Biocompatible repair strengthening silk fabric

[0001] Disease, aging, trauma or chronic wear often lead to tissue or organ damage. In the treatment of such abnormalities, the goal of many clinical procedures is to restore function. The patient often requires additional support beyond the self-healing capabilities of the body, such as surgery or implantation of a medical device. Such procedures are often needed to combat permanent disability and even death. The fields of biomaterials and tissue engineering provide new options for the gradual restoration of native tissue and organ functions by researching and developing temporary scaffolds, matrixes and constructs (i.e., devices) that initially support inefficient tissues or organs, but gradually allow the body's own tissue to grow and rebuild biologically and mechanically functional.

[0002] Tasks or requirements associated with the construction of such a scaffold are: (i) the ability to provide damaged or diseased tissue with immediate mechanical stabilization, (ii) to support the ingrowth of the cell and tissue into the device, (iii) to provide communication between the mechanical environment of the body and the developing tissue; this is achievable due to the appropriate mechanical and biological design of the device, (iv) degradation at such a rate that the ingrown cells and tissues have sufficient time for remodeling, thus creating a new autologically functional tissue that can save the patient's life. In some cases, the device should mimic the correct three-dimensional structure (e.g., bone skeleton) in the tissue it is to support. In other cases, the device can serve as a temporary ligation (e.g., a flat mesh to repair a hernia or a hemostat in the event of bleeding) in three-dimensional tissue (abdominal wall muscle in the case of a hernia). Regardless of the application, the current direction of the field of medical devices is aimed at completely restoring the body's functions by supporting the development of autologous tissues.

[0003] Unfortunately, most biomaterials currently available do not have mechanical integrity for applications requiring high loads (e.g. bones, ligaments, tendons, muscles) or adequate biological functionality; most biomaterials either degrade too quickly (e.g. collagen, PLA, PGA, or related copolymers) or are non-degradable (e.g. polyesters, metal), and in any case, functionally autologous tissue does not develop and the patient experiences disability. In some cases, the biomaterial may mistakenly redirect tissue differentiation and development (e.g., spontaneous bone formation, cancer) because it is not biocompatible with surrounding cells and tissue. Similarly, non-degradable biomaterials are usually associated with chronic inflammation, while such a response is actually harmful to the surrounding tissue (i.e. debilitating).

[0004] If properly designed, silk may offer new clinical options for developing a new class of medical devices, scaffolding and matrices. Silk has been shown to have the highest strength of all natural fibers and compete with mechanical properties with high performance synthetic fibers. Silks are also stable at high physiological temperatures and in a wide pH range and are insoluble in most aqueous and organic solvents. Silk is a protein, not a synthetic polymer, and degradation products (e.g., peptides, amino acids) are biocompatible. Silk is not of mammalian origin and leads to a much lower biological load than other comparable natural biomaterials (e.g. bovine or porcine collagen).

[0005] Silk, as the term is widely known in the art, means a textile product made of filaments secreted by an organism, such as a silkworm or spider. Silks produced by insects, namely (i) silkworms Bombyx mori and (ii) spider glands, usually Nephilia clavipes, are the most commonly studied forms of the material, but in nature there are hundreds to thousands of natural

EP 2 374 919 variants of silk. Fibroin is produced and secreted by two silkworm silk glands. When fibroin leaves the glands, it is covered with sericin, a glue-like substance. However, spider silk is valued (and distinguished from silkworm silk) as it is produced as a single fiber free of immunogenic impurities such as sericin.

[0006] Unfortunately, spider silk cannot be mass-produced because of the inability to tame spiders. However, spider silk, as well as other silks, can be cloned and produced recombinantly, but with extremely different results: Often, these processes introduce bioburden, are expensive, they cannot be used to obtain significant amounts of material, and result in highly variable properties and are neither strictly controlled nor reproducible.

[0007] As a result, for over 1000 years only silkworm silk has been used in biomedical applications. The silkworm species Bombyx mori produces silk fiber (known as "bave" - ​​silk fiber) and uses this fiber to build its cocoon. The silk fiber, as manufactured, contains two filaments (fibers) of fibroin or "broin", which are surrounded by a coating of resin known as sericin - the silk fiber fibroin has significant mechanical strength. When the silk fibers are collected for the production of yarn or textiles, including sutures, many fibers can be folded together and the sericin is partially dissolved and then solidified again to create a larger silk fiber structure for more than two broins mutually embedded in a sericin coating.

[0008] The term "fibroin" as used herein includes silkworm fibroin (ie from Bombyx mori) and fibroin-like fibers obtained from spiders (ie from Nephila clavipes). Alternatively, silk protein suitable for use in the present invention can be obtained from a solution containing silk engineered by genetic engineering, such as bacteria, yeast, mammalian cells, transgenic animals or transgenic plants. See, for example, WO 97/08315 and US Patent No. 5,245,012.

[0009] Silk fibers of silkworms traditionally available on the market for textile applications and as suture materials are often "degummed" and consist of many broins woven together to form a larger single multifilament fiber. De-resinizing refers here to the release of a sericin coating surrounding two broins by flushing or extraction in hot soapy water. This release allows braiding broin and producing larger individual multifilament fibers. However, complete extraction is often not achieved or desired. Reinforced silk often contains sericin or is re-coated with sericin and / or sericin impurities are introduced during braiding to clot a single multifilament fiber. A sericin coating protects brittle fibroin fibrils (only ~ 5 microns in diameter) against abrasion during traditional textile applications where highly efficient processing is required. In this regard, nourished silk, unless clearly indicated as being free of sericin, usually contains 10-26% (by weight) of sericin (see Tables 1 and 2).

[0010] It is generally concluded from literature references to "silk" that they focus on naturally occurring and only available "silk" (ie, sericin-coated fibroin fibers) that have been used for centuries in textiles and medicine. Medical grade silkworm silk is traditionally used in only two forms: (i) as intact silk sutures from which sericin has not been removed, and (ii) traditional more popular silk sutures, commonly called black silk braided suture, with whose sericin has been completely removed but replaced with a wax or silicone coating to provide a barrier between silk fibroin and tissue and body cells. Currently, the only medical application in which silk is still used is surgical thread ligation, especially because silk is still valued in surgery for its mechanical properties (e.g., knot strength and ease of use).

[0011] Despite the use of silk in its natural state as a suture material for thousands of years, the emergence of new biomaterials (collagen, synthetics) has made it possible to compare materials and identify problems with sericin. Silk, or more clearly defined as silkworm Bombyx mori, is not biocompatible. Sericin is antigenic and causes a strong immune, allergic or excessive T-cell type response (compared to a normal mild response to a "foreign body"). Sericin can be removed (rinsed / extracted) from silk fibroin, however, removing sericin from silk alters the ultrastructure of fibroin fibers, resulting in their exposure and resulting in loss of mechanical strength, which leads to brittle structure.

[0012] The extracted silk structures (ie, yarns, matrices) are particularly sensitive to abrasion and mechanical damage during standard textile procedures due to the multifilament nature of the smaller diameter fibroin fibers (~ 5 μm). The fragility of extracted fibroin is the reason that the use of silk in the design and development of medical devices, after extraction, usually (Perez-Rigueiro, J. Appl. Polymer Science, 70, 2439-2447, 1998) involves dissolving and reconstituting silk using standard methods (US Patent No. 5,252,285) to obtain a processable biomaterial. The inability to process silk fibroin extracted using modern methods and textile machinery prevented the use of non-dissolved, sericin-free fibroin as a medical device.

[0013] Additional restrictions on silk fibroin, whether extracted from silkworms, dissolved and reconstituted, or produced by insects other than silkworms include (i) the hydrophobic nature of silk, resulting directly from the beta-sheet conformation of the fibroin core protein crystal, which gives silk its strength, (ii) the absence of cell binding domains usually found in extracellular matrix mammalian proteins (e.g. RGD peptide sequences), and (iii) the smooth surface of silk fibroin. As a result, cells (e.g., macrophages, neutrophils) associated with inflammation and response of host tissues are unable to recognize silk fibroin as a degradable material. These cells will thus decide to encapsulate and delineate the foreign body (see Fig. 18A), thus limiting (i) silk fibroin degradation, (ii) tissue ingrowth, and (iii) tissue remodeling. Thus, silk fibroin filaments often induce a strong foreign body response (FBR) that is associated with chronic inflammation, peripheral granulomatosis, and scar encapsulation (Fig. 18A).

[0014] In addition to the biological defects of silk, the multifilament nature of silk (e.g., as a suture material), as well as the small size of fibroin filaments can lead to a tightly packed structure. As such, silk can degrade too quickly. Proteases (enzymes) produced by stimulated cells within the peripheral encapsulation can penetrate the implanted structure (see Fig. 11A and Fig. 11B), but cells that accumulate as new tissue (e.g. fibroblasts) that can strengthen the device (in this case, black plaited suture) during normal tissue remodeling, cannot. Therefore, the interior of untreated or unmodified fibroin products does not come into contact with foreign body and tissue responses (elongated and produced by fibroblasts) and as a result the device's ability to directly remodel tissue is limited. Host cell and tissue growth is limited and degradation is usually not possible.

[0015] For suture materials, it is believed that these problems can be solved by treating fibroin suture materials with cross-linking agents or coating them with wax, silicone or synthetic polymers, thereby protecting the material from the body. Coatings such as sericin, wax or silicone, designed to increase the mechanical stability of fibroin (by fighting its fragility while providing a barrier between the body and fibroin), reduce adhesion, recognition

EP 2 374 919 cells and tissue penetration and overgrowth and degradation of fibroin. As a result, silk is traditionally considered a non-degradable material.

[0016] Classification as non-degradable may be desirable if the silk is intended to be used as traditional suture suturing materials, ie, cell and tissue ingrowth into the device is not desirable. Therefore, the attachment and ingrowth of cells (which leads to matrix degradation and active tissue remodeling) is traditionally prevented by both the biological nature of silk and the mechanical design of the structure. In fact, the general belief that silk must be shielded from the immune system and the perception that silk is non-biodegradable results in limited silk use in surgery. Even in this area of ​​suture materials, silk has been replaced in most applications with synthetic, biodegradable or durable materials.

US 5849040 and US 6302922 describe a method of making woven or knitted material for use in textile clothing using raw silk with solid sericin.

US 5998615 refers to a material obtained from silk that has undergone a viscous treatment, for example using steam above the temperature at which the silk proteins are denatured.

US 6175053 describes a dressing material in which silk fibers are dissolved and reconstituted. WO 02/29141 relates to a method of producing a silk fibroin fabric in which the silk is treated in a soap bath at 95-98 ° C for 2-4 hours.

[0017] Therefore, there is a need to produce silkworm fibroin fibers with extracted sericin that are biocompatible, promote cell ingrowth, and are biodegradable. The present invention provides a biocompatible repair-reinforcing material as defined in claim 1.

SUMMARY [0018] The natural silk fibroin fiber constructs disclosed herein offer high strength, extended wear time, and stiffness and elongation at break, which properties closely match those of biological tissues. The fibers in the constructs are arranged in a non-random manner in one or more yarns. The fiber constructs are biocompatible (due to the extraction of sericin from silkworm silk fibers) and are essentially free of sericin. Fiber constructs are also non-immunogenic; i.e., they do not cause a significant allergic, antigenic or excessive response of the host T cells, reducing the detrimental effect on surrounding biological tissues, such as that which may accompany immune responses in another context. In addition, fiber constructs promote the ingrowth of cells around fibroin fibers and are biodegradable. [0019] The statement that the fiber construct is "substantially free" of sericin means that the fiber construct contains less than 20% by weight of sericin. Preferably, the fiber construct contains less than 10% by weight of sericin. Most preferably the fiber construct contains less than 1% by weight sericin (see Table 2). In addition, "substantially free" of sericin can be functionally defined as the content of sericin that does not cause significant allergic, antigenic or excessive host T cell response. Similarly, stating that the weight change after the second extraction is less than 3% would suggest that the first extraction "substantially removes" sericin from the construct and that the resulting construct was "substantially free" of sericin after the first extraction (see Table 2 and Fig. 1F ).

[0020] By the methods of the present disclosure, sericin is extracted from the construct much more accurately than the typical "degumming" procedures that are typical of traditional processing practices in the manufacture of silk textiles for non-surgical applications (see definition above). Figure 1A is a photo of a conditioned fiber where the fibroin filaments have been twisted together to form a larger fiber re-coated with sericin. This

EP 2 374 919 "nourished" fiber contains ~ 26%, by weight, of sericin. In a preferred embodiment, the silkworm fibroin fibers with extracted sericin retain the native structure of the proteins and have not been dissolved and reconstituted.

[0021] "Natural" silk fibroin fibers are produced by an insect, eg a silkworm or spider, and have a native, such as formed, protein structure. Preferably, the silk fibroin fiber constructs are not recombinant (i.e., they are not genetically modified) and have not been dissolved and reconstituted. In a preferred embodiment, fibroin fibers with extracted sericin include fibroin fibers obtained from silkworm Bombyx mori. In addition, the term "biodegradable" as used herein means that the fibers degrade within one year during continuous contact with body tissue. In addition, our data indicate (Fig. 13 AE, Fig. 18 AC and Fig. AD 19) that the rate of degradation can be influenced and enhanced by modifying the fibroin surface (Fig. 13 AD and Fig. 18 AC) as well as geometric configuration of the yarn and / or material (Fig. 19A-D). In one embodiment, the silk fibroin yarn lost 50% of its tensile strength within two weeks after in vivo implantation (Fig. 12) and 50% of its weight in about 30 to 90 days in vivo, depending on the implantation site ( Fig. 13 AD). In vivo selection of the implant site (e.g. intramuscular compared to subcutaneous) has been shown to have a significant effect on the rate of degradation (Fig. 13 AD).

