45] Figure 45 is a table summarizing the comparative information. [Figure 46] Figure 46 shows an embodiment of the method of the present disclosure. [Figure 47] Figure 47 is a diagram summarizing the properties and characteristics of one embodiment of the present disclosure. [Figure 48] Figure 48 is a diagram summarizing the properties and characteristics of one embodiment of the present disclosure. [Figure 49] Figure 49 is a diagram summarizing the properties and characteristics of one embodiment of the present disclosure. [Figure 50] Figure 50 is a diagram summarizing the properties and characteristics of one embodiment of the present disclosure. [Figure 51] Figure 51 is a diagram summarizing the properties and characteristics of one embodiment of the present disclosure. [Figure 52] Figure 52 is a diagram summarizing the properties and characteristics of one embodiment of the present disclosure. [Figure 53] Figure 53 is a diagram summarizing the properties and characteristics of one embodiment of the present disclosure. [Figure 54] Figure 54 is a diagram summarizing the properties and characteristics of one embodiment of the present disclosure. [Figure 55]Figure 55 is a diagram summarizing the properties and characteristics of one embodiment of the present disclosure. [Figure 56] Figure 56 is a diagram summarizing the properties and characteristics of one embodiment of the present disclosure. [Figure 57] Figure 57 is an SEM image of one embodiment of the present disclosure. [Figure 58] Figure 58 is an SEM image of one embodiment of the present disclosure. [Modes for carrying out the invention]

[0020] In one aspect, this disclosure relates to a biopolymer fiber containing collagen, which has the following characteristics Extreme tensile strength of approximately 1 MPa to approximately 1,700 MPa, Elastic modulus of approximately 10 MPa to approximately 20,000 MPa, Elongation at break of approximately 2 percent to 45 percent. Average fiber diameter: approximately 10 μm to 90 μm Having one or more of the following, After immersing in DPBS at room temperature for at least about 1 hour, maintain its strength. The filaments exhibit a regular longitudinal orientation structure.

[0021] In another aspect, the present disclosure relates to an implantable biopolymer scaffold for assisting the repair of soft tissue damage, comprising at least one biopolymer sheet containing biopolymer fibers, wherein the biopolymer contains collagen and has the following characteristics Extreme tensile strength of approximately 1 MPa to approximately 1,700 MPa, Elastic modulus of approximately 10 MPa to approximately 20,000 MPa, Elongation at break of approximately 2 percent to 45 percent. Average fiber diameter: approximately 10 μm to 90 μm Having one or more of the following, After immersing in DPBS at room temperature for at least about 1 hour, maintain its strength. The filaments exhibit a regular longitudinal orientation structure.

[0022] This fiber exhibits a regular longitudinal orientation structure, enabling cell invasion after implantation of this fiber and a device manufactured with this fiber according to the present invention into a subject.

[0023] In another aspect, the disclosure includes implantable biomolecular scaffolds for the repair or replacement of human body parts.

[0024] Biomolecular fibers are typically formed from collagen. In particular, telocollagen is usually obtained from any source (human, bovine, recombinant, jellyfish, etc.). Biomolecular fibers may be formed by blending collagen with bioacceptable polymers such as silk fibroin, other types of collagen (such as type II collagen), fibrin / fibrinogen, basement membrane proteins, hyaluronic acid, polyethylene oxide, polyethylene glycol, polycaprolactone, polyethylnene, polyhydroxybutyrate, PDLA, PDLLA and high molecular weight PDLLA, PLGA, and blends thereof.

[0025] In yet another aspect, the present disclosure relates to a method for producing a biopolymer fiber, comprising the step of dissolving collagen in an acid solution to create a collagen solution. In one embodiment of this method, the collagen is then fed at a first velocity through a first needle having a first diameter to achieve a first velocity, while simultaneously forming a coaxial flow by feeding a forming buffer at a second volumetric flow rate through a second needle coaxially surrounding the first needle and having a second diameter greater than the first diameter, forming a sheath around the collagen solution. The second volumetric flow rate of the forming buffer through the second needle is at least about twice the first volumetric flow rate of the collagen solution through the first needle.

[0026] Collagen and forming buffer are delivered coaxially through the reaction region containing the fibril-forming bath at a time and rate sufficient to form fibers, and the fibers are removed from the spool at a third rate greater than the first rate, usually twice the rate at which the fibers are extruded through the dehydration bath, sufficient to increase molecular orientation and reduce the diameter of the fibers. The fibers may then be crosslinked and dried.

[0027] In yet another aspect, the disclosure relates to an alternative method for producing biopolymer fibers. In this embodiment, a collagen solution is prepared and injected into a reaction region containing a fiber-forming bath, such as a forming buffer bath, at a rate and time sufficient to form fibers. The fibers are removed from the spool at a rate of about 2 to 10 times the injection rate to enhance molecular orientation and reduce fiber diameter. The fibers may then be crosslinked and dried.

[0028] In various embodiments of this disclosure, collagen or collagen and other suitable biopolymers are processed into biopolymers or collagen fibers. For ease of understanding, the features of this disclosure are described in relation to collagen. However, to obtain the types of fibers disclosed herein, collagen may be blended or mixed with suitable biopolymers in various combinations and proportions.

[0029] Throughout this specification, steps that are typically considered to be performed together during a standard manufacturing process, such as washing and drying, or immersion and drying, may be performed or repeated as necessary to achieve the desired results. For example, in one embodiment, the composition may be washed and dried before proceeding to the next step. In some embodiments, the material may be washed twice and dried twice before proceeding to the next processing step, or it may be washed twice and then proceeded to.

[0030] In other embodiments, the first washing or drying step may be optional. Therefore, materials that are normally washed and then dried may proceed directly to the drying step and then to the next processing step. Those skilled in the art will recognize situations in which these steps may be repeated or omitted.

[0031] Structures such as scaffolds made from these fibers enable intracellular proliferation. That is, cells of various types of animals into which the fibers (and devices made from the fibers) are embedded preferably align with the fibers of the scaffold and proliferate within the pores of the scaffold. The structures and scaffolds include single-layer and multi-layer products which may be used as substitutes for known repair functions, such as sutures used to reattach body parts, such as the opposing portion of a ruptured Achilles tendon. In addition to providing support structures for use in repairing torn or damaged tendons, embodiments of the present disclosure are also suitable for ligament repair. Thus, other exemplary ligaments for which a scaffold or support may be provided using the present invention include the ACL, MCL, PCL, UCL, and other human and animal ligaments. Other surgeries in which the products of the present disclosure are useful include superior capsular reconstruction as a treatment option for superior rotator cuff tears, in particular, where reconstruction of partial or complete tears is otherwise impossible or difficult. Multi-layer sheets may also be used to cover and reinforce the repair site.

[0032] In particular, embodiments of the present disclosure may be suitable for the repair of ligaments, tendons, and other soft tissues in all types of animals. The collagen fibers of the present disclosure may be used, for example, to rejoin torn ligaments and tendons, even if only partial tears are involved. Multiple fibers may be twisted, bundled, braided, woven, or otherwise arranged to improve the shape factor so that it is easier to manipulate than with a single fiber, for example during surgery. Improving the shape factor may facilitate the precise placement of the fibers or platform. Other shape factors may be constructed to serve as reinforcements or internal orthoses for torn natural body parts. Orthoses support joints by connecting one bone to another. Typically, orthoses form isometric joints that have the biomechanics and isometricity of the restored natural joint.

[0033] The effects of collagen fibers manufactured according to embodiments of this disclosure may be demonstrated, for example, by studying repairs performed on rabbits. In particular, reinforcements, internal orthoses, and stitched structures for rabbit knees are suitable for evaluating the properties and characteristics of the collagen fibers of this disclosure and structures manufactured with these fibers.

[0034] Figure 1 shows one embodiment of a system and method for producing collagen fibers. This system and method may be described as comprising four sections or production areas. A collagen solution is prepared in the first section, and collagen fibers are formed in the second section. The collagen fibers are then collected in the third section, and then post-processed in the fourth section, post-treatment, or final treatment to obtain wet or dry collagen fibers.

[0035] The steps of the system and method shown in Figure 1 may be classified into the following four categories. Category Name Steps Included 1. Preparation of collagen solution 105-120 2. Formation of collagen fibers 125-130 3. Collagen fiber recovery 135-150 4. Post-processing or final processing 155-180 As seen in step 105 of Figure 1, the collagen is mixed with an acidic solution and thoroughly stirred in step 110. In some embodiments, the acid is about 0.01 M to about 0.50 M acetic acid. In other embodiments, the acid is about 0.01 M to about 0.50 M hydrochloric acid. The solution may be degassed in step 115 and then centrifuged in step 120 to remove any remaining air bubbles. The resulting collagen solution is extruded through a needle, and a second needle coaxial with the first needle may be present to supply the forming buffer in step 125. The resulting fiber formation may be continued by the forming tube in step 130. The resulting product is the formed collagen fibers.

[0036] The fibers then proceed to the recovery system. In this system, the fibers are separated from the forming buffer in step 135 and dehydrated in step 140. The collagen fibers are recovered in step 145 and air-dried in step 150. Post-processing may then be performed in steps 155, 160, 165 and 170 as shown in the figure. The air-dried collagen fibers on the spool are immersed in the cross-linking solution in step 155, optionally washed in step 160, air-dried in step 165, and dried in step 170 to form dried fibers. As shown by the dashed line in Figure 1, the material may optionally be washed in step 160, dried in step 165, and returned to washing step 160.

[0037] Alternatively, collagen is injected into a forming solution bath to form fibers. In this system, a second needle for coaxial injection of the forming buffer is not required. The injected collagen is then introduced into a recovery system after dehydration in step 140. The fibers are then processed according to the remainder of the processing steps.

[0038] Figure 1 shows a general diagram of a system and method for carrying out embodiments of the present disclosure. Additional details and disclosures are included in the following specific aspects and embodiments of this specification.

[0039] In one embodiment, the present disclosure relates to a method for producing a biopolymer fiber, comprising the step of dissolving collagen in an acidic solution to prepare a collagen solution. The collagen is then delivered at a first volumetric flow rate through a first needle having a first diameter to produce a first velocity, while simultaneously a forming buffer is delivered at a second volumetric flow rate through a second needle coaxially surrounding the first needle and having a second diameter greater than the first diameter, forming a sheath around the collagen solution to create a coaxial flow. The second volumetric flow rate of the forming buffer through the second needle is at least twice the first volumetric flow rate of the collagen solution through the first needle.

[0040] Collagen and forming buffer are delivered coaxially through a reaction region containing a fibril-forming bath at a volumetric flow rate sufficient for sufficient time and to form fibers, and the fibers are extracted onto a spool at a third velocity greater than the first velocity. The third velocity is typically about twice the velocity at which the fibers are extruded through the dehydration bath, and is sufficient to increase molecular orientation and reduce the fiber diameter. The fibers are then crosslinked and dried.

[0041] In another embodiment, the disclosure relates to an alternative method for producing biopolymer fibers. A collagen solution is prepared and injected into a reaction region containing a fibril-forming bath, such as a forming buffer bath, at a rate and time sufficient to form fibers. A second needle to form a coaxial flow of the forming buffer is not required. Instead, the collagen fibers are injected directly into the fibril-forming bath and then transported through a dehydration bath. The fibers are transported by spooling them at a rate of about 2 to 4 times the injection rate to enhance molecular orientation and reduce fiber diameter. The fibers may then be crosslinked and dried.

[0042] An embodiment of Method 1300 is summarized in Figure 13. The collagen solution is prepared as shown in the figure. The collagen solution is prepared in step 1305. A biopolymer may be mixed with this collagen. The collagen is dissolved in an acidic solution to create a viscous solution. In step 1310, this solution is stirred to ensure thorough mixing. This mixed solution may contain trapped gases and therefore may be degassed one or more times in step 1315 using a degasser. The collagen solution may then be centrifuged in step 1320 as shown in the figure. Optionally, the degassing / centrifugation step may be repeated to reduce the volume of trapped gases in the solution, as shown by the dashed line in Figure 1 and feature 1316 in Figure 13.

[0043] The collagen solution thus prepared is formed into collagen fibers by coaxial extrusion with a forming buffer, which acts as a sheath for the fiber core, as shown in step 1325. The volumetric flow rate of the forming buffer is typically at least twice the volumetric flow rate of collagen formation. This configuration can suppress the formation of individual fibrils, stretch and direct the fibers, and smooth the fiber surface by imparting flow-induced crystallization to the fibers.

[0044] Next, the collagen fibers are recovered. Since the formation of the collagen fibers is complete in step 1330, the collagen is then separated from the formation buffer in step 1335 and dehydrated in the dehydration solution in step 1340. Next, in step 1345, the dehydrated collagen is recovered onto a rotating spool and the fibers are further stretched by rotating it at a speed exceeding the rate at which the fibers are supplied from the dehydration solution in step 1340, usually at about twice the speed. Finally, the recovered fibers are air-dried on the spool in step 1350.

[0045] In an alternative embodiment, the collagen solution is formed into collagen fibers by direct injection into the formation buffer. This omits step 1325. The fibers are collected, separated from the formation buffer, and dehydrated in a dehydrated solution in step 1340. The fibers are collected on a rotating spool in step 1345, and the rotating spool collects the fibers at a rate of approximately 2 to 4 times the formation rate.

[0046] Next, the air-dried fibers on the spool may be post-treated. The fibers may be cross-linked in a cross-linking solution in step 1355, and then rinsed in step 1360. The fibers are then air-dried in step 1365 and dried in step 1370 to obtain dried cross-linked collagen fibers.

[0047] The apparatus used for manufacturing collagen fibers is made of conventional constituent materials suitable for withstanding attacks by any of the raw materials used for manufacturing collagen fibers according to embodiments of this disclosure. Metals, plastics, and other materials have properties and characteristics suitable for withstanding attacks by raw materials, intermediates, solvents, and products during the manufacturing of collagen fibers.

[0048] Another aspect of this disclosure relates to collagen fibers, which have the following characteristics Extreme tensile strength of approximately 1 MPa to approximately 1,700 MPa, Elastic modulus of approximately 10 MPa to approximately 20,000 MPa, Elongation at break of approximately 4 percent to 12 percent. Average fiber diameter: approximately 16 μm to 70 μm Having one or more of the following, After immersion in biological fluid for approximately one hour, it maintains its strength at least.

[0049] These fibers exhibit a regular longitudinal orientation structure, which allows for the invasion of cells for proliferation.

[0050] The fibers of this embodiment are manufactured according to the methods of the embodiments of this disclosure. Collagen may be obtained from many sources and in various forms. The quality of the collagen fibers may depend on the quality of the raw materials used. In some embodiments, bovine collagen is typically used. Bovine collagen may be in its natural form or as a freeze-dried powder.

[0051] Bovine collagen 202 may be processed into a viscous solution 203 by dissolution in an acidic solution. Either a mineral acid such as hydrochloric acid or an organic acid such as acetic acid may be used to prepare the collagen solution. For example, in one embodiment, bovine type I collagen having intact terminal telopeptides may be dissolved in about 0.01 M acetic acid to about 0.5 M acetic acid 201 in a container 210 to prepare a viscous solution 203 containing about 16 mg / mL of collagen in the solution. The solution concentration may range from about 10 mg / mL of collagen to about 19 mg / mL of collagen in the solution. In another embodiment, lyophilized bovine type I dermis with terminal telopeptides attached is mixed with a mineral acid such as HCl having a concentration of about 0.01 M to about 0.5 M to prepare a solution having a collagen concentration of about 10 mg / mL to about 19 mg / mL of collagen in the solution, usually about 16 mg / mL of collagen in the solution.

