Microfluidic extrusion

Abstract

To provide a biopolymer fiber containing collagen.SOLUTION: The biopolymer fiber has an excellent ultimate tensile strength, modulus of elasticity and strain at break comparable to a native human tendon and ligament. The fiber can have a substantially circular, ovoid, square, rectangular, ribbon-like, triangular or irregular shape. The fiber exhibits an ordered, longitudinally oriented structure. The fiber enables infiltration of cellular growth. In addition to an implantable biopolymer scaffold and a suture thread containing the fiber, microfluidic and extrusion methods for producing the biopolymer fiber are provided.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of co - pending application Ser. No. 62 / 800,317, filed Feb. 1, 2019, the disclosure of which is hereby incorporated by reference in its entirety.

[0002] Statement Regarding Government Support The data presented in this application was supported, at least in part, under DARPA Contract No. HR0011 - 15 - 9 - 0006. The United States Government has certain rights in this invention.

Background Art

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

[0004] In particular, collagen fibrils bind to form strong connective tissues such as ligaments and tendons. Many efforts have been made to produce collagen - containing tissues for use in the body to replace damaged collagen body parts (especially including ligaments and tendons). A damaged site may be directly replaced with such an implantable device, or a scaffold may be provided to soft tissues such as damaged tendons and ligaments to ultimately help replace them.

[0005] Such products need to function in various difficult biomechanical environments that require dealing with multiple functional parameters. These parameters include, for example, compatibility with living tissues and body fluids, strength, flexibility, and biodegradability.

[0006] There is a need in the art for systems and methods to address the above-mentioned drawbacks of the prior art. SUMMARY OF THE INVENTION

[0007] In one aspect, the present disclosure relates to a biopolymer fiber containing collagen, and this fiber has 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, An average fiber diameter of about 10 μm to about 90 μm and has one or more of After being immersed in DPBS at room temperature for at least about 1 hour, it maintains its strength, and its filaments exhibit a regular longitudinal orientation structure.

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

[0009] In yet another aspect, the present disclosure relates to a transplantable biopolymer scaffold for assisting in the repair of soft tissue damage, which includes biopolymer fibers or bundles thereof.

[0010] The present disclosure also relates to a woven sheet-like support, patch, or appliance containing biopolymer fibers.

[0011] In yet another aspect, the present disclosure relates to a method for manufacturing biopolymer fibers. This method includes the step of dissolving collagen in an acid solution to create a collagen solution, While sending a collagen solution at a first rate through a first needle having a first diameter, a second needle that coaxially surrounds the first needle, has a second diameter that exceeds the first diameter, and forms a sheath around the collagen solution, is used to send a forming buffer at a second rate to form a coaxial flow, a step in which a second flow rate of the base buffer through the second needle is at least twice a first flow rate of the collagen solution through the first needle, a step of sending the collagen and the forming buffer flowing coaxially through a reaction zone including a fibril formation bath at a time and rate sufficient to form fibers, a step of dehydrating the collagen fibers at an extrusion rate, and a step of taking up the fibers at a third rate that exceeds the extrusion rate and is sufficient to enhance molecular orientation and reduce the fiber diameter.

[0012] In another aspect, the present disclosure relates to a method for manufacturing a biopolymer fiber. This method includes a step of dissolving collagen in an acid solution to prepare a collagen solution, a step of sending the collagen solution at a first rate through a first needle having a first diameter into a forming buffer, a step of sending the collagen and the forming buffer through a reaction zone including a fibril formation bath at a time and rate sufficient to form fibers, a step of dehydrating the collagen fibers at an extrusion rate, and a step of taking up the fibers at a rate from about two times to about ten times the extrusion rate and sufficient to enhance molecular orientation and reduce the fiber diameter.

[0013] In yet another aspect, the present disclosure is a method for manufacturing a biopolymer fiber, the method including a step of dissolving clinical-grade collagen in an acid solution to prepare a collagen solution, a step in which while sending the collagen solution at a first volumetric flow rate through a first needle to produce a first rate, a forming buffer is sent at a second rate into a tube that coaxially surrounds the first needle and forms a sheath around the collagen solution to form a coaxial flow, The step where the speed of the basic buffer solution is about 2 to about 20 times the first speed of the collagen solution through the first needle, and The step of coaxially flowing collagen and a forming buffer solution through a reaction region including a fibril forming bath at a time and speed sufficient to form fibers, and The step of dehydrating the collagen fibers at a certain extrusion speed, and The step of taking out the fibers at a third speed exceeding the extrusion speed, which is sufficient to enhance the orientation of the molecules and reduce the diameter of the fibers, relates to a method including the above steps.

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

[0015] In a further additional aspect, the present disclosure is a method for manufacturing a biopolymer fiber, comprising The step of dissolving clinical collagen in an acid solution to prepare a collagen solution, and The step of sending the collagen solution into a forming buffer solution at a first speed through a first needle having a first diameter, and The step of sending collagen and a forming buffer solution through a reaction region including a fibril forming bath at a time and speed sufficient to form fibers, and The step of dehydrating the collagen fibers at a certain extrusion speed, and The step of taking out the fibers onto a spool at a speed of about 2 times to about 12 times the extrusion speed, which is sufficient to enhance the orientation of the molecules and reduce the diameter of the fibers, in one or more stages, relates to a method including the above steps.

[0016] The present disclosure also includes aspects of providing a transplantable biopolymer scaffold for assisting in the repair of soft tissue damage including biopolymer fibers, and a method for assisting in the repair of soft tissue damage including the transplantation of the biopolymer scaffold.

[0017] Upon reviewing 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 included within this specification and this summary, are within the scope of the present invention, and are intended to be protected by the following claims.

[0018] The present invention can be understood more precisely by referring to the following drawings and description. The components in the drawings are not necessarily to scale; instead, emphasis is placed on illustrating the principles of the present invention. Further, in the drawings, like reference numerals indicate corresponding parts throughout the different drawings.

Brief Description of the Drawings

[0019]

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[0020] In one aspect, the present disclosure relates to a biopolymer fiber containing collagen, and this fiber has the following characteristics a tensile strength of about 1 MPa to about 1,700 MPa, a modulus of elasticity of about 10 MPa to about 20,000 MPa, an elongation at break of about 2% to about 45%, an average fiber diameter of about 10 μm to about 90 μm and has one or more of the above, maintains its strength after being immersed in DPBS at room temperature for at least about 1 hour, and its filaments exhibit a regular longitudinal orientation structure.

[0021] In another aspect, the present disclosure relates to a transplantable biopolymer scaffold for assisting in the repair of soft tissue damage, including at least one biopolymer sheet containing biopolymer fibers, and this biopolymer contains collagen and has the following characteristics a tensile strength of about 1 MPa to about 1,700 MPa, a modulus of elasticity of about 10 MPa to about 20,000 MPa, an elongation at break of about 2% to about 45%, an average fiber diameter of about 10 μm to about 90 μm and has one or more of the above, maintains its strength after being immersed in DPBS at room temperature for at least about 1 hour, and its filaments exhibit a regular longitudinal orientation structure.

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

[0023] In another aspect, the present disclosure includes a transplantable biopolymer scaffold for the repair or replacement of human body parts.

[0024] Biodegradable polymer fibers are usually formed of collagen. In particular, telocollagen is usually obtained from any source (human, bovine, recombinant, jellyfish, etc.). Silk fibroin, other types of collagen (such as type II collagen), fibrin / fibrinogen, basement membrane proteins, hyaluronic acid, polyethylene oxide, polyethylene glycol, polycaprolactone, polyethylene, polyhydroxybutyric acid, PDLA, PDLLA and high molecular weight PDLLA, PLGA, and blends thereof, etc. Biocompatible polymers may be blended with collagen to form biodegradable polymer fibers.

[0025] In yet another aspect, the present disclosure relates to a method for manufacturing biodegradable polymer fibers, which includes the step of dissolving collagen in an acid solution to prepare a collagen solution. In one embodiment of this method, collagen is then fed at a first rate through a first needle having a first diameter to exit at the first rate, while surrounding the first needle coaxially with a second needle having a second diameter greater than the first diameter and forming a sheath around the collagen solution, and a forming buffer is fed at a second volumetric flow rate through the second needle to form a coaxial flow. 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] The collagen and the forming buffer flowing coaxially are fed through a reaction zone including a fibril formation bath at a time and rate sufficient to form fibers, at a third rate greater than the first rate sufficient to enhance the orientation of the molecules and reduce the diameter of the fibers, usually twice the rate at which the fibers are extruded through a dehydration bath, and the fibers are wound onto a spool. The fibers may then be crosslinked and dried.

[0027] In yet another aspect, the present disclosure relates to an alternative method for manufacturing biopolymer fibers. In this embodiment, a collagen solution is prepared and injected into a reaction zone, which includes a fiber-forming bath such as a forming buffer bath, at a rate and for a time sufficient to form fibers. The fibers are taken up on a spool at a rate about two to about ten times the injection rate, which increases the orientation of the molecules and decreases the fiber diameter. The fibers may then be crosslinked and dried.

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

[0029] Throughout this specification, steps that would typically be carried out together during normal, typical manufacturing processes, such as washing and drying, or dipping and drying, may be carried out as necessary or repeated 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, dried twice, or washed twice and then dried before proceeding to the next processing step.

[0030] In other embodiments, the first washing or drying step may be optionally performed. Thus, a material that would typically be washed and then dried may proceed directly to the drying step and then move on to the next processing step. One of ordinary skill in the art will recognize situations where those steps may be repeated or omitted.

[0031] Constructs such as scaffolds made from these fibers enable intracellular colonization. That is, cells of various types of animals in which the fibers (and devices made from the fibers) are embedded preferably align with the scaffold fibers and proliferate within the pores of the scaffold. The constructs and scaffolds include single-layer and multi-layer products that may be used as alternatives for known repair functions, such as sutures used to reattach body parts such as opposing portions of a transected Achilles tendon. In addition to providing a support structure for use in repairing a torn or damaged tendon, embodiments of the present disclosure are also suitable for ligament repair. Thus, scaffolds or other exemplary ligaments for which the invention may be used to provide support may include the ACL, MCL, PCL, UCL, as well as other human and animal ligaments. Other surgeries for which the products of the present disclosure are useful include, for example, superior capsular reconstruction as a treatment option for a superior glenohumeral tendon avulsion, particularly where reconstruction of a partial or complete avulsion would otherwise be impossible or difficult. Also, a multi-layer sheet may 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. For example, during surgery, multiple fibers may be twisted, bundled, braided, woven together, or otherwise arranged to improve the shape factor for easier manipulation compared to manipulation with a single fiber. Improving the shape factor may facilitate the accurate placement of the fiber or platform. Other shape factors may be constructed that serve as reinforcement or internal fixtures for torn natural body parts. The fixture connects one bone to another to support the joint. Typically, the fixture forms an isometric joint having the biomechanics and isometry of the restored natural joint.

[0033] The effects of the collagen fibers produced in accordance with the embodiments of the present disclosure may be shown, for example, by studying the repair performed on rabbits. In particular, the reinforcement materials, interior fittings, and suture-like structures of rabbit knees are suitable for evaluating the properties and characteristics of the collagen fibers of the present disclosure and the structures produced from these fibers.

[0034] FIG. 1 shows an embodiment of a system and method for manufacturing collagen fibers. This system and method may be described as including four sections or manufacturing areas. The collagen solution is prepared in the first section, and the collagen fibers are formed in the second section. Next, the collagen fibers are collected in the third section, and then post-treated in the fourth section, the post-treatment or final treatment, to obtain wet collagen fibers or dry collagen fibers.

[0035] The steps of the system and method shown in FIG. 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 Recovery of Collagen Fibers 135 - 150 4 Post-treatment or Final Treatment 155 - 180 As seen in step 105 of FIG. 1, collagen is mixed with an acidic solution and stirred thoroughly in step 110. In some embodiments, the acid is acetic acid at about 0.01M to about 0.50M. In other embodiments, the acid is hydrochloric acid at about 0.01M to about 0.50M. The solution may be degassed in step 115 and then centrifuged in step 120 to remove residual air bubbles. The resulting collagen solution is extruded from a needle, and there may be a second needle coaxial with the needle that supplies a forming buffer in step 125. The resulting fiber formation may continue through 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 at step 135 and dehydrated at step 140. The collagen fibers are recovered at step 145 and air-dried at step 150. Then, post-treatment may be performed as shown in the figure at steps 155, 160, 165, and 170. The air-dried collagen fibers on the spool are immersed in a cross-linking solution at step 155, optionally washed at step 160, air-dried at step 165, and dried at step 170 to form dried fibers. As shown by the dashed line in Figure 1, the material may optionally be washed at step 160, dried at step 165, and returned to the 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 collagen thus injected is introduced into the recovery system via dehydration at step 140. The fibers are then processed according to the remaining part of the processing steps.

[0038] Figure 1 shows a general diagram of a system and method for implementing an embodiment 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 biopolymer fibers, which includes the step of dissolving collagen in an acidic solution to create a collagen solution. Then, while sending collagen at a first volumetric flow rate through a first needle having a first diameter to produce a first velocity, a second needle having a second diameter that coaxially surrounds the first needle and exceeds the first diameter is used to send a forming buffer at a second volumetric flow rate through the second needle to form a coaxial flow around the collagen solution. 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] Feed collagen and forming buffer flowing coaxially through a reaction zone containing a fibril-forming bath at a time and volumetric flow rate sufficient to form fibers, and take up the fibers on a spool at a third speed that exceeds a first speed. The third speed is typically about twice the speed at which the fibers are extruded through a dehydration bath and is sufficient to enhance molecular orientation and reduce the diameter of the fibers. The fibers are then crosslinked and dried.