[0022] "Textile quality silk" is a naturally occurring silk that has a sericin coating of greater than 19% - 28% by weight of the fiber. "Silk for suture applications" is silk that contains sericin ("natural silk for suture applications") or is coated with a hydrophobic composition such as beeswax, paraffin wax, silicone or synthetic polymer coating (black silk, braided surgical thread). The hydrophobic composition repels cells or inhibits the attachment of cells to coated fibers. Black braided silk is a silk suture material from which sericin has been extracted and replaced with an additional coating. Silk suture material is usually not biodegradable.

[0023] Due to the absence of a protective wax or other hydrophobic coating on the silk fibroin fibers, the described constructs are biologically (coupling of cell binding domains) and / or mechanically (increasing the silk surface and reducing the packing density), after implantation into body tissue to favor increased cell infiltration compared to silk of the quality of a textile product or silk suture material. As a result, silk fibroin constructs promote cell ingrowth and infiltration and improve cell attachment and spreading, which leads to degradation of the silk fibroin construct, thereby creating a fundamentally new biodegradable biomaterial for medical and tissue engineering applications. The ability of the fiber construct to support cell attachment and ingrowth / infiltration of cells and tissues to the construct, which in turn promotes degradation, can be further increased by modifying the fibroin surface (peptide coupling via RGD, chemical species modification and increasing hydrophilicity by plasma gas treatment) and / or mechanical design of the structure increasing the surface of the material, and thus increasing its susceptibility to those cells and enzymes that have the ability to degrade silk. Silk fibers are optionally coated with a hydrophilic composition, e.g. collagen or peptide composition, or mechanically combined with a biomaterial that promotes ingrowth of the cell and tissue to form a composite structure. The choice of biological material, amount and mechanical interaction (e.g., wrapping or plaiting around the silk fibroin core) can be used to change and / or improve the rate of cell growth and degradation of the construct.

[0024] The fibers in the construct are stacked together randomly into one or more yarns. Such a structure can be arranged in parallel, in the form of a weave, textured or spirally arranged (twisted, repeatedly threaded (e.g. wire rope)), forming a yarn. Yarn can be defined as

EP 2 374 919 consisting of at least one fibroin fiber. Preferably, the yarn consists of at least three co-held fibroin fibers. A yarn is a collection of fibers that are twisted or otherwise held together in a strand. An almost infinite amount of yarn can be generated by various means of making and joining fibers. Silk fiber has been described above, but the term fiber is a general term indicating that the structure is 100 times longer than its diameter.

[0025] When the fibers are twisted or otherwise interlaced to form yarn, they are twisted / braided enough to substantially block the relative positioning of the fibers and remove overhangs, but not enough to plastically deform the fibers (i.e. not exceeding the yield point of the material), which compromises their fatigue life (i.e., reduces the number of cycles before damage). Sericin-free fibroin fiber constructs may have ultimate tensile strength (UTS). ultimate tensile strength) at least 0.52 N / fiber (Table 1, 4) and stiffness between about 0.27 and about 0.5 N / mm per fiber. Depending on the fiber arrangement and hierarchy, we have shown that the UTS of fibroin constructs can be from 0.52 N / fiber to about 0.9 N / fiber. The fibroin constructs described here retained about 80% of their dry UTS values ​​and about 38% of their dry stiffness when tested wet (Table 5). Elongation at break between about 10% and about 50% were typical for both dry and wet tested fibroin constructs. Fibroin constructs typically achieved about 40 to 50% of their UTS value and had a fatigue life of at least 1 million cycles with a yarn load of about 20% of the ultimate tensile strength.

[0026] In one embodiment of the present invention, the stacked silkworm fibroin fibers with extracted sericin are twisted relative to each other at 0 to 11.8 twists per cm (see Tables 6 and 7). [0027] The number of layers in the geometrical arrangement of the fiber construct as well as the number of fibers / groups / bundles / strands / cords within the layer level, the method of interlacing at different levels, the number of levels and the number of fibers at each level can be varied to change the mechanical properties fiber construct (i.e. yarn) and thus the material (Tables 4 and 8). In one embodiment of the present invention, the fiber construct (i.e. yarns) is arranged in the form of a single-level hierarchical arrangement, whereby the single-level hierarchical arrangement comprises a group of parallel or interlaced yarns. Alternatively, the fiber (i.e., yarn) construct is arranged in the form of a two-level hierarchical arrangement, wherein the two-level hierarchical arrangement comprises a bundle of interwoven groups. In another embodiment of the present invention, the fiber construct (i.e. yarn) is arranged in the form of a three-level hierarchical arrangement, whereby this three-level hierarchical arrangement contains a strand of interlaced bundles. Finally, in another embodiment of the present invention, the fiber (i.e., yarn) construct is arranged in a four-level hierarchical arrangement, the four-level hierarchical arrangement comprising a string of interwoven strands.

[0028] Sericin can be removed from the fibroin fibers before being laid to the yarn or to a higher level in the hierarchical geometry of the fiber construct. After removing the sericin, the yarn is treated at low tensile values ​​(i.e., the force applied to the construct will never exceed the yield point of the material during any processing step) and with general caution and delicacy. Processing devices are also configured to limit friction materials and sharp angles in the guide elements that contact and direct the yarn during processing to protect delicate fibroin fibers from damage; one hour extraction periods are sufficient to extract sericin, but slow enough not to damage exposed filaments. Interestingly, when the silk fiber construct consisting of multiple fibers arranged in parallel was extracted under these conditions, a "single" larger sericin-free yarn was obtained (ie, the individual fibers cannot be separated from the construct again due to mechanical interactions between smaller fibroin filaments once exposed during extraction). In addition, as a result

Due to the mechanical interaction between sericin-free microfilaments, the extraction of twisted or repeatedly threaded yarns usually resulted in less "animated" yarns and systems. As a result of this phenomenon, a greater degree of flexibility was present in the design of the yarns and resulting materials, for example, higher levels of twisting values ​​per inch (TPI) could be used. twist per inch), which normally can create animated yarns that would be difficult to form into the material. An additional benefit of higher TPIs was the reduction of yarn and material rigidity (i.e. matrix flexibility can be increased) (Tables 6 and 7, Fig. 6A and Fig. 6B).

[0029] Many yarns are interlaced to produce material. Materials are generated by combining one or more individual yarns, the individual yarns being converted into textiles and medical device materials. In one embodiment of the present invention, the yarn is twisted at or below 30 twists per inch. Materials are made or formed by randomly joining yarns: weaving, knitting or sewing by sewing to produce complete materials. In one embodiment, this combination of yarn to form a material is done on a machine. However, it is very important to remember that the final material product is clearly dependent on the type of yarn used, thus, yarn design provides enormous potential to meet clinical needs. The material may be, but is not limited to, woven, knitted, warp knitted, bonded fabric, coated fabric, made on the harness, laminated, in the form of a net or combinations thereof.

[0030] It should be noted that textile braiding methods, in addition to the production of yarns, can also be used to produce materials such as flat braided material or larger circular weave (Fig. 4A). Conversely, weaving and knitting, two ways of forming the material, although not widely used, can also be used to make yarns. In such cases, the distinction between 'yarn' and 'material' is not entirely obvious, and uniformity should be used to make clear distinctions, ie yarn is usually more homogeneous in composition and structure than material.

[0031] In one embodiment of the present invention, many silkworm silk fibers can be arranged in a spiral (e.g., twisted or multiple threaded) or in parallel, at one hierarchical level or at many levels, extracted and used to obtain braided suture material for tissue ligation. In another embodiment, the interacting mechanically extracted fibroin filaments in a twisted or multiple-threaded configuration after extraction can be used as a medical suture material.

[0032] The nonwovens can be formed by randomly arranging multiple yarns or one yarn cut into many parts of short length. Non-limiting examples include hemostatic material or bone scaffolding. All materials can come from either a single yarn construct (homogeneous) or multiple yarn constructs (heterogeneous). The ability to design different silk fiber fibroin yarn structures, as described in detail below, significantly increases the material design possibilities if a heterogeneous material structure is considered.

[0033] In one embodiment of the present invention, the material is a composite of fibroin fibers or yarn with extracted sericin and one or more degradable polymers selected from the group consisting of collagens, polylactic acid or its copolymers, polyglycolic acid or its copolymers, polyanhydrides, elastin, glycosaminoglycans and polysaccharides. In addition, the material of the present invention can be modified to include material associated drug or cell adhesion factor (i.e., RGD). In one embodiment of the present invention, the material is treated with plasma gas or seeded with biological cells.

[0034] Additional aspects of the present disclosure relate to the repair of specific body tissues such as hernia, bladder tissues and suspensions, reconstruction of pelvic lining tissue, tissues

EP 2 374 919 peritoneal wall, vessels (e.g. arteries), muscle tissue (smooth muscles of the abdomen, heart), haemostatic factors and ligaments and tendons of the knee and / or shoulder blade, as well as other structures often damaged due to injury or chronic wear . Examples of ligaments and tendons that can be produced include, but are not limited to, anterior cruciate ligaments, posterior cruciate ligaments, rotational belt tendons, lateral medial ligaments of the elbow and knees, hand flexor tendons, lateral ankle ligaments and tendons and ligaments of the jaw or temporomandibular joint. Other tissues that can be produced by the methods of this disclosure include, but are not limited to, cartilage (both joints and menisci), bones, skin, blood vessels, stents for supporting and / or repairing vessels and generally soft connective tissue.

[0035] In other aspects, silkworm fibroin fibers, in the form of yarn or larger yarn constructs, referred to herein as devices, are stripped of sericin and processed into a material (e.g. woven, knitted, wet-laid nonwoven, plaited, stapled, etc.), sterilized and used as an implantable support or repair material that offers controlled durability (i.e., degradation) and controlled collagen and / or extracellular matrix deposition . The support or repair material can be used for any such purpose in the body, in particular it can be used to repair a hernia, reconstruct the walls of the body, in particular in relation to the chest and abdominal cavity, and to support, position or immobilize internal organs, including without limitation , bladder, uterus, intestines, urethra and ureters. Alternatively, silkworm fibroin fibers can be stripped of sericin and stacked into the nonwoven. Such a non-woven fabric can be used as an implantable support or repair material as above, but more specifically for applications where sponge formation would be useful.

[0036] Purified silk can be purified by any treatment method that removes sericin proteins present in native fibrils. Sericin is sufficiently removed when implants from purified silk induce only mild, transient reactions to a foreign body in the absence of an antigenic response (B-cells, T-cells), i.e. they are biocompatible. The reaction to foreign bodies is characterized by an internal layer of macrophages and / or giant cells with a secondary zone of fibroblasts and connective tissue. It has been shown that the degree of foreign body response is controlled by fibroin modification (Fig. 13 AD and Fig. 18 AC) and yarn design (Fig. 19 AD). Sericin can be removed from individual silkworm fibroin fibers, a group of silkworm fibroin fibers (i.e. yarns), oriented (e.g., parallel or twisted) or forming a material or other construct containing multiple yarns. The construct can then be sterilized and implanted in the body as a medical device.

[0037] Other features and advantages of the invention will become apparent from the following description of preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES [0038] Fig. 1A is a photo from a scanning electron microscope (SEM) of a single native 20/22 denier nourished and braided 20/22 silk fiber having a sericin coating.

[0039] Fig. 1B shows the SEM of the silk fibers of FIG. 1A subjected to extraction for 60 min. at 37 ° C. [0040] Fig. 1C illustrates the SEM of the silk fibers of FIG. 1A extracted for 60 minutes at 90 ° C and illustrates complete removal of the sericin coating.

[0041] Fig. 1D is a graph illustrating the ultimate tensile strength (UTS) and stiffness (N / mm for a 3 cm long matrix) as a function of extraction conditions.

[0042] Fig. 1E shows SEM of raw silk fibroin. Fig. 1F illustrates the first extraction at 90 ° for 60 min. Fig. 1G shows the second extraction under identical conditions. These illustrations show mechanical damage to the filaments that results in typical 3% weight loss after another

EP 2 374 919 extraction. Therefore, as long as the weight% loss does not change by more than 3% from the first to the second extraction (90 ° C, 1 h, standard detergent and salt), it is assumed that the extraction has gone completely. The tool in the form of 3% total weight loss reflects the variability of measurements, tests and mechanical damage resulting in the loss of mass yarn after the second extraction.

[0043] Fig. 2A is an exemplary 3-D model of yarn (multiple-threaded or twisted) showing its 5 hierarchy levels (single fiber level not shown). Depending on the number of fibers used at each level, the cord can be used either as a yarn for knitting a mesh to repair a hernia or as a cord used in parallel with other cords to form an anterior cruciate ligament (ACL).