[0052] In embodiments, the collagen is dissolved for at least about 14 hours, typically at least about 15 hours, and more commonly at least about 16 hours. In some embodiments, the collagen solution 301 is degassed in a degasser 300 to remove air bubbles from the collagen solution 301. A screen 304 ensures that collagen is not drawn out of the degasser through the gas outlet 303 of the degasser. The degasser 303 is typically operated at a pressure of about 0 psia to about 3 psia. The collagen solution may be subjected to a maximum of about 2 degassing cycles, typically about 1 to about 2 cycles. Degassing removes gas bubbles that may interfere with and hinder the extrusion of fibrous collagen.

[0053] Next, the degassed collagen may be further degassed using a centrifuge. In Figure 4, the centrifuge 400 is shown with its top 408 open so that the bowl 403 is visible. The tubes for the material to be centrifuged and the tubes used to balance the centrifuge are placed in the wells of the rotating bowl 409. The case 405 is robust enough to contain any debris in case any of the internal components fail during use.

[0054] Figure 5 shows a centrifuge 500 having a storage bowl 501 and a lid 508. The centrifuge rotates at high speed counterclockwise, as indicated by the motion arrow 505. Tube 502 shows a tube containing the collagen solution 503 before centrifugation. As shown in the figure, the collagen solution is homogeneous and otherwise has air bubbles trapped within the homogeneous collagen solution 512.

[0055] Centrifugation at relative centrifugal forces or g-values ​​of approximately 400 rcf to 4,000 rcf, typically 600 rcf to 1,000 rcf, and more commonly 700 rcf to 800 rcf, is suitable for reducing the volume of trapped bubbles to essentially zero within approximately 3 to 15 minutes, typically 4 to 10 minutes, and more commonly 5 to 7 minutes.

[0056] In some embodiments, a pair of related steps may be repeated by alternating between the steps. For example, the collagen may be subjected to one cycle in the degasser 303, then processed in the centrifuge 500 for 5 minutes, then returned to the degasser 303 for one cycle, and then centrifuged for 5 minutes. This alternative method may result in improved efficiency. This improvement in efficiency may be achieved by utilizing shorter processing times to achieve a given amount of bubbles or to achieve better results than linear processing.

[0057] Next, collagen is co-extruded with the solution to form collagen fibers. In some embodiments, the extrusion of the collagen solution in a coaxial fluid core may aid in the formation of collagen fibers.

[0058] In some embodiments of this disclosure, collagen is then introduced into the center of the coaxial flow needle, and the forming buffer is introduced into the outer needle. In this way, the forming buffer forms a sheath around the collagen. As shown in Figure 6, the collagen solution 650 is pumped through the pump 655 and introduced into the inner needle 603 as a collagen flow 601. Simultaneously, the forming buffer 660 is introduced into the outer needle 604 as a forming buffer flow 606. The outer needle 604 is coaxial with the inner needle 603 so that the forming buffer forms a sheath around the central core of the collagen. As the material exits the needle and flows into the reaction region containing the fibril-forming bath 701 (shown in Figure 7), the forming buffer 607 forms a sheath around the collagen fibers 602, which begin to form solid fibers.

[0059] The diameter of the resulting collagen fiber product is manufactured to be smaller than the inner diameter of the central needle by downstream processing. The diameter of the central needle may be larger than the target diameter of the finished fiber. In some embodiments, the inner diameter of the central needle is about 0.05 mm to about 100 mm, in some embodiments, the inner diameter of the central needle is about 0.1 mm to about 50 mm, in yet another embodiment, the inner diameter of the central needle is about 0.2 mm to about 20 mm, in yet another embodiment, the inner diameter of the central needle is about 0.3 mm to about 10 mm, and more generally, about 0.35 mm to about 5 mm. In some embodiments, the narrower range of the inner diameter of the central needle is, for example, about 0.03 mm to about 10 mm, typically about 0.10 mm to about 3 mm, more generally about 0.30 mm to about 1 mm, and even more generally about 0.35 mm to about 0.50 mm.

[0060] In some embodiments, the inner diameter of the central needle is approximately 0.38 mm to 0.44 mm, typically 0.39 mm to 0.43 mm, and more commonly 0.40 mm to 0.42 mm.

[0061] In some embodiments, the inner diameter of the surrounding coaxial outer needle for supplying the forming buffer is generally about 1.95 to 2.15 times the inner diameter of the central needle, generally about 2.00 to 2.10 times the inner diameter of the central needle, and more generally about 2.05 times the inner diameter of the central needle.

[0062] In embodiments, the formation buffer may be any solution to aid in the formation of collagen fibers. The formation buffer is typically a solution containing TES (2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid or N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid) with added salt and buffer.

[0063] In some embodiments of this disclosure, the formation buffer is WSB (a solution containing 30 mM TES, 4.14 mg / mL monosodium phosphate dihydrate, 12.1 mg / mL disodium phosphate heptahydrate, 135 mM NaCl, and 10 w / v percent PEG (polyethylene glycol)). Similar solutions may also be suitable.

[0064] The flow rates of the collagen solution and the forming buffer are adjusted so that the forming buffer sheath remains intact within the extrusion needle and the reaction region containing the fibril-forming bath. The rate of the forming buffer is also set to exceed the rate of the collagen solution to give the collagen fibers elongation and improve the fiber quality. In fact, this encourages the collagen to form relatively straight and continuous fibers without kinks and other physical morphological abnormalities. In some embodiments, the fibers may be substantially circular, oval, square, rectangular, ribbon-like, triangular, or irregular in shape.

[0065] In embodiments of this disclosure, the rate of the forming buffer within the needle reaction region, which includes the fiber-forming bath, is greater than the rate of the collagen solution. The forming buffer is used to neutralize the collagen solution and aid in fibril formation. Furthermore, a faster forming buffer is used to pull or stretch the collagen flow, thereby creating an elongation field that helps align the collagen monomers in a process called flow-induced crystallization. This alignment helps polymerize the collagen and increase the strength of the resulting product.

[0066] In some embodiments, the volumetric flow rate of the forming buffer in the needle is approximately 5 to 10 times the volumetric flow rate of the collagen solution in the needle, typically 7 to 9 times the volumetric flow rate of the collagen solution in the needle, and more generally 7.5 to 8.5 times the volumetric flow rate of the collagen solution in the needle. In particular, 8 times the volumetric flow rate of the collagen solution in the needle is effective.

[0067] In one embodiment, as shown in Figure 7, the collagen flow 702 and the forming buffer sheath 707 enter the reaction region of the reaction system 700, which includes the fibril-forming bath 701. The reaction region may have structures that form the reaction region 701, such as forming tubes. However, structures are not usually required. As this flow flows through the reaction region including the fibril-forming bath 701, the collagen fibers continue polymerization to form a collagen fiber product. The collagen fibers 752 and the forming buffer 702 flow out of the reaction region including the fibril-forming bath 701.

[0068] In embodiments, the rate of the collagen is adjusted to give the collagen a reaction or polymerization time of approximately 15 to 60 seconds, typically 20 to 50 seconds, and more commonly 25 to 40 seconds.

[0069] As shown in Figure 8, in this embodiment, at the end of the polymerization phase, as the flow exits the reaction region containing the fibril-forming bath 701, collagen fibers 852 are formed and separated from the forming buffer 808. Excess forming buffer flows into the deep dish 801. The dehydration system 800 is designed to receive the forming buffer 808 into the deep dish 801 and introduce the collagen fibers 852 into the dehydration bath 802.

[0070] The dewatering solution provides an opportunity to remove water from collagen fibers, reducing fiber diameter and aiding fibril formation. In embodiments, the dewatering solution comprises a Milli-Q aqueous solution containing about 10 percent ethanol to about 35 percent ethanol, typically containing about 15 percent ethanol to about 30 percent ethanol, and more commonly containing about 15 percent ethanol to about 25 percent ethanol. Those skilled in the art will recognize that Milli-Q water (also written as Milli-Q water) is highly purified water produced using equipment available from Millipore Sigma, Inc. (Burlington, Massachusetts, USA).

[0071] Collagen fibers 852 pass through the dehydration bath 802 for approximately 10 to 50 seconds, typically 15 to 45 seconds, and more commonly 20 to 40 seconds. Throughout this period, the collagen fibers 852 remain immersed in the dehydration bath 802. The volume of the dehydration bath 802 ranges from approximately 400 times the volume of the forming buffer per minute delivered by the pump to approximately 800 times the volume of the forming buffer per minute delivered by the pump, typically from approximately 450 times the volume of the forming buffer per minute delivered by the pump to approximately 750 times the volume of the forming buffer per minute delivered by the pump, and more commonly from approximately 500 times the volume of the forming buffer per minute delivered by the pump to approximately 700 times the volume of the forming buffer per minute delivered by the pump.

[0072] Figure 9 shows the end of the dehydration bath from which the dehydrated collagen fibers are removed. As seen in the embodiment shown in Figure 9, the dehydrated collagen fibers 930 are removed from the dehydration bath 802 at the position of the hook 910 connected to the ring 920. As shown in the figure, the hook 910 is held in the dehydration bath 802 by the ring 920. The hook 910 helps to remove the dehydration bath from the dehydrated collagen fibers. The dehydrated collagen fibers 930 are pulled upward in the direction of arrow 940 by the rotation of the spool 1001, as shown in Figure 10. Since the recovery rate of the spool 1001 (Figure 10) exceeds the extrusion flow rate of the collagen fibers 930, the hook 920 or a similar device is suitable to ensure that the collagen fibers remain immersed in the dehydration bath 802. As the dehydrated collagen fibers 930 are lifted above the height of the dehydration bath, it is possible to observe the fluid droplets 905 falling from the dehydrated collagen fibers 930.

[0073] Figure 10 shows the collection of the dewatered fibers into spool 1001. In this embodiment, spool 1001 is rotated clockwise by motor 1011, as indicated by arrow 1016. Spool 1001 is rotated at a speed that gives a stretch ratio of about 1.5 to about 3, typically about 1.75 to about 2.5, and more commonly about 1.90 to 2.20. Similarly, spool 1002 is rotated at the same speed. The stretch ratio is the ratio between the spool speed and the extrusion speed. Thus, there is tension on the first collagen fiber 1050, pulling the fiber upward at the position of hook 910. The fiber is then pulled over the wall of the dewatering bath 802 and then pulled out onto spool 1001.

[0074] Alternatively, in some embodiments of this disclosure, collagen is introduced directly into the fibril-forming bath without using the coaxial needle of Figure 6 to form a coaxial flow, as shown in Figure 7. Conversely, collagen fibers are formed when the collagen solution 852 is injected directly from the needle into the fibril-forming bath 870, and then processed in a manner similar to the coaxial formation method, which is an alternative embodiment of this disclosure. The size of the needle for collagen injection is selected similarly to the size of the needle for the coaxial injection method, and, as in other embodiments, the fibers are pulled through the fibril-forming bath and then drawn into the dehydration bath. However, the spool 1001 in Figure 10 is rotated at a speed that gives a draw-in speed of about 2 to 4 times the fiber-forming speed, usually about 2.5 to 3.5 times the fiber-forming speed, and more generally about 2.75 to 3.25 times the fiber-forming speed. Post-processing is then carried out as in other embodiments.

[0075] Arrow 1020 indicates the passage of time in some embodiments while the spool 1001 is moving parallel to the end of the dewatering bath 802 to form a single layer of fibers on the spool. This allows the spool to continue rotating at the same speed, and tension is applied to the fibers 1052 as the spool moves parallel until the spool 1002 is essentially full. Arrow 1030 indicates the passage of time until the fiber supply is exhausted. The spool 1055 may then be retrieved. The parallel movement speed may be adjusted to control the spacing between the fibers on the spool.

[0076] To ensure that tension is maintained on the fibers when the spool is rotated, the fibers are typically in contact with the entire surface of the spool, such as spool 1110 used in some embodiments, as shown in Figure 11. However, the spool does not need to have a continuous surface like spool 1110. In other embodiments, a number of rods could extend along the length of the spool. One such spool formed of multiple rods is spool 1120 in Figure 11, which includes a first rod 1125, a second rod 1126, and a third rod 1127. These rods provide a sufficient surface for winding collagen onto them.

[0077] The fibers of this disclosure may be chemically post-treated. Figure 12 shows possible post-treatment steps. In embodiments, the spool 1210 containing the collagen fibers is air-dried in 1220 for at least about 15 minutes, usually at least about 20 minutes, and more commonly at least about 30 minutes. The air-dried tube 1210 containing the fibers is then placed in a container for crosslinking. Typically, the container in embodiments of this disclosure minimizes the capacity of the crosslinking container to reduce the amount of crosslinking agent required. Thus, as shown in Figure 12, the cylinder 1230 contains the amount of crosslinking fluid required to cover the cylindrical spool 1210, as shown in 1231. In embodiments, the volume of crosslinking solution per meter of fiber is at least about 3 μL, usually at least about 4.5 μL, and more commonly at least about 6 μL.

[0078] The fibers of this disclosure may be functionalized to have amino groups, and may also contain amino groups that can crosslink with aldehydes, such as collagen. Typically, short-chain aldehydes are used, more commonly glyoxal (GLY) or other conventional crosslinking agents. For example, crosslinking agents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), genipine, glyceraldehyde, glutaraldehyde, o-dextran, and low and high molecular weight procyanidins may be used. Alternatively, if the fibers are functionalized with carboxyls, EDC and other carbodiimides may be used for crosslinking. Isocyanates react with both OH groups and amines. Therefore, isocyanate crosslinking agents may be used to crosslink OH groups to each other, for example, within functionalized PDLLA (linking OH groups to other OH groups), to improve the stability and strength of the medium. Isocyanates may also be used to bond the OH groups in functionalized PDLLA to collagen via the NH2 groups (i.e., amine groups) of collagen. Furthermore, photocrosslinking agents may also be used.

[0079] The following reaction sequence is an exemplary crosslinking reaction applicable to embodiments of the present disclosure. In each of these exemplary reactions, P = polymer (which is a fiber in these reactions).

[0080] [ka]

[0081] In particular, glyoxal provides suitable crosslinking in embodiments of the present disclosure. In embodiments of the present disclosure, a solution containing 10 mM glyoxal in a solution of 70 percent ethanol and 30 percent milli-Q water is used for crosslinking. The concentrations or proportions of these components may be varied to provide the desired degree of crosslinking and functionality.

[0082] In embodiments of this disclosure, a 0.25 mM EDC solution may be used as the crosslinking solution for the sheath.

[0083] In some embodiments, the tube and spool 1231 are rotated as schematically indicated by arrows 1235, as shown in Figure 12. For example, rollers may be used to rotate the tube and spool at about 1 RPM. The rotation is continued for a sufficient time to obtain the desired degree of crosslinking. In some embodiments, at least about 24 hours is sufficient to obtain the desired degree of crosslinking. Increasing the crosslinking time increases the bonding strength within the fibers and improves the stability of the resulting product. Therefore, in some embodiments, the material may be crosslinked for at least about 48 hours, and usually at least about 72 hours. It has been found that a crosslinking time of approximately one month further increases the crosslinking strength. The container may be moved in any way that ensures the entire coil is immersed in the crosslinking fluid.

[0084] In some embodiments, the spool containing the cross-linked collagen fibers 1211 is then removed from the tube and optionally placed in a Milli-Q aqueous rinse solution for about 10 minutes, as indicated by the rinse tank 1240 and arrow 1221. The rinsed spool and fibers 1212 are then placed in a bath 1250 containing 100 mM glycine for a time sufficient to inactivate any excess glyoxal. Ten minutes is usually sufficient. Removal of glyoxal helps to reduce the cytotoxicity of the fibers. Other cross-linking agents may be removed similarly, if necessary or appropriate.

[0085] In embodiments where the rinsing step is omitted, the spool and fibers 1213 are placed in glycine in a glycine bath 1250. The processing treatment for drying the fibers is carried out in the same manner as in embodiments where the rinsing step is performed.