[0041] In another embodiment, the present disclosure relates to an alternative method for manufacturing biopolymer fibers. A collagen solution is prepared and injected into a reaction zone containing a fibril-forming bath, such as a forming buffer bath, at a time and rate sufficient to form fibers. A second needle for forming a coaxial flow of the forming buffer is not necessary. Instead, the collagen fibers are injected directly into the fibril-forming bath and then conveyed through a dehydration bath. The fibers are conveyed by taking them up on a spool at a speed that is about two to about four times the injection speed that enhances molecular orientation and reduces the diameter of the fibers. The fibers may then be crosslinked and dried.

[0042] An embodiment of method 1300 is summarized in FIG. 13. The collagen solution is prepared as shown in the figure. Create the collagen solution at step 1305. A biopolymer may be mixed with this collagen. Dissolve the collagen in an acidic solution to create a viscous solution. Stir this solution at step 1310 to ensure sufficient mixing. This mixed solution may contain trapped gas and thus may be degassed one or more times with a degassing device at step 1315. Then, at step 1320, the collagen solution may be centrifuged as shown in the figure. Optionally, the degassing / centrifugation step may be repeated as shown by the dashed line in FIG. 1 and feature 1316 in FIG. 13 to reduce the volume of gas trapped in the solution.

[0043] The collagen solution thus prepared is formed into collagen fibers by coaxial extrusion with a forming buffer that functions as a sheath for the fiber core, as shown in step 1325. The volume flow rate of the forming buffer is typically at least twice the volume flow rate of collagen formation. This configuration may suppress the formation of individual fibrils, stretch and orient 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 completed in step 1330, the collagen is then separated from the forming buffer in step 1335 and dehydrated with a dehydrating solution in step 1340. Then, in step 1345, the dehydrated collagen is collected on a rotating spool and the fibers are further stretched by rotating the spool at a rate exceeding the rate at which the fibers are supplied from the dehydrating solution step 1340, typically at about twice the rate. The fibers thus collected are then 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 forming buffer. Thereby, step 1325 is omitted. The fibers are recovered, separated from the forming buffer, and dehydrated in a dehydrating 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 from about twice the forming rate to about four times the forming rate.

[0046] The fibers air-dried on the spool may then 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 construction materials suitable for withstanding attacks by any of the raw materials used in manufacturing collagen fibers according to embodiments of the present disclosure. Metals, plastics, and other materials have properties and characteristics suitable for withstanding attacks by raw materials, intermediates, solvents, and products during the manufacture of collagen fibers.

[0048] Another aspect of the present disclosure relates to collagen fibers, which fibers have one or more of the following characteristics a tensile strength of about 1 MPa to about 1,700 MPa, a modulus of elasticity of about 10 MPa to about 20,000 MPa, an elongation at break of about 4 percent to about 12 percent elongation, an average fiber diameter of about 16 μm to about 70 μm and maintain at least its strength after being immersed in a biological fluid for about 1 hour. The fibers exhibit a regular longitudinal orientation structure that allows infiltration of cell growth.

[0049] The fibers of this embodiment are manufactured according to the method of the embodiments of the present disclosure. Collagen may be obtained from many sources and in various forms. The quality of the collagen fibers may be related to the quality of the raw materials used. In some embodiments, bovine collagen is typically used. The bovine collagen may be in a natural form or in a lyophilized powder form.

[0050]

[0051] ​Bovine collagen 202 may be processed into a viscous solution 203 by dissolution in an acidic solution. To prepare the collagen solution, either a mineral acid such as hydrochloric acid or an organic acid such as acetic acid may be used. For example, in one embodiment, bovine type I collagen having intact terminal telopeptides is dissolved in about 0.01 M acetic acid to about 0.5 M acetic acid 201 in a container 210 to create 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 in the solution to about 19 mg / mL of collagen in the solution. In another embodiment, lyophilized bovine type I dermis with bound terminal telopeptides is mixed with a mineral acid such as HCl having a concentration of about 0.01 M to about 0.5 M to create a solution having a concentration of about 10 mg / mL of collagen 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, usually at least about 15 hours, more generally at least about 16 hours. In some embodiments, the collagen solution 301 is degassed within a degassing device 300 to remove air bubbles from the collagen solution 301. A screen 304 ensures that collagen is not drawn out of the degassing device through the gas outlet 303 of the degassing device. The degassing device 303 is typically operated at a pressure of about 0 psia to about 3 psia. The collagen solution may be exposed to a maximum of about 2 degassing cycles, usually about 1 to about 2 cycles. Degassing removes gas bubbles that could interfere with and prevent the extrusion of fibrous collagen.

[0053] The degassed collagen may then be further degassed in a centrifuge. In FIG. 4, the centrifuge 400 is shown with its top 408 opened so that the bowl 403 is visible. Tubes for the material to be centrifuged and tubes used to balance the centrifuge are placed in wells within the rotating bowl 409. The case 405 is sufficiently robust to contain any fragments in the event of a failure of any of the internal components during use.

[0054] FIG. 5 shows a centrifuge 500 having a storage bowl 501 and a lid 508. As shown by the arrow 505 of movement, the centrifuge rotates at high speed counterclockwise. Tube 502 represents a tube containing a collagen solution 503 before centrifugation. As shown in the figure, the collagen solution is homogeneous and has air bubbles trapped within a homogeneous collagen solution 512 except for that.

[0055] Centrifugation at a relative centrifugal force or g value of about 400 rcf to about 4,000 rcf, usually about 600 rcf to about 1,000 rcf, more generally about 700 rcf to about 800 rcf, is suitable for reducing the trapped air bubble volume to essentially zero within about 3 minutes to about 15 minutes, usually about 4 minutes to about 10 minutes, more generally about 5 minutes to about 7 minutes.

[0056] In some embodiments, a pair of related steps may be repeated by performing the steps alternately. For example, the collagen may be cycled once in the degassing device 303, then processed in the centrifuge 500 for 5 minutes, then returned to the degassing device 303 for one cycle, and then centrifuged for 5 minutes. Operating in this alternative way may result in improved efficiency. This efficiency improvement may be achieved using a shorter processing time to achieve a given amount of air bubbles or to achieve better results than a linear process.

[0057] The collagen is then co-extruded with the solution to form collagen fibers. Extrusion of the collagen solution in the core of the coaxial fluid may assist in the formation of collagen fibers in some embodiments.

[0058] In some embodiments of the present disclosure, collagen is then introduced into the center of the coaxial flow needle and a forming buffer is introduced into the outer needle. In this way, the forming buffer forms a sheath around the collagen. As shown in FIG. 6, the collagen solution 650 is pumped by a pump through the pump and introduced into the inner needle 603 as a collagen stream 601. At the same time, the forming buffer 660 is introduced into the outer needle 604 as a forming buffer stream 606. The outer needle 604 is coaxial with the inner needle 603 such that the forming buffer forms a sheath around the central core of the collagen. When the material exits the needle and flows into a reaction zone including a fibril formation bath 701 (shown in FIG. 7), the forming buffer 607 forms a sheath around the collagen fiber 602, and this collagen fiber 602 begins 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 from about 0.05 mm to about 100 mm, in some embodiments, the inner diameter of the central needle is from about 0.1 mm to about 50 mm, in still other embodiments, the inner diameter of the central needle is from about 0.2 mm to about 20 mm, in still other embodiments, the inner diameter of the central needle is from about 0.3 mm to about 10 mm, and more generally from about 0.35 mm to about 5 mm. In some embodiments, the narrower inner diameter range of the central needle is, for example, from about 0.03 mm to about 10 mm, typically from about 0.10 mm to about 3 mm, more generally from about 0.30 mm to about 1 mm, and even more generally from about 0.35 mm to about 0.50 mm.

[0060] In some embodiments, the inner diameter of the central needle is from about 0.38 mm to about 0.44 mm, typically from about 0.39 mm to about 0.43 mm, and more generally from about 0.40 mm to about 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 times the inner diameter of the central needle to about 2.15 times the inner diameter of the central needle, generally about 2.00 times the inner diameter of the central needle to about 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 an embodiment, the forming buffer may be any solution for assisting in the formation of collagen fibers. The forming buffer is typically a solution containing salts and a buffer added to TES (also known as 2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid or N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid).

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

[0064] The flow rate of the collagen solution and the flow rate of the forming buffer are adjusted so that the forming buffer sheath is maintained within the extrusion needle and within the reaction zone including the fibril formation bath. The rate of the forming buffer is also set to exceed the rate of the collagen solution so as to impart elongation to the collagen fibers and improve the quality of the fibers. In fact, this promotes the formation of relatively straight and continuous fibers without kinks and other physical shape abnormalities. In some embodiments, the fibers may be substantially circular, oval, square, rectangular, ribbon-shaped, triangular, or irregular in shape.

[0065] In embodiments of the present disclosure, the velocity of the forming buffer within the needle reaction zone including the fiber forming bath is greater than the velocity of the collagen solution. The forming buffer is used to neutralize the collagen solution and assist in fibril formation. Further, a higher velocity forming buffer is used to pull or stretch the collagen stream, creating an elongation field that aids in the alignment of collagen monomers in a process called flow-induced crystallization. This alignment polymerizes the collagen and helps increase the strength of the resulting product.

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

[0067] In an embodiment, as shown in FIG. 7, the collagen stream 702 and the forming buffer sheath 707 enter a reaction zone including the fibril forming bath 701 of the reaction system 700. The reaction zone may have a structure, such as a forming tube, that forms the reaction zone 701. However, typically, the structure need not be present. As this stream flows through the reaction zone including the fibril forming bath 701, the collagen fibers continue to polymerize to form a collagen fiber product. The collagen fibers 752 and the forming buffer 702 flow out of the reaction zone including the fibril forming bath 701.

[0068] In an embodiment, the velocity of the collagen is adjusted to give the collagen a reaction or polymerization time of from about 15 seconds to about 60 seconds, typically from about 20 seconds to about 50 seconds, and more generally from about 25 seconds to about 40 seconds.

[0069] As shown in FIG. 8, in the embodiment, when flowing out from the reaction region including the fibril forming bath 701 at the end of the polymerization period, collagen fibers 852 are formed and separated from the forming buffer solution 808. The excess forming buffer solution flows into the tray 801. The dehydration system 800 is designed to receive the forming buffer solution 808 in the tray 801 and introduce the collagen fibers 852 into the dehydration bath 802.

[0070] The dehydration solution provides an opportunity to remove water from the collagen fibers, reduce the fiber diameter, and assist in fibril formation. In an embodiment, the dehydration solution comprises a Milli-Q aqueous solution of about 10 percent ethanol to about 35 percent ethanol, usually a Milli-Q aqueous solution of about 15 percent ethanol to about 30 percent ethanol, and more generally a Milli-Q aqueous solution of about 15 percent ethanol to about 25 percent ethanol. Those skilled in the art recognize that Milli-Q water (also written as Milli-Q water) is highly purified water produced by an apparatus available from Millipore Sigma (Burlington, Massachusetts, USA).

[0071] The collagen fibers 852 pass through the dehydration bath 802 for about 10 seconds to about 50 seconds, usually about 15 seconds to about 45 seconds, and more generally about 20 seconds to about 40 seconds. Throughout that period, the collagen fibers 852 remain immersed in the dehydration bath 802. The volume of the dehydration bath 802 is from about 400 times the volume per minute of the forming buffer solution pumped to about 800 times the volume per minute of the forming buffer solution pumped, usually from about 450 times the volume per minute of the forming buffer solution pumped to about 750 times the volume per minute of the forming buffer solution pumped, and more generally from about 500 times the volume per minute of the forming buffer solution pumped to about 700 times the volume per minute of the forming buffer solution pumped.

[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 speed 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 for ensuring that the collagen fibers remain immersed in the dehydration bath 802. Since the dehydrated collagen fibers 930 are lifted above the height of the dehydration bath, it is possible to see the fluid droplets 905 falling from the dehydrated collagen fibers 930.

[0073] Figure 10 shows the recovery of the dehydrated fibers onto the spool 1001. In an embodiment, the spool 1001 is rotated in a clockwise direction by the motor 1011 as shown by the arrow 1016. The spool 1001 is rotated at a speed that gives a draw ratio of about 1.5 to about 3, usually about 1.75 to about 2.5, and more generally about 1.90 to 2.20. Similarly, the spool 1002 is rotated at the same speed. The draw ratio is the ratio between the spool speed and the extrusion speed. Thus, there is a tension on the first collagen fiber 1050 that pulls the fiber upward at the position of the hook 910. The fiber is then pulled over the wall of the dehydration bath 802 and then pulled out to the spool 1001.

