Multifilament collagen fiber bundle having tendon-like structure and method for producing same

Abstract

The method for producing a multifilament collagen fiber bundle involves treating a multifilament support polymer collagen fiber bundle, which has multiple collagen monofilaments supported on a support polymer, with a series of liquid media containing gradually decreasing concentrations of the support polymer, followed by removal of the support polymer from the multifilament support polymer collagen fiber bundle. The support polymer is more soluble than collagen in the liquid media, and the gradual removal of the support polymer allows sufficient time for the collagen molecules to self-assemble and form fibrils. The resulting multifilament collagen fiber bundle has a D-band structure similar to that of native collagen, but shifted by one-sixth of the D-band. This multifilament collagen fiber bundle can be UV-crosslinked in the absence of a photoinitiator, resulting in a crosslinked collagen product with excellent tensile strength and Young's modulus. This product is useful as a collagen-based biomaterial for tissue repair and regeneration.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application USSN 63 / 449,344, filed March 2, 2023, the entire contents of which are incorporated herein by reference. [Technical Field]

[0002] This application relates to a multifilament collagen fiber bundle and a method for producing a multifilament collagen fiber bundle. [Background technology]

[0003] Tendons are fibrous tissues composed primarily of collagen. Tendon ruptures and ruptures are common and can be caused by trauma, degenerative disease, or overuse (e.g., physical activity and exercise). For many patients suffering from debilitating tendon injuries, surgical treatment is the standard of care, which may require the use of artificial tendon scaffolds or the implantation of tendon fragments harvested from other parts of the body. While biodegradable synthetic biopolymer scaffolds offer suitable mechanical properties and degradation rates for tendon repair, natural biopolymers are a more desirable option due to their inherent biological activity and ability to mimic the biochemical and structural signals of the extracellular matrix (ECM). The ECM of most connective tissues is primarily composed of fibrogenic collagens, with type I collagen being the most abundant collagen type. Therefore, type I collagen-based materials have been developed to repair acute tendon injuries and promote the regeneration of native tendon tissue. Currently, the collagen-based scaffolds for tendon repair include Integr®. TM , TenoGlide TM , Zimmer TM Collagen Repair Patch, GRAFTJACKET TMRegenerative tissue matrices and other materials on the market retain some of the collagen hierarchical structure present in natural tissues. However, there is an important feature present in natural type I collagen ECM that is absent in most collagen-based biomaterials currently under development for tissue repair and regeneration: the hierarchical fibrillar structure of type I collagen. This structure provides tensile strength and contributes to the adhesion, alignment, proliferation, and differentiation of cells present in connective tissues such as tendons.

[0004] To promote collagen self-assembly into form factors suitable for tendon repair and tendon tissue engineering, various collagen spinning methods, including wet spinning and dry spinning, have been developed. Wet spinning can produce filaments with diameters ranging from 8 to 600 μm, with throughputs ranging from 1 to 10 m / h. Wet spinning has previously been used to produce collagen multifilament yarns consisting of six 80 μm-diameter single filaments. These wet-spun multifilament yarns were subsequently cross-linked with glutaraldehyde and exhibited properties similar to those of wet tendon collagen fibers, with a tensile strength of approximately 40 MPa. However, this method has a relatively low throughput, and the collagen monofilaments obtained by wet spinning have irregular cross-sectional shapes. To produce finer fibers with diameters ranging from the order of one micron to several hundred nanometers, collagen can be electrospun, usually in the presence of a polymer. For example, electrospinning has been used to produce collagen / polylactic acid nanofiber mats that can be wound into bundles to replicate the structure of tendon bundles. However, these techniques have so far had low throughput and only produced single or loosely aggregated monofilaments, limiting their applicability to tendon repair and tendon tissue engineering, which require relatively large quantities of high-tensile-strength materials. On the other hand, dry spinning of polymers dissolved in volatile solvents is an industrial-scale process capable of producing monofilaments at speeds of tens of meters per second. However, the solvents used in most dry spinning processes can denature proteins, including collagen. Contact drawing is a type of dry spinning method that uses water-soluble polymers, such as dextran or polyethylene oxide (PEO), as the polymer. The drawing process is initiated by inserting a nucleating element, such as a pin, into the solution and pulling back at a speed of approximately 1 m / s. Using contact drawing, aqueous collagen / polymer mixtures can be converted into composite filaments with diameters of 1–10 μm. They can then be processed into pure collagen fibers by eluting the polymer in a buffered aqueous solution. The resulting collagen fibers have been shown to promote cell orientation and growth.

[0005] There remains a need for a commercially useful process for producing multifilament collagen fiber bundles that have the hierarchical fiber structure of native collagen (eg, type I collagen).

[0006] A method for producing a multifilament collagen fiber bundle includes treating a polymer collagen fiber bundle, comprising a plurality of collagen monofilaments supported on a support polymer, with a series of liquid media of decreasing osmotic pressure to remove the support polymer from the polymer collagen fiber bundle, wherein the support polymer is more soluble than collagen in the liquid media.

[0007] A method for producing a multifilament collagen fiber bundle includes treating a polymer collagen fiber bundle, comprising a plurality of collagen monofilaments supported on a support polymer, with a series of liquid media with decreasing concentrations of the support polymer to remove the support polymer from the polymer collagen fiber bundle, the support polymer being more soluble in the liquid media than collagen.

[0008] The multifilament collagen fiber bundle comprises collagen fibers having a diameter of at least 0.3 μm, each collagen fiber comprising a collagen fibril having a diameter smaller than the collagen fiber, each collagen fibril comprising a collagen microfibril having a diameter smaller than the collagen fibril, each collagen microfibril comprising a bundle of collagen molecules, the collagen microfibrils forming a hierarchical D-band structure within the collagen fibril, and adjacent collagen microfibrils being shifted by 1 / 6 of the D-band compared to microfibrils within the same type of native collagen.

[0009] The method for producing a crosslinked multifilament collagen fiber bundle includes crosslinking the multifilament collagen fiber bundle with a chemical crosslinking agent or by light irradiation.

[0010] The crosslinked multifilament collagen fiber bundle comprises crosslinked collagen fibers having a diameter of at least 0.3 μm, each collagen fiber comprising a collagen fibril having a diameter smaller than the collagen fiber, each collagen fibril comprising a collagen microfibril having a diameter smaller than the collagen fibril, each collagen microfibril comprising a bundle of collagen molecules, the collagen microfibrils forming a hierarchical D-band structure in the collagen fibril, and adjacent collagen microfibrils being shifted by 1 / 6 of the D-band compared to microfibrils of the same type of native collagen.

[0011] The method for producing a crosslinked multifilament collagen fiber bundle is to add a total energy amount of 0.1 J / cm to the multifilament collagen fiber bundle. 2 This includes irradiating the above with ultraviolet light.

[0012] The crosslinked multifilament collagen fiber bundle comprises collagen fibers having a diameter of at least 0.3 μm, each collagen fiber comprising a collagen fibril having a diameter smaller than the collagen fiber, each collagen fibril comprising a collagen microfibril having a diameter smaller than the collagen fibril, each collagen microfibril comprising a bundle of collagen molecules, the collagen microfibrils forming a hierarchical D-band structure in the collagen fibril, adjacent collagen microfibrils being shifted by 1 / 6 of the D-band compared to microfibrils of the same type of native collagen, and phenylalanine residues on adjacent collagen molecules being covalently bonded to form crosslinks between adjacent collagen molecules.

[0013] The collagen fibers have a diameter of at least 0.3 μm and comprise collagen fibrils having a diameter smaller than the collagen fibers, each collagen fibril comprises collagen microfibrils having a diameter smaller than the collagen fibrils, each collagen microfibril comprises a bundle of collagen molecules, the collagen microfibrils form a hierarchical D-band structure within the collagen fibrils, and adjacent collagen microfibrils are shifted by 1 / 6 of the D-band compared to microfibrils in the same type of native collagen.