[0086] The spool containing collagen fibers 1214 from the glycine bath is then rinsed again with Milli-Q water in tank 1260. In this embodiment, 10 minutes is sufficient to remove the glycine. The spool and fibers 1215 are then air-dried in 1270 for about 1 hour, and then placed in drying chamber 1280 for about 24 hours. The dried, flexible fibers 1217 are then collected as shown in Figure 12.

[0087] Embodiments of this disclosure relate to method 1300 shown in Figure 13 for producing biopolymer fibers. In embodiments, collagen is dissolved in an acid solution (1305) to prepare a collagen solution (1310). In some embodiments, a biopolymer compatible with collagen is included. The collagen solution may then be degassed (1315) and then centrifuged (1320) to obtain a collagen solution.

[0088] Next, the collagen solution is co-extruded using the forming buffer as a sheath (1325). The collagen solution is delivered at a first velocity through a first needle having a first diameter, while the forming buffer is delivered at a second velocity through a second needle that coaxially surrounds the first needle and has a second diameter greater than the first diameter, forming a sheath around the collagen solution, thereby forming a coaxial flow. The second velocity of the basal buffer through the second needle is at least twice the first velocity of the collagen solution through the first needle.

[0089] In some embodiments, the inner diameter of the central needle is approximately 0.05 mm to approximately 100 mm; in some embodiments, the inner diameter of the central needle is approximately 0.1 mm to approximately 50 mm; in yet another embodiment, the inner diameter of the central needle is approximately 0.2 mm to approximately 20 mm; in yet another embodiment, the inner diameter of the central needle is approximately 0.3 mm to approximately 10 mm, and more generally, approximately 0.35 mm to approximately 5 mm. In some embodiments, the narrower range of the inner diameter of the central needle is, for example, approximately 0.03 mm to approximately 10 mm, typically approximately 0.10 mm to approximately 3 mm, more generally approximately 0.30 mm to approximately 1 mm, and even more generally approximately 0.35 mm to approximately 0.50 mm.

[0090] In some embodiments, the inner diameter of the central needle is approximately 0.38 mm to 0.44 mm, typically 0.39 mm to 0.43 mm, and more commonly 0.40 mm to 0.42 mm.

[0091] In some embodiments, the inner diameter of the surrounding coaxial outer needle for supplying the forming buffer is generally about 1.95 to 2.15 times the inner diameter of the central needle, generally about 2.00 to 2.10 times the inner diameter of the central needle, and more generally about 2.05 times the inner diameter of the central needle.

[0092] In embodiments, the formation buffer may be any solution to aid in the formation of collagen fibers. The formation buffer is typically a solution containing TES (2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid or N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid) with added salt and buffer.

[0093] In some embodiments of this disclosure, the formation buffer is WSB (a solution containing 30 mM TES, 4.14 mg / mL monosodium phosphate dihydrate, 12.1 mg / mL disodium phosphate heptahydrate, 135 mM NaCl, and 10 w / v percent PEG (polyethylene glycol)). Similar solutions may also be suitable.

[0094] In some embodiments, the volumetric flow rate of the forming buffer in the needle is approximately 5 to 10 times the volumetric flow rate of the collagen solution in the needle, typically 7 to 9 times the volumetric flow rate of the collagen solution in the needle, and more generally 7.5 to 8.5 times the volumetric flow rate of the collagen solution in the needle. In particular, 8 times the volumetric flow rate of the collagen solution in the needle is effective.

[0095] In embodiments, coaxially flowing collagen and forming buffer flow through a reaction region containing a fibril-forming bath at a rate and time sufficient to form fibers (1330). The formed collagen fibers are then separated from the forming buffer (1335) and placed in a dewatering solution (1340). The dewatering solution provides an opportunity to remove water from the collagen fibers, reducing their diameter and aiding in fibril formation. In embodiments, the dewatering solution contains a Milli-Q aqueous solution of about 10 percent ethanol to about 35 percent ethanol, typically a Milli-Q aqueous solution of about 15 percent ethanol to about 30 percent ethanol, and more commonly a Milli-Q aqueous solution of about 15 percent ethanol to about 25 percent ethanol.

[0096] In some embodiments, the fibers are drawn onto the spool at a third velocity greater than a first velocity, sufficient to enhance molecular orientation and reduce the fiber diameter (1345). This velocity is at least about twice the velocity at which the fibers typically flow through the dewatering bath.

[0097] In other embodiments, step 1325 is omitted, and coaxial sheath formation is not used. Instead, the collagen solution is injected into the forming buffer, and the fibers are stimulated through the forming buffer and dehydration solution by rotating the recovery spool, providing a pulling speed of approximately twice to four times the injection speed. The remainder of the steps, including any possible post-treatment, is then carried out.

[0098] In embodiments, after a short air-drying time in step 1350, the fibers are crosslinked in step 1355. Typically, crosslinking is carried out in a glyoxal solution with sufficient stirring time to achieve crosslinking. In embodiments, the fibers are left on the spool. To reduce the amount of crosslinking agent required, the capacity of the crosslinking container is generally minimized.

[0099] The crosslinking material may be any suitable crosslinking agent. In particular, glyoxal provides suitable crosslinking in embodiments of this disclosure. In embodiments of this disclosure, a solution containing 10 mM glyoxal in a solution of 70 percent ethanol and 30 percent milli-Q water is used for crosslinking. The concentrations or proportions of these components may be varied to provide the desired degree of crosslinking and functionality. In embodiments, the volume of the crosslinking solution per meter of fiber is at least about 3 μL, usually at least about 4.5 μL, and more commonly at least about 6 μL. Often, a crosslinking time of 24 hours is suitable to achieve the amount of crosslinking. However, typically, a crosslinking time of at least about 48 hours results in increased crosslinking, and at least about 72 hours provides further crosslinking.

[0100] Next, the spool containing the cross-linked collagen fibers is removed from the cross-linking container and, in some embodiments of this disclosure, is placed in a Milli-Q aqueous rinse solution for about 10 minutes. In other embodiments, rinsing of the spool is not necessary. Then, in step 1360, the spool and fibers are placed in a bath containing a 100 mM glycine bath for a time sufficient to inactivate any excess glyoxal. Ten minutes is usually sufficient. Removal of glyoxal may help reduce the cytotoxicity of the fibers.

[0101] If glyoxal is not used as a crosslinking agent, other processing steps may be employed. Those skilled in the art will recognize appropriate post-processing steps suitable for these other crosslinking systems.

[0102] Next, in the embodiment, the spool containing the collagen fibers from the glycine bath is rinsed again with Milli-Q water in step 1365. In the embodiment, 10 minutes is sufficient to remove the glycine. Then, in step 1270, the spool and fibers 1214 are air-dried for about 1 hour and then placed in a drying chamber for about 24 hours (1370). The dried, flexible fibers are collected.

[0103] In embodiments of this disclosure, the fibers produced are biopolymer fibers containing collagen. These biopolymer fibers have the following characteristics Extreme tensile strength of approximately 20 MPa to approximately 170 MPa, Elastic modulus of approximately 200 MPa to approximately 3,500 MPa, Elongation at break of approximately 4 percent to 12 percent. Average fiber diameter after drying: approximately 16 μm to approximately 70 μm Having one or more of the following, After immersion in biological fluids for approximately one hour, it maintains its strength at least.

[0104] These fibers exhibit a regular longitudinal orientation structure, which allows for the invasion of cells for proliferation.

[0105] In another aspect, the present disclosure relates to an implantable biopolymer scaffold for assisting in the repair of soft tissue damage or for the repair or replacement of human body parts. The scaffold comprises at least one biopolymer sheet containing biopolymer fibers, the biopolymer having the following characteristics Extreme tensile strength of approximately 20 MPa to approximately 170 MPa, Elastic modulus of approximately 200 MPa to approximately 3,500 MPa, Elongation at break of approximately 4 percent to 12 percent. Average fiber diameter after immersion in phosphate-buffered saline solution for approximately 1 hour, ranging from approximately 16 μm to 70 μm. It contains collagen and biopolymer fibers having one or more of the following: After immersion in biological fluids for approximately 24 hours, it maintains its strength at least.

[0106] These fibers exhibit a regular longitudinal orientation structure, which allows for the infiltration of cell proliferation. The sheet contains fibers arranged in a typical manner for ease of handling during use. For example, a single fiber would be extremely difficult to use due to its small diameter. Therefore, to provide fiber-containing products suitable for repairing or replacing body parts, it is necessary or appropriate to form a scaffold or structure larger than a single fiber. Thus, for example, several fibers can be knitted together to form a twisted yarn containing collagen fibers. Such a twisted yarn may be useful, for example, for suture ligament or tendon ruptures. These and other uses will become apparent to the user.

[0107] Throughout this disclosure, tests on the properties and characteristics of 10 randomly selected fibers will be conducted. Strength tests will be performed using 10 fibers and loads ranging from approximately 0.3 N to 2 N.

[0108] As noted herein, the stability of collagen fibers is maintained even after being placed in a biological solution for at least one hour. Furthermore, further crosslinking achieved by continuing the crosslinking time for at least about 48 hours, and even 72 hours, significantly reduces fiber swelling and maintains or increases load capacity.

[0109] The following embodiments are examples of embodiments of the present disclosure and are not intended to be limiting in any way.

[0110] Example 1 Type I bovine collagen with intact terminal telopeptides was removed from its packaging and mixed with 0.05 M acetic acid to prepare a viscous solution with a collagen concentration of 16 mg / mL. The collagen was dissolved in this solution for 16 hours, followed by several cycles of degassing. Excess air bubbles were removed by centrifugation at approximately 750 rcf for 5 minutes before and after degassing. The collagen was drawn into a 5 mL syringe and then attached to the central Luer fitting of a coaxial needle (collagen inlet inner diameter 0.41 mm, forming buffer inlet inner diameter 0.84 mm). The collagen syringe and coaxial needle were then placed on a syringe pump and pumped at 60 μL / min.

[0111] The pH of the formation buffer was adjusted to 8.0 ± 0.1 and placed in a covered beaker. The formation buffer was a solution containing 30 mM TES, 4.14 mg / mL monosodium phosphate dihydrate, 12.1 mg / mL disodium phosphate heptahydrate, 135 mM NaCl, and 10 percent w / v PEG (polyethylene glycol), and was also known as wet spinning buffer (WSB).

[0112] A tube was positioned at the bottom of the beaker and through a peristaltic pump, then attached to a coaxial external needle via a Luer fitting, thereby forming an outer sheath flow of collagen. The collagen solution was neutralized with forming buffer to aid fibril formation. The forming buffer was flowed at 500 μL / min. A faster forming buffer was also used to pull or stretch the collagen flow, creating an elongation field that aided the alignment of collagen monomers in a process called flow-induced crystallization. This alignment facilitated easier polymerization of collagen and increased the strength of the final product.

[0113] A collagen stream and a forming buffer stream were placed in a reaction region containing a fibril-forming bath, where the fibers were given time to polymerize and form long chains. The reaction region containing the fibril-forming bath ended at the entrance of a dehydration bath, which received the used forming buffer in a reservoir and moved the fibers approximately 45 cm through 20% ethanol and 80% Milli-Q water. This bath helped remove water from the collagen fibers, reducing their diameter and aiding fibril formation. This bath was 2.5 cm wide and held approximately 300 mL of solution.

[0114] After the fibers moved through the bath, they were then wound onto a 50mm diameter spool, 300mm in length, rotated at approximately 5 RPM, thereby giving a stretch ratio (ratio between spool speed and extrusion speed) of approximately 2. This stretch ratio helped to further enhance molecular orientation, reducing fiber diameter and ultimately increasing strength. The parallel movement speed of the spool was adjusted to vary the spacing between fibers.

[0115] The spool was air-dried for at least 15 minutes before being placed in a cylindrical tube for crosslinking. The inner diameter of this tube was close to the outer diameter of the spool to reduce the amount of crosslinking agent required for complete immersion. 120 mL of a 10 mM glyoxal solution of 70 percent ethanol and 30 percent MilliQ water was prepared and poured into the tube. The spool was then placed in this tube. The tube and spool were then placed on a roller at approximately 1 RPM for 24 hours.

[0116] After 24 hours, the spool was removed from the tube and placed in a Milli-Q bath for 10 minutes. Next, the spool was placed in a 100 mM glycine bath for 10 minutes to inactivate excess glyoxal and help reduce cytotoxicity, followed by a final 10-minute Milli-Q water bath to remove any remaining glycine. The spool and fibers were then air-dried for approximately 1 hour and then placed in a drying chamber for 24 hours.

[0117] After drying, the fibers were dry and flexible, which facilitated processing into useful shapes for scaffolding. The average diameter of the obtained fibers was 25 μm, and the tensile strength after half an hour of immersion in PBS was approximately 100 MPa. PBS, also known as phosphate-buffered saline, is a buffer widely used in biological research. PBS is an aqueous salt solution containing disodium hydrogen phosphate and sodium chloride, and some formulations also contain potassium chloride and potassium dihydrogen phosphate. This buffer helps maintain a constant pH. The osmotic and ion concentrations of this solution are compatible with those of the human body (i.e., isotonic).

[0118] Amino acids, calcium chloride, potassium chloride, magnesium sulfate, sodium chloride and monosodium phosphate, glucose, and vitamins such as folic acid, nicotinamide, riboflavin and B12. 12 A 7-day stability test at 37°C in DMEM, a synthetic cell culture medium containing [the specified ingredient], showed a decrease of approximately 25% from its original strength. DMEM also contains iron and phenol red for pH indication.

[0119] This embodiment illustrates the manufacture of a fiber within the scope of the claims, according to a method within the scope of the claims. This fiber is used to manufacture a scaffold within the scope of the claims for the repair or replacement of a human body part.

[0120] Example 2 Type I bovine collagen with intact terminal telopeptides was removed from the package and mixed with 10 mM hydrochloric acid to prepare a viscous solution with a collagen concentration of 16 mg / mL. The collagen was dissolved in this solution for 16 hours, followed by centrifugation at 733 rcf for 5 minutes. Excess air bubbles were removed by degassing for 2 minutes, and then the solution was centrifuged again at 733 rcf for 10 minutes. The collagen was drawn into a 20 mL syringe and then attached to the Luer fitting at the center of a coaxial needle (collagen inlet inner diameter 0.41 mm). The collagen needle was then placed on a syringe pump and pumped at 50 μL / min.

[0121] The pH of the formation buffer was adjusted to 8.0 ± 0.1 and placed in a long bath. The formation buffer was a solution containing 30 mM TES, 4.14 mg / mL monosodium phosphate dihydrate, 12.1 mg / mL disodium phosphate heptahydrate, 135 mM NaCl, and 10 percent w / v PEG (polyethylene glycol), and was also known as wet spinning buffer (WSB).

[0122] The collagen solution was neutralized using a fibril-forming buffer to aid fibril formation. The collagen was pumped into the fibril-forming buffer and guided through the bath. The collagen-forming buffer contained a reaction region that gave the fibers time to polymerize and form long chains. The reaction region containing the fibril-forming bath ended at the entrance of a dehydration bath of 20% ethanol and 80% Milli-Q water, through which the fibers were guided. This bath helped remove water from the collagen fibers, reducing their diameter and aiding fibril formation. Each bath was 2.5 cm wide and held approximately 300 mL of solution.

[0123] After the fibers moved through the bath, they were then wound onto a 50 mm diameter spool, 300 mm in length, rotating at approximately 10 RPM, thereby giving a stretch ratio (ratio between spool speed and extrusion speed) of at least approximately 2. This stretch ratio helped to further enhance molecular orientation, reducing fiber diameter and ultimately increasing strength. The parallel movement speed of the spool was adjusted to vary the spacing between fibers.

[0124] The spool was air-dried for at least 15 minutes and no more than 1 hour before being placed in a cylindrical tube for crosslinking. The inner diameter of this tube was close to the outer diameter of the spool to reduce the amount of crosslinking agent required for complete immersion. 120 mL of a 10 mM glyoxal solution of 70 percent ethanol and 30 percent MilliQ water was prepared and poured into the tube. The spool was then placed in this tube. The tube and spool were then placed on a roller at approximately 1 RPM for at least 24 hours and up to 72 hours.