[0074] Alternatively, in some embodiments of the present disclosure, the collagen is introduced directly into the fibril-forming bath without using the coaxial needle of FIG. 6 to form a coaxial flow as shown in FIG. 7. Conversely, when the collagen solution 852 is directly injected from the needle into the fiber formation bath 870, collagen fibers are formed, and then the processing proceeds in the same manner as the coaxial formation method, which is an alternative embodiment of the present disclosure. The size of the needle for collagen injection is selected in the same manner as the size of the needle for the coaxial injection method. As in other embodiments, the fibers are pulled through the fibril formation bath and then drawn into the dehydration bath. However, the spool 1001 in FIG. 10 is rotated at a take-up speed that provides a take-up speed of about 2 times to about 4 times the fiber formation speed, usually about 2.5 times to about 3.5 times the fiber formation speed, and more generally about 2.75 to 3.25 times the fiber formation speed. Then, post-treatment is performed in the same manner as in other embodiments.

[0075] Arrow 1020 indicates the passage of time in some embodiments while the spool 1001 is translating parallel to the end of the dehydration bath 802 so as to form a single layer of fibers on the spool. Thereby, the rotation of the spool continues at the same speed, and tension is applied to the fibers 1052 as the spool translates until the spool 1002 is essentially full. The time arrow 1030 indicates the passage of time until the fiber supply runs out. Then, the spool 1055 may be retrieved. The translation speed may be adjusted to adjust the spacing between the fibers on the spool.

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

[0077] The fibers of the present disclosure may be chemically post-treated. Figure 12 shows possible post-treatment steps. In an embodiment, a spool 1210 containing collagen fibers is air-dried at 1220 for at least about 15 minutes, usually at least about 20 minutes, more generally at least about 30 minutes. The air-dried tube 1210 containing the fibers is then placed in a container for crosslinking. Typically, the containers in embodiments of the present disclosure minimize the volume 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 necessary to cover the cylindrical spool 1210 as shown at 1231. In an embodiment, the volume of the crosslinking solution per meter of fiber is at least about 3 μL, usually at least about 4.5 μL, more generally at least about 6 μL.

[0078] The fibers of the present 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 generally glyoxal (GLY) or other conventional crosslinking reagents. For example, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), genipin, glyceraldehyde, glutaraldehyde, o-dextran, and crosslinking agents such as low molecular weight procyanidin and high molecular weight procyanidin may also be used. Alternatively, if the fibers are functionalized with carboxyl groups, EDC and other carbodiimides may be used for crosslinking. Isocyanates react with both OH groups and amines. Therefore, isocyanate-based crosslinking agents may be used to crosslink OH groups with each other, for example, within functionalized PDLLA (linking OH groups with other OH groups), to improve the stability and strength of the medium. Isocyanates may also be used to bind OH groups in functionalized PDLLA to collagen via the NH 2 groups (i.e., amine groups) of collagen. Additionally, photo-crosslinking agents can also be used.

[0079] The following reaction sequences are exemplary crosslinking reactions that can be used in embodiments of the present disclosure. In each of these exemplary reactions, P = polymer (the fiber in these reactions).

[0080]

Chemical formula

[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 concentration or ratio of these components may be varied to provide the desired degree of crosslinking and functionality.

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

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

[0084] In some embodiments, a spool containing crosslinked collagen fibers 1211 is then removed from the tube and optionally placed in milli-Q water rinse solution for about 10 minutes as shown by 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 excess glyoxal. Typically, 10 minutes is sufficient. Removal of glyoxal helps reduce the cytotoxicity of the fibers. Other crosslinking agents may be removed as well, if desired or appropriate.

[0085] In embodiments where the rinse step is omitted, the spool and fibers 1213 are placed in glycine in glycine bath 1250. The processing to dry the fibers is performed in the same manner as in the rinse step embodiments.

[0086] The spool containing collagen fibers 1214 is then rinsed again with milli-Q water in tank 1260. In embodiments, 10 minutes is sufficient to remove the glycine. The spool and fibers 1215 are then air dried at 1270 for about 1 hour and then placed in a drying chamber 1280 for about 24 hours. The dried flexible fibers 1217 of FIG. 12 are then recovered.

[0087] Embodiments of the present disclosure relate to a method 1300 of FIG. 13 for manufacturing biopolymer fibers. In embodiments, collagen is dissolved in an acid solution (1305) to create a collagen solution (1310). In some embodiments, a compatible biopolymer is included with the collagen. 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 with the forming buffer as a sheath (1325). While sending the collagen solution at a first rate through a first needle having a first diameter, a second needle having a second diameter that surrounds the first needle coaxially and exceeds the first diameter and forms a sheath around the collagen solution is used to send the forming buffer at a second rate to form a coaxial flow. The second rate of the base buffer through the second needle is at least twice the first rate of the collagen solution through the first needle.

[0089] In some embodiments, the inner diameter of the central needle is from about 0.05 mm to about 100 mm, in some embodiments, the inner diameter of the central needle is from about 0.1 mm to about 50 mm, in still other embodiments, the inner diameter of the central needle is from about 0.2 mm to about 20 mm, in still other embodiments, the inner diameter of the central needle is from about 0.3 mm to about 10 mm, and more generally from about 0.35 mm to about 5 mm. In some embodiments, the narrower inner diameter range of the central needle is, for example, from about 0.03 mm to about 10 mm, usually from about 0.10 mm to about 3 mm, more generally from about 0.30 mm to about 1 mm, and even more generally from about 0.35 mm to about 0.50 mm.

[0090] In some embodiments, the inner diameter of the central needle is from about 0.38 mm to about 0.44 mm, usually from about 0.39 mm to about 0.43 mm, and more generally from about 0.40 mm to about 0.42 mm.

[0091] In some embodiments, the inner diameter of the surrounding coaxial outer needle for supplying the forming buffer is generally from about 1.95 times the inner diameter of the central needle to about 2.15 times the inner diameter of the central needle, generally from about 2.00 times the inner diameter of the central needle to about 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 an embodiment, the forming buffer may be any solution for assisting in the formation of collagen fibers. The forming buffer is usually a solution containing salts and a buffer, and TES (also known as 2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid or N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid).

[0093] In some embodiments of the present disclosure, the forming buffer is WSB (a solution containing 30 mM of TES, 4.14 mg / mL of sodium dihydrogen phosphate dihydrate, 12.1 mg / mL of disodium hydrogen phosphate heptahydrate, 135 mM of NaCl, and 10 w / v percent of 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 from about 5 times the volumetric flow rate of the collagen solution in the needle to about 10 times the volumetric flow rate of the collagen solution in the needle, usually from about 7 times the volumetric flow rate of the collagen solution in the needle to about 9 times the volumetric flow rate of the collagen solution in the needle, and more generally from about 7.5 times the volumetric flow rate of the collagen solution in the needle to about 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 an embodiment, the coaxially flowing collagen and forming buffer flow through a reaction region including a fibril formation bath at a time and rate sufficient to form fibers (1330). The formed collagen fibers are then separated from the forming buffer (1335) and placed in a dehydration solution (1340). The dehydration solution provides an opportunity to remove water from the collagen fibers, reduce the fiber diameter, and assist in fibril formation. In an embodiment, the dehydration solution contains a milliQ aqueous solution of from about 10 percent ethanol to about 35 percent ethanol, usually a milliQ aqueous solution of from about 15 percent ethanol to about 30 percent ethanol, and more generally a milliQ aqueous solution of from about 15 percent ethanol to about 25 percent ethanol.

[0096] In some embodiments, the fiber is withdrawn from the spool at a third speed that exceeds the first speed and is sufficient to enhance the orientation of the molecules and reduce the diameter of the fiber (1345). This speed is typically at least about twice the speed at which the fiber flows through the dehydration 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, the collection spool is rotated, and the forming buffer and dehydration solution are agitated by providing a pulling speed of the fiber at a speed from about twice the injection speed to about four times the injection speed. Then, the remaining portion of the steps including possible post-treatment is performed.

[0098] In embodiments, after a short air-drying time at step 1350, the fiber is crosslinked at step 1355. Typically, crosslinking is performed by stirring in a glyoxal solution for a time sufficient to achieve crosslinking. In embodiments, the fiber remains on the spool. Generally, the volume of the crosslinking container is minimized to reduce the amount of crosslinking agent required.

[0099] The crosslinking material may be any suitable crosslinking agent. 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 milliQ water is used for crosslinking. The concentration or ratio 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, typically at least about 4.5 μL, and more generally at least about 6 μL. In many cases, 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 an increase in crosslinking, and a time of at least about 72 hours provides further crosslinking.

[0100] Next, a spool containing crosslinked collagen fibers is removed from the crosslinking container and, in some embodiments of the present disclosure, placed in milli-Q water rinse solution for about 10 minutes. In other embodiments, the spool need not be rinsed. Next, in step 1360, the spool and fibers are placed in a bath containing a 100 mM glycine bath for a time sufficient to inactivate excess glyoxal. Usually 10 minutes is sufficient. Removal of glyoxal may help reduce the cytotoxicity of the fibers.

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

[0102] Next, in an embodiment, the spool containing the collagen fibers from the glycine bath is rinsed again with milli-Q water in step 1365. In an embodiment, 10 minutes is sufficient to remove the glycine. Next, 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 recovered.

[0103] In embodiments of the present disclosure, the fibers produced are biopolymer fibers containing collagen. This biopolymer fiber has the following characteristics A tensile strength of about 20 MPa to about 170 MPa, An elastic modulus of about 200 MPa to about 3,500 MPa, An elongation at break of about 4 percent to about 12 percent, An average fiber diameter after drying of about 16 μm to about 70 μm And has one or more of Maintains at least its strength after immersion in biological fluid for about 1 hour.

[0104] This fiber exhibits a regular longitudinal orientation structure, which allows infiltration of cell growth.

[0105] In another aspect, the present disclosure relates to a transplantable biopolymer scaffold for assisting in the repair of soft tissue damage or for the repair or replacement of a human body part. The scaffold includes at least one biopolymer sheet containing biopolymer fibers, and the biopolymer has the following characteristics a tensile strength of about 20 MPa to about 170 MPa, a modulus of elasticity of about 200 MPa to about 3,500 MPa, an elongation at break of about 4 percent to about 12 percent, an average fiber diameter after immersion in a phosphate buffered saline aqueous solution for about 1 hour of about 16 μm to about 70 μm and includes collagen and biopolymer fibers having one or more of the above, and maintains at least its strength after immersion in body fluid for about 24 hours.

[0106] These fibers exhibit a regular longitudinal orientation structure, which allows for the infiltration of cell growth. The sheet includes 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. Thus, it is necessary or appropriate to form a scaffold or structure larger than a single fiber to provide a fiber-containing product suitable for the repair or replacement of a body part. Thus, for example, several fibers can be braided together to form a twisted thread containing collagen fibers. Such a twisted thread may be useful, for example, in suturing a ligament or tendon rupture. These and other uses will become apparent to the user.

[0107] Throughout the present disclosure, tests regarding properties and characteristics are performed on 10 randomly collected fibers. The strength test is performed using 10 fibers and a load of about 0.3 N to about 2 N.

[0108] As pointed out in this specification, the stability of the collagen fibers is maintained even after being placed in a biological solution for at least 1 hour. Further, additional cross-linking achieved by continuing the cross-linking time for at least about 48 hours and even up to 72 hours significantly reduces the swelling of the fibers and maintains or increases the load capacity.

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

[0110] Example 1 Type I bovine collagen with intact telopeptides was taken out of the package and mixed with 0.05 M acetic acid to create 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. Before and after degassing, centrifugation was performed at approximately 750 rcf for 5 minutes to remove excess bubbles. The collagen was aspirated into a 5 mL syringe and then attached to the Luer fitting in the center of a coaxial needle (inner diameter of the collagen inlet 0.41 mm, inner diameter of the forming buffer inlet 0.84 mm). The collagen syringe and the coaxial needle were then placed on a syringe pump and pumped at 60 μL / min.

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

[0112] The tube was placed through the bottom of the beaker and through a peristaltic pump, and then attached to a coaxial outer needle through a luer fitting, thereby forming an outer sheath flow of collagen. A forming buffer was used to neutralize the collagen solution and assist in 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 assisted in the alignment of collagen monomers in a process called flow-induced crystallization. This alignment assisted in the easier polymerization of collagen and increased the strength of the final product.

[0113] The collagen flow and the forming buffer flow were introduced into a reaction region containing a fibril formation bath, where the fibers were given time to polymerize and form long chains. The reaction region containing the fibril formation bath received the used forming buffer in a reservoir and ended at the inlet of a dehydration bath that moved the fibers approximately 45 cm through 20% ethanol and 80% milliQ water. This bath helped remove water from the collagen fibers, reduce their diameter, and assist in fibril formation. This bath was 2.5 cm wide and held approximately 300 mL of solution.

[0114] After the fibers had moved through the bath, the fibers were then wound around a 50 mm diameter spool that was 300 mm in length and rotated at approximately 5 RPM, thereby giving an elongation ratio (the ratio between the spool speed and the extrusion speed) of approximately 2. This elongation ratio helped further enhance the orientation of the molecules, reduce the fiber diameter, and ultimately increase the strength. The translational speed of the spool was adjusted to vary the spacing between the fibers.

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

[0116] After 24 hours, the spools were removed from the tubes and placed in Milli-Q bath for 10 minutes. Then, the spools were placed in 100 mM glycine bath for 10 minutes to inactivate the excess glyoxal and help reduce cytotoxicity, followed by a final 10-minute immersion in Milli-Q water bath to remove the remaining glycine. Next, the spools and fibers were 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. This facilitates the processing into a useful shape for constructing scaffolds. The average diameter of the obtained fibers was 25 μm, and the tensile strength after a half-hour 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 depending on the formulation, it may also contain potassium chloride and potassium dihydrogen phosphate. This buffer helps maintain a constant pH. The osmolarity and ionic concentration of this solution are compatible with (i.e., isotonic to) the osmolarity and ionic concentration of the human body.