[0044] Fig. 2B is a schematic representation of the generation of a two-level hierarchical twisted or multiple yarn thread containing 36 fibers before being twisted in parallel to form an ACL matrix or used to generate fabric or knitwear for tissue engineering and tissue repair (e.g., hernia mesh). The schematic representation visually defines two very popular forms of material forming: 'weaving' and 'knitting'.

[0045] Fig. 2C illustrates a single yarn string with geometry that is spirally arranged around a central axis and consists of two levels of twisting hierarchy. When six strings are used in parallel (e.g., Matrix 1), the yarn has mechanical properties similar to a native ligament.

[0046] Fig. 2D illustrates a single string of yarn with geometry that is arranged spirally around a central axis and consists of three levels of twisting hierarchy. When six strings are used in parallel (e.g., Matrix 2), the matrix has mechanical properties similar to a native ligament.

[0047] Fig. 3A shows load curves as a function of elongation for five samples (n = 5) of Matrix 1 formed of six parallel silk fibroin strings shown in Fig. 2A.

[0048] Fig. 3B is a graph of failure cycles relative to UTS, 1680N and 1200N loads (n = 5 for each load) imaging Matrix 1 fatigue data. Regression analysis of Matrix 1 fatigue data when extrapolated to physiological load levels (400 N) to predicting the number of cycles to failure in vivo, indicates a matrix lifetime of 3.3 million cycles.

[0049] Fig. 3C shows load curves as a function of elongation for three samples (n = 3) Matrix 2 (n = 3) formed from six parallel silk fibroin strings shown in Fig. 2B.

[0050] Fig. 3D is a graph of damage cycles relative to UTS, loads 2280N, 2100N and 1800N (n = 3 for each load) imaging Matrix 2 fatigue data. Regression analysis of Matrix 2 fatigue data when extrapolated to physiological load levels (400 N) to predict the number of cycles to failure in vivo, indicates a matrix lifetime of 10 million cycles. [0051] Fig. 4A presents photos of many yarns and forms of materials produced in our laboratories. Several different yarn structures, including different types of weaves (i, ii, iv), flat weave (iii), weave with different diameter or convergence (v), larger (~ 250 fibers) two-level multiple threaded bundle (vi), parallel twisted and glued (yarn consisting of 24-12-fibers textured yarn (vii), various twisted yarns (viii-xi) and parallel twisted and combined yarns consisting of 24-12-fiber two-tier multiple yarns (xii).

[0052] Fig. 4B is a graph of load curves as a function of elongation for (I) weave (48 fibers, 4-drum braider using twisted extracted 12 filament yarn) and textured yarns (48 fibers in total) and (II) twisted in comparison for multiple yarns, 12 filaments in all samples were 3 cm long.

[0053] Fig. 4C is a graph of fatigue data for small yarns, 3 cm long, compared to 3B and 3D for (I) small yarn multi-threaded with 36 fibers and (II) small textured yarn with 60 fibers).

[0054] Fig. 5A provides strength and stiffness data for a yarn of 36 fibers as a function of the 6 different deformation factors at which they were tested (N = 5 per group).

[0055] Fig. 5B is load curves as a function of elongation for a yarn of 36 fibers, 3 cm in length, tested at 2 of 6 different deformation factors. The data show the result of test procedures (here, specifically the deformation coefficients) on the indicated mechanical properties (e.g. UTS) of the yarn structure.

[0056] Fig. 6A is a UTS graph as a function of turns per inch (TPI); trend lines were generated to extrapolate data to 4th degree polynomial - TPI from 0-15 is shown. A maximum was observed, which indicates an ordered structure whose individual filaments work in unison.

[0057] Fig. 6B is a stiffness plot (for a 3 cm long sample) versus twists per inch (TPI); trend lines were generated to extrapolate data to a 5th polynomial - TPI from 0-15 is shown. A maximum was observed, indicating that TPI can be used as a tool to design specific UTS or rigidity.

[0058] Fig. 7A illustrates the SEM of extracted silk fibroin prior to seeding with cells.

[0059] Fig. 7B illustrates SEM of bone marrow stromal cells inoculated and attached to silk fibroin immediately after inoculation.

[0060] Fig. 7C illustrates the SEM of bone marrow cells attached and spread on silk fibroin 1 day after inoculation.

[0061] Fig. 7D illustrates SEM of bone marrow stromal cells inoculated on silk fibroin 14 days after inoculation forming an intact extracellular matrix layer.

[0062] Fig. 8A illustrates a 3 cm long silk fibroin cord shown in Fig. 2C and inoculated with bone marrow stromal cells grown for 14 days under static conditions and stained with MTT to show even coverage of the matrix with cells after a period of growth.

[0063] Fig. 8B illustrates a silk control fiber strand MTT dyed 3 cm silk cord. [0064] Fig. 9A is a graph illustrating the proliferation of bone marrow stromal cells on Matrix 1 from silk fibroin determined by total cellular DNA over a period of 21 days of culture, indicating a significant increase in cell proliferation after 21 days of culture.

[0065] Fig. 9B is a bar graph illustrating the proliferation of bone marrow stromal cells on Silk Matrix 2 determined by total cellular DNA over a 14 day culture period, indicating a significant increase in cell proliferation after 14 days of culture.

[0066] Fig. 10 shows the ultimate tensile strength of an extracted silk fiber construct that was inoculated with bone marrow stromal cells or was not inoculated within 21 days of culture under physiological growth conditions.

[0067] Fig. 11A is a graph of UTS as a function of in vitro enzymatic degradation; no loss of strength was observed in the negative control, PBS. Silk lost 50% of its strength after 21 days of breeding. A 1mg / ml solution of Protease XIV from Sigma was used.

[0068] Fig. 11B is a graph of weight loss as a function of in vitro enzymatic degradation, no loss of strength was observed in the PBS negative control. After 41 days of culture, a 50% weight loss was observed.

[0069] Fig. 12 is a graph of loss of UTS as a function of in vitro degradation after implantation of an RGD modified matrix into an unloaded rat subcutaneous model for 10, 20 and 30 days. After ~ 10 days, a 50% loss of strength was observed in vivo under no-load conditions.

[0070] Fig. 13A shows histological sections from 12 (0) x 3 (8) non-modified and RGD modified sericin-free silk fibroin matrices 30 days after subcutaneous implantation in a Lewis rat. Row I is H - E staining at 40X, row II is H - E staining at 128x, row III is tricolor collagen staining at 128x, row IV is collagen cut from row III images to allow estimation

Collagen ingrowth and row V are pixels associated with the cross sections of the remaining silk fibroins cut out to allow quantification of degradation. After qualitative assessment, in the subcutaneous environment, both the untreated and modified groups supported cell ingrowth and collagen deposition in the matrix itself with limited encapsulation on the periphery.

[0071] Fig. 13B quantitatively shows a 36% reduction in cross-sectional area of ​​RGD-modified silk 30 days after subcutaneous implantation indicating a significant improvement in the host's ability to degrade the surface of the modified silk fibroin matrix compared to untreated controls.

[0072] Fig. 13C quantitatively shows a significant 63% increase in collagen deposition in RGD modified fibroin matrices compared to untreated controls, again demonstrating the ability of the modified silk matrix to support the host cell and grow into tissue.

[0073] Fig. 13D shows H-E staining of extracted yarn from 36 fibroin fibers implanted intramuscularly into the abdominal wall of Lewis rat. Pictures of both unmodified and modified RGD matrices are shown at 40X and 128X magnification. The results qualitatively show that RGD modification dramatically increased cell number and tissue infiltration in 30 days in vivo. In contrast to the black silk thread in the weave or natural silk thread, no encapsulation or plasma cells were observed. Compared to subcutaneous implants, little was observed until there was no cell infiltration and collagen deposition in untreated controls, indicating the effect of the implant site, beyond surface modification.

[0074] Fig. 13E is a numerical representation of in vivo weight loss for two different groups of modifications compared to untreated controls. RGD modification followed by plasma gas modification significantly (p <0.05) increases the degree of degradation 90 days after intramuscular implantation. However, as expected, degradation appears to be more aggressive in the subcutaneous environment relative to the inside muscle.

[0075] Fig. 14 shows gel electrophoretic analysis of amplification of selected markers over time by RT-PCR. The gel shows activation in both types of collagen I and III by the growth of bone marrow stromal cells on Matrix 2 for 14 days of culture, with expression levels normalized to the GAPDH primary metabolism gene. Type II collagen (as a cartilage marker) and bone sialoprotein (as an indicator of bone formation) were not detected indicating a specific differentiation reaction of Bone Marrow Stromal Cells (BMSC) against ligaments when grown with Matrix 2.

[0076] Figs. 15A and 15B show a single Matrix 1 cord (not inoculated at implantation) six weeks after implantation in vivo and used for reconstruction of the medial collateral ligament (MCL) on a rabbit model. Fig. 15A shows a fibroin fiber matrix 1 surrounded by host progenitor cells and tissue ingrowth into the matrix and around individual fibroin fibers visualized by hematoxylin and eosin staining. FIG. 15B shows the ingrowth of collagen tissue into the matrix and around individual fibroin fibers visualized by tricolor staining.

[0077] Figs. 16A, 16B and 16C show bone marrow stromal cells inoculated and grown on collagen fibers for 1 day (Fig. 16A) and 21 days (Fig. 16B), RT-PCR (Fig. 16C) and electrophoretic analysis in collagen I and III expression gel with respect to GAPDH primary metabolism gene expression: a = collagen I, day 14, b = collagen I, day 18, c = collagen III, day 14, d = collagen III, day 18, e = GAPDH , days 14, f = GAPDH, day 18. Type II collagen (as a cartilage marker) and bone sialoprotein (as an indicator of bone formation) was not detected indicating a ligament-specific differentiation response.

[0078] Fig. 17 illustrates quantitative real-time RT-PCR on day 14 on which a ratio of collagen 1 to collagen III transcript obtained, normalized to GAPDH 8.9: 1.

[0079] Fig. 18A and Fig. 18B are H-E dyed cross-sections of 6 bundles (A) 2-0 black braided silk thread and (B) modified silk with RGD on the surface (36 fibers / bundle), 30 days after intramuscular implantation. 18C is RGD modified silk pre-inoculated with bone marrow stromal cells 4 weeks before implantation. FIG. 18A shows a typical and extensive foreign body response to commercially available (Ethicon, Inc) black braided silk thread where no cell ingrowth or infiltration can be observed. Fig. 18B illustrates the ability of modified silk to promote cell and tissue ingrowth. Figures 18A, 18B and 18C show tissue response to silk fiber constructs that are wax coated (Fig. 18A), stripped of sericin and coated with RGD (Fig. 18B) and stripped of sericin and inoculated with mature progenitor stem cells (Fig. 18C).

[0080] Figs. 19A-D are H-E dyed cross-sectional images at a magnification of 40X (upper row, Fig. 19A and Fig. 19B) and 128X (lower row, Figs. 19C and 19D) of two yarns (4x3x3 and 12x3), each containing the same number of fibers, but having a different arrangement of characteristic hierarchies, after implantation on a rat model for 30 days. The results indicate that the pattern and structure of the yarn may affect the extent of cell and tissue ingrowth, as for a 12x3 yarn construct left to ingrow, while 4x3x3 appears to impede ingrowth.

[0081] Fig. twenty A, B and C show photos (A) of a non-woven fabric from a single wet-laid fiber subjected to extraction after obtaining the material (the fibers can first be extracted and formed into a non-woven data not shown), (B) knit fabric made from a chain with a mesh chain using a yarn of 12 fibers extracted after fabrication, and (C) fabric made from pre-extracted yarn of 12 fibers with pre-extracted yarn of 36 fibers running in the direction of thread.

[0082] Fig. 21 is a block diagram of various methods and sequences that can be used to create a biocompatible and biodegradable silk fibroin matrix. For example, extracting a single fiber, twisting into a yarn and knitting into materials or braiding a yarn, stranding a twisted yarn, forming a material, and then extracting. There are almost an infinite number of combinations, but they will all depend on the yarn hierarchy, number of fibers per level and TPI per level, as shown in Tables 4, 6, 7 and 8.

DETAILED DESCRIPTION [0083] In the methods described in more detail below, the silk fibroin fibers are laid in a parallel position, the fibers may remain in a strictly parallel position, or may be twisted or otherwise interlaced to form yarn. The yarn can contain any number of hierarchies, starting from the level of fibers and spreading through the levels of beams, strands, cord, etc. Interlacing can be ensured at any level. In addition, sericin is extracted from silk fibers at any point in the hierarchy, up to the point where the number of fibers exceeds that at which the extraction solution can pass through the yarn. The maximum number of silkworm fibroin fibers (20/22 den, in commercial form) that can be combined and successfully extracted is about 50 (Table 4). These yarns can then be used as a fiber construct and can be incorporated into the material for use, e.g., in forming a soft tissue mesh for repairs such as hernia repair, reconstruction of the abdominal lining and bladder suspensions. The creation of fiber constructs in the context of example applications will be discussed below.