[0125] After 24 hours or up to 72 hours, the spool was removed from the tube, air-dried for approximately one hour, and then placed in a drying chamber for 24 hours.

[0126] After drying, the fibers were dry and flexible. This facilitates processing into useful shapes for constructing scaffolds. The average wet diameter of the obtained fibers was 30 μm, and the tensile strength after half an hour of immersion in PBS was approximately 120 MPa. PBS, also known as phosphate-buffered saline, is a buffer widely used in biological research. PBS is an aqueous salt solution containing disodium hydrogen phosphate and sodium chloride, and depending on the formulation, also contains potassium chloride and potassium dihydrogen phosphate. This buffer helps maintain a constant pH. The osmotic and ionic concentrations of this solution are compatible with those of the human body (i.e., isotonic).

[0127] Amino acids, calcium chloride, potassium chloride, magnesium sulfate, sodium chloride and monosodium phosphate, glucose, and vitamins such as folic acid, nicotinamide, riboflavin and B12. 12 A 7-day stability test at 37°C in DMEM, a synthetic cell culture medium containing [the specified ingredient], showed a decrease of approximately 25% from its original strength. DMEM also contains iron and phenol red for pH indication.

[0128] This embodiment illustrates the manufacture of a fiber within the scope of the claims, according to a method within the scope of the claims. This fiber is used to manufacture a scaffold within the scope of the claims for the repair or replacement of a human body part.

[0129] Additional disclosures and comparative information In embodiments of this disclosure, clinical-grade atelocollagen and telocollagen may be used to form microfluidic extruded collagen microfibers (which can then be crosslinked with a harmless biocrosslinking agent such as glyoxal or DL-glyceraldehyde (DLG)). These crosslinked fibers were significantly stronger than 50 other crosslinking strategies tested, exhibiting a hydrated ultimate tensile strength of nearly 300 MPa and a modulus greater than 3 GPa, exceeding the strength of natural human Achilles tendon and anterior cruciate ligament. Fibers crosslinked with glyoxal still retained 50% of their initial load-bearing capacity throughout 3–6 months in culture. Collagen fibers implanted in rats exhibited biocompatibility, promoting the production of aligned, new host-generated collagen growing along the fibers, and in the case of glyoxal crosslinking, promoting an increased pro-regenerative M2 macrophage response. Embodiments of this disclosure demonstrated significant improvements in healing compared to other crosslinked fibers, including conventional synthetic materials, making embodiments of this disclosure superior fibers for producing robust collagen sutures or for use in devices for ligament, tendon, or other soft tissue repair.

[0130] Attempts to create materials suitable for tendon and ligament repair have yet to result in the manufacture of adequate products. To date, autografts, allografts, and synthetic materials, such as sutures, braces, or grafts for soft tissue closure or joining, have been found to have significant clinical limitations. Allografts, such as dead, decellularized, and chemically treated implants, can be time-consuming to integrate, inflammatory, and potentially delay healing (Seon, Song, and Park, 2006). Synthetic grafts can degrade into acidic byproducts, damaging surrounding tissues (Taylor et al., 1994; van Sliedregt et al., 1994; Matsusue et al., 1995). Synthetic grafts often do not match the mechanical or material properties of tendons or ligaments (Hogan et al., 2015), and when used in joint spaces, can lead to stress riser generation and abrasive isometricity. Autografting increases surgical time and associated trauma (e.g., bleeding, infection risk) due to the need for another surgery to restore the patient's own tissue, leading to further trauma in the process (Chen et al., 2009; Perrone et al., 2017). Joint reconstruction using autografting or allogeneic transplantation further leads to a higher incidence and severity of premature osteoarthritis, thereby impacting quality of life (Leiter et al., 2014; Smith et al., 2014; Perrone et al., 2017). The increasing rate of post-traumatic osteoarthritis is a significant problem for veterans (Showery et al., 2016).

[0131] In the manufacture of ideal, robust biomaterials for tendon and ligament repair sutures and absorbable sutures, unaddressed needs remain. In attempts to improve biocompatibility, reduce inflammation, and decrease abrasion from robust synthetic materials, particularly for reconstructive surgery indications, synthetic non-absorbable sutures with collagen coatings (e.g., Collagen-Coated FiberWire®) are becoming available.

[0132] Cross-linked fiber extrusion from type I collagen has the potential to produce robust products. However, these products are often inadequate, exhibiting biological deficiencies, strength deficiencies, and other drawbacks. For example, most cross-linking agents are cytotoxic, using strong chemical agents that are foreign to the body and are not used in currently available products approved or authorized by the U.S. Food and Drug Administration (FDA), making their use for clinical applications more difficult. In addition to their possible use in enhancing ACL or AT repair, braided collagen fibers have potential for use as sutures in general surgery, ophthalmic surgery, and plastic / cosmetic surgery, given that they have been shown to possess high and uniform tensile properties, consistent uniform diameter, biocompatibility, and tunable absorption with regenerative capacity.

[0133] Embodiments of this disclosure relate to a novel microfluidic extrusion system for producing microfibers of clinical type I collagen in filamentous and thin ribbon-like structures. Embodiments of this disclosure meet stringent mechanical, biochemical, cytocompatibility, and biocompatibility criteria, and the fiber embodiments of this disclosure are characterized particularly for biomedical applications. Because embodiments of this disclosure exhibit medium- to large-scale molecular order necessary for producing useful products, these collagen fibers described herein have potential applications in tendon and ligament repair, wound closure, and other indications, where advanced collagen suture-based biomaterials have beneficial potential across the field of surgery in medicine.

[0134] Figure 14 schematically illustrates the production of collagen microfibers described in embodiments of this disclosure and possible biomedical applications of suitable products. In step 1401, freeze-dried collagen is dissolved in acid to obtain collagen molecules 1402. Extruded microfibers 1403 are twisted together with a spinneret 1404 to form twisted microfibers 1405. These microfibers contain aggregated molecular collagen 1406. This collagen may be spooled in step 1407.

[0135] Collagen may be used more appropriately in the form of a three-dimensional structure formed by twisting or braiding individual fibers. The braided fibers 1411 or twisted fibers 1405 may then be used to suture a tear 1415 in the anterior cruciate ligament (ACL) of the patient's knee 1412. Collagen ACL sutures 1414 may be used to repair the tear, and collagen skin sutures 1415 may be used to close the wound.

[0136] Any number of fibers may be combined to form bundles, whether or not they are twisted together, and these bundles may be combined into larger bundles. For example, a bundle may contain 2 to about 10,000 fibers, or about 4 to about 6,000 fibers, usually about 8 to about 4,000 fibers, and more generally about 12 to about 2,000 fibers. These bundles may then be twisted together or otherwise combined to form larger bundles. The bundles being combined do not need to have an equal number of fibers.

[0137] A bundle may also be described by the number of fibers in it. For example, a bundle of five fibers may be called a penta-fiber, an eight-fiber bundle would produce an octa-fiber, and so on. Systems and apparatus with other numbers of nozzles or extruders may be used to produce such bundles.

[0138] Figures 15 and 16 show a method for obtaining collagen fibers and a system in which the reaction can be carried out. In embodiments of this disclosure, up to 2% (w / v) of clinical-grade lyophilized telocollagen (Telo) or atelocollagen (Atelo) (Collagen Solutions, California) or methacrylated collagen (Advanced BioMatrix, California) was dissolved overnight with agitation in an acid up to 0.05 M (most commonly acetic acid or hydrochloric acid). The acidified collagen 1501 was then pumped through the center of a nozzle system, as shown in system 1500. This system may include a coaxially arranged conduit or needle 1503. A neutralizing alkali-forming phosphate buffer containing salts (sodium chloride, disodium phosphate, monosodium phosphate, and N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) and PEG (polyethylene glycol) were pumped through the system in the outer portion of the coaxial conduit 1503. The forming buffer flowed at a volume flow rate approximately 5 to 20 times, typically 8 to 15 times, and most commonly about 10 times, the rate at which the collagen was introduced, thereby stretching and partially aligning the protein and imparting mechanical strength to the resulting fibers 1504. The fibers became firmer as they passed through the forming tube before entering the bath 1505 of 20% aqueous ethanol. In addition to dehydrating the fibers, this bath helped remove residual forming buffer, thereby contributing to improved strength and stability of the resulting collagen microfibers. After dehydration 1506, the microfibers 1507 may be collected in a 2-bar device 1508. Other suitable collection devices may also be used.

[0139] In some embodiments, acidified collagen fibers may be formed by extrusion before being placed in the forming bath. For example, acidified collagen fibers may be formed using Spinaret 1404. In some embodiments, multiple syringes of acidified collagen may be formed simultaneously.

[0140] Figure 46 shows System 4600, which uses an array of syringes to form acidified collagen fibers. It may be considered that System 4600 is a high-throughput system. In some embodiments of this disclosure, clinical collagen is dissolved in an acid (acetic acid or a mineral acid such as HCl) in a sealed container made of a material inert to the acid and collagen. Polypropylene is one such material. The volume of collagen and acid is usually less than about 50% of the capacity of the sealed container to facilitate thorough mixing. The solution is stirred overnight or for about 16 to 30 hours, usually about 15 to 20 hours. The solution is then degassed by centrifugation.

[0141] Next, the degassed solution is placed into syringes. The number of syringes used is equal to the number of fibers to be formed simultaneously. System 4600 in Figure 46 shows the use of outlets from eight syringes mounted on a rotatable plate 4601. The plunger of each syringe is pressed into the syringe barrel by a plate (not shown) to ensure that essentially equal amounts of fibers are extruded. The acidified collagen is pushed through a first nozzle 4602 to form a first fiber 4612, through a second nozzle 4603 to form a second fiber 4613, through a third nozzle 4604 to form a third fiber 4614, through a fourth nozzle 4605 to form a fourth fiber 4615, through a fifth nozzle 4606 to form a fifth fiber 4616, and then pushed through the remaining nozzles. In some embodiments, not all nozzles are necessarily used. In embodiments of this disclosure, there may be more or fewer nozzles mounted on the rotatable plate 4601.

[0142] The fibers are collected by the guide 4630 and placed in the forming buffer bath 4640. The fibers are retained after extrusion. In some embodiments, the rotatable plate 4601 may rotate in either direction to produce twisted fibers. In system 4600, the rotatable plate 4601 may be rotated by the rotation of a drive plate 4620 engaged with a rosette notch 4621. Any suitable drive system may be used. In some embodiments, the rotatable plate 4601 is not rotated, and therefore the resulting bundle of fibers is not twisted. However, the bundle is maintained by tension from a tensioner until the fibers are dewatered and wound onto the collector. Typically, a grooved cylinder is a suitable collector, especially with respect to wet fibers.

[0143] Figure 16 shows details of the nozzle system 1600. Pump 1502 pumps acidified liquid collagen 1620 to the central needle of the twin-screw needle 1503. Buffer solution, also called sheath solution, is introduced from 1610 to the outer needle of the twin-screw needle 1503 in the direction of flow arrow 1611, thereby forming collagen microfibers 1504 as the polymerization process progresses. The collagen fluid is focused by the extensional flow of the sheath fluid 1611. The detailed diagram of the needle shows how the acidified liquid collagen 1620 moves in the direction of the slow arrow, and then its velocity increases as indicated by the fast arrow 1630 and the faster arrow 1640. Similarly, the sheath fluid moved in the directions of velocity arrows 1611, 1635, and 1645. The reagent flow proceeds in the direction of arrow 1650, where shading shows how the phosphate-containing buffer (sheath) fluid 1670 interacts with the collagen solution and removes water 1680 from the collagen flow.

[0144] After the microfibers 1507 were collected on device 1508, they were air-dried for half an hour and then crosslinked under various experimental conditions. The chemical agents used during extrusion and crosslinking are shown in Table 1 of Figure 17.

[0145] In situ crosslinking (chemical or enzymatic crosslinking) of the groups shown in Table 2 of Figure 18 was performed by dissolving the amount of each crosslinking agent shown in Figure 18 in an acidified collagen mixture for the time shown in Figure 18. The concentrations and times for crosslinking some materials were obtained from specific references shown in Figure 18. Figure 18 summarizes the comparison of strengths. The italicized conditions were selected for characterization after optimization of the recovery method. Microfibers from the in situ crosslinked collagen were then extruded into a 2-bar device and stored taut as shown in Figure 15. Figure 18 also shows that the crosslinking agents can be used in amounts of approximately 5 mM to approximately 500 mM, typically approximately 10 mM to approximately 500 mM, and more commonly approximately 25 mM to approximately 250 mM.

[0146] However, more generally, uncrosslinked microfibers may be collected on a solid spool 1110 (see Figure 11) having short-spacing grooves. The microfibers were collected directly into these grooves while remaining taut. Collection onto a spool is usually more efficient than with a two-bar device. The spool of uncrosslinked microfibers was chemically crosslinked in 70% aqueous ethanol, as used for the two-bar device. The tube containing the microfiber spool in the crosslinking agent solution was placed on a roller and rotated at 1 rpm, as shown in Figure 12, to ensure uniform crosslinking of the microfibers.

[0147] For chemical crosslinking after extrusion, uncrosslinked or in situ crosslinked taut collagen microfibers extruded through a 2-bar device 1508 or grooved roller 1110 were air-dried for half an hour, then immersed in a 70% ethanol solution of the crosslinking agent solution and placed on a low-speed rocker. The aqueous ethanol medium ensures that the microfibers remain dehydrated throughout the crosslinking time. After crosslinking, the microfibers were stored in a desiccator until further testing was performed.

[0148] In some embodiments, collagen fibers are wet or damp upon collection. In such cases, the fibers may tend to stick together, especially if they can come into contact with each other during collection. Therefore, in some embodiments, two-bar or multi-bar collector devices may be advantageous to use, as they allow the fibers to dry before they come into contact with other fibers. In some embodiments, grooved rollers are particularly useful for collecting wet fibers, as only one fiber is collected in each groove, preventing contact between fibers.

[0149] In some embodiments, the fibers may be dried by blowing gas (usually air) over them after they have left the dewatering bath and before they are recovered. The fibers are suspended between the dewatering bath and a collector, which may be a flat cylinder, a bobbin, or any other suitable collector. Since the fibers are dry, they do not need to be kept apart from each other.

[0150] In some embodiments, the fibers may be dried by passing air over them at room temperature in a speed bag at 0.25 m / s to about 10 m / s, typically about 1 m / s to about 4 m / s, more commonly about 2 m / s. The speed of the drying air should not be so high as to split, separate, or cut the fibers. The air is passed over for an approximate time it takes to dry the fibers, typically equal to the time it takes to move the fibers about 1 meter. The drying air may be driven by a blower in an open or circulating system. In some embodiments, a recovery device, such as a bobbin or flat cylinder, is used. The cylinder is rotated at a pull-in speed of about 1 to about 9 times the forming speed. In this environment, it is possible to produce fibers that are essentially infinitely long.

[0151] The dehydration heat treatment (DHT) of crosslinked microfibers involves dehydrating relaxed extruded microfibers under vacuum at 110°C for 1, 3, and 5 days, with or without further crosslinking with glyoxal as described above.

[0152] Methacrylated collagen was used for extrusion for UV-mediated crosslinking. The extruded microfibers were then exposed to a 365 nm UV light source for 20 minutes. These microfibers were then placed in a desiccator or further crosslinked with 70% aqueous ethanol containing 10 mM glyoxal.