[0118] Amino acids, calcium chloride, potassium chloride, magnesium sulfate, sodium chloride and sodium monophosphate, glucose, and vitamins such as folic acid, nicotinamide, riboflavin and B 12 The stability test in DMEM, a synthetic medium for cell culture containing the above components, at 37 °C for 7 days shows a decrease of approximately 25% in the original strength. DMEM also contains iron and phenol red for pH indication.

[0119] This example illustrates the production of fibers within the scope of the claims according to the method within the scope of the claims. Using these fibers, scaffolds according to the claims for the repair or replacement of human body parts are produced.

[0120] Example 2 The intact type I bovine collagen with terminal telopeptides was taken out of the package and mixed with 10 mM hydrochloric acid to create a viscous solution with a collagen concentration of 16 mg / mL. The collagen was dissolved in this solution for 16 hours and then centrifuged at 733 rcf for 5 minutes. It was degassed for 2 minutes to remove excess bubbles and then centrifuged again at 733 rcf for 10 minutes. The collagen was aspirated into a 20 mL syringe and then attached to the central luer fitting of a coaxial needle (inner diameter of the collagen inlet 0.41 mm). The collagen needle was then placed on a syringe pump and pumped at 50 μL / min.

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

[0122] The forming buffer was used to neutralize the collagen solution and assist in fibril formation. The collagen was pumped into the forming buffer and led 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 inlet of a dehydration bath of 20% ethanol and 80% milliQ water through which the fibers were led. This bath helped remove water from the collagen fibers, reduce their diameter and assist in fibril formation. Both baths were 2.5 cm wide and held approximately 300 mL of solution.

[0123] After the fibers had moved through the bath, they were then wound around a 50 mm diameter spool that was 300 mm long and rotated at approximately 10 RPM, thereby giving a draw ratio (the ratio between the spool speed and the extrusion speed) of at least approximately 2. This draw ratio helped further enhance the orientation of the molecules, reduce the fiber diameter and ultimately increase the strength. The translational speed of the spool was adjusted to vary the spacing between the fibers.

[0124] The spools were air-dried for at least 15 minutes and up to 1 hour before being placed in a cylindrical tube for cross-linking. The inner diameter of this tube was close to the outer diameter of the spools in order to reduce the cross-linking agent required for complete immersion. A 10 mM glyoxal solution of 70 percent ethanol and 30 percent Milli-Q water was prepared at 120 mL and poured into this tube. The spools were then placed in this tube. The tube and spools were then placed on a roller at approximately 1 RPM for at least 24 hours up to a maximum of 72 hours.

[0125] After 24 hours or a maximum of 72 hours, the spools were removed from the tube, air-dried for approximately 1 hour, and then placed in a drying chamber for 24 hours.

[0126] After drying, the fibers were dry and flexible. This facilitated processing into useful shapes for constructing scaffolds. The average wet diameter of the resulting fibers was 30 μm, and the tensile strength after a half-hour 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, sodium chloride, and, depending on the formulation, potassium chloride and potassium dihydrogen phosphate. This buffer helps maintain a constant pH. The osmolarity and ionic concentration of this solution are compatible with (i.e., isotonic to) the osmolarity and ionic concentration of the human body.

[0127] A 7-day stability test in DMEM, a synthetic medium for cell culture containing amino acids, calcium chloride, potassium chloride, magnesium sulfate, sodium chloride, monosodium phosphate, glucose, and the vitamins folic acid, nicotinamide, riboflavin, and B 12 showed a decrease of approximately 25% of the original strength. DMEM also contains iron and phenol red for pH indication.

[0128] This example illustrates the manufacture of fibers within the scope of the claims of this patent, according to a method within the scope of the claims of this patent. With these fibers, scaffolds according to the claims of this patent are manufactured for the repair or replacement of human body parts.

[0129] Additional disclosure and comparative information In embodiments of the present disclosure, clinical atelocollagen and telocollagen may be used to form microfluidic extruded collagen microfibers, which can then be crosslinked with harmless biocompatible crosslinking agents such as glyoxal or DL-glyceraldehyde (DLG). These crosslinked fibers were significantly stronger than the other 50 crosslinking strategies tested, showing a hydrated ultimate tensile strength of nearly 300 MPa and a modulus of over 3 GPa, exceeding the strength of natural human Achilles tendon and anterior cruciate ligament. Fibers crosslinked with glyoxal retained 50% of their initial load-bearing capacity throughout 3 to 6 months in culture. Collagen fibers implanted in rats showed biocompatibility, promoted the production of aligned new host-generated collagen growing along the fibers, and in the case of glyoxal crosslinking, promoted an increase in the regenerative-promoting M2 macrophage response. Embodiments of the present disclosure showed significant improvement in healing compared to other crosslinked fibers, including conventional synthetic materials, and made the embodiments of the present disclosure into excellent fibers for generating strong 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 not yet resulted in the production of an appropriate product. To date, for example, sutures, appliances or autografts, allografts and synthetic materials as soft tissue closures or ligatures have been found to have significant clinical limitations. Allografts such as cadaveric, decellularized, and chemically treated implants may take time to incorporate, are inflammatory, and may in some cases delay healing (Seon, Song and Park, 2006). Synthetic grafts may decompose into acidic by-products and damage surrounding tissues (Taylor et al., 1994; van Sliedregt et al., 1994; Matsusue et al., 1995). Synthetic grafts often do not conform to the mechanical or material properties of tendons or ligaments (Hogan et al., 2015) and, when used in joint fissures, may give rise to stress risers and consumptive anisometry. Autograft surgery increases the surgical time and associated trauma (e.g., bleeding, risk of infection) due to the need for another surgery to restore self-tissue and is a cause of further trauma in the process (Chen et al., 2009; Perrone et al., 2017). Joint reconstruction using autograft or allograft surgery further results in a higher incidence and severity of early-onset osteoarthritis, thereby affecting the quality of life (Leiter et al., 2014; Smith et al., 2014; Perrone et al., 2017). The rate of acceleration of post-traumatic osteoarthritis is an important issue for veterans (Showery et al., 2016).

[0131] In the production of ideal and strong biomaterials for tendon and ligament repair sutures and absorbable sutures, unaddressed needs remain. In attempts to improve biocompatibility, reduce inflammation, and in particular reduce wear from strong synthetic materials for orthopedic indications, synthetic non-absorbable sutures with a collagen coating (e.g., Collagen Coat FiberWire®) have become available.

[0132] The extrusion of cross-linked fibers from type I collagen can result in strong products. However, these products are insufficient and exhibit biological drawbacks, strength drawbacks, and other drawbacks. For example, most cross-linking agents are cytotoxic, use strong chemical agents that are foreign to the body, and are not used in currently noted products that have been approved or sanctioned by the US Food and Drug Administration (FDA), making their use for clinical applications more difficult. In addition to their possible use in enhancing the repair of the ACL or AT, braided collagen fibers have shown to have high and uniform tensile properties, consistent and uniform diameters, biocompatibility, and adjustable absorption with regenerative ability, and thus have the potential to be used as sutures for general surgery, ophthalmic surgery, and plastic and cosmetic surgery.

[0133] Embodiments of the present disclosure relate to a novel microfluidic extrusion system for manufacturing microfibers of clinical type I collagen in filaments and in thin ribbon-like structures. Embodiments of the present disclosure meet stringent mechanical, biochemical, and cell compatibility and biocompatibility criteria, and impart properties particularly for biomedical applications to the fiber embodiments of the present disclosure. Embodiments of the present disclosure exhibit the order at the molecular scale from medium to large scale necessary to produce useful products, and thus these collagen fibers described herein have potential uses in tendon and ligament repair, wound closure, and other indications where advanced collagen suture-based biomaterials have the potential to be beneficial across the surgical fields in medicine.

[0134] FIG. 14 schematically shows the manufacture of the collagen microfibers described in embodiments of the present disclosure and the possible biomedical uses of suitable products. At step 1401, lyophilized collagen is dissolved in acid to obtain collagen molecules 1402. Extruded microfibers 1403 are twisted with a spinneret 1404 to form twisted microfibers 1405. This microfiber contains aggregated molecular collagen 1406. This collagen may be spooled at step 1407.

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

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

[0137] The bundles may be represented by the number of fibers within the bundle. For example, a bundle of 5 fibers may be called a penta fiber, 8 fibers will produce an octa fiber, and so on. To manufacture such bundles, systems and apparatuses with other numbers of nozzles or extruders may be used.

[0138] Figures 15 and 16 show a method for obtaining collagen fibers and a system capable of carrying out the reaction. In embodiments of the present disclosure, up to 2% (w / v) of clinical freeze-dried telocollagen (Telo) or atelocollagen (Atelo) (Collagen Solutions, California) or methacrylated collagen (Advanced BioMatrix, California) was stirred and dissolved overnight in an acid up to 0.05 M (most commonly acetic acid or hydrochloric acid). As shown in system 1500, the acidified collagen 1501 was then pumped through the center of the nozzle system. This system may include a coaxial conduit or needle 1503. A neutralizing alkaline-forming phosphate buffer containing salts (sodium chloride, disodium phosphate, monosodium phosphate, and N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) and PEG (polyethylene glycol) was pumped through the system of the outer portion of the coaxial conduit 1503 1501. The forming buffer flowed at a volumetric flow rate of about 5 to about 20 times, usually about 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 a bath 1505 of 20% aqueous ethanol. In addition to dehydrating the fibers, this bath served to remove residual forming buffer, thereby contributing to the 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, the acidified collagen fibers may be formed by extrusion before being placed in the forming bath. For example, the acidified collagen fibers may be formed by the use of a spinneret 1404. In some embodiments, multiple syringes of acidified collagen may be formed simultaneously.

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

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

[0142] The fibers are collected by a guide 4630 and introduced into a forming buffer bath 4640. The fibers are captured 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 thus the resulting fiber bundle is not twisted. However, when dehydrating the fibers and until the fibers are wound onto a collector, the bundle is subjected to tension by a tensoner to maintain the fiber bundle. Ordinarily, a grooved cylinder is a suitable collector, especially for wet fibers.

[0143] FIG. 16 shows details of the nozzle system 1600. A pump 1502 pumps acidified liquid collagen 1620 into the central needle of a biaxial needle 1503. A buffer solution, also called a sheath solution, is introduced from 1610 into the outer needle of the biaxial needle 1503 in the direction of flow arrow 1611, thereby forming collagen microfibers 1504 as the polymerization process proceeds. The collagen fluid is converged by the sheath fluid 1611 of the extensional flow. The detailed view of the needle shows how the acidified liquid collagen 1620 moves in the direction of the low-speed arrow and then increases in speed as shown by the high-speed arrow 1630 and the even higher-speed arrow 1640. Similarly, the sheath fluid moves in the directions of velocity arrows 1611, arrow 1635, and arrow 1645. The flow of the reactant proceeds in the direction of arrow 1650, where the shading shows how the buffer (sheath) fluid 1670 containing phosphate interacts with the collagen solution and removes water 1680 from the collagen stream.

[0144] After the microfibers 1507 were collected on a device 1508, they were air-dried for half an hour and subsequently crosslinked under various experimental conditions. The chemicals used during extrusion and crosslinking are included in Table 1 of FIG. 17.

[0145] In situ crosslinking (chemical or enzymatic crosslinking) of the groups shown in Table 2 of FIG. 18 was performed by dissolving the amounts of each crosslinking agent shown in FIG. 18 in the acidified collagen mixture for the time shown in FIG. 18. The concentrations and times for crosslinking of some materials were obtained from the specific references shown in FIG. 18. FIG. 18 summarizes the strength comparison. The italicized conditions were selected for property evaluation after optimization of the recovery method. Microfibers from the in situ crosslinked collagen were then extruded into the 2-bar device and stored under tension as shown in FIG. 15. FIG. 18 also shows that the crosslinking agent can be used in amounts from about 5 mM to about 500 mM, usually from about 10 mM to about 500 mM, more generally from about 25 mM to about 250 mM.

[0146] However, more generally, the uncrosslinked microfibers may be collected on a solid spool 1110 (see FIG. 11) having grooves at short intervals. The microfibers were collected directly into these grooves while under tension. Collection onto the spool is usually more efficient than the 2-bar device. The spool of uncrosslinked microfibers was chemically crosslinked in 70% aqueous ethanol as used for the 2-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 FIG. 12 to ensure uniform crosslinking of the microfibers.

[0147] For chemical crosslinking after extrusion, the uncrosslinked or in situ crosslinked collagen microfibers extruded into the 2-bar device 1508 or the grooved roller 1110 were air-dried for half an hour, then immersed in a 70% ethanol solution of the crosslinking agent 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, the collagen fibers are wet or moist at the time of collection. In such cases, the fibers may tend to stick to each other, especially if they can come into contact at the time of collection. Thus, in some embodiments, a two-bar or multi-bar collector device may advantageously be used because it can dry the fibers before they contact other fibers. In some embodiments, a grooved roller is particularly useful for collecting wet fibers because only one fiber is collected in each groove and contact between the fibers is prevented.