[0084] Although much of the discussion that follows is directed to a silk-based matrix (i.e., construct, scaffolding) for the production of anterior cruciate ligament (ACL), many other tissues such as other ligaments and tendons, cartilage, muscles , bones, skin, blood vessels,

EP 2 374 919 can be obtained by means of a new matrix based on silk fiber. For ACL, coarse yarn (540-3900 fibers per yarn, before parallel braiding, see Table 8 and 11) has been described with multiple hierarchical levels of interleaving and corresponding physiological properties. In addition to the ACL matrix based on silk fiber, several smaller yarn configurations (1-50 silk fibers) (Table 1, 4 and 5) with appropriate physiological properties when combined for parallel or specific material formation, can serve as tissue matrices for targeted tissue formation ( Fig. 2AB). In addition to silk matrices for targeted tissue formation or modification, this work is particularly directly directed at the production of various matrix structures based on silk tissue supporting tissue, for targeted tissue repair (e.g. hernia, bladder suspension in urinary incontinence) (Fig. 2A-B and Fig. 20A-C).

[0085] The constructs (i.e. materials or yarns) may have a surface modified or inoculated with suitable cells (Figs. 7A-D, Figs. 8A-B and Figs. 16A-C) and, if necessary, exposed to appropriate mechanical stimulation to proliferate and differentiate into the desired ligament, tendon or other tissue according to the techniques described above.

[0086] In addition, the present invention is not limited to the use of bone marrow stromal cells to inoculate fiber and other progenitor cells and pluripotent stem cells, such as those present in bone, muscle and skin, for example, can also be used to differentiate in ligaments and other tissues.

[0087] Materials can also be formed from similar constructions of refined filaments and used in various applications. The materials can be divided into different classes, including fabrics, nonwovens, knitted fabrics and stapled fabrics, each of which includes many sub types. Each of these types can be useful as an implant in specific circumstances. In the discussion of these silk-based materials, we describe natural silk, e.g., from Bombyx mori as "fibroin fiber". These fibers should be at least one meter long and this length should be maintained throughout the process to facilitate their treatment during processing and incorporation into the material. Given that yarn can be defined as a set of twisted or otherwise held in a continuous strand and that a single fibroin fiber, as defined above, consists of many twisted broins, sometimes multiple cocoons, a single fibroin fiber can be determined as "yarn". Also, fibroin fibers are twisted together or otherwise interwoven to form "yarn". Yarns are used to weave or knit materials for use in the invention. In an alternative procedure, the silk yarns are split into smaller (5 mm to 100 mm) lengths or into silk fibroin filaments. These filaments can then be laid (wet) to form a nonwoven fabric (Fig. 20A).

[0088] When the fibers are formed into the material, the stress (force) exerted on the yarns (usually by machinery) is no greater than the yield strength of the yarn (Figs. 3A-D). Accordingly, the fibers are processed at lower speeds and less loads than the yarns that are typically used in, e.g., the production of textiles during the formation of the material, in such a way as to preserve the integrity of exposed delicate fibroin fibers. Similarly, the contact points between machinery and yarn are designed to avoid sharp corners and friction interactions to prevent destruction and fraying of fibers around the perimeter of the yarn (Figs. 4A-C).

[0089] Numerous uses of materials as implants are known in the field of medicine and surgery. One example is maintaining a hernia. For such repair, material, usually warp knit with the desired stitch (e.g. atlas stitch designed to prevent unraveling of the mesh during cutting), stitched (or sometimes stapled or glued) or simply laid in place without stretching, on the inner surface of the abdominal wall, after repair with conventional suture materials.

EP 2 374 919

One of the functions of warp knitted fabric is to provide short-term sustain for the repair. In a preferred embodiment of the present invention, fibroin fibers within the material promote cell ingrowth and subsequent cell ingrowth and subsequent tissue ingrowth into the material itself (Fig 13A and 13D) as well as through the material cracks formed during knitting and into the region requiring repair. This embodiment aims to permanently strengthen the damaged area during functional tissue ingrowth and remodeling, as the silk matrix degrades (Figures 13A, B and C).

[0090] Restorative-reinforcing materials are used in similar situations to repair or support any part of the abdominal wall, especially when repairing a hernia and reconstructing deep abdominal muscles, or to repair or support other walls and partitions in the body, e.g. in the chest or organs such as heart and bladder, especially after surgery to remove the tumor. Implantable materials can also be used to support blisters or other internal organs (including, but not limited to, the intestine, ureters and urethra and uterus) to keep them in their normal positions after surgery, damage or natural wear as a result of age or pregnancy or to put them in the right place. The "organ" here includes both "solid" organs such as the liver and ductal organs such as the small intestine or ureter. Materials, especially massive materials, such as some types of nonwoven fabric, or those that can be created by 3-dimensional knitting or braiding (Figs. 4A-C), can be used to replace defects remaining after surgery to provide a fiber construct for which they can migrate cells or to which cells can pre-attach (e.g. to increase repair speed). Places of use are defects in both soft and hard tissues such as bones. In other cases, materials are used to prevent adhesions or to prevent cell attachment and / or ingrowth, which can be achieved by modifying the surface of the silk fibroin matrix or by attaching a drug or agent to the matrix.

[0091] The silk fibroin based materials of the invention can be easily modified in a variety of ways to improve healing or in-situ repair. These modifications can be used alone or in combination. The silk-based fibroin materials of the present invention can be modified to support the attachment and spreading, ingrowth of cells and tissues, and remodeling and biodegradation of the device by using RGD peptide coupling or plasma gas radiation (Fig. 13A-E). Materials may be modified to incorporate cell adhesion factors, such as the well-known "RGD" (arginine-glycine-aspartic acid) peptide, or any of a variety of natural and synthetic attachment materials, such as serum, serous agents and proteins, including fibronectin , blood, bone marrow, groups, determinants, etc., known in the literature. Such materials may be any of the usual biochemical groups of such materials, including, without limitation, proteins, peptides, carbohydrates, polysaccharides, proteoglycans, nucleic acids, lipids, small organic molecules (less than about 2,000 Dalton) and combinations thereof. This modification of the plasma can improve the surface functionality of the material and / or the charge without affecting the mechanical properties of the major part of the materials. The materials can be irradiated with plasma gas after extraction of sericin, without losing the integrity of the silk fibroin fibers with the extracted sericin (Table 9).

[0092] In addition, the material can be treated to provide a drug. Drug attachment to the material may be covalent or covalent by degradable bonds, or by any type of binding (e.g., charge attraction) or absorption. Potentially any drug may be used, non-limiting examples of drugs include antibiotics, growth factors such as bone morphogenetic proteins (BMPs) or growth differentiation factors (GDFs), growth inhibitors, chemoattractants and nucleic acids for transformation, with or without encapsulating materials.

[0093] In another modification, cells may be added to the material prior to its implantation (Figs. 7A-D, Figs. 8A-B and Figs. 9A-B). Cells can be inoculated / absorbed into or into tissue. In addition, or in addition, cells can be cultured on the material as a first step towards tissue replacement or improvement. The cells may be of any type, but allogeneic cells, preferably "immunologically protected", "immunologically privileged" or parental cells are preferred, and autologous cells are particularly preferred. Cells are selected to be able to proliferate to the desired cell types on or in the fiber construct (Fig. 9A-B).

[0094] Another class of modification is the incorporation of other polymers into the material (e.g. in the form of fibers or gel) to provide specific structural properties or to modify the native surfaces of silk fibroin and its biological characteristics (see Fig. 16A-C: inoculation of collagen fibers with BMSC cells ). In one type of incorporation, the fibers or yarn of silk and other material are blended in the material production process. In another type, silk-based fibers, yarns or materials are coated or wrapped in a solution or wrapped in fibers of another polymer. Mixing can be carried out (i) randomly, for example by braiding (1 or multiple fibers) of both silk and polymer simultaneously in parallel before twisting, or (ii) in an arranged manner, such as for braiding, in which fibers or yarns fed into coarser yarn or material can alternate between the feeding stations creating a predictable product. Coating or wrapping, both depending on the desired effect, can be done by braiding or repeated threading around the central core, the core being a polymer, silk fibroin or composite. Alternatively, one yarn may be wrapped in a controlled manner with another polymer, wherein the wrapping yarn may be used to stabilize the structure. Any biocompatible polymer is potentially useful. Examples of suitable polymers include proteins, in particular structural proteins such as collagen and fibrin and degraded synthetic polymers such as polymers containing anhydrides, hydroxy acids, and / or carbonates. The coatings may be provided in the form of gels, in particular degradable gels, obtained from natural polymers or synthetic degradable polymers. Gels containing fibrin, collagen, and / or basement membrane proteins may be used. These gels can be used to deliver cells or ingredients or to shield surfaces against cell attachment. In addition, proteins or peptides may be covalently attached to the fibers or the fibers may be plasma modified in a charged gas environment (e.g. nitrogen) for the deposition of amino groups, each of these coatings promotes cell adhesion and ingrowth, as silk is naturally hydrophobic and these coatings make the fibers more hydrophilic.

[0095] Non-limiting examples of some of these embodiments are described in the examples below.

[0096] Wet stacking was chosen for prototype material creation because it is the simplest procedure. A nonwoven product (Fig. 20A) was made from a single silk fibroin fiber before extraction at material level. Accordingly, the product is a relatively inexpensive material and can be used in applications where its low tensile strength will be sufficient. When higher tensile strength is required, the nonwoven material can be combined with each other, as is well known in the case of materials and paper, or mineralized for bone repair. Alternatively, the silk yarn material produced by extracting sericin can be formed into various more complex yarns as described above. The size and pattern of the yarn can be used to control the porosity, regardless of the efficiency of the fibrinser. Yarns can also be knitted (Fig. 20B) or woven (Fig. 20C) to obtain material. One type of material of interest is a simple gauze-like mesh that can be used alone (e.g. as a hemostat), or to deliver cells or drugs (e.g., clotting factor) to a site where flexibility is important.

EP 2 374 919 [0097] If strength is important, warp knit (Fig. 20B), including known leotards and jerseys, with elasticity that can be controlled by the spiral yarn pattern used in the material and usually significant tensile strength, can be very useful for applications (e.g., hernia repair, bladder suspension, gout muscle reconstruction pelvis, etc.) requiring mechanical support for a significant length of time, such as a month.

[0098] For other applications, the material should have low elasticity and high strength. For such materials, a dense weave of thick yarns is appropriate, forming a material similar to standard fabrics (Fig. 20C). Such a material can optionally be supplemented with a coating or heat treatment to bind the intersecting yarn segments, thereby preventing both entanglement and stretching. Heat treatment cannot completely denature the silk protein. Optionally, materials can be stapled, glued or stapled in place, as is currently the case with polypropylene mesh. The implant, like any other type described herein, can be coated with a variety of materials to increase local treatment and the process of tissue ingrowth and / or coating to prevent fusion of the repair site with bowels.

[0099] In another option, the material, mesh, non-woven fabric, knitwear or other repair material can be made of non-extracted silk, and then the finished material can be extracted as described herein (Fig. 21) (for example, with an alkaline soap solution at elevated temperature) to remove immunomodulatory sericins from the material. As a further option, the extraction of sericin can take place at an intermediate stage, such as extraction from the yarn, bundle or strand obtained, provided that the number of fibers does not exceed that at which the extraction solution can penetrate the fibers (see Fig. 21 as an example of non-limiting possibilities).

[0100] The above discussion describes the production of yarn consisting of materials in which the most common forms of yarn in forming the materials discussed above would have been from twisting silkworm fibroin fibers together in an arranged manner and extracting sericin. As described, many yarn geometries and yarn forming methods can also be used (Tables 4, 5, 6, 7 and 8). Such methods may include forming non-twisted bundles of fibroin fibers bound together by wrapping the silk bundles with other materials, as discussed above. Each of these yarns may, as described above, be formed by blending silk fibers with other materials. Furthermore, still, the fibers can be interlaced, e.g., by repeated threading, twisting, braiding, forming a mesh, knitting, etc. (see Figs. 2A and B and 21). The term "interlaced" is used herein to indicate an arranged (ie non-random) repetitive structure in terms of how the fibers contact and interrelate.

[0101] Mixing can also be performed at higher stacking levels, such as using filaments of different materials to form a thicker yarn or using yarns of different materials for weaving or knitting. In any case, the final material will contain purified, essentially sericin-free silk as an essential ingredient, used for one or all of its strengths and biocompatibility and (e.g. long-term) degradation characteristics (Figures 11A-B). The other polymer or polymers are selected for their biocompatibility, supporting cell attachment or infiltration (or inhibition through rapid tissue formation at desired sites) (Figs. 16A-C), degradation profile and mechanical properties. Biodegradable polymers include any known biodegradable polymers including natural products such as proteins, polysaccharides, glycosaminoglycans, derivatized natural polymers, e.g. celluloses and synthetic biodegradable polymers and copolymers, including polyhydroxy acids, polyanhydrides, polycarbonates, certain polyamides and their copolymers and mixtures thereof. In particular, collagen and elastin are suitable proteins.

[0102] Constructs / matrices of silk-containing materials used for tissue repair can be treated to contain cells at the time of implantation (Figs. 7A-D, Figs. 8A-B, Figs. 9A-B and Figs. 18C ) to improve tissue results in vivo. The cells may be xenogeneic, more preferably allogeneic, and most preferably autologous. Each cell type is potentially useful, depending on the location and intended function of the implant. Pluripotent cells are preferred when appropriate differentiation signals are present or provided in the environment. Other cell types include osteogenic cells, fibroblasts, and cells such as tissue at the implant site.