[0153] Because single microfibers are too delicate to handle consistently, the mechanical properties of single microfibers were obtained using an "individual fiber" test method, which measures the ultimate tensile strength (UTS), modulus, and strain at break (%) by averaging the cross-sectional area of ​​individual microfibers on a cartridge with a known quantity of microfibers. The 2-bar recovery device 1508 yielded cylindrical microfibers, but the microfibers recovered on a solid grooved spool were thin ribbon-like. The width of ribbon-like collagen fibers is approximately 10 μm to 70 μm, typically 15 μm to 60 μm, and more commonly 20 μm to 50 μm. The thickness of ribbon-like collagen fibers is approximately 4 μm to 20 μm, typically 5 μm to 18 μm, and most commonly 6 μm to 17 μm.

[0154] The width was measured by analyzing images obtained at 10 different locations on three individual 1.5-inch microfibers using an inverted optical microscope such as the Axio Vert.A1 model (Zeiss, Germany) and ImageJ software (NIH shareware, Bethesda, Maryland). The thickness of the microfibers was measured using ImageJ software with cross-sectional images of the microfiber bundle obtained using a scanning electron microscope (SEM). To meet the stringent mechanical testing requirements that may be relevant to the in vivo performance of the embodiments of collagen microfibers of this disclosure, high-throughput methods for wet tensile testing of our microfiber samples, such as those disclosed by Gentleman et al., 2003, may be used.

[0155] A bath and sample holding system was used to perform wet tensile strength mechanical testing of bundles within the cartridges. This system allowed for 30-minute immersion, processing samples every 5 minutes during immersion of the extruded microfiber embodiments of this disclosure. The immersion fluid may be Gibco Dulbecco's phosphate-buffered saline (DPBS), available from Thermo Fisher Scientific. Typically, stress-strain curves were obtained by mechanically wet testing at room temperature at a tensile speed of 1 mm / s using uniaxial tensile testing on MTS Reference Model 42 (Eden Prairie, Minnesota) for a minimum of four cartridges. Results were obtained rapidly by performing individual fiber testing while optimizing processing parameters.

[0156] The bath and sample holding system includes a bath sufficiently filled to cover the material to be tested in the fluid. The fluid may be Gibco's Dulbecco's phosphate-buffered saline (DPBS). During the test, the sample holder was held in the fluid by the jaws at opposite ends of the tension tester. The test was performed by moving the jaws away from each other.

[0157] The UTS of the wetted embodiments of this disclosure is typically about 1 MPa to about 800 MPa, typically about 75 MPa to about 400 MPa, more commonly about 90 MPa to about 350 MPa, and even more commonly about 100 MPa to about 325 MPa. The modulus of the wetted embodiments of this disclosure is about 10 MPa to about 7,500 MPa, typically about 100 MPa to about 6,000 MPa, and more commonly about 1,000 to 4,000 MPa.

[0158] The UTS of the drying embodiments of this disclosure is typically about 25 MPa to about 1,900 MPa, typically about 100 MPa to about 1,800 MPa, more commonly about 5,000 MPa to about 1,700 MPa, and even more commonly about 1,200 MPa to about 1,700 MPa. The modulus of the drying embodiments of this disclosure is about 14,000 MPa to about 20,000 MPa, typically about 15,000 MPa to about 19,000 MPa, and more commonly about 15,500 to 18,500 MPa.

[0159] Comparative tests of specific embodiments of wet and dry fibers showed relative ranges of approximately 25–1650 MPa for dry fibers compared to approximately 1–755 MPa for wet fibers, approximately 15,950–18,600 MPa for dry fibers compared to approximately 10–7,200 MPa for wet fibers, approximately 9–14% elongation at break for dry fibers compared to approximately 2–41% for dry fibers, and approximately 10–70 μm average fiber diameter for dry fibers compared to approximately 14–82 μm for wet fibers.

[0160] The cross-sectional and longitudinal microstructural characteristics of uncrosslinked and crosslinked extruded microfibers were determined using SEM imaging. SEM imaging was performed using a Zeiss Evo 10 microscope (Zeiss) at a beam intensity of 10 kV. For cross-sectional imaging, microfiber bundles were immersed in DPBS for 30 minutes, dried on an SEM stub for 1 hour, sputter-coated, and then imaged.

[0161] For TEM, dried microfibers from the Telo GLY group (glyoxal-crosslinked telocollagen) were rehydrated with distilled water. These were then fixed at room temperature for 30 minutes in 2% glutaraldehyde (Electron Microscopy Sciences, Pennsylvania) and 4% paraformaldehyde (Alfa Aesar, Massachusetts). Subsequently, they were washed twice with cacodylate buffer (Electron Microscopy Sciences) for 10 minutes each. This was followed by incubation in 1% osmium tetroxide (Electron Microscopy Sciences) for 30 minutes, one wash with cacodylate buffer, and two washes with distilled water (10 minutes each). Dehydration was performed with a series of ascending concentrations of ethanol (one wash for 10 minutes at 30%, 50%, 70%, and 95%, respectively, and two washes for 10 minutes at 100%). Next, the microfibers were immersed twice for 10 minutes in a 1:1 mixture of ethanol and propylene oxide (Electron Microscopy Sciences), followed by a 10-minute treatment with 100% propylene oxide. These samples were left overnight in a 1:1 EPON 812:propylene oxide (Electron Microscopy Sciences) solution. EPON 812 is a glycerol-based aliphatic epoxy resin. The following day, the samples were immersed in a 4:1 EPON 812:propylene oxide solution for 4 hours, then transferred to 100% EPON 812 and incubated overnight. The next day, the samples were transferred to fresh EPON 812 resin, incorporated into bullet capsules (Electron Microscopy Sciences), and polymerized at 60°C for 12 hours. The molds were thinly sectioned and imaged using a TEM (model Jeol 1230, Jeol USA, Massachusetts). Alternative methods may be used to measure these values.

[0162] The ninhydrin test may be used to evaluate the amount of free amino groups in cross-linked microfibers. For this purpose, uncross-linked and cross-linked microfibers were cut to lengths of 14–16 cm. Simultaneously, 0.05% acetic acid solutions of various known concentrations of standard amino acids (glycine (Sigma-Aldrich)) were prepared according to the manufacturer's protocol. The microfiber samples and glycine solutions were heated in the ninhydrin solution (Sigma-Aldrich) for 20 minutes, followed by cooling to room temperature for at least 1.5 hours. Then, 95% ethanol was added to both the samples and the glycine standards. The absorbance of these samples was recorded at 570 nm using a UV-Vis spectrophotometer (SpectraMax i3, Molecular Devices, Old Dominion University, Norfolk, Virginia). Other testing methods may be used.

[0163] Calibration curves were created using absorbances of various known glycine concentrations. The amount of free amino groups in uncrosslinked (Mux) and crosslinked (Mc) microfibers was proportional to the absorbance of the solution, and this amount was determined from the glycine calibration curves created. Equation 1 below was used to calculate the degree of crosslinking.

[0164]

number

[0165] Single-fiber extruded microfluidic fibers, bundles of 150 microfibers (connected with coated Vicryl 4-0 sutures (Ethicon, New Jersey) and cut to a final size of 10 mm), or the cartridges used in the aforementioned mechanical tests were sealed in Tyvek pouches containing a STERRAD chemical indicator (4MD Medical Solutions, Lakewood, New Jersey) and sent to electron beam sterilization (Steri-Tek, Fremont, California) using a targeted dose of 20 KGy + 1-2 KGy.

[0166] Sterile glyoxal and DL-glyceraldehyde crosslinked microfibers were hydrated in tendinocyte growth medium for 30 minutes and placed in 24-well plates pre-coated with poly(2-hydroxyethyl methacrylate) (pHEMA) (Sigma-Aldrich). 25,000 human tendinocytes (ZenBio, North Carolina) (in 100 μl of tendinocyte growth medium) were seeded in triplicate onto the sterile microfibers. After seeding, the cells were allowed to adhere for 1 hour, followed by the addition of 500 μl of tendinocyte growth medium. After 12 days, the tendinocytes attached to the microfibers were stained with the live cell stain CellTracker® Green CMFDA (5-chloromethylfluorescein diacetate) (Thermo Fisher Scientific) according to the manufacturer's protocol. Next, the samples were fixed using 4% paraformaldehyde, followed by staining with the nuclear stain DAPI (Thermo Fisher Scientific), and the tendon cells attached to the microfibers were visualized using a confocal microscope (Zeiss Axio Observer Z1, Zeiss).

[0167] The cytotoxicity (or cell viability) of extruded microfiber embodiments against human tendon cells was evaluated using the CyQuant lactate dehydrogenase (LDH) cytotoxicity assay kit (Invitrogen) and the MTT assay kit (Sigma-Aldrich), following the manufacturer's protocols. In short, after determining the optimal seeding density for the assay, 7 × 10¹¹ microfibers were placed in each well of a 48-well plate. 3 Tendon cells were plated and grown for 24 hours in tendon cell growth medium in a humidified incubator maintained at 37°C and 5% CO2. Sterilized microfiber bundles were rinsed in cell culture medium for 10 minutes and placed on the tendon cells in each well. Tendon cells (cells only) grown on plastic were used as a positive control (for cell viability or viability). Dibutyldithiocarbamate zinc (ZDBC) film and 10 mM glyoxal solution were used as negative controls (for cell viability or viability). Ethicon Vicryl sutures were used to secure the extruded microfiber bundles, so the effect of ethicon Vicryl sutures was also evaluated in this experiment. Wells seeded with tendon cells without samples were prepared to assess maximal and spontaneous LDH release as described in the manufacturer's protocol. Samples were incubated for 7 days, and then the release of LDH into the medium was assessed. % cytotoxicity was calculated using an LDH assay according to the manufacturer's protocol. Next, % cell viability was calculated as 100-% cytotoxicity. In embodiments of this disclosure, % cell viability is at least about 94%, typically at least about 95%, more commonly at least about 96%, and most commonly at least about 97%. It is also possible to achieve 98% or 99% cell viability. % cell viability was calculated using the MTT assay according to the manufacturer's protocol. In embodiments of this disclosure, % cell viability is at least about 70%, typically at least about 80%, more commonly at least about 85%, and most commonly at least about 90%. Other suitable test methods are available.

[0168] The health and viability of viable tendon cells grown with the embodiments of the extruded microfibers of this disclosure were also evaluated using the AlamarBlue® assay (Bio-Rad, Hercules, California) according to the manufacturer's protocol.

[0169] Cross-linked microfiber bundle embodiments were subcutaneously implanted in rats. All surgical procedures were performed according to ISO 10993-6 and protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Old Dominion University (Norfolk, Virginia). In n=3, cross-linked collagen microfiber bundles (prepared and sterilized as described above) or collagen-coated FiberWire® (suture control) were subcutaneously implanted in female Sprague Dolly rats. The rats were anesthetized by isoflurane inhalation. The flank hairs were shaved, and Nair depilatory cream was applied to remove hair from the surgical site. A dorsal incision was made in the flank position, and a pocket for the implant was created using hemostatic forceps. After placing the scaffold in the pocket, the incision was closed with sutures. Four weeks later, the rats were humanely euthanized for tissue harvesting.

[0170] Microfiber explants extracted at 4 weeks were fixed with 4% paraformaldehyde (Alpha Acer) for 24 hours, and then transferred to DPBS (Thermo Fisher Scientific). The samples were sectioned to a thickness of 5 μm, and serial sections were stained with hematoxylin & eosin (HE) and Masson's trichrome (IDEXX, West Sacramento). The collagen composition of the tissue surrounding the implant was imaged using polarized light microscopy.

[0171] Immunolabeling was also performed on serial sections to detect the presence of CCR7(M1) and the phenotype of CD163(M2) macrophages in the native tissue surrounding our implants, using the standard protocol provided by the antibody manufacturer. Briefly, after deparaffinization, antigen collection (boiled in 10 mM citrate buffer at pH 6 for 20 minutes), permeabilization, and blocking with 2.5% horse serum, slides were stained for either CD163 (M2 macrophage phenotype) or CCR7 (M1 macrophage phenotype). Mouse anti-rat CD163 (#MCA342GA, BioRad, California), which is labeled as M2 macrophage, was diluted 1:30 and incubated overnight in a humidified room. After incubation, slides were washed with PBS and incubated with a 1:50 dilution of goat anti-mouse secondary antibody (#A-11005, Thermo Fisher Scientific) for 1 hour at room temperature in the dark. CCR7, an M1 macrophage label, was diluted 1:50 in PBS and incubated overnight (#MA5-31992, Thermo Fisher Scientific). The following day, after PBS washing (3 times), goat anti-rabbit fluorescent antibody (#A32740, Thermo Fisher Scientific) was applied to the slides at a concentration of 1:200 and left at room temperature in the dark for 1 hour. For primary control, serum-blocked slides were stained with either IgG mouse (1:30) (Thermo Fisher Scientific) and goat anti-mouse secondary antibody (1:50) or IgG rabbit (1:200) (Thermo Fisher Scientific) and goat anti-rabbit secondary antibody (1:200). For secondary control, serum-blocked slides were stained with secondary fluorescent antibody alone. All antibodies were diluted with blocking serum. All slides were stained with DAPI for 5 minutes, washed with PBS, mounted using a VectaMount (Vector Labs, California), and then visualized and analyzed.

[0172] Immunolabeled slides were examined and imaged using an inverted microscope (Axio Vert.A1 model, Zeiss). Fluorescence images were obtained of test slides and control slides (data not shown) under the same exposure conditions. Images of the test samples were evaluated. Quantitative analysis was performed to determine the number of cells expressing M1 only, M2 only, M1 and M2, and / or no M1 / M2 phenotype. Here, 4-5 regions were analyzed using high-power microscopy (40× magnification) for each image of approximately 20-30 μm at the interface between the implant and native tissue (2-3 cell layers) (3 images were analyzed per test sample). The total number of cells was measured by counting DAPI-stained nuclei. The number of cells positively labeled for each marker was also counted. The percentage of cells labeled with a specific marker was measured as the percentage of the total number of cells in that region.

[0173] Long-term stability testing was also performed on embodiments of this disclosure. Telo GLY microfibers were despooled by applying tension to the cartridges. After hydrating the six sterilized cartridges as described above and performing mechanical tests to obtain the mechanical properties of the microfibers, the remaining sterilized cartridges were incubated in petri dishes containing Eagle's Minimum Essential Medium (EMEM) (ATCC, Virginia) supplemented with 1% Gibco® Antibiotic-Antifungal Agent (ABAM) (Thermo Fisher Scientific) to suppress bacterial and fungal contamination in an incubator maintained at 37°C and 5% CO2. Throughout the experiment, it was confirmed that the cartridges were always immersed in sterile, uncontaminated medium, thereby maintaining hydration. At week 1, month 1, month 3, and month 6, the six immersed cartridges were removed and subjected to MTS testing. Simultaneously, the diameter of the microfibers was measured (as described above) to determine the degree of swelling of the microfibers over time.

[0174] Unpaired two-tailed t-tests were used to assess significant differences in properties or features between any two groups. Two-way ANOVA, followed by Tukey's post-hoc multiple comparison test, was also used to assess differences in UTS for the various crosslinking agent groups shown in Table 1 of Figure 17. Dunnett's multiple comparison test, following a standard one-way ANOVA, was also performed to assess differences in health and viability, as described in the additional details below. A priori, p-value < 0.05 was defined as significant. All tests were performed using GraphPad Prism 7. All parameters are shown as mean ± standard error (SEM).

[0175] Additional Examples Examples were obtained by carrying out embodiments of the products and methods of this disclosure. To consistently produce collagen microfibers for subsequent testing, a robust microfluidic extruder, as shown in Figures 15 and 16, was designed and used. This approach resulted in the continuous production of microfibers without crosslinking abnormalities.