[0014] The collagen fibers comprise atelomeric collagen microfibrils that form a hierarchical D-band structure in the collagen fibrils, with adjacent collagen microfibrils shifted by 1 / 6 of the D-band compared to microfibrils of the same type of native collagen, and polyethylene oxide (PEO) in an amount of 60% by weight or less based on the total weight of the collagen fibers.

[0015] Multifilament collagen fiber bundles and fibers are useful as collagen-based biomaterials, particularly for tissue repair and regeneration of connective tissues such as tendons. Thus, multifilament collagen fiber bundles and fibers are useful as collagen-based sutures, scaffolds for tendon repair, and in regenerative medicine as scaffolds to support the growth and differentiation of cells, such as tendon-forming stem cells.

[0016] Additional features will be described or will become apparent in the following detailed description. It should be understood that each feature described herein can be utilized in any combination with any one or more of the other described features, and that each feature does not necessarily depend on the presence of other features, unless otherwise apparent to one of ordinary skill in the art. [Brief explanation of the drawings]

[0017] For a clearer understanding, preferred embodiments will now be described in detail, by way of example, with reference to the accompanying drawings, in which:

[0018] [Figure 1] Figure 1 shows the fabrication method for PEO-collagen multifilament bundles. (A) After contacting the PEO-collagen solution with a pin array, the PEO-collagen monofilament is pulled over a frame, the monofilaments are gathered from both ends, twisted, and wound to obtain a compact multifilament. (B) Representative image of a wound multifilament approximately 9 cm long. (C) Representative image of a wound multifilament with adjustable diameter. (D) Epifluorescence image of a PEO-collagen-FITC-PEG / PEO-phalloidin-TRITCR multifilament bundle showing the tilt relative to the multifilament axis.

[0019] [Figure 2] Figure 2 shows optical microscope images of the washing process of PEO-collagen multifilaments. (A) After fabrication, multifilaments immersed directly in 1X PBS immediately begin to degrade. (B) Stepwise washing of PEO:1X PBS.

[0020] [Figure 3] Figure 3 shows wide-angle X-ray scattering (WAXS) patterns of (A) PEO multifilament, (B) PEO-collagen multifilament, (C) PEO-collagen multifilament washed with PEO:1X PBS solution, (D) PEO-collagen multifilament washed with PEO:10X PBS solution, and (E) rat tail tendon. The arrows indicate the orientation of the multifilaments. (F) shows the azimuthally averaged WAXS profile.

[0021] [Figure 4] Figure 4 shows small-angle X-ray scattering (SAXS) patterns of (A) PEO-collagen multifilament, (B) PEO:PEO-collagen multifilament washed with 1X PBS, (C) PEO:PEO-collagen multifilament washed with 10X PBS, and (D) rat tail tendon. The arrows indicate the orientation of the multifilament. (E) shows the SAXS profile.

[0022] [Figure 5] Figure 5 shows scanning electron micrographs (SEM) of PEO-collagen multifilament bundles washed with PEO:10X PBS. (A) 2000x magnification. (B) 50,000x magnification. (C) Enlarged panel of (B). (D) Profile extracted between the two arrows in (C).

[0023] [Figure 6] Figure 6 shows transmission electron micrographs (TEM) of PEO collagen monofilaments washed with (A) 1X PBS and (B-C) PEO:10X PBS solutions.

[0024] [Figure 7] Figure 7 shows collagen fiber models. (A) A model based on the microfibril structure of type I collagen. (B) The corresponding electron density profile along the microfibril. (C) In (A), the collagen fiber model is offset by one-sixth of the D band. (D) The corresponding electron density profile constructed using the density profile in (C).

[0025] [Figure 8] Figure 8 shows (A) the stress-strain curves in the wet state, (B) the ultimate tensile strength (UTS), (C) Young's modulus, and (D) the swelling ratio of PEO:collagen multifilament bundles without crosslinking after washing with 10X PBS solution, and after crosslinking with 0.4 J / cm2 UVC, 0.8 J / cm2 UVC, 2 J / cm2 UVC, and 10 J / cm2 UVC in the dry state and 1% glutaraldehyde after washing. Error bars indicate standard deviation. The dashed lines in (B) and (C) show the UTS and Young's modulus of rat tail, respectively. (YP Kato, DL Christiansen, RA Hahn, SJ Shieh, JD Goldstein, FH Silver, Biomaterials 1989, 10, 38)

[0026] [Figure 9] Figure 9 shows the WAXS patterns of (A) a PEO-collagen multifilament washed with PEO:10X PBS solution and (B) a PEO-collagen multifilament washed with PEO:10X PBS solution after 10 J / cm2 UVC irradiation. (C) WAXS profile. The arrow indicates the direction of the multifilament. (D) ATR-FTIR spectrum.

[0027] [Figure 10] FIG. 10 is a graph showing (A) the thermogravimetry (TG) curve and (B) the differential thermogravimetry (DTG) curve of a PEO collagen multifilament, and (B) the differential thermogravimetry (DTG) curve of a PEO collagen multifilament after stepwise washing with PEO:10X PBS.

[0028] [Figure 11] Figure 11 shows an optical image of a more complex 3D braided structure formed by braiding three PEO collagen multifilaments. DETAILED DESCRIPTION OF THE INVENTION

[0029] Multifilament collagen fiber bundles are produced from polymer collagen fiber bundles. Polymer collagen fiber bundles include collagen monofilaments supported on a support polymer. Removal, or extraction, of the support polymer from the collagen to obtain a multifilament collagen fiber bundle can be achieved by selectively removing the support polymer, leaving only the collagen. In some embodiments, selective removal of the support polymer can be achieved by a liquid medium in which the support polymer is more soluble than the collagen, such that the liquid medium selectively dissolves the support polymer but not the collagen. The collagen is preferably insoluble in the liquid medium, e.g., at a concentration of less than about 0.01 mg collagen per mL of liquid medium.

[0030] However, we found that removing the supporting polymer all at once inhibits the self-assembly of collagen into physically integrated collagen fibers. To overcome this problem, we found that sequentially subjecting the polymer-collagen fiber bundle to a supporting polymer removal process involving a liquid medium in which the supporting polymer concentration is gradually decreased allows the supporting polymer to be gradually removed from the polymer-collagen fiber bundle, providing sufficient time for the collagen to self-assemble into physically integrated multifilament collagen fiber bundles. Furthermore, the multifilament collagen fiber bundles produced by this process possess a hierarchical structure, and the average axial period throughout the multifilament collagen fiber bundle exhibits a D-band structure very similar to that of native collagen, except for a shift in amino acid position relative to that of adjacent collagen molecules across the axial span of the D-band structure. The shifted D-band structure statistically aligns more phenylalanine units between adjacent collagen molecules, allowing photocrosslinking (e.g., ultraviolet C crosslinking) to be used to crosslink collagen molecules through phenylalanine units without the need for photoinitiators or chemical crosslinkers, which in some embodiments can provide crosslinked multifilament collagen fiber bundles with tensile properties that meet the requirements for tendon replacement (i.e., Young's modulus in the range of 450-2,000 MPa and ultimate tensile strength (UTS) in the range of 25-148 MPa).

[0031] The polymer collagen fibers in the polymer collagen fiber bundle can be produced by any suitable fiber-forming process, such as contact drawing or other dry spinning methods, wet spinning, or electrospinning. In some embodiments, contact drawing (e.g., multi-pin contact drawing) is used to produce a large number of polymer collagen monofilaments (fibers) from a press-stranded composition containing a supporting polymer and collagen. The polymer collagen monofilaments can be any length. In some embodiments, the length of the polymer collagen monofilaments is up to 50 cm, e.g., 0.5 to 20 cm. The diameter of the polymer collagen monofilaments is at least 0.3 μm. In some embodiments, the diameter is at least 0.5 μm or at least 1 μm. In some embodiments, the diameter is in the range of 0.3 to 50 μm. This process is easily scalable, does not require specialized equipment or hazardous materials, and can produce thousands of aligned polymer collagen monofilaments at room temperature. The polymer collagen monofilaments are then consolidated into a tightly packed polymer collagen fiber bundle, for example, by twisting or rolling.