[0149] In some embodiments, the fibers may be dried by blowing a gas (usually air) onto the fibers after they leave the dehydration bath and before they are collected. The fibers are suspended between the dehydration bath and the 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 at room temperature over the fibers at a speed of 0.25 m / second to about 10 m / second, usually about 1 m / second to about 4 m / second, and more typically about 2 m / second, in a speed bag at room temperature. The speed of the drying air should not be so high as to break, separate, or cut the fibers. The air is passed for a time approximately equal to the time it takes to dry the fibers, usually the time it takes for the fibers to move about one meter. The drying air may be moved by a blower in an open system or a circulation system. In some embodiments, a collection device, such as a bobbin or a flat cylinder. The cylinder is rotated at a draw speed of about 1 to about 9 times the formation speed. In this environment, fibers of essentially infinite length can potentially be produced.

[0151] The dehydration heat treatment (DHT) of the crosslinked microfibers involves dehydrating the relaxed extruded microfibers under vacuum at 110 °C for 1, 3, and 5 days, regardless of the presence or absence of further crosslinking with glyoxal as described above.

[0152] For ultraviolet radiation (UVR)-mediated crosslinking, methacrylated collagen was used for extrusion. The extruded microfibers were then exposed to a 365 nm irradiation 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] Since a single microfiber is too delicate to handle consistently, the mechanical properties of single microfibers were obtained using an "individual fiber" test method that averages the cross-sectional area of individual microfibers on a cartridge and a known amount of microfibers to measure the ultimate tensile strength (UTS), modulus, and strain at break (%). The 2-bar recovery device 1508 resulted in cylindrical microfibers, but the microfibers recovered on a solid-grooved spool were in the form of thin ribbons. The width of the ribbon-shaped collagen fibers is from about 10 μm to about 70 μm, usually from about 15 μm to about 60 μm, and more generally from about 20 μm to about 50 μm. The thickness of the ribbon-shaped collagen fibers is from about 4 μm to 20 μm, usually from about 5 μm to about 18 μm, and most commonly from about 6 μm to about 17 μm.

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

[0155] A bath and sample holding system was used to perform a wet tensile strength mechanical test on the bundles within the cartridge. This system was capable of performing a 30-minute immersion while processing the samples every 5 minutes during the immersion of the extrusion microfiber embodiments of the present disclosure. The immersion fluid may be Gibco Dulbecco's phosphate buffered saline (DPBS) available from Thermo Fisher Scientific. Typically, at least four cartridges were mechanically wet tested at room temperature at a tensile rate of 1 mm / s using a uniaxial tensile test on an MTS reference model 42 (Eden Prairie, Minnesota) to obtain stress-strain curves. Individual fiber tests were performed while optimizing the processing parameters to obtain results quickly.

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

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

[0158] The UTS of the dry embodiments of the present disclosure is typically from about 25 MPa to about 1,900 MPa, usually from about 100 MPa to about 1,800 MPa, more generally from about 5000 MPa to about 1,700 MPa, and even more generally from about 1,200 MPa to about 1,700 MPa. The modulus of the dry embodiments of the present disclosure is from about 14,000 MPa to about 20,000 MPa, usually from about 15,000 MPa to about 19,000 MPa, and more generally from about 15,500 to 18,500 MPa.

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

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

[0161] For TEM, dry microfibers from the Telo GLY group (telocollagen crosslinked with glyoxal) were rehydrated using distilled water. These were then fixed in 2% glutaraldehyde (Electron Microscopy Sciences, Pennsylvania) and 4% paraformaldehyde (Alfa Aesar, Massachusetts) at room temperature for 30 minutes. Subsequently, washing was performed twice (10 minutes each wash) using cacodylate buffer (Electron Microscopy Sciences). Following this, incubation was carried out in 1% osmium tetroxide (Electron Microscopy Sciences) for 30 minutes, washed once with cacodylate buffer, and washed twice (10 minutes each) with distilled water. Dehydration was carried out with a series of ascending concentrations of ethanol (washed once for 10 minutes each at 30%, 50%, 70% and 95%, and twice for 10 minutes at 100%). The microfibers were then 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 1:1 EPON 812:propylene oxide (Electron Microscopy Sciences). EPON 812 is a glycerol-based aliphatic epoxy resin. The next day, the samples were immersed in 4:1 EPON 812:propylene oxide for 4 hours and 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 mold was sectioned thinly and imaging was performed using TEM (model number Jeol 1230, Jeol USA, Massachusetts). Alternative methods for measuring these values may be used.

[0162] The ninhydrin test may be used to evaluate the amount of free amino groups in the cross-linked microfibers. For this purpose, uncross-linked and cross-linked microfibers were each cut to a length of 14 - 16 cm. At the same time, 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 a ninhydrin solution (Sigma-Aldrich) for 20 minutes and then cooled to room temperature for at least 1.5 hours. Then, 95% ethanol was added to each of the samples and glycine standards. The absorbance of these samples was recorded at 570 nm using an ultraviolet-visible spectrophotometer (SpectraMax i3, Molecular Devices, Norfolk, Virginia, Old Dominion University). Other methods of testing may be used.

[0163] A calibration curve was created using the absorbances of various known glycine concentrations. The amount of free amino groups in the uncross-linked sample (Mux) and cross-linked (Mc) microfibers is proportional to the absorbance of the solution, and such amount was determined from the created glycine calibration curve. Equation 1 below was used to calculate the degree of cross-linking.

[0164] [Number] Differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to measure whether amide bonds characteristic of type I collagen were present. The microfiber tests were performed using a differential scanning calorimeter (DSC2500, TA Instruments, Delaware), and the FTIR spectroscopy was performed with Platinum ATR (Brucker, Billerica, Massachusetts). Using the FTIR spectrum, wavelengths of 1235 cm -1 , 1560 cm -1The presence of three major peaks of amide bonds characteristic of type I collagen at 1650 cm was confirmed. Uncrosslinked and crosslinked microfibers were compared to the starting material by evaluating peak shifts using Essential FTIR bioinformatics software (Operant, Madison, WI).

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

[0166] Sterilized glyoxal and DL-glyceraldehyde crosslinked microfibers were hydrated in tenocyte growth medium for 30 minutes and placed into 24-well plates pre-coated with poly(2-hydroxyethyl methacrylate) (pHEMA) (Sigma-Aldrich). Twenty-five thousand human tenocytes (ZenBio, Research Triangle Park, NC) in (100 μl of tenocyte growth medium) were seeded in triplicate onto the sterilized microfibers. After seeding, the cells were allowed to attach for 1 hour, followed by the addition of an additional 500 μl of tenocyte growth medium. After 12 days, tenocytes 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. The samples were then fixed using 4% paraformaldehyde and subsequently stained with the nuclear stain DAPI (Thermo Fisher Scientific), and tenocytes attached to the microfibers were visualized using a confocal microscope (Zeiss Axio Observer Z1, Zeiss).

[0167] The cytotoxicity (or cell viability) of the 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) according to the manufacturer's protocols. Briefly, after measuring the optimal seeding density for the assay, 7×10 3 tendon cells were plated in each well of a 48-well plate and grown for 24 hours in tendon cell growth medium in a humidified incubator maintained at 37°C and 5% CO 2 . The sterilized microfiber bundles were rinsed in cell culture medium for 10 minutes and placed on top of the tendon cells in each well. Tendon cells grown on plastic (cells only) were used as a positive control (for cell survival or viability). Zinc dibutyldithiocarbamate (ZDBC) film and 10 mM glyoxal solution were used as negative controls (for cell survival or viability). Ethicon Vicryl suture was used to tie off the extruded microfiber bundles, so the effect of Ethicon Vicryl suture was also evaluated in this experiment. As described in the manufacturer's protocol, wells seeded with tendon cells without samples were prepared to evaluate maximum and spontaneous LDH release. After incubating the samples for 7 days, the release of LDH into the medium was evaluated. The % cytotoxicity was calculated using the LDH assay according to the manufacturer's protocol. Then, the % cell survival was calculated as 100 - % cytotoxicity. In embodiments of the present disclosure, the % cell viability is at least about 94%, usually at least about 95%, more generally at least about 96%, and most generally at least about 97%. It is also possible to achieve 98% or 99% cell viability. The % cell viability was calculated using the MTT assay according to the manufacturer's protocol. In embodiments of the present disclosure, the % cell viability is at least about 70%, usually at least about 80%, more generally at least about 85%, and most generally at least about 90%. Other suitable test methods can be used.

[0168] The health and viability of tendon cells grown with the extrusion microfiber embodiments of the present disclosure were also evaluated using the AlamarBlue™ assay (Bio-Rad, Hercules, CA) according to the manufacturer's protocol.

[0169] Crosslinked microfiber bundle embodiments were implanted subcutaneously in rats. All surgeries were performed according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the facilities of Old Dominion University (Norfolk, VA) in accordance with ISO 10993-6. At n = 3, crosslinked collagen microfiber bundles (prepared and sterilized as described above) or collagen-coated FiberWire® (suture control) were implanted subcutaneously in female Sprague-Dawley rats. The rats were anesthetized by isoflurane inhalation. The hair on the flanks was shaved and Nair hair removal cream was applied to remove hair from the surgical site. A dorsal incision was made at the flank site, and hemostatic forceps were used to create a pocket for the implant. After placing a scaffold in the pocket, the incision was closed using sutures. Four weeks later, the rats were humanely euthanized for tissue harvesting.

[0170] Microfiber explants removed at week 4 were fixed with 4% paraformaldehyde (Alpha Aesar) 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 from IDEXX (West Sacramento). Polarization microscopy was used to image the collagen composition of the tissue around the implant.

[0171] Using the standard protocol provided by the antibody manufacturer, we also performed immunolabeling on serial sections to detect the presence of CCR7 (M1) and the phenotype of CD163 (M2) macrophages in the native tissue surrounding our implants. Briefly, after deparaffinization, antigen retrieval (boiling in 10 mM citrate buffer, pH 6 for 20 minutes), permeabilization, and blocking with 2.5% horse serum, the slides were stained for either CD163 (M2 macrophage phenotype) or CCR7 (M1 macrophage phenotype). Mouse anti-rat CD163 (#MCA342GA, BioRad, California), an M2 macrophage marker, was diluted 1:30 and incubated overnight in a humid chamber. After incubation, the slides were washed with PBS and incubated for 1 hour at room temperature in the dark with a 1:50 dilution of goat anti-mouse secondary antibody (#A-11005, Thermo Fisher Scientific). CCR7, an M1 macrophage marker, was diluted 1:50 in PBS and incubated overnight (#MA5-31992, Thermo Fisher Scientific). The next day, after the PBS wash step (3 times), a 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 the primary control, slides blocked with serum 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 the secondary control, slides blocked with serum were stained with the secondary fluorescent antibody only. All antibodies were diluted in blocking serum. All slides were stained with DAPI for 5 minutes to stain the nuclei, washed with PBS, mounted using VectaMount (Vector Labs, California), and visualized and analyzed.

[0172] The immunolabeled slides were examined and imaged using an inverted microscope (Axio Vert. A1 model, Zeiss). Fluorescent images of the test slides and control slides (data not shown) were acquired under the same exposure conditions. The images of the test samples were evaluated. Quantitative analysis was performed to determine the number of cells expressing only M1, only M2, M1 and M2, and / or without the M1 / M2 phenotype. Here, for approximately 20 - 30 μm images (3 images were analyzed for each test sample) at the interface between the implant and the native tissue (2 - 3 cell layers), 4 - 5 regions were analyzed using a high-power microscope field (40× magnification). The total number of cells was measured by counting the DAPI-stained nuclei. The number of cells positively labeled for each marker was also counted. The percentage of cells labeled with the specific marker was measured as a percentage of the total number of cells in that region.

[0173] For the embodiments of the present disclosure, long-term stability tests were also conducted. The Telo GLY microfibers were unspooled by applying tension to the cartridge. Six cartridges sterilized as described above were hydrated and subjected to mechanical tests to obtain the mechanical properties of the microfibers, and then placed in an incubator maintained at 37°C and 5% CO 2 To inhibit bacterial and fungal contamination in the incubator, the remaining parts of the sterilized cartridges were incubated in Petri dishes containing Eagle's minimum essential medium (EMEM) (ATCC, Virginia) supplemented with 1% Gibco® antibiotic-antifungal (ABAM) (Thermo Fisher Scientific) to maintain hydration. Throughout the experiment, it was confirmed that the cartridges were always immersed in a sterile and uncontaminated medium, thereby maintaining hydration. At 1 week, 1 month, 3 months, and 6 months, the six immersed cartridges were taken out and subjected to an MTS test. At the same time, the diameter of the microfibers was measured (as described above) to measure the degree of swelling of the microfibers over time.

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

[0175] Additional Examples Examples were obtained by performing embodiments of the products and methods of the present disclosure. To consistently generate collagen microfibers for subsequent testing, the robust microfluidic extrusion device of Figures 15 and 16 was designed and used. This approach resulted in the generation of continuous microfibers without crosslinking abnormalities.

[0176] A wide range of conventional, novel, and combined crosslinking conditions for strengthening and stabilizing collagen microfibers were screened. Table 2 of Figure 18 shows a list of crosslinkers and the average UTS of 50 types of crosslinked microfibers compared to uncrosslinked microfibers using the aforementioned test methods. This data showed that various crosslinker / crosslinking protocols (in situ or post-extrusion crosslinking, range of crosslinker concentrations, and crosslinking times) affected the UTS of the microfibers to some extent. Crosslinking conditions that showed significantly high average UTS among all conditions tested with those crosslinkers were marked with a star in Table 2 of Figure 18 (p < 0.01).