[0176] A wide range of conventional, novel, and combined crosslinking conditions were screened to strengthen and stabilize collagen microfibers. Table 2 in Figure 18 lists the crosslinking agents and shows the average UTS of 50 types of crosslinked microfibers compared to uncrosslinked microfibers using the aforementioned test methods. This data showed that various crosslinking agents / crosslinking protocols (in situ or post-extrusion crosslinking, range of crosslinking agent concentrations, and crosslinking time) affected the microfiber UTS to varying degrees. Crosslinking conditions that showed significantly higher average UTS among all conditions tested with those crosslinking agents are marked with an asterisk in Table 2 in Figure 18 (p<0.01).

[0177] As shown in Figure 18, crosslinking procedures using post-extrusion chemical agents such as glyoxal (10 mM post-extrusion for 72 hours, 121.2 ± 7 MPa) and DL-glyceraldehyde (25 mM post-extrusion for 72 hours, 128 ± 12 MPa) produced microfibers exhibiting a UTS nearly 20 times greater than uncrosslinked microfibers (6.1 ± 1 MPa). In particular, crosslinking of microfluidic microfibers using this extruder with EDC and EDC / NHS resulted in UTS values ​​of 16.6 ± 2 MPa and 30.2 ± 1 MPa, respectively, which were significantly lower than the glyoxal and DL-glyceraldehyde groups mentioned above. In situ crosslinking with chemical crosslinking agents such as choline tartrate (1 mM or 100 mM), EGCG (200 mM and 1 mM), and D-sorbitol (200 mM) resulted in a significant reduction in UTS (p<0.01) compared to uncrosslinked microfibers. Physical crosslinking techniques such as DHT after extrusion (3 days, 16.2±1 MPa) also resulted in stronger microfibers than uncrosslinked microfibers, but were weaker than the chemical crosslinking groups using glyoxal and DL-glyceraldehyde mentioned above. UVR treatment of methacrylated collagen microfibers after extrusion (1.9±0.2 MPa) also resulted in significantly weaker fibers than uncrosslinked collagen microfibers (p<0.01).

[0178] Since extruded microfibers using glyoxal had the highest UTS, additional crosslinking was performed on several in situ (L-lysine or D-sorbitol) or alternative crosslinked fibers (DHT and UVR) using 10 mM glyoxal at various time points. Additional crosslinking using glyoxal increased the UTS in all of these groups, but the most significant increase (p<0.01) was observed in the L-lysine (10 mM, 2 hours) / glyoxal (10 mM, 24 hours) (96.9±5 MPa) and UVR (0.3 hours) / glyoxal (10 mM, 24 hours) (86.6±10 MPa) groups.

[0179] Next, referring to Figures 19, 20, and 21, the mechanical properties of typical microfibers from the crosslinking agent groups tested in Table 2 of Figure 18 were compared to values ​​reported for human ACL (Noyes and Grood, 1976; Peters et al., 2018), Achilles tendon (Wren et al., 2001), and dermis (Gallagher et al., 2012). Figure 19 shows UTS(MPA) in Graph 1900. Graph 2000 in Figure 20 summarizes the modulus I MPa, and Graph 2100 in Figure 21 relates to the strain % at fracture. ACL values ​​are shown at lines 1930, 2030, and 2130. AT values ​​are shown at lines 1910, 2010, and 2110, and dermal values ​​are shown at lines 1920, 2020, and 2120. Each result is for a single microfiber. The results shown in Figure 19 reveal that the average UTS of collagen microfibers in some cross-linking groups, particularly with and without 10 mM L-lysine, and with 10 mM glyoxal and 25 mM DL-glyceraldehyde in situ, were greater than the UTS reported for human ACL, AT, and dermis.

[0180] These charts demonstrate that, as mentioned above, the mechanical properties of the extruded microfibers can be adjusted to match, and / or exceed, the mechanical properties of the human anterior cruciate ligament (ACL), human Achilles tendon (AT), and human dermis by altering the crosslinking scenario. Data were collected from at least four identical replicate tests, and error bars indicate the standard error.

[0181] Examples 3 to 6 Comparative Examples 1, 2, and 3 The four crosslinking conditions shown in italics from the primary screening, as shown in Table 2 of Figure 18, Figures 19, 20, and 21, take into account significant requirements such as mechanical performance, processing time, and / or cost for further evaluation. The following fibers are exemplified herein:

[0182] Example 3 is telocollagen (Telo GLY) crosslinked with 10 mM glyoxal for 72 hours. Example 4 is telocollagen (Telo DLG) crosslinked with 25 mM DL-glyceraldehyde for 24 hours. Example 5 is atelocollagen (Atelo GLY) crosslinked with 10 mM glyoxal for 24 hours. Example 6 is atelocollagen (Atelo DLG) crosslinked with 25 mM DL-glyceraldehyde for 72 hours. Comparative Example 1 is telocollagen (Telo EDC) crosslinked with 0.25 mM EDC for 24 hours. These groups were compared with uncrosslinked microfibers (Comparative Example 2) and dried Telo GLY fibers (Comparative Example 3). The Telo EDC group (Comparative Example 1) was used for comparison because it is a harmless crosslinking agent widely used in this field (Cornwell et al., 2007; Enea et al., 2011; Ahmad et al., 2015). Furthermore, to further optimize the material properties, a high pull-in recovery rate (higher recovery rate compared to the raw material supply) to the grooved solid spool 1110 was used compared to the 2-bar device 1508 to produce the thin ribbon-like microfibers shown in Figure 22.

[0183] Figure 22 shows images of Telo GLY microfibers from Example 3 depicting their microstructural features. Frame A is an optical microscope image of a single dry extruded crosslinked microfiber. Frames B and C are SEM images of a single dry microfiber at various magnifications. Frame D shows a cross-section of bundled microfibers immersed in PBS for 30 minutes. Frame D reveals structural details and evidence that the extrusion and subsequent crosslinking strategy using the embodiment of the novel microfluidic apparatus shown in Figures 15 and 16 described herein produced a consistent, uniform, thin, ribbon-like microfiber, as indicated by arrow 2206. Arrows 2201 and 2202 in Frame B show gaps and ridges along the longitudinal axis of the dry microfiber. Arrows 2204 and 2205 in Frame C show the fibrous subfiber structure of the wet microfiber. Frames E, F, and G are longitudinal TEM images of the extruded microfiber.

[0184] Optimization of the crosslinking chemistry and changes in the recovery method led to significant differences in mechanical properties, as summarized in Figures 23, 24, 25, 26, 27, and 28. Tension tests were performed in the aforementioned bath and sample holding systems. Using the width and thickness of ribbon-shaped collagen microfibers immersed in DPBS in Figures 23 and 24, measured from typical images such as those shown in Example 3, the improved UTS in Graph 2500 of Figure 25 and the modulus in Graph 2600 of Figure 26 were calculated in Graph 26. Examples 3, 4, 5, and 6 were tested in the same manner as Comparative Examples 1, 2, and 3.

[0185] As shown in Figure 2402, the wet Atelo GLY (39.2±1 μm) and Telo EDC (46.4±2 μm) ribbon-shaped collagen microfibers were significantly wider than the wet, uncrosslinked (34.1±2 μm) ribbon-shaped collagen microfibers (p<0.05). Similarly, as shown in Figures 233, Figures 2301 and 2303, the wet Atelo GLY ribbon-shaped collagen microfibers were significantly thicker than the uncrosslinked ribbon-shaped collagen microfibers (9.2±0.5 μm) (11.9±0.5 μm) (p<0.01). The thickness of Telo GLY (11.1±0.5 μm), Telo DLG (8.6±0.2 μm), and Atelo DLG (10.9±0.4 μm) and the width of Telo GLY (36.1±0.7 μm), Telo DLG (35.4±0.8 μm), and Atelo DLG (31.1±1 μm) of ribbon-shaped collagen microfibers immersed in DPBS were similar to those of uncrosslinked ribbon-shaped collagen fibers. The largest change in UTS, shown in Graph 2500 of Figure 25, was observed in uncrosslinked ribbon-shaped collagen fibers, with the average UTS confirmed at Indication 2504 and the modulus confirmed at Indication 2604 in Graph 2600 of Figure 26 increasing from 6.1±1 MPa and 119.8±23 MPa, respectively, to 35.8±3 MPa and 701±53 MPa. Ribbon-shaped collagen microfibers from groups such as Telo GLY (121±7 MPa UTS and 1103±63 MPa modulus to 299±15 MPa and 3431±86 MPa, respectively) and Atelo DLG (128 MPa UTS and 1734±79 MPa modulus to 231±18 MPa and 3408±185 MPa, respectively) showed at least a twofold increase in average UTS as shown in Graph 2700 in Figure 27 and modulus as shown in Graph 2800 in Figure 28. There was no change in strain (%) at fracture for any of the groups tested.

[0186] A significant increase in the tensile properties was observed in all extruded ribbon-shaped collagen microfibers from grooved solid spools. The uncrosslinked ribbon-shaped collagen microfiber group showed the highest magnification changes in average UTS and modulus compared to the other crosslinking agent groups. For each of Examples 3 to 6 and Comparative Examples 1 to 3, Graph 2700 in Figure 27 shows a significant magnification change in UTS compared to the data shown in Figures 19, 20, and 21, and Graph 2800 in Figure 28 shows a significant magnification change in modulus compared to the data shown in Figures 19, 20, and 21.

[0187] Unpaired two-sided t-tests were used to assess significant differences between any two groups in Figures 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 37, 38, and 39. Two-way ANOVA, followed by Tukey's post-hoc multiple comparison test and unpaired two-sided t-tests, was used to assess differences in UTS for the various crosslinking agent groups in Table 1 of Figure 17. Standard one-way ANOVA, followed by Dunnett's multiple comparison test, was used to assess differences in Figures 31, 32, and 33, as described in the additional details below. A priori, p-value < 0.05 was defined as significant. All tests were performed using GraphPad Prism 7. All parameters are shown as mean ± standard error (SEM).

[0188] The results are shown as mean ± standard error, which represents three replicated trials from two or more individual experiments. Display 2301 indicates p<0.05. Display 2402 and Display 2502 indicate p<0.01. Display 2303 indicates p<0.005. Display 2504 and Display 2604 indicate p<0.0001.

[0189] In embodiments of this disclosure, the microstructure of the microfibers was measured using optical microscopy, SEM, and TEM imaging. Other types of imaging may be used. In Example 3, glyoxal-crosslinked telocollagen microfibers were characterized. Optical microscopy imaging shown in frame A of Figure 22 and SEM imaging in frame B of Figure 22 supported the homogeneous width of the dry microfibers along the longitudinal axis. Frame B of Figure 22 and high-magnification SEM (frame C of Figure 22) imaging of the longitudinal section revealed the parallel orientation of ridges and gaps within the dry microfibers, as shown in frame D of Figure 22. Frame D of Figure 22 highlights the SEM-based cross-sectional features of an extruded crosslinked microfiber bundle immersed in DPBS. These images reveal the microstructural features of the smooth outer surface with a clear fibrous subfiber structure, as indicated by arrow 2206. This indicates that the extruded crosslinked microfibers are consistent, thin ribbon-like. Further evidence that the molecular-to-nanoscale collagen orientation in natural connective tissue is reproduced in our cross-linked microfibers is revealed by TEM imaging of frames E, F, and G in Figure 22.

[0190] To biochemically evaluate the degree of crosslinking, the ninhydrin test, which is a biochemical and biophysical characterization of the crosslinked microfiber embodiments, was used. The results are shown in Graph 2901 of Figure 29. The Telo GLY (86±1%) microfibers of Example 3 and the Atelo DLG (82±3%) microfibers of Example 6 showed significantly higher degrees of crosslinking compared to the Atelo GLY (68±4%) of Example 5 and the Telo DLG (59±6%) of Example 4, which highlights that prolonged crosslinking improved the crosslinking efficiency.

[0191] The primary and secondary protein structures of the extruded collagen microfibers were also evaluated. SDS-PAGE analysis of the acidified starting material confirmed the presence of collagen α, β, and γ primary chains. However, the microfibers could not be dissolved in acid, so collagen in the acid extract of the microfibers could not be detected.

[0192] Biophysical characterization of the extruded microfibers using differential scanning calorimetry (DSC) revealed a non-significant increase in melting temperature between the uncrosslinked microfiber group and the crosslinked microfiber group, as shown in graph 2902 of figure 29 for the same samples tested in graph 2901. However, the average melting temperature of the extruded microfibers (74 ± 3 °C) (line 2930 in graph 2902) was significantly higher than that of human AT (line 2920 in graph 2902) (60 °C) (Wiegnad, Patczai, and Lorinczy, 2017), which indicates greater overall structural stability (Sanchez-Ruiz, 1995). The FTIR spectra in graph 2903 of figure 29 showed no significant peak shifts in the amide I (~1650 cm -1 ) region, amide II (~1560 cm -1 [[ID=⑧]]) region, amide III (~1235 cm -1 ) region, amide A (~3285 cm -1 ) region, and amide B (~2917 cm -1 ) region, indicating no change in the secondary structure of the microfibers after extrusion and after the crosslinking process used in this disclosure. The data in graph 2901 are shown as mean ± standard error and represent three replicate tests from two individual experiments. In graph 2901, indication 2910 shows p < 0.05.

[0193] As shown in illustrations 3001 and 3002 of Figure 30, and in Figures 31, 32, and 33, the cell adhesion, metabolic activity, and cytotoxicity of the extruded microfibers of the embodiments of this disclosure were also measured. These figures include Examples 3 to 6, Comparative Example 2, and other samples. Using human tendon cells, the cytocompatibility of collagen fibers was evaluated as described above. The adhesion of tendon cells to elongated Telo GLY microfibers (Example 5) is shown in illustration 3001 of Figure 30. Approximately 70% of the tendon cells seeded on the Telo GLY microfibers remained attached after 12 days. As summarized in Figure 31 for Examples 3 to 6, Comparative Example 1, and other samples, no significant changes in the metabolic activity of tendon cells were observed for more than 7 days by AlamarBlue fluorescence compared to the positive control (group of cells only). However, the metabolic activity of cells growing with microfibers from the selected fiber group was significantly higher than that of the negative control (10 mM glyoxal chemical agent and ZDBC film) (p<0.05). As shown in Figure 32, when assayed with the MTT reagent, the viability of tendinocytes incubated with microfibers was 75%–85% compared to tendinocytes grown on plastic (100%). The negative control (10 mM GLY chemical agent and ZDBC film) showed significantly lower tendinocyte viability (p<0.005) than the "cells only," Telo DLG (Example 4), Atelo GLY (Example 5), and Telo GLY (Example 3) groups. When using the LDH assay (Figure 33), similar results were observed, except that all extruded microfiber groups except Atelo DLG and Telo DLG induced tendinocyte viability similar to the "cells only" group. As shown in Figure 33, label 3303, at the end of the 7-day period, the 10 mM GLY chemical agent group had no tendon cells (ND) sufficient to test for LDH release into the culture medium. This was compared to commercially available coated Ethicon Vicryl sutures, which are typically recommended for wound closure.Embodiments of the microfibers described herein were shown to exhibit significantly lower cytotoxicity (p<0.005) than the sutures when using both LDH and MTT assays (Figures 32 and 32). Overall, the cytocompatibility of the extruded microfibers was demonstrated using multiple assays.

[0194] In particular, images 3001 and 3002 show typical confocal images of human tendinocytes attached to Telo GLY microfibers (Example 3), showing cytoplasmic elongation and elongated nuclei, respectively, using DAPI (arrow 3005) and a live cell stain (CMFDA, indicated by arrow 3003). Figure 31 shows no significant change in metabolic activity of human tendinocytes incubated with cross-linked microfibers after assaying with AlamarBlue and incubation for 7 days, compared to the cell-only group. Metabolic activity was significantly lower in tendinocytes incubated with negative controls (ZDBC film and 10 mM GLY chemical) and Vicryl sutures compared to the microfiber group. The MTT assay results summarized in Figure 32 revealed that the viability of tendinocytes incubated with the microfiber group was reduced compared to the cell-only group, but showed a significant increase compared to the negative controls. On the other hand, the LDH assay results shown in Figure 33 show a significant decrease in cell viability not only in the negative control but also in the Atelo DLG (Example 6) and Telo DLG (Example 4) microfiber groups. Both the MTT and LDH assays were performed 7 days after incubation in tendon cells. All data in Figures 32 and 33 are normalized to the cell-only group. Indication 3303 (ND) indicates that the 10 mM glyoxal chemical treatment group had significant growth inhibition with insufficient cell counts to detect LDH at the end of the assay. In these figures, indications 3101 and 3301 show p<0.05, indication 3202 shows p<0.01, indications 3103 and 3203 show p<0.005, and indications 3204 and 3304 show p<0.0001).