[0103] Although silk from Bombyx mori and other classic silkworms has been described, any source of silk or silk-derived proteins may be used in the invention as long as it does not cause more than mild foreign body reactions during implantation (i.e., it is biocompatible) (see Figs. 18B and C). These include, without limitation, silkworms, spiders and breeding cells, especially genetically modified cells and transgenic plants and animals. The silk produced by cloning can be derived from the full or partial gene sequence of native silk lines or from synthetic genes encoding silk-like sequences.

[0104] While in many cases only one type of material will be used to form a medical device or prosthesis, in some cases it may be useful to use two or more types of material in a single device. For example, when repairing a hernia, it is desirable for the tissue contact side of the repair material to attract cells, while the peritoneal surface should repel cells to prevent adhesions. This effect can be achieved by having one layer of silk that does not attract cells and another layer that attracts them (for example, the untreated layer and the layer containing RGD, as in the example below). Another example involves forming bladder suspensions. The chassis base should be fitted and slightly flexible and have a long life expectancy. However, the surface of the suspension closest to the bladder should have as little texture as practicable. According to the invention, this can be achieved by placing in the suspension, where it contacts the bladder, a thin but tightly woven layer of nonwoven or knitted fabric made of small diameter yarn (e.g., monofilament). The non-woven fabric should be as small (denier) as possible. Many other situations that require two or more types of material are possible.

[0105] Examples of the structures described above were made and evaluated in a series of tests. In the first example, the material was formed from refined silk fibers. First, raw silk was processed into purified fibroin fibers. The crude silkworm fibers were extracted in an aqueous 0.02 M Na2CO3 solution and 0.3% w / v IVORY soap solution for 60 minutes at 90 ° C. The obtained fibers were washed with water to complete the extraction of glue-like sericin protein. The resulting fiber suspension was wet-laid on a sieve, needle punched and dried (Fig. 20A). The felt material obtained felt a bit like wool and was very porous. It was sufficiently interconnected by entanglement and needling so that it could be processed and cut to the desired shape.

[0106] In another example, the purified silk fibroin fibers were treated with cell attracting factors (Table 9). First, the yarns were prepared by twisting the cleaned silk fibroin fibers together. Some yarns were made of filaments that were derivatized with RGD peptide to attract cells, using the procedures described in Sofia et al., J. Biomed. Mater. Res. 54: 139-148, 2001. Sections of treated and untreated yarns (braided black silk thread) were implanted into the abdominal wall of rats (Figs. 18A-C). 30 days after implantation, the black braided threads contained small bundles of fibrils, with infiltration of cells between the bundles of fibrils, but not within them. In contrast, RGD-treated filament bundles were extensively controlled by

And the host cells were swollen and not compact (Figures 13A-E, 18B), but were not yet significantly degraded (Figures 13A-E).

[0107] This example illustrates the use of derivatization to regulate the rate of degradation of implanted silk fibroin fibrils as well as illustrates the ability of derivatized fibrils to incorporate cells into a material-like structure. Of course, greater specificity for inclusion can be obtained by using more specific attractants. Similar techniques (chemical derivatization) or simpler methods such as absorption, adsorption, coating and soaking can be used to deliver other materials to the implant site.

[0108] Each of the samples listed in the Tables below was prepared as described above, wherein the sericin was removed within 60 minutes at 90 ° / -2 ° C. It has been found that applying a temperature in this range for a sufficient period of time provides the fibers from which sericin is substantially removed (Fig. 1A-C, Table 1, 2, 3) (to produce a fiber construct that is essentially free of sericin so as not to generate a significant immune response and not significantly impede the biodegradation of the fiber) while maintaining essential mechanical integrity of fibroin (Table 1). It should be noted that when the temperature reaches 94 ° C (Table 1), no radical effect on UTS is observed, however, the stiffness is significantly reduced indicating the heat sensitivity of silk at 94 ° C and above. The fibers in each group were straightened manually (i.e. laid in parallel) by pulling the ends of the fibers, alternatively, straightening can easily be carried out by an automated process. The applied force was slightly higher than required to straighten the group.

[0109] The sample geometry designations in all Tables include the following constructs: number of fibers (TPI at the fiber level in the S direction) x number of groups (TPI at the group level in the Z direction) x number of beams (TPI at the level of the beam in the S direction) x number strands (TPI at the level of the strand in the Z direction) x, etc., where the samples are twisted between levels unless otherwise indicated. Twist-to-inch designation, e.g. 10s x 9z TPI, reflects (number of strands of fibers / inch in the group) x (number of turns in the group / inch in the bundle). In each sample, the twist pitch is generally higher than is usually seen in ordinary yarns that are twisted at a low pitch value, which is only to keep the fibers together. Increased turn pitch (i.e. increasing the number of turns per inch) reduces tensile strength, but also additionally reduces stiffness and increases the value of elongation at break of the construct.

[0110] Ultimate tensile strength (UTS), percentage elongation at break (% yield) and stiffness were all measured with an INSTRON 8511 servo-hydraulic material testing machine with FAST-TRACK software that deforms the sample at high speeds at ~ 100% of the sample length per second using stress-to-break analysis. In other words, until it ruptures, every second, the sample is stretched to double its length, which greatly limits the sample's ability to relax and unwind before breaking. However, Figs. 5A-B illustrates the effect that the strain ratio can have on the observed mechanical properties, under either wet or dry test conditions, which has been shown (Figs. 6A-B) that has a great impact on UTS and silk matrix rigidity. If the comparison is made between data sets, consistency is required. The data obtained was analyzed using Instron Series IX software. The ultimate tensile strength is the maximum stress on the resulting stress / strain curve, and the stiffness is the slope of the stress / strain curve up to the yield point. Unless otherwise specified, at least N = 5 was used to generate mean values ​​and standard deviation for all study groups. Standard statistical methods were used to determine if there were statistically significant differences between groups, e.g. Student's t-test, one-way ANOVA.

[0111] Fibroin fibers in the tests in all the above Tables and Figures (and in the present description) are native (ie, these fibers are not dissolved and reformed), dissolution and change in fiber composition results in a different fiber structure with different properties mechanical after reforming. Surprisingly, these samples show that the yarn from silk fibroin fibers, from which sericin has been completely or almost completely removed, can be characterized by high strengths and other mechanical properties that make the yarn suitable for various biomedical applications (Table 4, Fig. 2A-D and Figs. 20A-C), for example for forming a fiber construct or supporting ligament replacement, hernia repair or reconstruction of pelvic lining tissue. Previously, fibroin was thought to require dissolution and extrusion to reformulate the fiber to provide the desired mechanical properties. It has generally been found that fatigue strength is lost in such reformed fibroin fibers. The methods of the present invention allow sericin to be removed without significant loss of strength (Tables 1 and 4, Figs. D-3A and 4A-B).

[0112] In Table 8, samples 1 and 2 compare the properties of the 3-fiber group (sample 1) with those of the 4-fiber group (sample 2). Sample 2 had a square fiber configuration while sample 1 fibers had a triangular configuration. As shown in the Table, the addition of additional fiber in sample 2 reduced the stiffness on the fiber of the sample demonstrating the ability to control the properties of yarn and material using a hierarchical structure.

[0113] Table 4 shows the effect of different geometry configurations of multifilament and twisted fiber constructs. Note, in particular, samples 7 and 8 contain the same number of fibers and the same number of geometric levels. The geometry of the twisted fiber of sample 8 offers higher UTS values ​​and greater stiffness, while the geometry of multiple strands of sample 7 has less strength and less stiffness. For samples 7-9, the multiple strand geometry of sample 7 has the highest strength-to-stiffness ratio; for use as an ACL fiber construct, a high strength-to-stiffness ratio (i.e., having high strength and low stiffness) is desired.

[0114] Tables 1 and 4 show the effect of the extraction of sericin on fibers. All samples were immersed in the extraction solution as described in Table 1. Samples 1-5 were immersed in a bath at room temperature at 33 ° C and 37 ° C. These temperatures are considered too low to ensure significant extraction of sericin. Samples 6-9 were extracted at 90 ° C, at which it is believed that complete desiccation of sericin can be achieved, but at different times. Similarly, sample 10 was extracted at a slightly higher temperature of 94 ° C. These data suggest that 30 to 60 min at 90 ° C is sufficient to remove sericin significantly (see Tables 2 and 3) and that 94 ° C may damage the structure of silk proteins, as demonstrated by a drastic decrease in stiffness.

[0115] Finally, samples 11 to 16 have comparable multiple strand geometries; sample fibers, 12, 14 and 16 were extracted, while sample fibers 11, 13 and 15 were not. As can be seen in the Table, extraction appears to have little effect on the (high) fiber ultimate tensile strength.

[0116] The fibers of sample 10 of Table 4 were subjected to a curling shrink procedure with the fibers twisted in one direction and then in the opposite direction rapidly; the fibers were then heated to block the torsion structure and tested in an non-extracted state. The strength and rigidity of the yarns obtained were relatively lower than most other non-extracted test yarns. However, Tables 6 and 7 show the remarkable ability of fibroins, after extraction, to withstand up to 30 TPI. Table 6 shows the ordering effect of TPI on silk matrices probably due to the ordering of the multifilament structure after extraction.

[0117] Fig. 10 shows the properties of groups of parallel fibroin fibers inoculated and unvaccinated under culture conditions for 21 days. These three samples showed very similar mechanical properties, thus reflecting little if any degradation of the silk matrix in vitro due to cell growth on it or due to time. The stiffness values ​​are probably much lower in this experiment compared to other samples, which is the result of a 21-day incubation period prior to mechanical testing (see Table 5).

[0118] Table 4, all samples 14-16 are braided samples. Sample fibers 14 were braided from eight drums, with a spool mounted on each drum, with two fibers drawn from each spool. Sample fibers 15 were pulled out of 16 drums, with a spool mounted on each drum, again two fibers were pulled out of each spool. Finally, sample 16 was formed from 4 yarns, each yarn contained 3 groups of four twisted fibers (providing a total of 12 fibers in the yarn); each yarn was pulled from a separate spool and drum.

[0119] Table 9 shows the effect of surface modification. The assay, "PBS" reflects the fact that the samples were immersed in phosphate buffered saline for approximately 24 hours before testing. The effect of exposing the sample to physiological saline was measured and provided an indication that the fiber construct could retain its mechanical properties and essentially preserve the natural protein structure in physiological saline (e.g., within the human body). The "RGD" assay reflects the fact that the samples were immersed in Arg-Gly-Asp (RGD) saline solution for approximately 24 hours prior to testing. RGD can be used in a construct to attract cells to the construct, thereby promoting cell growth on it. Accordingly, any effect of RGD on the mechanical properties of the construct is also interesting, although no significant degradation of the construct has been noticed. Therefore, these tests provide evidence that prolonged exposure to saline solution or sterilization with ethylene oxide or to RGD solution results in little, if any, degradation of the material properties of the fiber constructs. Although, data related to samples 28 and 29, for which the geometric hierarchy has been extended to a higher level, reveal that UTS / fiber decreases as higher levels are reached (and generally the number of fibers increases). However, this is the effect of a hierarchical pattern (Table 8) rather than surface modification.

[0120] Table 4, samples 18 to 23 were stretched under a constant force of 6 pounds for 1, 2, 3, 4, 5 and 6 days, respectively, before testing to assess the effect of stretching on mechanical properties over time. From the data obtained, it does not appear that the changes in the material properties of the construct are significant, if at all, when the stretching procedure is extended for a longer period of time. Sample 25 was also "pre-stretched" (after twisting) using 6 pounds of force for a day before testing; for comparison, sample 24 that had the same geometrical configuration was not pre-stretched. Samples 24 and 25, respectively, illustrate the effect of pre-stretching the construct on removing clearance in the structure, which results in a slight decrease in both UTS and elongation at break.

[0121] The silk-based construct serves as a matrix for infiltrating or already infiltrated cells or for inoculation with cells such as progenitor cells, fibroblast ligaments or tendons or muscles that can proliferate and / or differentiate to form the anterior cruciate ligament (ACL) or other the right kind of tissue. The new silk fiber construct was designed based on fibers having one of many yarn geometries, such as multiple threads or an interlaced structure, such as twisted yarn, weave, net-like yarn or knit-like yarn. The yarn exhibits mechanical properties that are identical or nearly identical to those of natural tissue such as the anterior cruciate ligament (see Table 4, 1 below) and simple differences in fiber structure and geometry arrangement can result in any desired tissue type (see Table 10 , below). Alternatively, many yarns can be formed into a material or other construct that

EP 2 374 919 is implanted either for positioning or supporting the organ. In addition, the construct can be used to fill internal cavities after surgery to prevent tissue adhesion or promote cell attachment or ingrowth.

[0122] Pluripotent bone marrow stromal cells (BMSCs) that are isolated and cultured as described in the following example can be seeded on a silk fiber construct and grown in a bioreactor under static conditions. Cells seeded on the fiber construct, if directed correctly, will be subject to differentiation specific for the ligament and tendon to form viable and functional tissue. In addition, the histomorphological properties of tissue resulting from bioengineering generated in vitro from pluripotent cells within a fiber construct are exposed to a direct application of mechanical force to the fiber construct during tissue generation. This discovery is a significantly new view on the relationship between mechanical and biochemical interactions and cell immobilization and cell differentiation methods, and is applicable to the production of a variety of ligaments, tendons and tissues in vitro from pluripotent cells.