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

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

[0179] Next, referring to FIGS. 19, 20, and 21, the mechanical properties of typical microfibers from the crosslinker groups tested in Table 2 of FIG. 18 were compared with 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). FIG. 19 shows, in graph 1900, the UTS (MPA). The graph 2000 in FIG. 20 summarizes the modulus in MPa, and the graph 2100 in FIG. 21 relates to the strain % at break. The ACL values are shown by lines 1930, 2030, and 2130. The AT values are shown by lines 1910, 2010, and 2110, and the dermis values are shown by lines 1920, 2020, and 2120. Each result relates to a single microfiber. The results shown in FIG. 19 revealed that the average UTS of collagen microfibers of several of the crosslinking groups, particularly 10 mM glyoxal with and without 10 mM L-lysine in situ and 25 mM DL-glyceraldehyde, was above the UTS reported for human ACL, AT, and dermis.

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

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

[0182] Example 3 is telocollagen (Telo GLY) cross-linked with 10 mM glyoxal for 72 hours. Example 4 is telocollagen (Telo DLG) cross-linked with 25 mM DL-glyceraldehyde for 24 hours. Example 5 is atelocollagen (Atelo GLY) cross-linked with 10 mM glyoxal for 24 hours. Example 6 is atelocollagen (Atelo DLG) cross-linked with 25 mM DL-glyceraldehyde for 72 hours. Comparative Example 1 is telocollagen (Telo EDC) cross-linked with 0.25 mM EDC for 24 hours. These groups were compared with uncrosslinked microfibers (Comparative Example 2) and dry 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 draw recovery (high 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 FIG. 22.

[0183] FIG. 22 shows an image of the Telo GLY microfibers of Example 3 depicting the microstructural features. Frame A is an optical microscope image of a single dry extruded cross-linked microfiber. Frames B and C are SEM images of a single dry microfiber at various magnifications. Frame D shows a cross-section of the bundled microfibers immersed in PBS for 30 minutes. Frame D reveals the structural details and evidence that the extrusion followed by the cross-linking strategy described herein using the novel microfluidic device embodiments shown in FIGS. 15 and 16 produced a consistent and 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 microfibers.

[0184] The optimization of the crosslinking chemistry and the change in the recovery method led to significant differences in the mechanical properties, as summarized in FIGS. 23, 24, 25, 26, 27, and 28. Tensile tests were performed in the aforementioned bath and sample holding system. In frames A, B, and C of FIG. 22, using the width and thickness of the ribbon-shaped collagen microfibers immersed in DPBS of FIGS. 23 and 24, measured from a typical image as shown in Example 3, the improved UTS of graph 2500 in FIG. 25 and the modulus of graph 2600 in FIG. 26 were calculated. Examples 3, 4, 5, and 6 were tested in the same manner as Comparative Examples 1, 2, and 3.

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

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

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

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

[0189] For the embodiments of the present disclosure, the microstructure of the microfibers was measured using optical microscopy, SEM, and TEM imaging. Other types of imaging may also be used. In Example 3, the glyoxal-crosslinked telocollagen microfibers were characterized. Optical microscopy imaging shown in Frame A of FIG. 22 and SEM imaging in Frame B of FIG. 22 confirmed the uniform width of the dried microfibers along the longitudinal axis. Imaging of the longitudinal fragment in Frame B of FIG. 22 and high-magnification SEM (Frame C of FIG. 22) revealed the parallel orientation of the ridges and gaps within the dried microfibers, as shown in Frame D of FIG. 22. Frame D of FIG. 22 highlights the SEM-characterized cross-section of the extruded crosslinked microfiber bundle immersed in DPBS. These images reveal the microstructural features of the smooth external surface with a distinct fibrous subfiber structure, as indicated by arrow 2206. This indicates that the extruded crosslinked microfibers are in the form of consistent thin ribbons. Further evidence that the collagen orientation from the molecular to the nanoscale in native connective tissue is reproduced in our crosslinked microfibers is revealed by the TEM imaging in Frames E, F, and G of FIG. 22.

[0190] To biochemically evaluate the degree of crosslinking, the ninhydrin test, a biochemical and biophysical characterization of the crosslinked microfiber embodiments, was used. The results are shown in graph 2901 of FIG. 29. The Telo GLY (86 ± 1%) and Atelo DLG (82 ± 3%) microfibers of Example 3 showed a significantly higher degree of crosslinking compared to Atelo GLY (68 ± 4%) of Example 5 and Telo DLG (59 ± 6%) of Example 4, highlighting that longer crosslinking times 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, since the microfibers could not be dissolved in acid, collagen in the acid extract of the microfibers could not be detected.

[0192] The biophysical characterization of the extruded microfibers using differential scanning calorimetry (DSC) measurements revealed a non-significant increase in the melting temperature between the uncrosslinked and crosslinked microfiber groups, 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 ) 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, display 2910 indicates p < 0.05.

[0193] The cell attachment, metabolic activity, and cytotoxicity of the extruded microfibers of the embodiments of the present disclosure were also measured, as shown in illustrations 3001 and 3002 of illustration 3000 in Figure 30 and in Figures 31, 32, and 33. These figures include Examples 3 to 6, Comparative Example 2, and other samples. Using human tendon cells, the cell compatibility of the collagen fibers was evaluated as described above. The attachment of tendon cells to the elongated Telo GLY microfibers (Example 5) is shown in illustration 3001 of FIG. 30. Approximately 70% of the tendon cells seeded on the Telo GLY microfibers remained attached after 12 days. As summarized in FIG. 31 for Examples 3 - 6, Comparative Example 1, and other samples, no significant change in the metabolic activity of tendon cells was observed by AlamarBlue fluorescence for more than 7 days compared to the positive control (the cell-only group). However, the metabolic activity of the cells growing with the microfibers from the selected fiber groups was significantly higher (p < 0.05) than the metabolic activity of the negative control (10 mM glyoxal chemical agent and ZDBC film). As shown in FIG. 32, when assayed using the MTT reagent, the viability of tendon cells incubated with the microfibers was 75% - 85% compared to tendon cells grown on plastic (100%). The negative control (10 mM GLY chemical agent and ZDBC film) showed significantly lower (p < 0.005) tendon cell viability than the "cells only", Telo DLG (Example 4), Atelo GLY (Example 5), and Telo GLY (Example 3) groups. Similar results were observed when using the LDH assay (FIG. 33), except that all extrusion microfiber groups except Atelo DLG and Telo DLG induced tendon cell viability similar to the "cells only" group. As shown in display 3303 of FIG. 33, at the end of 7 days, there were no tendon cells (ND) sufficient to test for LDH release into the medium in the 10 mM GLY chemical agent group. It was compared to a commercially available coated Ethicon Vicryl suture commonly recommended for wound closure. The microfiber embodiments of the present disclosure were shown to have significantly lower cytotoxicity (p < 0.005) than this suture when using both the LDH and MTT assays (FIGS. 32 and 32). Overall, multiple assays were used to demonstrate the biocompatibility of the extrusion microfibers.

[0194] In particular, Images 3001 and 3002 show typical confocal images of human tendon cells attached to Telo GLY microfibers (Example 3) showing cytoplasmic elongation and elongated nuclei, using DAPI (arrow 3005) and a live cell stain (CMFDA, indicated by arrow 3003), respectively. Figure 31 shows that there is no significant change in the metabolic activity of human tendon cells incubated with cross-linked microfibers for 7 days, assayed using AlamarBlue, compared to the cell-only group. The metabolic activity was significantly lower in tendon cells incubated with the negative control (ZDBC film and 10 mM GLY agent) and Vicryl suture compared to the microfiber group. The MTT assay results summarized in Figure 32 revealed that the viability of tendon cells incubated with the microfiber group decreased compared to the cell-only group, but showed a significant increase compared to the negative control. On the other hand, the LDH assay results shown in Figure 33 show a significant decrease in cell survival in the negative control as well as the Atelo DLG (Example 6) and Telo DLG (Example 4) microfiber groups. Both the MTT and LDH assays were performed 7 days after incubation with tendon cells. All data in Figures 32 and 33 are normalized to the cell-only group. (ND) in Display 3303 indicates that the 10 mM glyoxal agent-treated group had significant growth inhibition with insufficient cell numbers to detect LDH at the end of the assay. In these figures, Display 3101 and Display 3301 indicate p < 0.05, Display 3202 indicates p < 0.01, Display 3103 and Display 3203 reveal p < 0.005, and Display 3204 and Display 3304 reveal p < 0.0001).

[0195] To evaluate the biocompatibility of the embodiments of the extruded microfibers of the present disclosure, sterilized microfiber bundles of four selected crosslinker groups of Examples 3 to 6 (Atelo DLG, Telo DLG, Telo GLY, and Atelo GLY) were subcutaneously implanted into rats according to ISO 10993-6. The microfiber bundles transplanted from each of the four crosslinker groups in FIGS. 23, 24, 25, 26, 27, and 28, and the suture control (collagen-coated FiberWire®) group induced distinct host tissue reactions characterized by varying degrees of cell infiltration, neovascularization, collagen deposition, and tissue remodeling, as shown in FIGS. 34, 35, and 36. Among them, the glyoxal-crosslinked microfiber group (Telo(GLY) (Example 3) or Atelo(GLY) (Example 5)) showed a weaker inflammation-inducing reaction compared to the DL-glyceraldehyde (Telo DLG (Example 4) or Atelo DLG (Example 6)) crosslinked group. The typical HE staining images of the Telo GLY (Example 3) group shown in the transverse image 3401 and longitudinal image 3402 of FIG. 34 showed significantly higher cell infiltration compared to the suture control shown in FIG. 36, including the transverse image 3601 and longitudinal image 3602. The suture control in FIG. 36 induced a strong inflammatory reaction at the 4th week compared to the microfiber implant.

[0196] In the image 3501 of FIG. 35, the deposition of newly formed collagen in the native tissue around the Telo(GLY) (Example 3) microfiber implant was visualized by Masson's trichrome staining. The longitudinally sectioned Masson's trichrome-stained fragment is shown in the image 3502 of FIG. 35, and the polarized imaging of the image 3602 in FIG. 36 shows the deposition of tissue-freshly formed collagen around the microfibers.

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

[0198] Figures 34, 35, and 36 are typical images of subcutaneous implants in rats of the Telo GLY (Example 3) group at week 4. Images 3401 and 3501 show microfiber m identified by arrow 3410 on stained slides showing transverse sections of HE of FIG. 34 and Masson's trichrome of FIG. 35. Inserted image 3490 shows the entire fragment of the HE implant, and inserted image 3495 shows the portion shown in image 3401. Similarly, inserted image 3590 shows the entire fragment of the Masson's trichrome implant, and inserted image 3595 shows the portion shown in image 3501. Both images 3401 and 3501 show significant cell infiltration. Arrow 3420 in FIG. 34 indicates a blood vessel within the implant. Image 3601 shows negligible cell infiltration in the collagen-coated FiberWire® control sample using HE staining. Image 3402 shows HE, and image 35032 shows Masson's trichrome staining of a longitudinal section of the microfiber implant. Polarized image 3602 in FIG. 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 native tissue around the implant. The legend "suture control" in image 3601 indicates the collagen-coated FiberWire®.

[0199] Using immunostaining, the degree of macrophage polarization in the native tissue surrounding the microfiber implants from four crosslinker groups was measured. Figures 37 and 38 are representative immunofluorescence images showing the expression patterns of the CCR7 (M1) (image 3700 in Figure 37) and CD163 (M2) (image 3800 in Figure 38) macrophage phenotypes in the native rat tissue surrounding the 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 the graph 3900 of Figure 39, the glyoxal crosslinker groups (Telo(GLY) (Example 3) and Atelo(GLY) (Example 5)) showed a significantly higher percentage (about 40%) of macrophages expressing the M1 and M2 phenotypes compared to the DL-glyceraldehyde crosslinker 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 the M2-only phenotype (6%), while the rest of the groups showed negligible M2-only phenotypes (Atelo GLY (0.2%), Telo DLG (0%), and Atelo DLG (0%) (Figure 39)). The percentage of cells with the M1 phenotype was significantly higher in the DL-glyceraldehyde crosslinker groups (Telo DLG (64%) and Atelo DLG (58%)) compared to the glyoxal crosslinker groups (Telo GLY (24%) and Atelo GLY (19%)). As described above, appropriate control staining showed negligible non-specific background staining (not shown). Sectioning artifacts of the suture control samples and significant background staining made it difficult to perform this analysis on these samples.

[0200] Typical immunofluorescence images 3700 and immunofluorescence image 3800 show an example of the host macrophage response at the fourth week to the microfibers Telo(GLY) (Example 3) indicated by arrow 3840. Yellow arrows 3710 and yellow arrow 3810 show examples of cells expressing both M1 and M2. Orange arrows 3720 and orange arrow 3820 show examples of cells expressing only M1. White arrows 3730 and white arrow 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, only M1, only M2, or no M1 / M2 phenotype for four groups of crosslinked microfibers. The results from this analysis show the initiation of the regenerative promoting M2 macrophage phenotype in all microfiber groups tested. The fiber group crosslinked with glyoxal showed a higher percentage 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 M2-only macrophages. In graph 3900, display 3901 reveals that p < 0.05, display 3902 indicates that p < 0.01, and display 3903 shows that p < 0.005).