[0195] To evaluate the biocompatibility of the embodiments of the extruded microfibers of this disclosure, sterile microfiber bundles from four selected crosslinking agent groups (Atelo DLG, Telo DLG, Telo GLY, and Atelo GLY) of Examples 3-6 were subcutaneously implanted into rats according to ISO 10993-6. Microfiber bundles implanted from each of the four crosslinking agent groups, as shown in Figures 23, 24, 25, 26, 27, and 28, and the suture control (Collagen-Coated FiberWire®) group, induced distinct host tissue responses characterized by varying degrees of cellular infiltration, neovascularization, collagen deposition, and tissue remodeling, as shown in Figures 34, 35, and 36. In particular, the glyoxal-crosslinked microfiber group (Telo(GLY) (Example 3) or Atelo(GLY) (Example 5)) showed a weaker pro-inflammatory response compared to the DL-glyceraldehyde (Telo DLG (Example 4) or Atelo DLG (Example 6)) crosslinked group. Typical HE-stained images of the Telo GLY (Example 3) group, shown in cross-sectional image 3401 and longitudinal image 3402 in Figure 34, showed significantly higher cell infiltration compared to the suture control shown in Figure 36, including cross-sectional image 3601 and longitudinal image 3602. The suture control in Figure 36 induced a stronger inflammatory response at week 4 compared to the microfiber implant.

[0196] In Image 3501 of Figure 35, the deposition of newly formed collagen in the natural tissue around the Telo(GLY) (Example 3) microfiber implant was visualized by Masson's trichrome staining. A longitudinal section stained with Masson's trichrome is shown in Image 3502 of Figure 35, and the polarized imaging in Image 3602 of Figure 36 shows the histologically newly formed collagen deposition around the microfiber.

[0197] As seen in the high-magnification cross-sectional image of the HE-stained section (yellow arrow in image 3401 of Figure 34), blood vessels and capillaries were identified within and around the microfiber implant.

[0198] Figures 34, 35, and 36 are typical images of rat subcutaneous implants in the Telo GLY (Example 3) group at week 4. Images 3401 and 3501 show microfiber m, identified by arrow 3410, on stained slides showing cross-sectional fragments of HE in Figure 34 and Masson's trichrome in Figure 35. Inset image 3490 shows the entire fragment of the HE implant, and inset image 3495 shows the portion shown in image 3401. Similarly, inset image 3590 shows the entire fragment of the Masson's trichrome implant, and inset image 3595 shows the portion shown in image 3501. Both images 3401 and 3501 show significant cellular infiltration. Arrow 3420 in Figure 34 points to a blood vessel within the implant. Image 3601 shows negligible cellular infiltration in the control sample, collagen-coated FiberWire®, using HE staining. Image 3402 shows HE staining, and Image 35032 shows Masson trichrome staining of a longitudinal section of a microfiber implant. Polarized image 3602 in Figure 36 shows oriented microfibers and newly formed collagen around the implant. Images 3402, 3502, and 3602 show the formation of new oriented collagen in the natural tissue around the implant. The legend "Suture control" in Image 3601 refers to collagen-coated FiberWire®.

[0199] Immunostaining was used to measure the degree of macrophage polarization in native tissue surrounding microfiber implants from four crosslinking agent groups. Figures 37 and 38 are typical immunofluorescence images showing the expression patterns of CCR7(M1) (Image 3700 in Figure 37) and CD163(M2) (Image 3800 in Figure 38) macrophage phenotypes in native rat tissue surrounding Telo GLY (Example 3) microfiber implants at week 4. Figure 38 shows the quantification of the percentage of macrophages expressing both M1 and M2, M1 only, M2 only, or no M1 / M2 phenotype. As shown in Graph 3900 of Figure 39, the glyoxal crosslinking groups (Telo(GLY) (Example 3) and Atelo(GLY) (Example 5)) showed a significantly higher percentage (approximately 40%) of macrophages expressing M1 and M2 phenotypes compared to the DL-glyceraldehyde crosslinking groups (Telo DLG (Example 4) and Atelo DLG (Example 6)). Furthermore, between the Telo(GLY) (Example 3) group and the Atelo(GLY) (Example 5) group, the Telo GLY implant induced a small subset of cells expressing only the M2 phenotype (6%), while the remainder of the group showed a negligible M2-only phenotype (Atelo GLY (0.2%), Telo DLG (0%), and Atelo DLG (0%) (Figure 39)). Compared to the glyoxal crosslinking group (Telo GLY (24%) and Atelo GLY (19%)), the proportion of cells exhibiting the M1 phenotype was significantly higher in the DL-glyceraldehyde crosslinking group (Telo DLG (64%) and Atelo DLG (58%)). As previously mentioned, appropriate control staining resulted in negligible nonspecific background staining (not shown). Sectioning artifacts and significant background staining in suture control samples made it difficult to perform this analysis on these samples.

[0200] Typical immunofluorescence images 3700 and 3800 show examples of the host macrophage response at week 4 to the microfiber Telo(GLY) (Example 3), indicated by arrow 3840. Yellow arrows 3710 and 3810 show examples of cells expressing both M1 and M2. Orange arrows 3720 and 3820 show examples of cells expressing only M1. White arrows 3730 and 3830 show cells expressing only the M2 phenotype. Arrow 3840 points to the microfiber bundle indicated by m. Graph 3900 in Figure 39 shows the percentage of cells expressing M1 and M2, M1 only, M2 only, or no M1 / M2 phenotype for four groups of cross-linked microfibers. Results from this analysis show the initiation of the phagocytic M2 macrophage phenotype in all microfiber groups tested. The glyoxal-crosslinked fiber group showed a higher proportion of cells with M1 and M2 phenotypes compared to the DL-glyceraldehyde-crosslinked fiber group. Furthermore, the Telo GLY group had a small but significant subset of macrophages that were M2 only. (In Graph 3900, Indicator 3901 shows p<0.05, Indicator 3902 shows p<0.01, and Indicator 3903 shows p<0.005).

[0201] The effects of long-term hydration reactions in culture media on the mechanical properties and degree of swelling of embodiments of the microfibers of this disclosure were measured. Since the Telo(GLY) (Example 3) microfibers exhibited optimal mechanical properties, cytocompatibility, and biocompatibility, this group was further tested for long-term stability by mimicking physiological conditions in vitro. Incubation in EMEM (Eagle's Minimum Essential Medium) resulted in a 53% increase in microfiber width at 6 months (from 36.4±1.1 μm (day 0) to 56.0±1.6 μm (month 6)), as shown in Graph 4000 of Figure 40. This graph shows the degree of swelling of the hydrated microfibers over time. This swelling is accompanied by a significant decrease in mechanical properties. Graph 4100 of Figure 41 shows that the average force at break decreased by 54% from its initial value at 6 months. The mean UTS (Graph 4200 in Figure 42) and modulus (Graph 4300 in Figure 43) also decreased by 82% from the baseline at 6 months. There was no significant change in break elongation (%) between day 0 and 6 months of incubation (Graph 4400 in Figure 44).

[0202] Thus, Figures 40, 41, 42, 43, and 44 demonstrate that Telo GLY microfibers are stable and did not dissolve up to 6 months when incubated under conditions mimicking the biological environment in vitro (37°C, humidified incubator maintained at 5% CO2, and sterile cell culture medium).

[0203] As can be seen from these figures, the mechanical stability of Telo GLY microfibers incubated under tension in sterile EMEM, evaluated at week 1, month 1, month 3, and month 6 in a humidified incubator at 37°C and 5% CO2, compared to day 0, showed that at the end of month 6, the Telo GLY microfibers had swollen by 50% (Figure 40), the breaking strength had decreased by 60% (Figure 41), the UTS had decreased by 80% (Figure 42), and the modulus had decreased by 80% (Figure 43). However, there was no significant change in % strain at break at the end of month 6 (Figure 44). All values ​​shown in these figures are normalized to a value of 1 on day 0. The continuous lines in these figures were drawn visually to serve as a guide for the reader only. Data are shown as mean ± standard error, which represents at least 5 repeated tests.

[0204] SDS-PAGE was used to compare collagen starting materials (lyophilized telocollagen or atelocollagen) with uncrosslinked and crosslinked microfibers. The collagen starting materials readily dissolved in 50 mM HCl after overnight agitation. However, the extruded microfibers did not dissolve at a concentration of 0.5 mg / ml and therefore did not show a band. To confirm the presence or absence of collagen in the acid extracts of the microfibers, these extracts were electrophoresed on gradient gels (3%–8%) (Invitrogen) with solutions of the starting materials and a pre-stained molecular weight marker (HiMark, Invitrogen, California). The gels were stained with SimplyBlue (trademark) (Invitrogen, California) and then destained by rinsing with deionized water. The gels were then projected with white light, and all visible protein bands were examined. Thus, SDS-PAGE revealed that extruded fibers from the group with maximum UTS and uncrosslinked fibers showed resistance to acid hydrolysis compared to acidified starting materials exhibiting type I collagen fingerprints with characteristic bands in the monomer region of approximately 115 kDa, the dimer region of approximately 230 kDa, and the trimmer region of approximately 460 kDa.

[0205] Figure 45 summarizes some of the mechanical tensile properties of the highest-performing (hydrated) cross-linked collagen fibers published in the literature, compared to embodiments of the present disclosure.

[0206] In summary, this disclosure relates to a novel microfluidic extrusion process for producing type I collagen microfibers with precision, consistency, and scalability as biocompatible fibers for use in indications ranging from natural sutures to engineered connective tissue. This disclosure demonstrates that embodiments of the bio-produced glyoxal-crosslinked telocollagen microfibers of this disclosure exhibit superior dry and wet tensile properties compared to conventional crosslinked collagen extruded microfibers (Paul and Bailey, 2003; Caruso and Dunn, 2004; Zeugolis, Paul and Attenburrow, 2009; Enea et al., 2011).

[0207] Much of the prior research has either failed to report whether tensile tests were performed on hydrated fibers, presented questionable and misleading results for dry fibers, or failed to disclose how the fibers were moistened if they were sufficiently hydrated. However, the results of the embodiments of this disclosure provide optimized dry and hydrated properties of crosslinked fibers, along with detailed test methods that are important for comparison and for growth in this field.

[0208] The retrieval of fibers into grooved drums resulted in significant changes to the structural and mechanical hydration properties of all cross-linked microfibers (see Figures 22, 23, 24, 25, 26, 27, and 28). Mechanically, this improved strength may be related to tempering, thinning, and improved molecular orientation of the formerly fibrous ribbons, resulting in stronger fiber tensile properties than the ACL, Achilles tendon, dermis, or any other soft connective tissue.

[0209] In embodiments of this disclosure, the measurement of the degree of efficiency of the crosslinking mechanism is important. Insufficient crosslinking may result in lower tensile strength, while excessive use of chemical crosslinking agents may lead to crosslinking agent residues on the microfiber surface, causing cytotoxicity. The ninhydrin test (Figure 36) revealed that the group with the highest degree of crosslinking was the 72-hour crosslinking group (Telo GLY and Atelo DLG), which was also associated with a significant increase in tensile strength. The chemical structure of aldehyde-based crosslinking involves the formation of Schiff base-type compounds with functional amino groups in collagen, resulting in strong molecular bonds (Fathima et al., 2004).

[0210] Chemical analysis of embodiments of extruded microfibers revealed that they exhibited even greater resistance to acid hydrolysis. The microfluidics instruments or apparatus disclosed herein produced microfibers with higher chemical stability than lyophilized starting materials, suggesting dense packing of collagen molecules within the microfibers, resulting in a stable higher-order structure, and also suggesting a low internal moisture content. Such higher-order structures have been reported in natural connective tissue (Benjamin, Kaiser and Milz, 2008; Wang, Guo and Li, 2012). The integrity of the secondary structure in the extruded microfibers was confirmed by FTIR analysis shown in Figure 29, which also suggests that neither the extrusion process nor the crosslinking technology denatured the collagen.

[0211] While crosslinking collagen in biomimetics may help improve tensile properties, the degradation of chemical agents used for crosslinking (e.g., glutaraldehyde) can be toxic (Gough, Scotchford, and Downes, 2002; Umashankar, Kumari, and Mohanan, 2012). Among other chemical agents with relatively low cytotoxicity, EDC or EDC / NHS are common basic research choices for collagen microfibers as crosslinking agents (Enea et al., 2011; Ahmad et al., 2015; Shepherd et al., 2015). However, improvements in tensile strength have been minimal, and due to the widely known toxic effects of these classic crosslinking agents, they have been largely unsuitable for use in connective tissue repair. In this study, we demonstrate the development of mechanically superior extruded collagen microfibers that are highly cytocompatible (see Figures 30, 31, 32, and 33) and biocompatible in vivo (as shown in Figures 34, 35, and 36), chemically crosslinked with either glyoxal or glyceraldehyde, according to the standard ISO 10993 testing generally required for USFDA approval. Furthermore, the glyoxal-crosslinked collagen microfibers exhibit resistance to acid hydrolysis, revealing their microstructural features down to almost the molecular level, maintaining stability in cell culture medium for at least 6 months, and the capacity of a single microfiber maintaining approximately 30% to 50% of its initial load-bearing capacity, typically around 40%, and maintaining a greater UTS than natural ACL (see Figures 40, 41, 42, 43, and 44).

[0212] Enhancement of ACL or Achilles tendon suture repair using collagen-based microfibers or collagen-based braided sutures in wound healing as described herein requires collagen-based materials that not only mechanically maintain tissue but also promote tissue remodeling at a reasonable rate (Dunn, Avasarala, and Zawadsky, 1993). In vitro and / or in vivo biocompatibility testing is important to demonstrate the effects of these chemically crosslinked microfibers on cytotoxicity, inflammatory response, and regenerative response. Embodiments of extruded microfiber bundles of this disclosure were cytocompatible and showed minimal toxicity to human tendon cells. Microfluidic extruded microfibers of this disclosure are intended to further support human tendon cell adhesion and are envisioned to have an elongated shape, as observed in connective tissue (Benjamin, 2010). Biocompatibility is defined as the ability of an implant to "locally induce and guide non-fibrous wound healing, reconstruction, and tissue integration" (Ratner, 2011). Cross-linked microfiber bundle implants showed a low (glyoxal group) to moderate (glyceraldehyde group) inflammatory response at 4 weeks after subcutaneous implantation in rats, and the glyoxal-telocollagen group showed the initiation of a pro-regenerative response. Furthermore, long-term stability data and rat tissue images showed microfiber stability up to at least 6 months in vitro and at least 4 weeks in vivo. Thus, embodiments of this disclosure can maintain strength for at least approximately 1 month in vivo, at least approximately 3 months in vitro, and up to approximately 6 months.