[0123] A fiber construct comprising silk fibers with multiple strand geometry is illustrated in Figs. 2C and 2D. The fiber construct has a hierarchy in the sense of how the fibers are grouped in parallel and twisted and how the resulting group is grouped and twisted etc. across multiple levels of the hierarchy, as explained below. The silk fibers are first stretched in parallel by, for example, a rack having spring clamps that serve as the fiber attachment. The frame can be immersed in the sericin extraction solution so that the clamps can maintain a constant stretching of the fiber during extraction, rinsing and drying.

[0124] The extraction solution may be an alkaline soap or detergent solution and is kept at a temperature of about 90 ° C. The rack is immersed in the solution for a period of time (e.g. at least 0.5 to 1 hour, depending on the solution flow and mixing conditions), which is sufficient to remove all (+/- 0.4% remaining, by weight) or essentially all fiber sericin (allowing for possible trace residues). After extraction, the rack is removed from the solution, and the fibers are rinsed and dried. Computer controlled twisting machines, each of which fixes fibers or fiber constructs around the perimeter of the disk and rotates the disk around the central axis to twist the fibers (e.g. by multiple threads) or fiber constructions twisted around each other according to standard processes used in the textile industry, but with a higher pitch level for turns (e.g., from about 0 to about 11.8 turns per cm) than is generally generated in traditional fibers. However, the speed of multiple stranding and twisting should not be high enough to cause plastic deformation of the fibers as a result of the tension of the fibers in the balloon obtained when the yarn is released from the supply spool before twisting or repeated threading.

[0125] Extraction can be carried out at any level of the construct, provided that the solution can pass through the construct to remove sericin from all fibers. It is believed that the upper limit of the number of fibers in a compact system through which the solution can still penetrate completely is about 20-50 fibers. Although, of course, these fibers can be arranged in one group of 20 parallel fibers or, for example, in 4 groups of 5 parallel fibers each, these groups can be twisted or even a construct comprising an even higher level, such as 2 bundles of 2 groups of 5 fibers in which groups and bundles can be twisted. By increasing the number of hierarchical levels in the structure, you can also enlarge the voids, and thus potentially increase the maximum number of fibers from which sericin can be completely extracted from 20 to 50 fibers.

[0126] Since sericin is, in some cases, removed from the construct after grouping the fibers and after forming the higher level construct, it is not necessary to apply wax or any other type of mechanically protective coating to the fibers or also to create a barrier for

EP 2 374 919 prevents contact with sericin on the fibers, and the construct can be completely free of coatings (in particular being free of coatings that are not completely degraded by the body or cause an inflammatory reaction).

[0127] As described in the examples below, the mechanical properties of silk fibroin (as shown in Figs. 1A, 1B and 1C) were characterized and using a theoretical calculation model, geometries were formed to form suitable matrices for ACL engineering (see Fig. 1D) . The six-string construct was chosen for use as an ACL replacement to increase matrix surface area and increase support for tissue ingrowth. The two geometric construct hierarchies for ACL repair include:

1: 1 matrix, ACL yarn = 6 parallel strings; 1 cord = 3 twisted strands (3 twists / cm); 1 strand = 6 twisted bundles (3 twists / cm); 1 bundle = 30 parallel washed fibers; and Matrix 2: 1 ACL yarn = 6 parallel strings; 1 cord = 3 twisted strand (2 twists / cm); 1 strand = 3 twisted bundles (2.5 twists / cm); 1 bundle = 3 groups (3 turns / cm); 1 group = 15 parallel extracted silk fibroin fibers.

[0128] The number of fibers and geometries for Matrix 1 and Matrix 2 were selected so that the silk prostheses are similar to the ACL biomechanical properties in terms of ultimate tensile strength, linear stiffness, yield strength and% elongation at break, serving as a reliable starting point for development tissue engineering ACL. The effects of increasing the number of fibers, the number of levels, and the number of turns on each of these biomechanical properties are shown in Table 8 and Tables 6 and 7, respectively. [0129] The ability to generate two matrices with different geometries, each of which results in mechanical properties that mimic ACL properties indicate that there are many different geometric configurations to achieve the desired mechanical properties. Alternative geometries for any desired ligament or tendon tissue may include any number, combination or arrangement of cords, strands, bundles, groups and fibers (see Table 10, below) that result in a fiber construct with usable mechanical properties that mimic the desired ligaments or tendons. For example, one (1) ACL prosthesis may have any number of parallel strings, provided that it contains means for securing the final fiber of the construct in vitro or in vivo. In addition, different numbers of torsion levels (where one level is referred to as a group, bundle, strand or string) can be used for a given geometry, provided that the fiber construct results in the desired mechanical properties. In addition, there is a considerable degree of freedom in the design of the geometry and positioning of the fiber construct in the field of ACL prosthesis engineering, so the developed theoretical calculation model can be used to predict the fiber construct pattern of the desired ligament or tendon tissue (see example below). For example, when many smaller matrix beams are desired (e.g. 36 fibers in total) with only two hierarchy levels promoting ingrowth, a TPI of 8-11 or ~ 3-4 turns per cm is required and can be predicted using a model without the need for experimental work.

[0130] In this regard, a change in geometry (i.e., the number of strings used to make the prosthesis or the number of fibers in the group) can be used to generate matrices that are used in most ligaments and tendons. For example, in the case of smaller ligaments and tendons of the hand, the geometry and arrangement used to generate a single Matrix I cord (or two cords or three cords, etc.) may be appropriate provided that the positioning of the fiber construct results in mechanical properties appropriate to the specific physiological environment . In particular, to accommodate a smaller ligament or tendon compared to Matrix 1 or Matrix 2, fewer fibers per level can be used to generate smaller bundles or strands. Multiple beams can be used in parallel. For larger ligaments, such as ACL, it may be desirable to have more

EP 2 374 919 smaller bundles twisted at higher TPI values ​​to reduce stiffness and promote ingrowth, and then have fewer larger beams where ingrowth cannot occur and thus limited matrix degradation.

[0131] The invention, however, is not limited to multiple thread geometry that has been described, and may use any geometry or combination of geometries (e.g., parallel, twisted, braided, mesh-like) resulting in ACL-like fiber construct mechanical properties ( ie. ultimate tensile strength greater than 2000 N, linear stiffness between 100-600 N / mm for native ACL or commonly used replacement implants (such as patella ligaments between 26-30 mm long) or to the desired ligament and tendon to be manufactured . Fiber number and geometry of both Matrix 1 and Matrix 2 were selected to generate mechanically appropriate ACL matrices or other desired ligament or tendon matrices [e.g. posterior cruciate ligament (PCL)]. For example, a single string of Matrix 1 six-string construct was used to reconstruct the medial collateral ligament (MC) in a rabbit (see Fig. 15A and Fig. 15B). The mechanical properties of the six-string silk constructs Matrix 1 and Matrix 2 are described in Table 10 and Fig. 3A-3D, as further described in the example below. Additional geometries and associated mechanical properties are shown in Table 11 as an example of a significant degree of design freedom that would result in a fiber construct applicable in ACL tissue engineering in accordance with the methods described herein.

[0132] Preferably, the silk fiber based on the fiber construct may consist of only silk. The types and sources of silk are: silk from silkworms such as Bombyx mori and related species; spider silks, like Nephila clavipes; silks of genetically modified bacteria, yeast, mammalian cells, insect cells and transgenic plants and animals; silks obtained from cells grown from silkworms or spiders; native silks; cloned total or partial native silk sequences and silks obtained from synthetic genes encoding silk or silk-like sequences. In raw form, native silk fibroins obtained from Bombyx mori silkworms are coated with an adhesive-like protein called sericin, which is extracted from the fibers completely or almost completely before the fibers that make up the fiber construct are inoculated with cells.

[0133] The fiber construct may comprise a composite of: (1) silk and collagen fibers, (2) silk and collagen foam or mesh or sponge, (3) silk fibroin fibers and silk mesh or foam sponge, (4) silk and biodegradable polymers [ for example. cellulose, cotton, gelatin, polylactide, polyglycol, poly (lactide-co-glycolide), polycaproloactone, polyamides, anhydrides, polyamino acids, polyorthoesters, polyacetals, proteins, degradable polyurethanes, polysaccharides, policanoacrylates, glycosaminates, e.g. , etc.), polysaccharides (native, recycled or genetically modified versions: e.g., hyaluronic acid, alginates, xanthanes, pectin, chitosan, chitin, and the like), elastin (native, processed or genetically modified and chemically modified versions) and collagen (native, recycled or genetically modified versions) or (5) silk and non-biodegradable polymers (e.g., polyamide, polyester, polystyrene, polypropylene, polyacrylate, polyvinyl, polycarbonate, polytetrafluoroethylene or nitrocellulose material. The composite generally enhances the properties of the construct fiber, such as porosity, degradability, and also enhances cell seeding, proliferation, differentiation or tissue development. Figures 16A, 16B and 16C illustrate the ability of collagen fibers to promote BMSC growth and ligament specific differentiation.

[0134] The fiber construct may also be treated to increase cell proliferation and / or tissue differentiation on it. Exemplary fiber construct treatments that promote cell proliferation and tissue differentiation include, but are not limited to, metals, radiation, crosslinking, and chemical

EP 2 374 919 surface modifications [e.g. RGD peptide coating (Arg-Gly-Asp), fibronectin coating for growth factor coupling] and physical surface modifications.

[0135] A second aspect of the present invention relates to mechanically and biologically functional ACL formed from a new fiber construct based on silk fiber and autologous and allogeneic (depending on the recipient of the tissue) bone marrow stromal cells (BMSC) inoculated on the construct fiber. The silk fiber-based fiber construct induces stromal cell differentiation towards the ligament line without the need for any mechanical stimulation during bioreactor culture. BMSCs inoculated on the silk fiber basis and grown in a Petri dish began to attach and spread (see Figs. 7A-D); cells proliferate to coat the construct fibers (see Figures 8A-B, Fig. 9A and Fig. 9B) and differentiate, as demonstrated by expressing ligament-specific markers (see Fig. 14). Cartilage (type II collagen) and bone markers (bone sialoprotein) were not expressed (see Fig. 14). Data illustrating the expression of ligament-specific markers are provided in the example below. [0136] Another aspect of the present invention relates to an ex vivo ACL production method. Cells capable of differentiating into ligament cells are cultured under conditions simulating the movements and forces ACL experiences in vivo during embryonic development to mature ligament function. Specifically, under sterile conditions, pluripotent cells are seeded into a three-dimensional silk-based fiber construct to which the cells may adhere, and which preferably has a cylindrical shape. The three-dimensional silk-based fiber construct used in the method serves as a pre-fiber construct that is supplemented and possibly even replaced with the extracellular components of the fiber construct produced by differentiating cells. The use of a new silk-based fiber construct may enhance or accelerate ACL development. For example, a new silk fiber-based fiber construct may be designed to have specific mechanical properties (e.g., increased tensile strength) so that it can withstand high forces before strengthening the extracellular components of the fiber construct (e.g., collagen and tenascin) ). Other beneficial properties of the new silk-based fiber pre-construct include, without limitation, biocompatibility and biodegradability.

[0137] Pluripotent cells can be inoculated into the pre-fiber construct before or after forming the fiber construct, depending on the particular fiber construct used and the method of forming the fiber construct. Homogeneous seeding is usually preferred. In theory, the number of cells seeded does not limit the final ligament produced, however, optimal inoculation can increase the rate of generation. Optimal amounts of graft will depend on the particular culture conditions. The fiber construct can be inoculated from about 0.05 to 5 times the physiological cell density of the parent ligament.

[0138] The method uses one or more types of pluripotent cells. These cells have the ability to differentiate into different cell types in response to appropriate differentiation signals and to express ligament specific markers. More specifically, the method uses cells, such as bone marrow stromal cells, which have the ability to differentiate into tendon and ligament tissue cells. If the ligament obtained using bioengineering is to be transplanted into the patient's body, the cells should come from a source that is compatible with the intended recipient. Although the recipient will be essentially human, there are also applications in veterinary medicine. Cells can be taken from the recipient (autologous), although compatible donor cells can also be used to produce allogeneic ligaments. For example, when producing allogeneic ligaments (e.g., using cells from another human body, such as donor bone marrow stromal cells derived from donor bone marrow or ACL fibroblasts isolated from donor ACL tissue), human anterior cruciate ligament fibroblast cells isolated from intact donor ACL tissue

EP 2 374 919 (e.g., from a corpse or total knee transplant), torn ACL tissue (e.g., taken during surgery from a patient undergoing ACL reconstruction) or bone marrow stromal cells. Determining compliance is within the scope of a qualified practitioner's resources.