[0201] The influence of the long-term hydration reaction of the microfiber embodiments of the present disclosure on the mechanical properties and degree of swelling in a medium was measured. Since the Telo(GLY) (Example 3) microfibers exhibited optimal mechanical properties, cell compatibility, 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 an increase in the microfiber width of 53% at 6 months (from 36.4 ± 1.1 μm (day 0) to 56.0 ± 1.6 μm (6 months)), as shown by graph 4000 in FIG. 40. This graph shows the degree of swelling of the wet microfibers over time. This swelling is accompanied by a significant decrease in mechanical properties. Graph 4100 in FIG. 41 shows that the average force at break decreased by 54% from its initial value at 6 months. The average UTS (graph 4200 in FIG. 42) and modulus (graph 4300 in FIG. 43) also decreased by 82% from the starting point at 6 months. There was no significant change in the elongation at break (%) between day 0 and 6 months of incubation (graph 4400 in FIG. 44).

[0202] Thus, FIGS. 40, 41, 42, 43, and 44 show that the Telo GLY microfibers are stable and do not dissolve until 6 months when incubated in a humidified incubator maintained at 37 °C, 5% CO 2 in a sterile cell culture medium under conditions mimicking the biological environment in vitro.

[0203] As can be seen from these figures, in a humidified incubator at 37 °C, 5% CO 2Evaluated at 1 week, 1 month, 3 months, and 6 months, the mechanical stability of Telo GLY microfibers incubated under tension in sterile EMEM was such that, at the end of 6 months compared to day 0, the Telo GLY microfibers swelled 50% (Figure 40), the breaking strength decreased by 60% (Figure 41), the UTS decreased by 80% (Figure 42), and the modulus decreased by 80% (Figure 43). However, at the end of 6 months, there was no significant change in the strain at break (%) (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 only to serve as a guide to the reader. Data are shown as mean ± standard error, which represents at least 5 replicate tests.

[0204] SDS-PAGE was used to compare the collagen starting materials (lyophilized telocollagen or atelocollagen) with the uncrosslinked and crosslinked microfibers. The collagen starting materials readily dissolved in 50 mM HCl after overnight stirring. However, the extruded microfibers did not go into solution at a concentration of 0.5 mg / ml and thus did not show bands. To confirm the presence or absence of collagen in the acid extracts of the microfibers, these extracts were electrophoresed on a gradient gel (3% - 8%) (Invitrogen) together with solutions of the starting materials and a prestained molecular weight marker (HiMark, Invitrogen, California). The gels were stained with SimplyBlue™ (Invitrogen, California) and then rinsed with deionized water to destain them. The gels were then projected under white light and all visible protein bands were examined. Thus, SDS-PAGE has revealed that the extruded fibers from the group with the maximum UTS and uncrosslinked fibers showed resistance to acid hydrolysis compared to the acidified starting materials that showed characteristic bands in the monomer region at approximately 115 kDa, the dimer region at approximately 230 kDa, and the trimer region at approximately 460 kDa of type I collagen fingerprint.

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

[0206] In summary, the present disclosure relates to a novel microfluidic extrusion process for manufacturing type I collagen microfibers having accuracy, consistency, and scalability as biocompatible fibers for use in indications ranging from natural sutures to engineered connective tissues. The present disclosure demonstrates that embodiments of the biofabricated glyoxal cross-linked telocollagen microfibers of the present disclosure exhibit superior dry and wet tensile properties compared to conventional cross-linked collagen extruded microfibers (Paul and Bailey, 2003; Caruso and Dunn, 2004; Zeugolis, Paul, and Attenburrow, 2009; Enea et al., 2011).

[0207] Many prior studies have not reported whether tensile testing was performed on hydrated fibers, or have presented equivocal and confusing results for dry fibers, or have not disclosed how the fibers were hydrated if they were fully hydrated, but the results of the embodiments of the present disclosure herein provide the dry and hydration properties of optimized cross-linked fibers, along with important detailed test methods for comparison and for the growth of this field.

[0208] The retrieval of fibers onto 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 the tempering, thinning, and improved molecular orientation of the ribbons into fibers that result in fiber tensile properties stronger than those of the ACL, Achilles tendon, dermis, or any other soft connective tissue.

[0209] In embodiments of the present disclosure, measuring the degree of efficiency of the crosslinking mechanism is emphasized. Insufficient crosslinking may result in lower tensile strength, but overuse of the crosslinking agent of the chemical agent may cause cytotoxicity and may result in residues of the crosslinking agent on the surface of the microfibers. The ninhydrin test (Figure 36) revealed that the groups with the maximum degree of crosslinking were the 72-hour crosslinking groups (Telo GLY and Atelo DLG), which was also associated with a significant increase in tensile strength. The chemical structure of crosslinking with aldehydes involves the formation of Schiff base-type compounds with functional amino groups in collagen, which results in strong molecular bonds (Fathima et al., 2004).

[0210] Chemical analysis of embodiments of the extruded microfibers revealed that they were more resistant to acid hydrolysis. The microfluidics devices or apparatuses disclosed herein produced microfibers with higher chemical stability than the lyophilized starting materials, which suggests a close packing of collagen molecules within the microfibers that results in a stable higher-order structure and also suggests a low internal moisture content. Such higher-order structures have been reported in native connective tissues (Benjamin, Kaiser and Milz, 2008; Wang, Guo and Li, 2012). The integrity of the secondary structure in the extruded microfibers was confirmed from the FTIR analysis shown in Figure 29, which also suggests that neither the extrusion process nor the crosslinking technique denatured the collagen.

[0211] Crosslinking of collagen in biomimetics may help improve tensile properties, but the degradation of chemical agents used for crosslinking (such as glutaraldehyde) may exhibit toxicity (Gough, Scotchford and Downes, 2002; Umashankar, Kumari and Mohanan, 2012). Among other chemical agents that show slightly lower cytotoxicity, EDC or EDC / NHS as crosslinking agents are common basic research options for collagen microfibers (Enea et al., 2011; Ahmad et al., 2015; Shepherd et al., 2015). However, little improvement in tensile strength was observed, and due to the well-known toxic effects of these classical crosslinking agents, they were hardly suitable for use in connective tissue repair. In this study, we developed highly cell-compatible (see Figures 30, 31, 32 and 33) and in vivo biocompatible (as shown in Figures 34, 35 and 36) mechanically excellent extruded collagen microfibers chemically crosslinked with either glyoxal or glyceraldehyde in accordance with the standard ISO 10993 tests generally required for USFDA approval. Furthermore, collagen microfibers crosslinked with glyoxal showed resistance to acid hydrolysis, revealed microstructural features down to nearly the molecular level, maintained stability in cell culture medium for at least 6 months, and the ability of single microfibers maintained approximately 30% to about 50%, usually about 40% of the initial load-bearing capacity and maintained a UTS greater than that of native ACL (see Figures 40, 41, 42, 43 and 44).

[0212] The enhancement of suture repair of the ACL or Achilles tendon using the collagen-based microfibers or collagen-based braided sutures described herein for wound healing requires a collagen-based material that not only mechanically maintains the tissue but also promotes tissue remodeling at a reasonable rate (Dunn, Avasarala and Zawadsky, 1993). In vitro and / or in vivo biocompatibility studies demonstrating the effects of these chemically cross-linked microfibers on cytotoxicity, inflammatory response and regenerative response are important. Embodiments of the extruded microfiber bundles of the present disclosure are cell-compatible and showed minimal toxicity to human tendon cells. The microfluidic extruded microfibers of the present disclosure further support the attachment of human tendon cells and are expected to have an elongated shape as seen in connective tissue (Benjamin, 2010). Biocompatibility has been 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-telopeptide collagen group showed the initiation of a regenerative-promoting response. Furthermore, long-term stability data and rat tissue images showed the stability of the microfibers up to at least 6 months in vitro and up to at least 4 weeks in vivo. Thus, embodiments of the present disclosure can maintain strength for at least about 1 month in vivo, at least about 3 months in vitro, and up to about 6 months at most.

[0213] Macrophages are a heterogeneous population of mononuclear cells that are activated in the host, for example, as a response to tissue injury during material implantation (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 the host's potential to overcome inflammatory signals and transition towards tissue repair and remodeling in response to a surgical implant. Macrophage phenotypes are widely characterized as M1 (or "classical" activation) with inflammatory signals and M2 (or "alternative" activation) with immunomodulatory or tissue remodeling properties (Mills et al., 2000). However, it is important to note that activated macrophages have the plasticity to easily switch phenotypes from M1 to M2 and from M2 to M1. This plasticity is induced by changes in the local microenvironment (Porcheray et al., 2005; Stout et al., 2005). As a result, macrophages can also adopt migratory properties of both M1 and M2 phenotypes (Brown and Badylak, 2013). In embodiments of the present disclosure, the proportion of cells exhibiting M1, M1 and M2, or M2 phenotypes was measured. These measurements suggested the following. (1) At 4 weeks post-implantation, the glyoxal cross-linked group had more cells with M1 and M2 or M2-only phenotypes, suggesting that the tissue remodeling response was initiated by the host at 4 weeks. This suggests that the microfibers from the glyoxal group are mostly biocompatible. To our knowledge, such a detailed analysis of the immune response has not been done using cross-linked collagen microfibers.

[0214] The incorporation of collagen into sutures for wound healing has been a challenge. However, the present disclosure provides methods and apparatus for the manufacture of effective products. As a control for the studies herein, a collagen-coated FiberWire® non-absorbable suture (the only collagen-based synthetic suture available on the market) was used. This FiberWire® showed limited cellular infiltration with little ingrowth or regeneration of native tissue around the implant. In contrast, embodiments of the glyoxal-crosslinked collagen microfibers of the present disclosure in the form of suture-like bundles showed significant cellular infiltration with newly formed collagen in the surrounding tissue, which suggests regenerative healing.

[0215] Embodiments of the present disclosure demonstrate that microfluidic extrusion of clinically quality type I collagen fibers crosslinked with glyoxal exhibits exemplary tensile strength, structural stability, cell compatibility, and biocompatibility that exceed those of pure collagen produced by other reported biomanufacturing processes. The use of glyoxal to stabilize the collagen fibers provides a clinically meaningful, safe, and effective method for the biomanufacture of additional collagen microfibers. These optimized collagen microfibers can be readily processed into a variety of biomedical applications, including surgical sutures, internal fixtures for ligaments, ligaments made in tissue engineering, tendons, and other strong fibrous tissues, designed to significantly improve human health.

[0216] Example 7 A collagen solution and a forming buffer were prepared. A sufficient amount of clinical freeze-dried atelocollagen (Symatese, France) to create a solution with a concentration of 1.6% (w / v) was dissolved in 0.05 M acetic acid in a polypropylene sealed container. This solution was stirred at 180 rpm overnight at room temperature. To ensure uniform mixing, the total volume of the solution was kept less than half of the container's capacity. The next day, the acidified collagen mixture was spin-down in a centrifuge at 730 g for 5 minutes. The solution was degassed for 2 minutes and then spin-down at 730 g for 10 minutes to remove air bubbles. The obtained acidified atelocollagen was aspirated into eight 20 mL syringes (Hsw® Norm-Ject® sterile Luer-lock syringes, VWR) and used directly in the high-power collagen microfiber extrusion device shown in Figure 46.

[0217] To prepare the forming 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 MilliQ water. This mixture was placed in a glass beaker on a stirring plate and stirred at 400 rpm overnight at room temperature. The next day, the pH of this solution was adjusted to 8 by adding 10 M sodium hydroxide (Sigma-Aldrich), and then the solution was filtered using a 0.45 μm filter.

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

[0219] Figure 46 shows a part of system 4600 where acidified atelocollagen is processed. A syringe array pump was attached to cooperate with a rotatable plate 4601 and all eight syringes. Fiber bundle samples were produced with and without twisting. The fiber bundles moved through a forming bath and became strong when the buffer solution neutralized the acid to form fibrils. Then, the twisted and untwisted bundles were placed in a 20% aqueous ethanol dehydration bath, where water was removed to further strengthen the fibers. A tensioning rig applied a constant tension to the fiber bundles until the bundles adhered to a grooved spool (not shown) at the end of the bath. The tension in the fibers helped to stretch and build the collagen for strength and stability. Then, the spooled collagen was dried, cross-linked with glyoxal, and used to create a 3D graft.

[0220] For post-extrusion chemical cross-linking, the uncross-linked pins and the tensioned 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 cross-linking agent in a large acrylic tube, and then placed on a rocker at 1 rpm. The aqueous ethanol medium ensured that the microfibers remained dehydrated throughout the cross-linking time. After cross-linking, the microfibers were stored in a desiccator until further testing was performed.