[0032] As described above, in some embodiments, the method for producing a multifilament collagen fiber bundle includes exposing a polymer collagen fiber bundle to a series of liquid media containing a gradually decreasing concentration of support polymer. The series can include a process in which the support polymer concentration is continuously decreased (i.e., the liquid medium is continuously replaced or diluted with a medium containing a gradually decreasing concentration of support polymer), a discontinuous process in which the support polymer concentration is decreased at regular time intervals, or a combination thereof. In either continuous or discontinuous processing, successive processing steps (i.e., washing steps) are used, including liquid media containing a gradually decreasing concentration of support polymer. The concentration reduction can be achieved by complete replacement of the liquid medium in successive steps, with each successive batch of liquid medium containing a lower concentration of support polymer. Alternatively, or in addition, the concentration reduction can be achieved by continuous or time-interval dilution of the liquid medium without complete replacement of the liquid medium in each step. A combination of serial dilution and replacement can also be used. The polymer collagen fiber bundle is subjected to at least two processing steps, with the final processing step containing a lower concentration of support polymer than the initial processing step. Two, three, four, five, six or more successive processing steps may be used.

[0033] The initial concentration of the support polymer in the liquid medium will vary depending on the type of support polymer. However, an initial concentration of 10% by weight or less, based on the total weight of the liquid medium, is generally suitable. In some embodiments, a concentration of 0.25 to 5% by weight is used. In some embodiments, a concentration of 0.75 to 2% by weight is used.

[0034] Successive concentrations of the support polymer in the liquid medium are lower than the initial concentration. For example, the second processing step can include a liquid medium in which the concentration of the support polymer ranges from 0 to 0.75 wt. %, based on the total weight of the liquid medium in the second processing step. In some embodiments, a concentration of 0.35 to 0.75 wt. % is used in the second processing step. If a third processing step is used, the liquid medium can include a concentration of the support polymer ranges from 0 to 0.35 wt. %, based on the total weight of the liquid medium in the second processing step. In some embodiments, a concentration of 0.1 to 0.35 wt. % is used in the second processing step. In a continuous process, the concentration of the support polymer in successive steps can be points along a successively decreasing concentration. In some embodiments, the successive processing steps include successively halving the concentration of the support polymer in each successive processing step. Regardless of the number of processing steps utilized, or whether successive reductions in support polymer concentration are employed, the final processing step may involve a liquid medium that contains some support polymer (i.e., a concentration of support polymer greater than 0 wt %) or a liquid medium that is free of support polymer (i.e., a liquid medium with a concentration of support polymer of 0 wt %).

[0035] In some embodiments, each processing step is performed for a period of time during which the support polymer is slowly removed from the polymer-collagen fiber bundle until the concentration of the support polymer in the liquid medium reaches an undesirable level. The duration of each step depends, in part, on the solubility of the support polymer in the liquid medium used in that step. Generally, the duration of each step ranges from 1 to 24 hours. Early processing steps, where the support polymer concentration is higher and more support polymer is removed from the polymer-collagen fiber bundle, are generally performed for shorter periods of time than later processing steps, where the support polymer concentration is lower and less support polymer must be removed from the polymer-collagen fiber bundle. In some embodiments, early processing steps are performed for 1 to 8 hours, e.g., 2 to 6 hours, and later processing steps are performed for 3 to 24 hours, e.g., 8 to 18 hours. These time ranges are general; later processing steps may be performed for shorter periods of time, while earlier processing steps may be performed for longer periods of time.

[0036] Because collagen is insoluble in water, the liquid medium can include an aqueous medium in which collagen is insoluble or poorly soluble, but the support polymer is soluble or at least dissolves faster than collagen. When an aqueous medium is used, processing the polymer-collagen fiber bundles includes hydrating the polymer-collagen fiber bundles. In some embodiments, one or more of the aqueous media includes a buffer. In some embodiments, one or more of the aqueous media can include water without a buffer. In some embodiments, one or more of the aqueous media can include a biologically useful medium, such as cell culture medium, an alcohol / water mixture, or the like. Examples of buffers include phosphate-buffered saline (PBS), TRIS, HEPES, PIPES, MES, MOPS, imidazole, or mixtures thereof.

[0037] The process of removing the support polymer can be carried out at any suitable temperature that does not denature the collagen and does not freeze the liquid medium, hi some embodiments, the temperature at which this process is carried out is in the range of 20-37°C.

[0038] Collagen is a major structural protein of the extracellular matrix found in various mammalian connective tissues (e.g., cartilage, bone, tendon, ligament, and skin). Examples of collagen include type I collagen (skin, tendon, blood vessels, organs, and bone), type II collagen (cartilage), type III collagen (reticular collagen), type IV collagen (basement membranes, basement membrane layers secreted by epithelial cells), and type V collagen (cell surfaces, hair, and placenta). In some embodiments, the collagen comprises type I collagen. In some embodiments, the collagen is atelomeric. Atelomeric collagen has had its telomeric peptides removed, for example, by pepsin solubilization. Collagen is a linear polymer with a diameter of approximately 1 nm. Multiple collagen molecules, for example, five collagen molecules, are bundled together to form collagen microfibrils. Microfibrils have a diameter of 4-5 nm. Multiple microfibrils, for example, seven microfibrils, can be bundled together to form fibrils. The diameter of the fibrils ranges from 10 to 100 nm, e.g., 30 to 50 nm. Multiple fibrils can be bundled together adjacent to one another to form fibers. The collagen fibers produced by this process can have a diameter of at least 0.3 μm. In some embodiments, the diameter is at least 0.5 μm or at least 1 μm. In some embodiments, the diameter is in the range of 0.3 to 50 μm, e.g., 1 to 10 μm.

[0039] The supporting polymer provides a scaffold for collagen to assemble and maintain stability during collagen chain formation. Examples of supporting polymers include dextran, polyethylene oxide (PEO), polyvinyl acetate (PVA), polyethylene glycol (PEG), hydroxypropyl cellulose, poly(2-ethyl-2-oxazoline), poly(4-styrenesulfonic acid-co-maleic acid), poly(acrylic acid), poly(diallyldimethylammonium chloride), poly(methacrylic acid), poly(methyl vinyl ether-alt-maleic acid), poly(vinylpyrrolidone) (PVP), low-density polyethylene (LDPE), high-density polyethylene (HDPE), polypropylene (PP), polyvinyl chloride (PVC), polystyrene (PS), nylon, polytetrafluoroethylene, thermoplastic polyurethanes, crosslinked polymers thereof, and copolymers thereof. In some embodiments, the supporting polymer comprises polyethylene oxide (PEO). In some embodiments, only one type of supporting polymer is used in the polymer collagen fiber bundle, while in other embodiments, two or more types of supporting polymers are used in the polymer collagen fiber bundle.

[0040] A support polymer may be present in the fibers. In some embodiments, the support polymer is present in the fibers in an amount of 60% by weight or less, based on the total weight of the fibers. In some embodiments, the support polymer is present in an amount of 0.0001 to 50% by weight. In some embodiments, the support polymer before being removed from the fibers is present in an amount of 40 to 60% by weight, e.g., 47 to 53% by weight. In some embodiments, the support polymer after being removed from the fibers is present in an amount of less than 20% by weight, e.g., 0.0001 to 20% by weight or 0.0001 to 5% by weight.

[0041] The product obtained by the method for producing a multifilament collagen fiber bundle is a multifilament collagen fiber bundle containing collagen fibers with a diameter of at least 0.3 μm. Each collagen fiber contains collagen fibrils with a smaller diameter than the collagen fibers. Each collagen fibril contains collagen microfibrils with a smaller diameter than the collagen fibrils. Each collagen microfibril contains a bundle of collagen molecules. The collagen microfibrils form a hierarchical D-band structure in the collagen fibril. Adjacent collagen microfibrils are offset by 1 / 6 of the D-band compared to microfibrils of the same type of native collagen.