[0139] Ligaments or tendons, including, but not limited to, posterior cruciate ligament, rotational belt tendons, lateral medial ligament of the elbow and knee joint, hand flexor tendons, lateral ankle ligaments and tendons and ligaments of the jaw or temporomandibular joint other than the temporomandibular joint , cartilage, bone and other tissues can be constructed from a fiber construct in accordance with the methods of the present disclosure. In this way, the cells to be inoculated into the fiber construct are selected according to the tissue to be produced (e.g., pluripotent or desired tissue). Cells inoculated onto the fiber construct as described herein may be autologous or allogeneic. The use of autologous cells effectively creates an allograft or autograph for implantation in a recipient.

[0140] To form ACL, cells, such as bone marrow stromal cells, are reported to be seeded on a fiber construct. Bone marrow stromal cells are a type of pluripotent cells and are also referred to in the field as mesenchymal stem cells or simply as stromal cells. As stated, the source of these cells may be autologous or allogeneic. In addition, mature or embryonic stem or pluripotent cells may be used if the appropriate environment (in vivo or in vitro) of inoculation on a silk fiber-based fiber construct can replicate ACL or any other desired ligament or tissue in the extracellular fiber construct composition (e.g. protein, glycoprotein content), arrangement, structure or function.

[0141] Fibroblast cells can also be inoculated into the inventive fiber construct. Because fibroblast cells are often referred to as pluripotent cells, fibroblasts are intended to include mature human ACL fibroblasts (autologous or allogeneic) isolated from ACL tissue, fibroblasts from other ligament tissues, tendon tissue fibroblasts, from neonatal foreskin, from umbilical cord blood or from any cell, whether mature or pluripotent, mature differentiated or genetically modified, yes, that when grown in an appropriate environment (in vivo or in vitro), and inoculated onto a silk-based fiber construct, ACL or any other desired ligament or tissue may be reconstituted in the extracellular fiber construct composition (e.g. protein, glycoprotein content), alignment, structure or function.

[0142] Each of the surfaces of the cylindrical fiber construct is attached to trailers through which a range of forces can be applied to the fiber construct. To facilitate the delivery of force to the fiber construct, the entire surface of each respective plane of the fiber construct may contact the surface of the respective trailers. Trailers with a shape that reflects the location of the trailer (e.g. cylindrical) is best suited for this method. After fixing, the cells in the anchored construct are cultured in conditions suitable for cell growth and regeneration. The fiber construct during culture is subjected to one or more mechanical forces applied by attached trailers (e.g., by displacement of one or both attached trailers). Mechanical forces are applied throughout the culture to mimic the conditions that native ACL or other tissues experience in vivo.

[0143] Trailers should be made of a material suitable for securing the construct fiber and the obtained attachment should be strong enough to withstand the applied mechanical forces. In addition, trailers can be made of a material that is suitable for attaching the extracellular fiber construct that is produced by differentiating cells. Attachments support the ingrowth of bone tissue (both in vitro and in vivo), fixing the developing ligaments. Some examples of suitable fastening materials include, without limitation, hydroxyapatite, coral

EP 2 374 919

Goinopra, demineralized bone, bone (allogeneic or autologous). Fastening materials may also include titanium, stainless steel, high density polyethylene, DACRON and TEFLON.

[0144] Alternatively, the attachment material can be made or further enhanced by laminating the selected material by infusion with a factor that promotes binding of the ligament fiber construct or binding of the bone fiber construct or both. The term infusion is considered to include any application method that appropriately distributes the agent to the attachment (e.g. coating, permeation, contact). Examples of such factors include, without limitation, laminin, fibronectin, any extracellular protein fiber construct that promotes adhesion, silk, agents that contain arginine glycine-aspartate (RGD) peptide binding regions or RGD peptides as such. Growth factors or bone morphogenetic protein can also be used to increase attachment adhesion. In addition, trailers can be pre-inoculated with cells (e.g. stem cells, ligament cells, osteoblasts, osteogenic progenitor cells) that adhere to the attachments and bind the fiber construct to produce a reinforced attachment of the fiber construct both in vitro and in vivo.

[0145] An exemplary attachment system is disclosed in co-application USSN 09 / 950,561. The fiber construct is attached to the trailers by contact with the surface of the trailer or alternatively by actual penetration of the fiber construct material through the trailer material. Because the force applied to the fiber construct by the trailers determines the ultimately produced ligament, the size of the ultimately produced ligament is partly dictated by the size of the attachment location of the attachment. Use attachments of a size appropriate to the desired final ligament. An example of the shape of an ACL trailer is a cylinder. However, other shapes and sizes of the trailer will also function properly. For example, trailers may be of a size and composition suitable for direct placement in bone channels in the femur and tibia of a recipient constructed using ligament bioengineering.

[0146] Optionally, trailers can only be used temporarily during in vitro cultivation, and then removed while the fiber structure itself is implanted in vivo.

[0147] Furthermore, a new silk fiber-based fiber construct can be inoculated with BMSC and grown in a bioreactor. There are currently two types of growth environment that can be used in accordance with the methods of the present invention: (1) in an in vitro bioreactor device system, and (2) in vivo the knee joint, which serves as a "bioreactor" because it provides a physiological environment. including progenitor cells and stimulants (both chemical and physical) necessary for the development of viable ACL for a given fiber construct with adequate biocompatibility and mechanical properties. The bioreactor device provides optimal culture conditions for obtaining ligaments in the field of differentiation and production of extracellular fiber construct (ECM), and which thus provides ligaments with optimal mechanical and biological properties prior to implantation in the recipient. In addition, when the silk-based fiber construct was inoculated and cultured with cells in vitro, the Petri dish can be considered a bioreactor in an environment in which there are proper conditions for cell growth and regeneration, i.e., a static environment.

[0148] Cells can be grown on a fiber construct without applying any mechanical forces, i.e. in a static environment. For example, the silk fiber-based fiber construct itself, without applying mechanical forces or in vitro stimulation when inoculated and cultured with BMSC, induces these cells to proliferate and express ligament and tendon specific markers (see examples described herein) . The knee joint can serve as a physiological growth and development environment that can provide cells and normal environmental signals (chemical and physical) to

EP 2 374 919 construct a fiber so that ACL develops technically. Therefore, the knee joint (as such as a bioreactor) and the fiber construct (unvaccinated, vaccinated and undifferentiated in vitro, or vaccinated and differentiated in vitro prior to implantation) will result in the development of ACL or other desired tissue depending on the type cell inoculated onto the fiber construct and anatomical location of the fiber construct implantation. FIG. 15 AB illustrates the effect of the medial collateral ligament on the medial collateral ligament (MCL) when only a non-vaccinated silk fiber-based fiber construct with MCL-appropriate mechanical properties was implanted in vivo for 6 weeks. Conditions suitable for cell growth and regeneration are preferably present for the construction of the desired ligament or tissue when the cells are grown under static conditions without the use of mechanical stimulation or in a dynamic environment, for example in a bioreactor device.

[0149] In the experiments described in the examples below, mechanical stimulation has been shown to affect the morphology and cellular structure of progenitor cells within the resulting tissue. The extracellular components of the fiber construct secreted by the cells and the arrangement of the extracellular fiber construct throughout the tissue were also significantly influenced by the forces applied to the fiber construct during tissue generation. When generating tissue in vitro, cells and the extracellular fiber construct are positioned along the load axis, which reflects the native ACL arrangement in vivo, which also occurs along different load axes generated by natural motion and knee function. These results suggest that physical stimuli that cells of developing tissue, such as ACL, experience in nature play a significant role in the differentiation of progenitor cells and tissue formation. They also indicate that this function can be effectively duplicated in vitro by mechanical surgery to produce similar tissue. The more the forces generated by the mechanical procedure resemble the forces that ACL experiences in vivo, the more the resulting tissue will resemble native ACL.

[0150] While in vitro mechanical stimulation of the fiber construct using a bioreactor is used, there is independent but competitive regulation of both cyclic and rotational deformations that are applied to one attachment relative to other trailers. Alternatively, the fiber construct alone can be implanted in vivo, inoculated with ACL cells from the patient, and exposed in vivo to mechanical signaling by the patient.

[0151] When the fiber construct is seeded with cells prior to implantation, the cells are cultured within the fiber construct under conditions suitable for cell growth and differentiation. During the culturing process, the fiber construct may be subjected to one or more mechanical forces by the movement of one or both of the attached trailers. Mechanical tensile, compressive, torsion and shear forces and combinations thereof are applied in appropriate combinations, sizes and frequencies to mimic the mechanical stimuli ACL experiences in vivo.

[0152] Various factors will affect the amount of force that can be tolerated by a fiber construct (e.g., fiber construct composition, cell density). The strength of the fiber construct is expected to change during tissue development. Accordingly, the applied mechanical forces or deformations will increase, decrease or remain constant in size, duration, frequency and diversity during the ligament manufacturing period so as to suit the strength of the fiber construct during use.

[0153] During ACL production, the more accurate the intensity and combination of stimuli applied to the fiber construct during tissue development, the more the ligament obtained will resemble native ACL. Regarding the natural functions of ACL, two issues should be considered when developing an in vitro mechanical force application scheme that closely mimics the in vivo (1) different types

EP 2 374 919 movement that ACL experiences and ACL reactions to knee movements, and (2) the range of mechanical stress that the ligament experiences. Specific combinations of mechanical stimuli are generated from the natural movements of the knee structure and transmitted to native ACL.

[0154] Briefly describing knee movements, the combination of the tibia and femur by ACL provides six degrees of freedom when considering the movement of both bones relative to each other. The tibia can move in three directions and can rotate about the axis of each of these three directions. The knee is limited before reaching the full ranges of these six degrees of freedom due to the presence of ligaments and covering fibers and the knee surfaces themselves (Biden et al., "Experimental Methods Used to Evaluate Knee Ligament Function," Knee Ligaments: Structure, Function, Injury and Repair , ed. D. Daniel et al., Raven Press, pp. 135-151, 1990). Small translational movements are also possible. The ACL attachment sites are responsible for its knee stabilizing effects. ACL acts as the main stabilizer of anterior-tibial translation and as an additional stabilizer for angulation of valgus valgus and tibial rotation (Shoemaker et al., "The Limits of Knee Motion," Knee Ligaments: Structure, Function, Injury and Repair, ed. D. Daniel et al. ., Raven Press, pp. 1534-161, 1990). Therefore, ACL is responsible for knee stabilization in three out of six possible degrees of freedom. As a result, ACL has developed a specific fiber arrangement and overall structure to perform these stabilizing functions. These conditions are simulated in vitro to produce tissue with similar structure and fiber arrangement.

[0155] The range of mechanical stress that ACL experiences can be combined in a similar manner. ACL is subject to cyclic loads of approximately 400 N from one to two million cycles per year (Chen et al. J. Biomed. Mat. Res. 14: 567-586, 1980). When developing surgical ACL replacement, linear stiffness ~ 182 N / mm), final deformation (100% ACL) and energy absorbed at failure (12.8 Nm) are also taken into account (Woo et al., The tensile properties of human anterior cruciate ligament (ACL) and ACL graft tissues, Knee Ligaments: Structure, Function, Injury and Repair, edited by D. Daniel I et al. Raven Press, pp. 279-289, 1990).

[0156] The examples section below details the production of a prototype constructed using bioengineering anterior cruciate ligament (ACL) ex vivo. Mechanical forces that mimic a subset of mechanical stimuli experienced by native ACL in vivo (rotational and linear deformation) were applied in combination, and the resulting ligament that was formed was tested to determine the effect of applied forces on tissue development. Exposure of the developing ligament to physiological loads during in vitro formation induced cells to adopt a specific orientation along the load axis as well as to generate extracellular matrix along the axis. These results indicate that the introduction of complex multidimensional mechanical forces into the scheme to produce a more complex load axis connection that mimics the native ACL environment creates a ligament constructed by bioengineering methods that more closely resembles native ACL.

[0157] Various mechanical forces that can be used include, without limitation, stretching, compression, torsion and shear. These forces are applied in combinations that simulate the forces that ACL experiences during the natural movements and functions of the knee joints. These movements include, but are not limited to, extension and flexion of the knee, as determined in the coronary and sagittal plane, and flexion of the knee. Optimally, the combination of applied forces mimics the mechanical stimuli experienced by the anterior cruciate ligament in vivo as accurately as possible experimentally. It is expected that the differentiation of specific patterns of force application during ligament generation will have an impact on the rate and outcome of tissue development, with experimental determination of optimal conditions. Potential variables in the diagram include, among others: (1) deformation rate, (2) deformation percentage, (3) type

EP 2 374 919 deformations (e.g. translational and rotational), (4) frequency, (5) number of cycles in a given pattern, (6) number of different patterns (7), duration at extreme points of ligament deformation (8), pressure force and (9) various combinations of forces. There is a wide variety of variants. The scheme of applied mechanical forces can generate spirally arranged fibers similar to those in the native ligament, as described below.

[0158] Bundles of fibers from a native ligament are arranged in a spiral arrangement. The method of attachment and the need for the knee joint for ~ 140 ° of flexion rotation caused that the native ACL achieved 90 ° twist, and the peripheral fiber bundles developed a spiral arrangement. This unique biomechanical feature allows ACL to withstand very high loads. In the functional aspect of ACL, this spiral arrangement of fibers allows the anterior-posterior and posterior-anterior fibers to remain relatively isometric relative to each other at each degree of bending, in this way the load can be evenly distributed over all fiber bundles at any degree of knee bending, stabilizing the knee in all areas of joint movement. Mechanical forces that simulate a combination of knee flexion and knee extension can be applied to the developing ligament to produce an engineered ACL that has the same spiral arrangement. The mechanical device used in the experiments shown in the examples below provides control of deformation and deformation rate (both translational and rotational). The mechanical device will monitor the actual load experienced by the growing ligament, "teaching" the ligament during monitoring and increasing the load regime.