[0213] Macrophages are a heterogeneous mixture of mononuclear cells activated in the host in response to tissue damage, for example, during implantation of materials (Mosser, 2003; Gordon and Taylor, 2005). Macrophage phenotypic polarization at the interface between the implant and host tissue (Kasner et al., 2009; Brown et al., 2012) is important for determining pro-inflammatory signaling and the host's potential to overcome transitions toward tissue repair and remodeling in response to surgical implants. Macrophage phenotypes are broadly characterized as M1 (or "classical" activation) with pro-inflammatory signaling and M2 (or "selective" activation) with immunomodulatory or tissue remodeling properties (Mills et al., 2000). However, it is important to note that activated macrophages possess plasticity, allowing them to easily switch phenotypes from M1 to M2 and from M2 to M1. This plasticity is caused by changes in the local microenvironment (Porcheray et al., 2005; Stout et al., 2005). This allows macrophages to adopt transitional properties for both M1 and M2 phenotypes (Brown and Badylak, 2013). In embodiments of this disclosure, the percentage of cells exhibiting M1, M1 and M2, or M2 phenotypes was measured. These measurements suggested that: (1) at 4 weeks post-transplant, the glyoxal crosslinking group had more cells with M1 and M2 or M2-only phenotypes, suggesting that a tissue remodeling response is initiated by the host at 4 weeks. This suggests that microfibers from the glyoxal group are largely biocompatible. To our knowledge, such a detailed analysis of the immune response has not been performed using crosslinked collagen microfibers.

[0214] Incorporating collagen into sutures for wound healing has been a challenge. However, this disclosure provides methods and apparatus for the manufacture of an effective product. For the studies described herein, collagen-coated FiberWire® nonabsorbable sutures (the only collagen-based synthetic sutures available on the market) were used as a control. This FiberWire® showed limited cellular infiltration with little or no endoproliferation or regeneration of natural tissue around the implant. In contrast, embodiments of the glyoxal-crosslinked collagen microfibers of this disclosure, in the form of suture-like bundles, showed significant cellular infiltration with newly formed collagen in the surrounding tissue, suggesting regenerative healing.

[0215] Embodiments of this disclosure demonstrate that microfluidic extrusion of clinical-grade type I collagen fibers crosslinked with glyoxal exhibits exemplary tensile strength, structural stability, cytocompatibility, and biocompatibility compared to pure collagen produced by other previously reported biofabrication processes. The use of glyoxal to stabilize the collagen fibers provides a clinically significant, safe, and effective method for the biofabrication of additional collagen microfibers. These optimized collagen microfibers can be readily fabricated for a diverse range of biomedical applications, from surgical sutures and internal orthotics for ligaments to tissue engineering-created ligaments, tendons, and other robust fibrous tissues, designed to significantly improve human health.

[0216] Example 7 Collagen solution and forming buffer were prepared. A sufficient amount of clinical-grade lyophilized atelocollagen (Symatese, France) to prepare a 1.6% (w / v) solution was dissolved in 0.05 M acetic acid in a sealed polypropylene container. This solution was stirred overnight at 180 rpm at room temperature. To ensure uniform mixing, the total volume of the solution was kept below half the capacity of the container. The following day, the acidified collagen mixture was spun down in a centrifuge at 730 g for 5 minutes. The solution was degassed for 2 minutes and then spun down at 730 g for 10 minutes to remove air bubbles. The resulting acidified atelocollagen was drawn into eight 20 mL syringes (Hsw® Norm-Ject® sterile Luer lock syringes, VWR) and used directly in a high-power collagen microfiber extruder as shown in Figure 46.

[0217] To prepare the formation buffer, 10 gm of PEG (polyethylene glycol) (35 kDa, ChemCruz), 0.686 gm of TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) (Sigma-Aldrich), 0.790 gm of sodium chloride (Sigma-Aldrich), 0.414 gm of monosodium phosphate (Baker Analyzed), and 1.21 gm of disodium phosphate (Sigma-Aldrich) were added to 100 ml of Milli-Q water. This mixture was placed in a glass beaker on a stirring plate and stirred at 400 rpm overnight at room temperature. The following day, the pH of the solution was adjusted to 8 by adding 10 M sodium hydroxide (Sigma-Aldrich), and the solution was then filtered using a 0.45 μm filter.

[0218] On the day of extrusion, 800 ml of Milli-Q water was mixed with 200 ml of ethanol (Fisher Scientific) to obtain a 20% ethanol solution for the dehydration bath.

[0219] Figure 46 shows part of system 4600 in which acidified atelocollagen is processed. A syringe array pump was mounted to work with a rotatable plate 4601 and all eight syringes. Fiber bundle samples were prepared with and without twisting. The fiber bundles moved through a forming bath and became firm as the buffer solution neutralized the acid and formed fibrils. The twisted and untwisted bundles were then placed in a 20% aqueous ethanol dehydration bath, where water was removed and the fibers were further strengthened. Constant tension was applied to the fiber bundles with a tensioning rig until the bundles adhered to a grooved spool (not shown) at the end of the bath. The tension in the fibers helped to pull and build the collagen for strength and stability. The spooled collagen was then dried, crosslinked with glyoxal, and used to create 3D grafts.

[0220] For chemical crosslinking after extrusion, uncrosslinked, taut collagen fiber bundles were collected on a large grooved spool, air-dried for half an hour, then immersed in a 70% ethanol solution of the crosslinking agent in a large acrylic tube, and then placed in a 1 rpm rocker. The aqueous ethanol medium ensures that the microfibers remain dehydrated throughout the crosslinking time. After crosslinking, the microfibers were stored in a desiccator until further testing was performed.

[0221] The chemical crosslinking agent used was glyoxal, a dialdehyde, at a concentration of 10 mM. The chemical structure of crosslinking using aldehydes involves the formation of Schiff base-type compounds with functional amino groups in collagen, which results in strong molecular bonds.

[0222] The mechanical properties of the single fiber bundles produced in this way were obtained using an "individual fiber" test method that averages the cross-sectional area of the individual fiber bundles on the cartridge and a known amount of fiber bundles and measures the ultimate tensile strength (UTS), modulus, and strain (%) at break. The fiber diameter was measured by analyzing images obtained at 10 different locations on three fiber bundles with an individual length of 1.5 inches using an inverted microscope (Axio Vert.A1 model, Zeiss, Germany) and ImageJ software (NIH shareware, Bethesda, Maryland).

[0223] Figures 47 to 51 show the results of mechanical tests of fiber bundles under various test conditions, i.e., for non-twisted fibers and twisted fibers. There were no significant differences between fiber types. Figures 52 to 56 show the results of mechanical tests of non-twisted fiber bundles after cross-linking with glyoxal at various times. The significance level at the peak load (Figure 53) and UTS (Figure 55) was p < 0.01( ** ), and the significance level at the modulus (Figure 54) and UTS (Figure 55) was p < 0.001( **** ).

[0224] Figure 57 is a cross-sectional image of a microfiber bundle obtained using a scanning electron microscope (SEM). Figure 57 shows the structure of two multi-fiber bundles. The first bundle 5701 and the second fiber bundle 5702 contain eight fibers. The first bundle 5701 clearly shows the first fiber 5710, the second fiber 5720, and the third fiber 5730. Figure 57 also clearly shows the second end 5721 of the second fiber 5720, the third end 5721 of the third fiber 5730, and the fourth end 5739 of a fiber that cannot be clearly identified otherwise. The surface 5705 is the end of all the fibers in the first fiber bundle.

[0225] The second fiber bundle 5702 shows the fifth fiber 5740 and the fifth end 5741, the sixth fiber 5750 and the sixth end 5751, the seventh fiber 5760 and the seventh end 5761, and the eighth end 5749 of a fiber that cannot be clearly identified otherwise. The surface 5706 is the end of all the fibers in the second fiber bundle 5702.

[0226] Figure 58 is an SEM image of an octa fiber bundle.

[0227] While various embodiments of the present invention have been described, the description is illustrative and not limiting, and it will be apparent to those skilled in the art that many more other embodiments and forms are possible within the scope of the present invention. Accordingly, the present invention is limited only by the appended claims and their equivalents. Furthermore, various modifications and changes can be made within the scope of the appended claims.

[0228] References All documents cited herein, including the following articles and patents, are incorporated in their entirety by reference. PCT Application No. 1PCT / US2018 / 000119, attached hereto as Appendix A; and PCT Application No. PCT / US2018 / 57412, attached hereto as Appendix B; Ahmad, Z. et al. (2015) 'Effect of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide concentrations on the mechanical and biological characteristics of cross-linked collagen fibers for tendon repair', Regenerative Biomaterials, 2(2), pp. 77-85. doi: 10.1093 / rb / rbv005. Benjamin, M. (2010) The structure of tendons and ligaments, Regenerative Medicine and Biomaterials for the Repair of Connective Tissues. Woodhead Publishing. doi: 10.1533 / 9781845697792.2.351. Benjamin, M., Kaiser, E. and Milz, S. (2008) ‘Structure-function relationships in tendons: A review’, Journal of Anatomy, 212, pp. 211-228. doi: 10.1111 / j.1469- 7580.2008.00864.x. Bokor, D. J. et al. 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Claims

1. A biopolymer fiber containing collagen, having the following characteristics: an ultimate tensile strength of about 1 MPa to about 1,700 MPa; an elastic modulus of about 10 MPa to about 20,000 MPa; an elongation at break of about 2 percent to about 45 percent; Average fiber diameter of about 10 μm to about 90 μm and maintains its strength after immersion in DPBS at room temperature for at least about 1 hour; Fibers exhibiting a regular longitudinally oriented structure.

2. the ultimate tensile strength is from about 1 MPa to about 800 MPa; the elastic modulus is from about 10 MPa to about 7,500 MPa; the average fiber diameter is about 10 μm to about 30 μm; The biopolymer fiber of claim 1 .

3. the ultimate tensile strength is from about 25 MPa to about 1,700 MPa; the elastic modulus is from about 15,000 MPa to about 29,000 MPa; an elongation at break of about 7 percent to about 20 percent; The biopolymer fiber of claim 1 .

4. The biopolymer fiber of claim 1 , wherein the collagen comprises clinical collagen, atelocollagen, telocollagen, recombinant collagen, or a blend thereof.

5. The biopolymer fiber of claim 1 , wherein the collagen further comprises one or more biocompatible polymers.

6. 10. The biopolymer fiber of claim 1, further retaining a strength of greater than about 60 MPa after 6 months in DBPS at room temperature or after implantation in a subject.

7. 10. The biopolymer fiber of claim 1, which is crosslinked with a crosslinking agent comprising glyoxal, DL-glyceraldehyde, or a combination thereof.

8. 10. The biopolymer fiber of claim 1, further comprising attached tenocytes, wherein the tenocytes maintain at least about 75% cell viability and at least about 95% cell survival after about 7 days of incubation at conventional mammalian cell culture conditions of temperature, pH, and humidity.

9. 10. The biopolymer fiber of claim 1, having a substantially circular, oval, square, rectangular, ribbon-like, triangular, or irregularly shaped cross section.

10. 10. The biopolymer fiber bundle of claim 1, comprising from 2 to about 10,000 fibers.

11. An implantable biopolymer scaffold for assisting the repair of soft tissue injuries, comprising a biopolymer fiber according to any one of claims 1 to 9 or a bundle of biopolymer fibers according to claim 10.

12. A woven sheet-like support, patch or appliance comprising the biopolymer fiber according to any one of claims 1 to 9.

13. dissolving collagen in an acid solution to form a collagen solution; simultaneously directing the collagen solution at a first velocity through a first needle having a first diameter and directing a formation buffer at a second velocity through a second needle coaxially surrounding the first needle and having a second diameter greater than the first diameter to form a sheath around the collagen solution, forming a coaxial flow; the second flow rate of the basal buffer solution through the second needle is at least twice the first flow rate of the collagen solution through the first needle; passing said coaxially flowing collagen and formation buffer through a reaction zone containing a fibril-forming bath for a time and at a rate sufficient to form fibers; dehydrating the collagen fibers at an extrusion rate; removing the fiber onto a spool at a third speed above the extrusion speed sufficient to increase molecular orientation and reduce the diameter of the fiber; 1. A method for producing a biopolymer fiber, comprising:

14. dissolving collagen in an acid solution to form a collagen solution; passing the collagen solution at a first velocity through a first needle having a first diameter into a formation buffer; passing the collagen and formation buffer through a reaction zone containing a fiber formation bath for a time and at a rate sufficient to form fibers; dehydrating the collagen fibers at an extrusion rate; removing the fiber onto a spool at a rate from about 2 times the extrusion rate to about 10 times the extrusion rate sufficient to increase molecular orientation and reduce the diameter of the fiber; 1. A method for producing a biopolymer fiber, comprising:

15. 15. The method of claim 13 or claim 14, further comprising degassing the collagen solution before transferring the collagen solution to the formation buffer.

16. dissolving clinical collagen in an acid solution to form a collagen solution; simultaneously directing the collagen solution at a first volumetric flow rate through a first needle to create a first velocity and directing a formation buffer at a second velocity into a tube coaxially surrounding the first needle to form a sheath around the collagen solution, thereby creating a coaxial flow; a velocity of the basal buffer solution that is about 2 to about 20 times the first velocity of the collagen solution through the first needle; passing collagen and a formation buffer coaxially through a reaction zone containing a fibril-forming bath for a time and at a rate sufficient to form fibers; dehydrating the collagen fibers at an extrusion rate; withdrawing the fiber at a third speed above the extrusion speed sufficient to increase molecular orientation and reduce the diameter of the fiber; 1. A method for producing a biopolymer fiber, comprising:

17. 17. The method of claim 16, further comprising collecting the fibers in a bar collector or flat cylinder.

18. The method of claim 16 further comprising collecting the fiber on a grooved spool.

19. dissolving clinical collagen in an acid solution to form a collagen solution; extruding the solution through a nozzle into a guide that directs the extrusion solution into a flowing bath of forming buffer to form fibers; dehydrating the formed fibers in the forming buffer bath; recovering the fibers; 1. A method for producing a biopolymer fiber, comprising:

20. 20. The method of claim 19, further comprising drying the dewatered fibers by directing air at the fibers for a time sufficient to dry the fibers before collecting the fibers.

21. 20. The method of claim 19, further comprising crosslinking the fibers with a crosslinking agent comprising glyoxal, DL-glyceraldehyde, or a combination thereof, and drying the crosslinked fibers.

22. dissolving clinical collagen in an acid solution to form a collagen solution; passing the collagen solution at a first velocity through a first needle having a first diameter into a formation buffer; passing the collagen and formation buffer through a reaction zone containing a fiber formation bath for a time and at a rate sufficient to form fibers; dehydrating the collagen fibers at an extrusion rate; removing the fiber onto a spool in one or more stages at a rate from about 2 times the extrusion rate to about 12 times the extrusion rate sufficient to increase molecular orientation and reduce the diameter of the fiber; 1. A method for producing a biopolymer fiber, comprising:

23. 23. The method of claim 22, further comprising cross-linking the fibers with a cross-linking agent comprising glyoxal, DL-glyceraldehyde, or a combination thereof, and drying the cross-linked fibers.

24. A biopolymer fiber produced by the method according to any one of claims 13 to 23.

25. 25. An implantable biopolymer scaffold for assisting in the repair of soft tissue injuries, comprising the biopolymer fiber of claim 24.

26. 26. A method for assisting the repair of soft tissue damage comprising the implantation of a biopolymer scaffold according to claim 25.

27. 27. The method of claim 26, wherein the soft tissue is selected from the group comprising ligaments, tendons, tendon-ligament attachments, bone, muscle, muscle-tendon junctions, connective tissue including skin, fascia, viscera, and eyes.

28. A suture comprising the biopolymer fiber of claim 24.

29. 30. The suture of claim 28, which is absorbable.

30. 25. An internal prosthesis comprising the biopolymer fiber of claim 24, which when implanted in a subject, supports, reinforces, augments, or co-bears the mechanical loads of ligaments or tendons in a joint, such as the anterior cruciate ligament, Achilles tendon, and rotator cuff.

31. 25. An endoprosthesis comprising the biopolymer fiber of claim 24, which, when implanted in a subject, supports an injured joint by connecting one bone to another, optionally restoring biomechanics and isometry to levels substantially comparable to those of a healthy, natural joint.

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