[0221] The chemical cross-linking agent used was glyoxal, a dialdehyde at a concentration of 10 mM. The chemical structure of cross-linking with 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 a single fiber bundle 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 the fiber bundle 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, each 1.5 inches in length, 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 levels for peak load (Figure 53) and UTS (Figure 55) were p < 0.01( ** ), and the significance levels for modulus (Figure 54) and UTS (Figure 55) were 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 otherwise be clearly identified. 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 otherwise be clearly identified. 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] Although various embodiments of the present invention have been described, the description is for illustrative purposes only and is not limiting. It will be apparent to those skilled in the art that many other embodiments and implementations within the scope of the present invention are possible. Therefore, the present invention is limited only by the appended claims and their equivalents. Also, various modifications and changes can be made within the scope of the appended claims. The spirit and scope of the present invention lie within the appended claims, but also potentially within the following [Provisional Claims], which existed as claims at the time of filing of this application and a part of which has been deleted by amendment. The matters described in this [Provisional Claims] are considered to be included in the disclosure of this specification. [Provisional Claims] [Provisional Claim 1] A biopolymer fiber containing collagen, having the following characteristics A tensile strength of about 1 MPa to about 1,700 MPa, A modulus of elasticity of about 10 MPa to about 20,000 MPa, An elongation at break of about 2 percent to about 45 percent, An average fiber diameter of about 10 μm to about 90 μm having one or more of A fiber that maintains its strength and exhibits a regular longitudinal orientation structure after being immersed in DPBS at room temperature for at least about 1 hour. [Provisional Claim 2] The tensile strength is about 1 MPa to about 800 MPa, The modulus of elasticity is about 10 MPa to about 7,500 MPa, The average fiber diameter is about 10 μm to about 30 μm, The biopolymer fiber according to Provisional Claim 1. [Provisional Claim 3] The tensile strength is about 25 MPa to about 1,700 MPa, The modulus of elasticity is about 15,000 MPa to about 29,000 MPa, An elongation at break of about 7 percent to about 20 percent, The biopolymer fiber according to preliminary claim 1. [Preliminary claim 4] The biopolymer fiber according to preliminary claim 1, wherein the collagen comprises clinical collagen, atelocollagen, telocollagen, recombinant collagen, or a blend thereof. [Preliminary claim 5] The biopolymer fiber according to preliminary claim 1, wherein the collagen further comprises one or more biocompatible polymers. [Preliminary claim 6] The biopolymer fiber according to preliminary claim 1, which further retains a strength of more than about 60 MPa after 6 months at room temperature in DBPS or after transplantation into a subject. [Preliminary claim 7] The biopolymer fiber according to preliminary claim 1, which is crosslinked by a crosslinking agent comprising glyoxal, DL-glyceraldehyde, or a combination thereof. [Preliminary claim 8] The biopolymer fiber according to preliminary claim 1, which further comprises attached tendon cells, and the tendon cells maintain at least about 75% cell viability and at least about 95% cell survival after incubation for about 7 days at the temperature, pH, and humidity of conventional mammalian cell culture conditions. [Preliminary claim 9] The biopolymer fiber according to preliminary claim 1, which has a cross-section that is substantially circular, oval, square, rectangular, ribbon-shaped, triangular, or irregular in shape. [Preliminary claim 10] A bundle of biopolymer fibers according to preliminary claim 1, comprising from 2 to about 10,000 fibers. [Preliminary claim 11] A transplantable biopolymer scaffold for assisting in the repair of soft tissue damage, comprising the biopolymer fiber according to any one of preliminary claims 1 to 9 or a bundle of biopolymer fibers according to preliminary claim 10. [Preliminary claim 12] A woven sheet-like support, patch, or appliance comprising the biopolymer fiber according to any one of preliminary claims 1 to 9. [Preparatory Claim 13] Dissolving collagen in an acid solution to prepare a collagen solution, while sending the collagen solution at a first rate through a first needle having a first diameter, coaxially surrounding the first needle with a second needle having a second diameter greater than the first diameter and forming a sheath around the collagen solution, and sending a forming buffer at a second rate through the second needle to form a coaxial flow, wherein the second flow rate of the forming buffer through the second needle is at least twice the first flow rate of the collagen solution through the first needle, the step; Sending the coaxial flowing collagen and forming buffer through a reaction zone including a fibril forming bath at a time and rate sufficient to form fibers, Dehydrating the collagen fibers at a certain extrusion rate, Taking up the fibers onto a spool at a third rate greater than the extrusion rate sufficient to enhance molecular orientation and reduce the diameter of the fibers, a method for producing a biopolymer fiber. [Preparatory Claim 14] Dissolving collagen in an acid solution to prepare a collagen solution, Sending the collagen solution into a forming buffer at a first rate through a first needle having a first diameter, Sending the collagen and forming buffer through a reaction zone including a fibril forming bath at a time and rate sufficient to form fibers, Dehydrating the collagen fibers at a certain extrusion rate, Taking up the fibers onto a spool at a rate from about twice the extrusion rate to about ten times the extrusion rate sufficient to enhance molecular orientation and reduce the diameter of the fibers, A method for producing a biopolymer fiber. [Preparatory Claim 15] The method according to preparatory claim 13 or preparatory claim 14, further comprising degassing the collagen solution before sending the collagen solution into the forming buffer. [Preparatory Claim 16] The step of dissolving clinical collagen in an acid solution to prepare a collagen solution; While sending the collagen solution at a first volumetric flow rate through a first needle to generate a first velocity, a step of sending a forming buffer at a second velocity into a tube that coaxially surrounds the first needle to form a sheath around the collagen solution to form a coaxial flow, wherein 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; The 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; The step of dehydrating the collagen fibers at a certain extrusion rate; The step of taking out the fibers at a third velocity exceeding the extrusion rate, sufficient to enhance the orientation of the molecules and reduce the diameter of the fibers; A method for manufacturing biopolymer fibers, comprising: [Preparatory Claim 17] The method according to preparatory claim 16, further comprising collecting the fibers on a bar collector or a flat cylinder. [Preparatory Claim 18] The method according to preparatory claim 16, further comprising collecting the fibers on a grooved spool. [Preparatory Claim 19] The step of dissolving clinical collagen in an acid solution to prepare a collagen solution; The step of extruding the solution through a nozzle into a flowing bath of a forming buffer to a guide for forming fibers; The step of dehydrating the fibers formed in the forming buffer bath; The step of collecting the fibers; A method for manufacturing biopolymer fibers, comprising: [Preparatory Claim 20] The method according to preparatory claim 19, further comprising drying the dehydrated fibers by sending air to the fibers for a time sufficient to dry the fibers before collecting the fibers. [Preparatory Claim 21] The method according to preliminary claim 19, further comprising crosslinking the fibers with a crosslinking agent comprising glyoxal, DL-glyceraldehyde or a combination thereof, and drying the crosslinked fibers. [Preliminary claim 22] Dissolving clinical collagen in an acid solution to prepare a collagen solution; Feeding the collagen solution into a forming buffer at a first rate through a first needle having a first diameter; Feeding the collagen and the forming buffer through a reaction zone comprising a fiber-forming bath at a time and rate sufficient to form fibers; Dehydrating the collagen fibers at an extrusion rate; Taking up the fibers onto a spool at a rate from about 2 times to about 12 times the extrusion rate, sufficient to increase the molecular orientation and decrease the diameter of the fibers, in one or more steps; A method for producing a biopolymer fiber, comprising: [Preliminary claim 23] The method according to preliminary claim 22, further comprising crosslinking the fibers with a crosslinking agent comprising glyoxal, DL-glyceraldehyde or a combination thereof, and drying the crosslinked fibers. [Preliminary claim 24] A biopolymer fiber produced by the method according to any one of preliminary claims 13 to 23. [Preliminary claim 25] A transplantable biopolymer scaffold for assisting in the repair of soft tissue damage, comprising the biopolymer fiber according to preliminary claim 24. [Preliminary claim 26] A method for assisting in the repair of soft tissue damage, comprising the transplantation of the biopolymer scaffold according to preliminary claim 25. [Preliminary claim 27] The method according to preliminary claim 26, wherein the soft tissue is selected from the group consisting of ligaments, tendons, tendon-ligament attachments, bone, muscle, myotendinous junctions, connective tissues including skin, fascia, internal organs, and eyes. [Preliminary claim 28] A suture comprising the biopolymer fiber according to auxiliary claim 24. [Auxiliary claim 29] The suture according to auxiliary claim 28, which is absorbable. [Auxiliary claim 30] An orthosis comprising the biopolymer fiber according to auxiliary claim 24, which, when implanted into a subject, supports, reinforces, increases, or jointly bears the mechanical load on ligaments or tendons in joints such as the anterior cruciate ligament, Achilles tendon, and aponeurosis. [Auxiliary claim 31] An orthosis comprising the biopolymer fiber according to auxiliary claim 24, which, when implanted into a subject, supports an injured joint by connecting one bone to another bone, and optionally restores biomechanics and isometry to a level substantially comparable to that of a healthy natural joint.

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Claims

1. 1. A biocompatible extruded collagen fiber comprising: the fiber comprises a plurality of extruded subfibers, the subfibers being crosslinked with glyoxal; The fibers exhibit a regular longitudinally oriented structure.

2. 10. The fiber of claim 1 having one or more of the following characteristics: (a) an ultimate tensile strength of 1 MPa to 1,700 MPa; (b) a modulus of elasticity between 10 MPa and 20,000 MPa; (c) an elongation at break of 2 percent to 45 percent; (d) an average fiber diameter of 10 μm to 90 μm; (e) maintains its integrity after immersion in Dulbecco's phosphate buffered saline (DPBS) at room temperature for at least 1 hour; (f) retain 50% of its initial load-bearing capacity after 3 months of culture; (g) Promotes the upregulation of pro-regenerative M2 macrophage responses in vivo.

3. 10. The fiber of claim 1, wherein the fiber comprises telocollagen having a degree of cross-linking of at least 85 percent or atelocollagen having a degree of cross-linking of at least 65 percent.

4. said ultimate tensile strength being in the range of 25 MPa to 1,700 MPa; the elastic modulus is in the range of 15,000 MPa to 29,000 MPa; or 3. The fiber of claim 2, wherein the elongation at break is in the range of 7% to 20%.

5. 2. The fiber of claim 1, wherein the collagen is selected from the group consisting of clinical grade collagen, atelocollagen, telocollagen, recombinant collagen, and combinations thereof.

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

7. 10. The fiber of claim 1, wherein the fiber maintains a strength of greater than 60 MPa after immersion in Dulbecco's phosphate buffered saline (DPBS) at room temperature for at least six months.

8. 10. The fiber of claim 1, further comprising attached tenocytes, said tenocytes maintaining at least 75% cell viability and at least 95% cell survival after 7 days of incubation.

9. 10. The fiber of claim 1, wherein the fiber has a substantially circular, oval, square, rectangular, ribbon-like, triangular, or irregular shaped cross-section.

10. 10. The fiber of claim 1, wherein there is a plurality of said fibers, said plurality of fibers being associated by a bundled, braided, or interwoven form factor.

11. The fiber of claim 10 , wherein the shape factor is a bundle.

12. The fiber of claim 11 , wherein the collagen fibers in the bundles are twisted.

13. The fiber of claim 11 , wherein the collagen fibers in the bundles are untwisted.

14. The fiber of claim 10 , wherein the form factor is braided.

15. The fiber of claim 10 , wherein the shape factor is woven.

16. 11. The fiber of claim 10, wherein the collagen is selected from the group consisting of clinical grade collagen, atelocollagen, telocollagen, recombinant collagen, and combinations thereof.

17. 11. The fiber of claim 10, further comprising attached tenocytes, said tenocytes maintaining at least 75% cell viability and at least 95% cell survival after 7 days of incubation.

18. 11. The fiber of claim 10, wherein the fiber maintains a strength of greater than 60 MPa after immersion in Dulbecco's phosphate buffered saline (DPBS) at room temperature for at least six months.

19. 11. The fiber of claim 10, wherein the fiber maintains a strength of greater than 60 MPa six months after implantation in a subject.

20. The fiber of claim 10 , wherein the collagen further comprises one or more biocompatible polymers.

21. a plurality of biocompatible extruded collagen fibers, the fibers being associated by a bundle form factor; the fiber comprises a plurality of extruded subfibers, the subfibers being crosslinked with glyoxal; The fibers exhibit a regular longitudinally oriented structure, The extruded collagen fibers are a plurality of fibers having one or more of the following characteristics (a)-(c): (a) an ultimate tensile strength of 1 MPa to 800 MPa; (b) a modulus of elasticity between 10 MPa and 7,500 MPa; (c) Average fiber diameter between 10 μm and 30 μm.

22. 22. The plurality of fibers of claim 21, wherein the fibers in the bundle are twisted.

23. 22. The plurality of fibers of claim 21, wherein the fibers are untwisted.

24. 22. The plurality of fibers of claim 21, wherein the collagen is selected from the group consisting of clinical grade collagen, atelocollagen, telocollagen, recombinant collagen, and combinations thereof.

25. 22. The plurality of fibers of claim 21, further comprising attached tenocytes, said tenocytes maintaining at least 75% cell viability and at least 95% cell survival after 7 days of incubation.

26. 25. The plurality of fibers of claim 24 comprising between 2 and 10,000 fibers.

27. 22. The plurality of fibers of claim 21, wherein the fibers maintain a strength of greater than 60 MPa after immersion in Dulbecco's phosphate buffered saline (DPBS) at room temperature for at least six months.

28. 22. The plurality of fibers of claim 21, wherein the fibers maintain a strength of greater than 60 MPa six months after implantation into a subject.

29. 22. The plurality of fibers of claim 21, wherein the collagen further comprises one or more biocompatible polymers.

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