[0042] The multifilament collagen fiber bundles produced by this process are not exactly the same as native collagen. Multifilament collagen fiber bundles have a band structure (D-band), but this D-band structure differs from native collagen. If the D-band is modeled as a cross-section of the horizontally oriented collagen molecular layer in the bundle cut vertically, it is found that collagen molecules are missing at 1 / 6 the distance (approximately 11 nm) compared to native collagen. If the collagen molecular layer in the bundle is cut diagonally, the D-band can be defined as a missing collagen molecule every 67 nm.

[0043] It has been found that additives added during fiber formation are not extracted by the liquid medium during the process. Therefore, it is possible to incorporate one or more additives into multifilament collagen fiber bundles by introducing one or more additives into the polymer collagen fibers during fiber formation. In some embodiments, when fibers are produced using a contact drawing method, one or more additives are included in the press-stranded composition and are incorporated into the polymer collagen fibers as the fibers are drawn from the press-stranded composition. In some embodiments, the one or more additives comprise functional additives commonly used in biochemistry, e.g., cell culture. In some embodiments, functional additives include sugars, growth factors, hormones, extracellular matrix proteins (ECM) (e.g., fibronectin, laminin, etc.), enzymes, cytokines, chemokines, antibodies (e.g., monoclonal antibodies), anti-inflammatory agents, steroids, immunosuppressants, chemotherapeutic agents, lipids, hyaluronic acid, liposomes, micro / nanocapsules, genetic material (e.g., DNA (e.g., plasmids), RNA (e.g., mRNA, interfering RNA), nucleotides), amino acids, extracellular vesicles, whole cells, metal ions, non-metal ions, nanoparticles (e.g., carbon nanotubes), etc. Functional additives include, for example, citric acid, other natural health products, other small molecule pharmaceuticals (e.g., minocycline, riluzole, dalfampridine, escitalopram, deoxygedunin, 7,8-dihydroflavone, quercetin, dexamethasone, tacrolimus), and combinations thereof. The functional additive is preferably a pharmaceutically active agent, a precursor to a pharmaceutically active agent, or a combination thereof.

[0044] Crosslinking of multifilament collagen fiber bundles can be achieved by contacting the multifilament collagen fiber bundles with chemical crosslinkers (e.g., glutaraldehyde, carbodiimide, genipin, transglutaminase, lysyl oxidase, and other enzymes), physical crosslinking by dehydrothermal treatment, or photocrosslinking with light of an appropriate wavelength (e.g., ultraviolet (UV) light with a wavelength of 100-400 nm, especially UVC light with a wavelength of 100-280 nm).

[0045] The shifted D-band structure of multifilament collagen fiber bundles statistically places more phenylalanine units between adjacent collagen molecules, making it possible to crosslink collagen molecules by photocrosslinking via phenylalanine units without the need for photoinitiators or chemical crosslinkers, and in some embodiments, providing crosslinked multifilament collagen fiber bundles with improved mechanical properties and / or adjusting the mechanical properties of crosslinked multifilament collagen fiber bundles by adjusting the total amount of light energy. The shifted D-band structure allows for the use of a higher total energy amount than that of native collagen, resulting in an increased crosslink density when photocrosslinking is used compared to photocrosslinking of native collagen. In some embodiments, the crosslink density of crosslinked multifilament collagen fibers is greater than 1 μm 3 In some embodiments, the crosslink density ranges from 1 million to 26 million crosslinks per μm 3 In some embodiments, the crosslink density ranges from 2 million to 26 million crosslinks per μm 3 The range is 3.5 million to 26 million crosslinking points per polymer. The range is 5.5 million to 26 million crosslinking points per polymer. The range is 7 million to 26 million crosslinking points per polymer.

[0046] Thus, in some embodiments, a method for producing a crosslinked multifilament collagen fiber bundle comprises administering a total energy dose of 0.1 J / cm to the multifilament collagen fiber bundle. 2The total energy dose is 0.1-100J / cm 2 In some embodiments, the total energy dose ranges from 5 to 100 J / cm. 2 In some embodiments, the total energy dose ranges from 5 to 50 J / cm. 2 Photocrosslinking is preferably performed when the multifilament collagen fiber bundle is dry. UVC radiation with a wavelength of 250-260 nm (e.g., 254 nm) is well suited for photocrosslinking adjacent phenylalanine units. Photocrosslinking can be performed with or without a photoinitiator, but this process allows photocrosslinking without a photoinitiator, resulting in a crosslinked multifilament collagen fiber bundle with high crosslink density and good mechanical properties.

[0047] Crosslinked multifilament collagen fiber bundles retain the D-band structure of multifilament collagen fiber bundles, and phenylalanine residues on adjacent collagen molecules are covalently bonded to form crosslinks between adjacent collagen molecules.

[0048] In some embodiments where photocrosslinking is used, the crosslinked multifilament collagen fiber bundles have an ultimate tensile strength of 1 MPa or greater and / or a Young's modulus of 20 MPa or greater. In some embodiments, the ultimate tensile strength and / or Young's modulus may even exceed that of multifilament collagen fiber bundles crosslinked with chemical crosslinkers. Higher total energy doses (e.g., 5 J / cm) may also be used. 2 Above, especially 10J / cm 2When photocrosslinked (at or above), the crosslinked multifilament collagen fiber bundles approach tendon-like mechanical performance. In some embodiments, the ultimate tensile strength is up to 250 MPa. In some embodiments, the ultimate tensile strength is in the range of 10-200 MPa. In some embodiments, the ultimate tensile strength is in the range of 25-150 MPa. In some embodiments, the Young's modulus is up to 3,000 MPa. In some embodiments, the Young's modulus is in the range of 50-2,500 MPa. In some embodiments, the Young's modulus is in the range of 450-2,000 MPa.

[0049] Collagen molecules contain repeating phenylalanine residues at a length scale of D / 6, where d is the length scale of collagen's characteristic density band, approximately 67 nm. When two phenylalanine residues on adjacent collagen molecules are in close proximity, they can be crosslinked by UVC irradiation. This crosslinking process, combining contact-stretching polymer-collagen fiber binding with a treatment process to extract the supporting polymer, produces collagen fibers that retain remarkable order and D / 6 density bands even after UVC irradiation. Therefore, this process for producing multifilament collagen fiber bundles aligns collagen molecules, allowing for better alignment of phenylalanine residues and photocrosslinking. Furthermore, the photocrosslinking process is highly tunable, and increasing the total UV energy dose increases the degree of crosslinking, thereby increasing fiber strength and stiffness. This is contrary to what is known about collagen. While the literature indicates clear limits for physical crosslinking of collagen, this process and product overcomes these limits and does not require a photoinitiator. [Example]

[0050] Experimental procedure Preparation of PEO-collagen solution (pressurized composition) PEO powder (8 MDa, Sigma-Aldrich) was dissolved in type I collagen solution (approximately 6 mg / mL in 0.01 M HCl, Collagen Solutions) at a weight ratio of 1:1 to prepare PEO-collagen. The type I collagen used was pepsin-solubilized collagen with telomerase peptides removed, and the collagen was atelomeric. Small PEO aggregates were dispersed as much as possible by stirring with a metal spatula, and the solution was transferred into two syringes connected with a dual-lock syringe tip. The PEO-collagen solution was transferred between the two syringes through the syringe tips at least 100 times to ensure uniform dissolution of the PEO. The PEO-collagen solution was then stored at 4 °C and, after allowing air bubbles to disperse, transferred to a -20 °C freezer. The frozen PEO-collagen solution was lyophilized at -80 °C using an FD8508 freeze dryer (Ilshin BioBase). The lyophilized PEO-collagen sponge was chopped with tweezers and dissolved in 20 mM acetic acid (diluted glacial acetic acid, ASC grade, VWR). To ensure complete dissolution of the PEO and collagen, the mixture was again stirred with a metal spatula and transferred to two syringes as described above. The resulting PEO-collagen solution was stored at 4 °C overnight and then returned to room temperature for contact drawing. The concentration of the lyophilized PEO-collagen in acetic acid was 6.75 wt%.

[0051] Assembly, hydration and cross-linking of collagen multifilaments Contact drawing of PEO-collagen fibers was performed using a multi-pin array designed for fiber nucleation and a substrate tool designed to hold a viscous PEO-collagen solution, as previously described (see WO2022 / 032387, published February 17, 2022, the entire contents of which are incorporated herein by reference). Briefly, the PEO-collagen solution was applied to a flat, rectangular substrate tool. Next, a 30 x 55 array of cylindrical pins, each 0.6 mm in diameter, 5 mm in height, and 1.6 mm center-to-center, was placed on the substrate tool. The pin array was then contacted with the viscous solution and subsequently pulled away from the rectangular plate to produce multiple PEO-collagen monofilaments. This drawing process was repeated 30 times. The monofilaments were then collected on a frame, their ends gathered together, twisted by hand 10 times, and wound into a tightly integrated multifilament bundle. The imparted twist was visualized by epifluorescence imaging of multifilament bundles formed from solutions of PEO-collagen / FITC-PEG (0.05 wt%, MW 10 kg / mol, Creative PEGWorks) and PEO / phalloidin-TRITC (0.05 wt%, MW 1231.40 g / mol, Sigma-Aldrich). A Nikon Eclipse Ti optical microscope (Nikon Instruments) was used. Monofilaments were formed from each of these solutions using two sets of pin arrays and a rectangular plate. Fluorescently labeled multifilaments were fabricated by one drawing with PEO-collagen / FITC-PEG followed by one drawing with PEO / phalloidin-TRITC. This process was repeated 10 times for a total of 20 fiber drawing runs. All processes were performed at 27 ± 2 °C and 28 ± 2% relative humidity.

[0052] After fiber consolidation, the PEO-collagen multifilament bundles were mounted on a custom 3D-printed stand and immersed in graded mixtures of PEO:1X PBS or PEO:10X PBS. The hydration protocol was as follows: 1 wt% PEO:PBS for 4 hours; 0.5 wt% PEO:PBS for 4 hours; 0.25 wt% PEO:PBS for 16 hours; 100% PBS for 8 hours; 100% PBS for 16 hours; and water for 3 hours. The dried collagen multifilament bundles were then immersed in a crosslinking solution of 1.0 wt% glutaraldehyde at room temperature for 1.5 hours or exposed to UVC irradiation (UVP Crosslinker CL-3000, Analytik Jena). UVC (wavelength 254 nm) was used with a total energy of 0.4 J / cm. 2 , 0.8J / cm 2 , 2J / cm 2 , 10J / cm 2 Irradiated with.

[0053] Wide-angle and small-angle X-ray scattering (WAXS and SAXS) WAXS and SAXS patterns were acquired for pure PEO, PEO collagen, hydrated PEO collagen multifilaments, and rat tail tendon. WAXS patterns were acquired using a D8 Advance X-ray diffractometer (Bruker) operating at a wavelength of 0.15406 nm. Data were recorded over the 0-50° (2θ) range with a sample-to-detector distance of 70 mm and an exposure time of 600 seconds. WAXS frames were integrated from 2D images into 1D powder patterns by azimuthal averaging using the Fit2D software package developed at the European Synchrotron Radiation Facility (ESRF). All multifilaments were positioned vertically in front of the instrument. SAXS measurements were performed using a SAXSpoint 2.0 (Anton Paar) equipped with a copper source and an Eiger detector positioned 575.6533 mm from the sample. The X-ray exposure time was 30 minutes per frame, for a total of four frames. The sample was oriented perpendicular to the X-ray beam. The two-dimensional images were converted into one-dimensional meridian profiles using FIT2D software.

[0054] Scanning electron microscope (SEM) The surface morphology of collagen multifilament bundles was evaluated by scanning electron microscopy using a Sigma 300 VP field emission SEM (Zeiss). Prior to imaging, multifilaments were fixed with 2.5% glutaraldehyde in 1X PBS for 2 hours and then washed three times for 10 minutes with 1X PBS. Next, the multifilaments were washed with distilled water. The multifilaments were then dehydrated using a graded ethanol treatment (50% ethanol for 10 minutes, 70% ethanol for 10 minutes twice, 95% ethanol for 10 minutes twice, 100% ethanol for 10 minutes twice, and 100% ethanol for 10 minutes once). Subsequently, critical point drying was performed using an EM CPD300 system (Leica Microsystems). The samples were removed from the critical point dryer and attached to SEM stubs using carbon tape. An ultrathin gold / palladium (80 / 20) layer was then applied using a sputter coater EM ACE600 (Leica Microsystems). Multifilaments were observed at an accelerating voltage of 5 kV and a working distance of 7.5–11.5 mm. High-magnification scans (50,000x) were used to examine the collagen fiber structure within the monofilaments. Low-magnification scans (2,000x) were used to observe the arrangement of the monofilaments.

[0055] Transmission electron microscope (TEM) PEO collagen monofilaments were captured on a 400-mesh copper grid coated with a Formvar carbon film (Electron Microscopy Sciences). The monofilaments were washed on the grid with one drop of 1X PBS for 2 minutes, followed by three drops of water for 1 minute each. Alternatively, the grid was washed with a gradient of PEO:10X PBS (1 wt% PEO:PBS for 1 hour, 0.5 wt% PEO:PBS for 1 hour, 0.25 wt% PEO:PBS for 1 hour, and 100% PBS for 2 hours), followed by one drop of water for 5 seconds, one drop of 1% glutaraldehyde solution for 1 minute, and three drops of water for 5 seconds each. In both cases, the grid was stained with 2% uranyl acetate solution for up to 30 seconds. Images were then observed using a JEM-1230 transmission electron microscope (JEOL) equipped with a Hamamatsu Photonics ORCA-HR digital camera at an accelerating voltage of 80 kV.

[0056] Mechanical property evaluation The mechanical properties of collagen multifilament bundles, including UTS and Young's modulus, were evaluated using a Mark-10 F105 tensile tester (Mark-10 Corporation) equipped with 0.5 N and 10 N force sensors at a crosshead speed of 6 mm / min and a gauge length of 10 mm. The multifilament bundles were mounted on a custom-made 3D-printed frame, and both ends were secured with superglue (Elmer Products). Bright-field images of the multifilament bundles were acquired using a 4x objective lens on a Nikon Eclipse Ti microscope. The diameter of the multifilament bundles was measured using ImageJ, and the cross-sectional area was calculated to determine the tensile stress. The mounted multifilaments were immersed in water for 1.5 hours before diameter measurement.

[0057] Swelling analysis To quantify the swelling of multifilaments, five 10 mm long multifilaments were selected. The dry multifilament diameter was measured from images taken with a Nikon Eclipse Ti microscope equipped with a 10x objective. The multifilaments were hydrated in water for 1.5 hours, and the diameter was measured again. The swelling ratio was calculated according to Equation 1.

[0058]

number

[0059] Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) ATR-FTIR analysis was performed using a Nicolet iZ10 MX integrated FTIR microscope (Thermo Fisher Scientific) to record the vibrational modes of functional groups derived from hydrated PEO collagen multifilaments. A 1 cm long multifilament bundle was placed on an ATR slide and fixed at both ends with adhesive tape. The spectrum of each sample was recorded in ATR mode using a slide-on ATR objective equipped with a conical germanium crystal. All measurements were performed in the 700–4000 cm range. -1 Range: 8cm resolution -1 , acquired with an aperture of 100 × 100 μm.

[0060] Thermogravimetric analysis (TGA) The thermal stability of PEO-collagen multifilaments and PEO:collagen multifilaments washed with 10X PBS solution was measured under argon atmosphere at a heating rate of 10°C / min up to 600°C. The weight percentage change and decomposition temperature of the multifilaments were recorded on an SDT-Q600 (TA) thermogravimetric analyzer.

[0061] Results and Discussion: Using a contact drawing method with a multi-pin tool and a flat plate containing a viscous polymer-collagen solution, we were able to fabricate thousands of aligned PEO-collagen monofilaments in approximately 1-second contact-draw cycles (Figure 1, Panel A, Step 1). After each contact, a series of liquid bridges form from each pin, which are then stretched into monofilaments by elongational flow. These monofilaments are collected on a frame and allowed to dry. Unlike other fiber formation methods that may require nozzles, high-voltage power supplies, flow rate control, and complex mechanical control, contact drawing only requires that the polymer chains form entanglements in solution, that the drawing be fast enough to prevent deentanglement, and that the pin array be spaced far enough apart to prevent the liquid bridges from merging during fiber drawing.

[0062] Production of collagen multifilament bundles Thirty contact-drawing operations were required to produce approximately 50,000 monofilaments within a 12 x 8 cm frame. The monofilaments were consolidated by bringing the ends together and twisting them simultaneously clockwise and counterclockwise (Figure 1, Panel A, steps 2 and 3). The twisted multifilament was then gently rolled on a flat plastic surface (Figure 1, Panel A, step 4). This process yielded well-consolidated multifilament structures at least 9 cm long. The diameter could be adjusted by varying the number of monofilaments forming each bundle (Figure 1, Panel B and Figure 1, Panel C). A typical multifilament bundle formed from 50,000 monofilaments had an average diameter of 190 μm and a twist angle of 6° ± 2° (Figure 1, Panel D).

[0063] When multifilament bundles were directly immersed in 1X PBS, the monofilaments broke and dispersed (Figure 2, panel A). One explanation for this result is that immersing multifilaments directly in PBS generates osmotic shock due to the large difference in PEO concentration between the multifilaments and the surrounding PBS. Therefore, to promote collagen self-assembly, remove PEO, and preserve the structure of the multifilament bundle, a hydration protocol was developed in which PEO-collagen multifilaments were washed with decreasing PEO concentrations in PBS. Washing multifilaments with decreasing PEO concentrations in PBS reduces osmotic shock at each wash step. Therefore, multifilament bundles were washed in a graded PEO series buffered with 1X or 10X PBS (Figure 2, panel B). This graded hydration approach maintains the twist angle of the multifilament bundle and allows sufficient time for collagen molecules to self-assemble into fibrils.

[0064] Molecular orientation and packing of collagen in multifilament bundles. Figure 3, Panel F shows the azimuth-averaged WAXS profiles of a pure PEO multifilament bundle (Figure 3, Panel A), a PEO-collagen multifilament bundle (Figure 3, Panels B–D), and a rat tail tendon (Figure 3, Panel E). The WAXS pattern of the pure PEO multifilament bundle indicates that the PEO chains are oriented along the multifilament axis (Figure 3, Panel A). The corresponding WAXS profile (Figure 3, Panel F) shows two high-intensity diffraction peaks at 19.14° and 23.27°, which can be assigned to the (120) and (112) planes of crystalline PEO, respectively. These sharp peaks arise from the alignment of the polyether chains due to van der Waals interactions between PEO chains. This indicates that the multi-pin contact drawing technique and all the manufacturing processes used in this study to fabricate the collagen multifilament bundle did not affect the formation of PEO crystals. As shown in Figure 3, Panel B, two spots appear near the beam center for the PEO-collagen multifilament bundle. A small peak centered at a 2θ angle of 7.68° (d = 1.151 nm) (Figure 3, Panel F) indicates the presence of collagen and is attributed to the intermolecular lateral packing of collagen triple helices. Addition of collagen does not alter the alignment of PEO chains. When PEO-collagen multifilament bundles were hydrated in serial dilutions of PEO:1X PBS or PEO:10X PBS to remove the PEO, several new peaks appeared, indicating the presence and alignment of collagen triple helices along the multifilament direction (Figure 3, Panel C and Figure 3, Panel D and Figure 3, Panel F). The peaks at (2θ = 31.19°, d = 0.287 nm) and (2θ = 20.29°, d = 0.437 nm) are assigned to the inter-residue distance along the triple helix axis and amorphous scattering originating from the disordered portion of the collagen molecule, respectively. The WAXS pattern of the multifilament bundle after PEO hydration is indistinguishable from the well-aligned triple helices present in rat tail tendon ( Figure 3 , panel E and Figure 3 , panel F).The main difference between tendon and multifilament collagen chains is a slight increase in the intermolecular lateral packing distance between collagen molecules (tendon: 2θ = 8.16°, d = 1.08 nm; multifilament: 2θ = 7.84°, d = 1.127 nm) (Figure 3, panel F).

[0065] To gain a deeper understanding of the supramolecular structure of collagen molecules within the multifilament bundle, we used SAXS analysis. Figure 4, Panels A through D, show the SAXS patterns of a PEO-collagen multifilament, a PEO-collagen multifilament washed with PEO:1X PBS solution, a PEO-collagen multifilament washed with PEO:10X PBS solution, and a rat tail. The SAXS profiles for all these conditions are shown in Figure 4, Panel E. The PEO-collagen multifilament did not exhibit the periodic SAXS pattern shown in Figure 4, Panel A, indicating that the presence of PEO chains prevented collagen molecules from self-assembling into tendon-like packing during fiber formation. Stepwise washing of the PEO-collagen multifilament with PEO:1X PBS or PEO:10X PBS revealed the presence of four arcs with d-spacings of 11.2 nm, 9.65 nm, 8.47 nm, and 7.41 nm, respectively (Figure 4, Panel B and Figure 4, Panel C). No other arcs with smaller angles were observed, as in the rat tail tendon sample (Figure 4, panel D). However, the observed tails can be indexed as the sixth, seventh, eighth, and ninth D-band repeats of the 67.2 nm D-band repeat. This is slightly longer than the 64.8 nm D-band repeat measured in rat tail tendon. A possible explanation for this finding is that after washing, the multifilaments contain collagen microfibrils with 67.2 nm D-band repeats offset by one-sixth of the repeat. Such an arrangement would produce collagen fibers with an axial period of 11.2 nm. A similar structure has previously been observed by electron microscopy in type I collagen fibers formed at pH 7.

[0066] Collagen multifilament structure Scanning electron microscopy (SEM) confirmed that the collagen multifilament structure was maintained after hydration (Figure 5, Panel A). Each collagen monofilament contained numerous closely packed fibrils with diameters of 16 × 5 nm (n = 222) (Figure 5, Panel B). Each fiber exhibited a band-like pattern with a repeat period of 10 × 3.8 nm (n = 84) (Figure 5, Panel C), consistent with the axial period of 11.2 nm observed by SAXS. The visibility of this spacing in the SEM image varied along the length of each collagen fiber, with short segments showing clear periodicity (Figure 5, Panel D). In contrast, after a 2-minute wash in 1X PBS, no band-like structure was observed by transmission electron microscopy (TEM) (Figure 6, Panel A). These rapidly washed collagen monofilaments showed no axial repeats, but rather well-aligned collagen fibrils. This is consistent with the idea that axial repeats emerge after collagen molecules laterally aggregate into microfibrils and fibrils. After stepwise washing for 1 h each, collagen fibrils were still present, and the appearance of a banding pattern along the monofilament axis was observed (Figure 6, Panel B and Figure 6, Panel C). However, the quality and length of the axial repeats varied from monofilament to monofilament (compare Figure 6, Panel B with Figure 5, Panel C), with some monofilaments exhibiting axial repeats in the 5-7 nm range, equivalent to 1 / 11 of the D-band repeats (Figure 6, Panel C).

[0067] A model of the organization of collagen fibrils within a collagen multifilament is shown in Figure 7. This model is based on the rat tail tendon microfibril structure obtained by Orgel et al. (JPRO Orgel, TC Irving, A. Miller, TJ Wess, Proc. Natl. Acad. Sci. USA 2006, 103, 9001). Figure 7A shows a stick model of six parallel microfibrils, each with a quarter-wave alternating molecular arrangement, and Figure 7B shows predicted electron density profiles along the microfibril axis. Figure 7, panel C, shows a collagen fibril model constructed using the density profile of a quarter-wave-shifted microfibril (Figure 7, panel D). As a result of the microfibril offset, the D-band repeat (67 nm) is almost completely removed and replaced with a prominent (11 nm) repeat. It should be noted that the electron density fluctuations are primarily due to the edges of the overlapping region where the N- and C-terminal domains are located (blue and red markers on the stick model in Figure 7). Furthermore, sequence analysis of type I collagen reveals that most amino acids in the sequence have repeats of approximately 39 residues, including one-sixth of the D-band repeats, i.e., phenylalanine. This means that in the model shown in Figure 7, phenylalanine residues on the surface of adjacent microfibrils are more likely to be in close proximity compared to phenylalanine residues in standard D-band fibrils. Phenylalanine and tyrosine are the only two naturally occurring amino acids that can be photocrosslinked to other phenylalanines or tyrosines, respectively, by UVC irradiation without a photoinitiator. In other words, washed collagen multifilament bundles are likely to be susceptible to UVC crosslinking based on their molecular structure.

[0068] Thermogravimetric analysis (TGA) The thermal degradation of PEO-collagen multifilaments proceeds in a three-stage degradation process, as evidenced by the three peaks observed in differential thermogravimetry (DTG) (Figure 10, Panel B). The first peak, appearing at 58°C in the TG and DTG curves, is attributed to the evaporation of unbound water (G. Ramanathan, et al. J. Biomater. Tissue Eng. 2014, 4, 203). The second peak, appearing at 310°C in the DTG curve, is due to the decomposition of collagen (BH Leon-Mancilla, et al. J. Appl. Res. Technol. 2016, 14, 77). The third peak, appearing at 411°C, corresponds to the decomposition of PEO within the multifilaments (NS Vrandecic, et al. Thermochim. Acta 2010, 498, 71).

[0069] To estimate the weight percent of PEO in the washed multifilament, the area of ​​the PEO peak at 411 °C in the DTG curve (Figure 10, panel B) was estimated and compared with the areas of the peaks at 310 °C and 411 °C in the DTG curve (Figure 10, panel B). Using this method, which is a modification of ASTM E1131-20 "Standard Test Method for Compositional Analysis by Thermogravimetry," the PEO content was determined to be approximately 2.5 wt%.

[0070] Effect of cross-linking on the tensile properties of collagen multifilament bundles To investigate the effect of crosslinking on ultimate tensile strength (UTS) and Young's modulus (E), collagen multifilament bundles hydrated in a PEO:10x PBS solution were treated with UVC irradiation or 1.0% glutaraldehyde. UVC irradiation produces zero-length crosslinks without a photoinitiator, mimicking in vivo enzymatic crosslinks formed by the activity of lysyl oxidase. Glutaraldehyde, on the other hand, exhibits excellent mechanical properties and biodegradability.

[0071] UVC treatment dramatically increased the UTS and Young's modulus of the multifilament bundle in a dose-dependent manner (Figure 8A-C). This indicated that the number of cross-links increased with UVC dose. At the highest UVC dose tested (10 J / cm), 2 ), the UTS and Young's modulus values ​​exceeded those of the 1.0% glutaraldehyde-treated collagen multifilament bundles. Compared to the uncrosslinked collagen multifilament bundles, the values ​​were 10 J / cm 2 At a UVC dose of 10 J / cm, the UTS increased 22-fold to 38.5 ± 0.8 MPa (Figure 8B), which is within the lower limit of values ​​reported for mammalian tendons. This UTS is similar to that of wet-spun glutaraldehyde-crosslinked collagen multifilaments (40 MPa) and to that reported for rat tail tendon fibers (39 MPa). A similar trend was observed for Young's modulus, which increased 19-fold compared to uncrosslinked collagen multifilament bundles at a UVC dose of 10 J / cm. 2 The swelling reached 638 ± 95 MPa at 10 J / cm (Figure 8, panel C), exceeding that of wet-spun glutaraldehyde-crosslinked collagen multifilaments (281 MPa). The swelling rate was inversely proportional to the UVC dose (Figure 8, panel D). The uncrosslinked collagen multifilament bundle swelled by 572.7 ± 141.8% within 1.5 h after incubation, but only at 10 J / cm. 2 The UVC-crosslinked multifilaments showed only 120.1 ± 4.3% (Figure 8, panel D). A statistical analysis comparing the UTS, Young's modulus, and swelling ratio under various crosslinking conditions is shown in Table 1. [Table 1]

[0072] This is not the first attempt to crosslink collagen materials using UV without the use of a photoinitiator. Previous studies have shown that collagen crosslinking and collagen denaturation compete during UV treatment. In other words, the main limitation of UV in other collagen materials is a radiation dose of 1 J / cm. 2In order to evaluate the change in collagen structure, Figure 9 shows the results of irradiation of 10 J / cm2 on a PEO-collagen multifilament bundle washed in a stepwise prepared PEO:10X PBS solution and a PEO-collagen multifilament bundle washed in a stepwise prepared PEO:10X PBS solution. 2 Figure 9 shows the WAXS patterns and ATR-FTIR spectra after UVC irradiation. From the WAXS patterns in Figure 9, panels A to C, it is clear that there is no change in peak shape or intensity between the uncrosslinked and crosslinked PEO collagen multifilament bundles, indicating that the spacing of the collagen triple helices and the distance between residues along the triple helix axis remain unchanged after UV crosslinking.

[0073] The ATR-FTIR spectrum shown in panel D of Figure 9 shows the peak at 1635 cm -1 , 1542cm -1 , 1238cm -1 The absorption bands at 3305 cm are those of amide I, amide II, and amide III, respectively. These are thought to be due to the presence of collagen in the multifilament. These absorption bands are related to the C=O stretching vibration, the NH bending vibration of the polypeptide chain, and the combination of the CN stretching vibration and the NH bending vibration, respectively. -1 The absorption band of amide A was observed at 3077 cm. This is due to the NH stretching vibration. Finally, -1 A weak absorption band of amide B was present at 100 nm, which is attributed to the C-N stretching vibration of the peptide bond. After UVC treatment, there was no visible change in the position or relative intensity of either band (Figure 9, panel D). This provided further evidence that the chemical composition of the collagen multifilament bundles was not altered by the UVC crosslinking process.

[0074] Typically, crosslinks formed during UVC irradiation result from the formation of free radicals on aromatic amino acid residues such as phenylalanine and tyrosine, which absorb strongly in that wavelength range and account for less than 2% of the amino acid residues in collagen I molecules. These two residues are thought to react first with water molecules bound to the collagen molecule in the dry state to form phenoxyl and tyrosyl radicals that can react with adjacent side chains of the same type to form covalent bonds. UVC irradiation is typically 10 J / cm². 2 The lack of molecular-level structural and chemical changes within the UVC-exposed multifilaments, combined with the observed improvement in tensile properties (Figure 8), suggests that UVC treatment may generate new C-C or C-N bonds within the multifilaments. In addition to these qualitative findings, the presence of amide III and 1450 cm bonds was also observed for both crosslinked and non-crosslinked collagen multifilament bundles. -1 Absorption ratio (1238cm -1 / 1450cm -1 ) was calculated, which allows us to evaluate the degree of triple helix structure preservation. A ratio of approximately 1.0 indicates that the triple helix structure is properly folded, whereas for denatured collagen (gelatin), this ratio is approximately 0.6. Here, the ratios for crosslinked and non-crosslinked collagen multifilaments were 1.002 and 0.996, respectively. This further confirms that UVC treatment did not disrupt the triple helix structure within collagen. Taken together, these results support the structural model prediction that a one-sixth shift in the D band of microfibrils within multifilaments brings normally distant phenylalanine residues into sufficient proximity to allow efficient UVC crosslinking without molecular damage.

[0075] Collagen monofilaments were fabricated by drawing an entangled polymer solution consisting of acid-solubilized collagen and polyethylene oxide (PEO) using a multi-pin contact drawing method. This method simultaneously produced thousands of PEO collagen monofilaments, measuring up to 35 cm in length and 1–5 μm in diameter, which were then assembled on a frame and consolidated into multifilament bundles resembling tendon bundles. Next, the multifilament bundles were hydrated with graded concentrations of PEO and PBS, promoting the assembly of collagen fibrils within each monofilament while maintaining the structure of the multifilament bundle. Wide-angle X-ray scattering (WAXS) and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) confirmed the presence of collagen in the hydrated multifilament bundles and the formation of an ordered collagen structure. The hierarchical structural characteristics were further investigated by small-angle X-ray scattering (SAXS), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). SAXS and electron microscopy results are consistent with collagen fibrils containing microfibrils, with a periodicity of 11 nm, offset by exactly one-sixth of the microfibril D-band spacing. Sequence analysis predicted that in this structure, phenylalanine residues are sufficiently close together within and between microfibrils for ultraviolet C (UVC) crosslinking to occur. Phenylalanine crosslinking does not occur spontaneously. Consistent with this analysis, the maximum tensile strength and Young's modulus of hydrated collagen multifilament bundles crosslinked by UVC irradiation increased nonlinearly with total UVC energy, reaching values ​​in the range of those of natural tendons. ATR-FTIR and WAXS analysis showed that the chemical and molecular structure of hydrated collagen multifilaments was maintained after UVC crosslinking, further supporting the concept of phenylalanine-specific crosslinking. This fabrication method reproduces tendon structure across multiple length scales and allows for tuning of tensile properties using only collagen molecules and without any chemical additives other than PEO, which is removed during the hydration process.

[0076] The novel features will be apparent to those skilled in the art upon review of this specification. However, it should be understood that the scope of the claims should not be limited by the embodiments, but should be given the broadest interpretation consistent with the language of the claims and the entire specification. All documents mentioned in this specification are incorporated herein by reference.

Claims

1. 1. A method for producing a multifilament collagen fiber bundle, comprising treating a polymer collagen fiber bundle comprising a plurality of monofilaments of collagen supported on a support polymer with a series of liquid media having decreasing concentrations of the support polymer, thereby removing the support polymer from the polymer collagen fiber bundle, wherein the support polymer has a higher solubility in the liquid media than the collagen.

2. 2. The method of claim 1, further comprising producing the polymer collagen fiber bundle by contact-stretching the support polymer and the collagen from a pressed composition containing the support polymer and the collagen to form polymer collagen fibers, and bundling a plurality of the polymer collagen fibers together to form the polymer collagen fiber bundle.

3. 3. The method of claim 1 or claim 2, wherein the treating comprises hydrating, and the liquid medium comprises an aqueous medium in which the support polymer is more soluble than the collagen.

4. 4. The method of claim 3, wherein at least one of the aqueous media in the series of liquid media comprises a buffer.

5. 5. The method of any one of claims 1 to 4, wherein the series of liquid media comprises a series of aqueous media, the series of aqueous media including a first aqueous medium, the first aqueous medium comprising no more than 10 wt% of the support polymer, based on the total weight of the first aqueous medium.

6. The method of claim 5, wherein the first aqueous medium comprises 0.25 to 5% by weight of the support polymer.

7. 5. The method of any one of claims 1 to 4, wherein the series of liquid media comprises a first aqueous medium comprising 0.75 to 2 wt. % of the support polymer, a second aqueous medium comprising 0.35 to 0.75 wt. % of the support polymer, and a third aqueous medium comprising 0.1 to 0.35 wt. % of the support polymer, all weight percentages being based on the total weight of the respective aqueous medium.

8. The method of any one of claims 1 to 7, wherein the series of liquid media includes a final aqueous medium that does not contain the support polymer.

9. The method of any one of claims 5 to 8, wherein the polymer-collagen fiber bundles are treated in the aqueous medium of the series of liquid media for 1 to 24 hours.

10. 9. The method of any one of claims 1 to 8, wherein the concentration of the support polymer in the liquid medium is successively decreased, at least in part, as the liquid medium is successively replaced or diluted with other liquid media having progressively lower concentrations of the support polymer.

11. 1. A multifilament collagen fiber bundle comprising collagen fibers having a diameter of at least 0.3 μm, wherein each collagen fiber comprises a collagen fibril having a diameter smaller than said collagen fiber, each collagen fibril comprises a collagen microfibril having a diameter smaller than said collagen fibril, each collagen microfibril comprises a bundle of collagen molecules, said collagen microfibrils form a hierarchical D-band structure within said collagen fibril, and adjacent collagen microfibrils are offset by one-sixth of a D-band compared to microfibrils in native collagen of the same type.

12. 12. The multifilament collagen fiber bundle of claim 11, wherein the collagen molecules are type I, type III, or type V collagen.

13. The multifilament collagen fiber bundle according to claim 11 or 12, wherein the collagen molecules are atelomeric.

14. The multifilament collagen fiber bundle according to any one of claims 11 to 13, further comprising one or more additives.

15. A method for producing a crosslinked multifilament collagen fiber bundle, comprising: applying a crosslinked multifilament collagen fiber bundle according to any one of claims 11 to 14 to a fiber bundle of 0.1 J / cm ２ irradiating with ultraviolet light at a total energy dose of at least 100 wt. %.

16. The total energy dose is 5 J / cm ２ ~100 J / cm ２ The method of claim 15, wherein the range is

17. 17. The method of claim 15 or claim 16, carried out without a photoinitiator.

18. 1. A crosslinked multifilament collagen fiber bundle comprising collagen fibers having a diameter of at least 0.3 μm, wherein each collagen fiber comprises a collagen fibril having a diameter smaller than said collagen fiber, each collagen fibril comprises a collagen microfibril having a diameter smaller than said collagen fibril, each collagen microfibril comprises a bundle of collagen molecules, said collagen microfibrils form a hierarchical D-band structure within said collagen, and adjacent collagen microfibrils are offset by one-sixth of a D-band compared to microfibrils in native collagen of the same type.

19. 20. The crosslinked multifilament collagen fiber bundle of claim 18, wherein the collagen molecules are atelomeric.

20. 20. The crosslinked multifilament collagen fiber bundle of claim 18 or claim 19, further comprising one or more additives.

21. The crosslinked multifilament collagen fiber bundle according to any one of claims 18 to 20, having an ultimate tensile strength of 1 MPa or more and / or a Young's modulus of 20 MPa or more.

22. A method for producing a multifilament collagen fiber bundle, comprising treating a polymer collagen fiber bundle comprising a plurality of monofilaments of collagen supported on a support polymer with a series of liquid media of decreasing osmotic pressure to remove the support polymer from the polymer collagen fiber bundle, wherein the support polymer has a higher solubility in the liquid media than the collagen.

23. A collagen fiber having a diameter of at least 0.3 μm, comprising collagen fibrils of a smaller diameter than said collagen fiber, each collagen fibril comprising collagen microfibrils of a smaller diameter than said collagen fibril, each collagen microfibril comprising a bundle of collagen molecules, said collagen microfibrils forming a hierarchical D-band structure within said collagen fibril, wherein adjacent collagen microfibrils are offset by one-sixth of a D-band compared to microfibrils in native collagen of the same type.

24. 24. The collagen fiber of claim 23, wherein the collagen molecules are atelomeric.

25. Collagen fibers, atelomeric collagen microfibrils that form a hierarchical D-band structure within the collagen fibrils, wherein adjacent collagen microfibrils are offset by one-sixth of a D-band compared to microfibrils in the same type of native collagen; and polyethylene oxide (PEO) in an amount of 60% by weight or less, based on the total weight of the collagen fibers; Contains collagen fibers.

26. The collagen fiber according to claim 25, wherein the amount of PEO is in the range of 0.0001 to 50% by weight.

27. The collagen fibers of any one of claims 23 to 26, further comprising one or more additives.

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