[0159] Another aspect of the present disclosure relates to the anterior cruciate ligament constructed by bioengineering, prepared by the methods described above. A ligament constructed using bioengineering produced by these methods is characterized by cellular orientation and / or a folding pattern of the fiber construct towards mechanical forces applied during generation. The ligament is also characterized by the production / presence of extracellular components of the fiber construct (e.g. type I and type III collagen and fibronectin and tenascin C proteins along the mechanical load axis experienced during culture. Bundles of ligament fibers may be arranged in a spiral arrangement, as discussed above.

[0160] The above methods using the new silk fiber-based fiber construct are not limited to ACL production, but can also be used to produce other ligaments and tendons in the knee (e.g. posterior cruciate ligament) or other parts of the body (e.g. hand, wrist, ankle, elbow, jaw and shoulder blades), such as, but not limited to, posterior cruciate ligament, rotational belt tendon, medial lateral ligament of the elbow and knee, hand flexor tendons, lateral ankle ligaments and tendons and ligaments mandible or temporomandibular joint. All movable joints in the human body have specialized ligaments that connect the joint ends of the bones in the joint. Each ligament in the body has a specific structure and arrangement, which are dictated by its function and environment. Various body ligaments, their location and functions are listed in Anatomy, Descriptive and Surgical (Gray, H., Eds. Pick, TP, Howden, R., Bounty Books, New York, 1977). By determining the physical stimuli experienced by a given ligament or tendon and by introducing forces that mimic those stimuli, the above-described method of producing ACL ex vivo can be adapted to the production of bioengineered ligaments and tendons ex vivo that simulate any organ ligament or tendon.

[0161] The specific type of ligament or tendon that will be produced is pre-determined prior to tissue generation, because several aspects of the method vary depending on the specific in vivo conditions experienced by the native ligament or tendon. The mechanical forces to which the developing ligament or tendon is exposed during cell culture are determined for the type of ligament or tendon that is being cultured. Specific conditions can be established by examining the native ligament or

EP 2 374 919 tendon and its surroundings and functions. One or more mechanical forces experienced by the ligament or tendon in vivo are applied to the fiber construct during cell culture in the fiber construct. The practicing specialist will know that the ligament and tendon that is superior to the currently available can be produced by applying a subset of forces experienced by the native ligament or tendon. Optimally, however, a full range of in vivo forces are applied to the fiber construct in appropriate sizes and combinations to produce a final product that is most similar to a native ligament or tendon. These forces include, without limitation, the forces described above for producing ACL. Since the applied mechanical forces depend on the type of ligament or tendon, and the final size of the ligament or tendon will be affected by the trailers used, therefore the optimal composition of the attachment, size and location of the fiber construct for each type of ligament or tendon will be determined by a specialist practicing in this field. The type of cell inoculated on the fiber construct is of course determined by the type of ligament or tendon to be produced.

[0162] Using methods similar to those described above for the production of ligaments or tendons ex vivo, other types of tissue can be produced ex vivo. The methods described above can also be used to produce a variety of tissue engineered products, which in most of their functions include mechanical deformation, such as muscles (e.g. smooth muscles, skeletal muscles, heart muscle), bones, cartilage, spinal disk and some types of blood vessels . Bone marrow stromal cells have the ability to differentiate into these as well as other tissues. The geometry of the silk fiber construct or the composite fiber construct can be easily adapted to the appropriate anatomical geometric configuration of the desired tissue type. For example, silk fibroin fibers can be reformed in a cylindrical tube to restore arteries. [0163] The results presented in the examples below indicate that growth in an environment that mimics the specific mechanical environment of a given type of tissue will induce appropriate cell differentiation so as to produce tissue resulting from bioengineering that largely resembles native tissue. The ranges and types of mechanical deformation of the fiber construct can be extended to obtain a wide range of structural tissue arrangement. The cell culture environment may reflect the in vivo environment that native tissue and the cells it contains experiences throughout the entire period of embryonic development to mature cell function in the native tissue as accurately as possible. Factors to consider when designing specific culture conditions to produce a given tissue include, without limitation, composition of the fiber construct, methods of cell immobilization, method of attaching the fiber or tissue construct, specific applied forces and cell culture medium. The specific scheme of mechanical stimulation depends on the type of tissue to be produced and is determined by changing the application of mechanical forces (e.g. only stretching, only twisting, a combination of stretching and twisting, with and without shearing, etc.), force amplitude (e.g. angle or swing), touch frequency and duration, and duration of stimulation and rest periods.

[0164] An ex vivo method for producing a particular type of tissue is to adapt the method of producing ACL described above. The components needed include pluripotent cells, a three-dimensional fiber construct to which the cells may adhere, and many attachments that have a surface suitable for attaching the fiber construct. When pluripotent cells (e.g. stromal bone marrow cells) are inoculated into the three-dimensional fiber construct by uniformly immobilizing the cells within the fiber construct. The number of inoculated cells is also not limited, however, inoculation of the fiber construct with high density cells can accelerate tissue generation.

[0165] Specific applied forces should be determined for each type of tissue produced by testing native tissue and mechanical stimuli in vivo. The type of tissue experiences

EP 2 374 919 characteristic forces, which are dictated by the location and function of the tissue in the body. For example, it is known that cartilage experiences in vivo shear and compression / stretching combinations; the bone experiences compression.

[0166] Additional stimuli (e.g., chemical stimuli, electromagnetic stimuli) may also be included in the above-described methods for producing ligaments, tendons and other tissues that are bioengineering products. It is known that cell differentiation is influenced by environmental chemical stimuli often produced by surrounding cells, such as secreted growth or differentiation factors, cell-cell contact, chemical gradients, and specific pH values, to name a few. Other, more unique stimuli are experienced by more specialized types of tissue (e.g. electrical stimulation of the heart muscle). The use of such tissue-specific stimuli (e.g., 1-10 ng / ml transforming growth factor beta-1 (TGF-βΙ), alone or together with appropriate mechanical forces, is intended to facilitate cell differentiation into tissue that better approximates specific natural tissue.

[0167] Tissues prepared by the methods described above provide an unlimited pool of tissue equivalents for the surgical implantation of a compatible recipient, in particular for the replacement or repair of damaged tissues. Engineering-derived tissues can also be used in in vitro studies, function of normal or pathological tissues, e.g., for in vitro testing of cell and tissue response levels to molecular, mechanical or genetic manipulations. For example, tissues based on normal or transfected cells can be used to assess the tissue's response to mechanical or biochemical stimuli, determine the function of specific genes or gene products that can be overexpressed or excluded, or to investigate the effects of pharmacological agents. Such studies are likely to provide a better view of ligaments, tendons and tissue development, normal and pathological function, and ultimately lead to fully functional tissue substitutes that are engineering products, in part based on existing tissue engineering methods, as well as a new look at cell differentiation and tissue development and the use of mechanical regulatory signals in combination with cell-derived and exogenous biochemical factors to improve the structural and functional properties of tissues.

[0168] Engineering production of tissues such as tendons and ligaments also has application possibilities, such as collecting bone marrow stromal cells from individuals at high risk for tissue damage (e.g., ACL rupture) prior to damage. These cells can either be stored until use or inoculated into appropriate fiber constructs and cultured and differentiated in vitro using mechanical stimuli to produce various prosthetic tissues by bioengineering that will be in reserve until used by the donor. The use of prostheses created by bioengineering of living tissues that are better adapted to the biological environment in vivo, and which ensure the maintenance of the required physiological load, for example, the dynamic balance of a normal, fully functional ligament, should reduce the rehabilitation time of the recipient of the prosthesis from months to weeks, in particular if the tissue was pre-cultured and stored. Benefits include rapid recovery of functional activity, shorter hospital stays and fewer problems with tissue rejection and failure.

[0169] Additional aspects of the present invention are further illustrated in the following examples. It will be apparent to those skilled in the art that many modifications, both materials and methods, can be used without departing from the scope of the invention.

[0170] In the first example, the raw Bombyx mori silkworm fibers, shown in FIG. 1A, were extracted to remove sericin, an adhesive-like protein coating native silk fibroin (see Figs. 1A-C). The appropriate number of fibers in each group were laid in parallel and

EP 2 374 919 was extracted with an aqueous 0.02 M Na2CO3 solution and 0.3% (w / v) IVORY soap solution for 60 minutes at 90 ° C, followed by thorough washing with water to extract glue-like sericin proteins.

[0171] The Costello equation for three-strand spiral rope geometry was derived to predict the mechanical properties of a silk fiber-based construct. The derived model is a series of equations that in combination take into account the properties of the extracted silk fiber material and the geometric hierarchy of the desired fiber construct to calculate the total strength and rigidity of the fiber construct as a function of the angle of inclination for a given level of geometric hierarchy.

[0172] The properties of the single silk fiber material include fiber diameter, modulus of elasticity, Poisson's ratio and ultimate tensile strength (UTS). Geometric hierarchy can be defined as the number of torsion levels at a given fiber construct level. Each level (e.g., group, bundle, strand, string, ligament) is further determined by the number of fiber groups twisted around each other and the number of fibers in each group of the first twisted level, where the first level is defined as the group, the second level as the bundle, the third as a strand and the fourth as a cord, and the fifth as a ligament.

[0173] The model assumes that each composite group of multiple fibers acts as a single fiber with an effective radius determined by the number of individual fibers and their respective radius, i.e. the model omits friction between individual fibers due to its limited role in a given relatively high angle of inclination.

[0174] For more detailed analysis, two usable geometries (Matrix 1 and Matrix 2) of the geometrical configurations of the multifilament construct (see Table 10, above) calculated to obtain mechanical properties mimicking those of native ACL were obtained. The six-string construct was chosen for use as an ACL replacement. The matrix configurations are as follows: Matrix 1: 1 ACL prosthesis = 6 parallel cords; 1 cord = 3 twisted strands (3 twists / cm); 1 strand = 6 twisted bundles (3 twists / cm), 1 bundle = 30 parallel washed fibers and 2: 1 matrix ACL = 6 parallel strings; 1 cord = 3 twisted strands (2 twists / cm); 1 strand = 3 twisted bundles (2.5 twists / cm), 1 bundle = 3 groups (3 twists / cm), 1 group = 15 parallel extracted silk fibroin fibers. The amount of fibers and geometry was selected so that the silk prostheses have similar biomechanical properties to ACL in terms of UTS, linear stiffness, yield strength and% elongation at break (see Table 10 above), thus serving as a permanent starting point for ACL development by tissue engineering.

[0175] The mechanical properties of silk fibroin were characterized using the Instron 8511 servo-hydraulic stretching / compression system with Fast-Track software (Instron Corporation, Canton, Massachusetts, USA) (see Fig. 1D). Individual stress-breaking and fatigue analyzes were performed on single silk fibers, extracted fibroin and cords of a specific arrangement. For characterization, both fiber and fibroin were arranged in parallel spiral geometries of Matrix 1 (see Fig. 2C) and Matrix 2 (see Fig. 2D). Individual strain-add tests were carried out at a strain rate of 100% / sec; histograms of force versus elongation were generated, and data were analyzed using Instron Series IX software. Both Matrix 1 and Matrix 2 obtained ACL-like mechanical and fatigue properties in terms of UTS, linear stiffness, yield strength, and elongation at break (see Table 10 and Figures 3A-D). [0176] Fatigue analyzes were performed using the Instron 8511 stretch / compression servo-hydraulic system with WaveMaker software on single strings of both Matrix 1 and Matrix 2. The data was extrapolated to represent the 6-string LMA prosthesis, which is shown in Figures 3B and 3D. The ends of the cord were embedded in epoxy to generate 3 cm

EP 2 374 919 length construct between trailers. Cycles to break at 1680 N and 1200 N UTS (n = 5 for each load) for Matrix 1 (see Fig. 3B) and at 2280 N, 2100 N and 1800 N UTS (n = 3 for each load) for Matrix 2 ( see Fig. 3D) was determined using the sine H wave equation at 1 Hz generated by WaveMaker 32 software version 6.6 (Instron Corp.). Fatigue tests were performed in neutral phosphate buffered saline (PBS) at room temperature. [0177] Complete removal of sericin was observed after 60 minutes at 90 ° C, as determined by SEM (see Figures 1A-C). Removal of sericin from silk fibers changes the fiber ultrastructure, resulting in a smoother fiber surface, and the underlying silk fibroin (shown in Figures 1A-C) is exposed with an average diameter in the range between 20-40 μm. Offset fibroin showed a significant 15.2% decrease in tensile strength (1.033 + / -0.042 N / fiber to 0.876 + / -0.1 N / fiber) (p <0.05, paired Student's t-test) (see Fig. 1D). The mechanical properties of the optimized silk matrix (see Figs. 2A-D and Figs. 3A-D) are summarized in Table 11 and Fig. 3A (for Matrix 1) and Fig. 3C (for Matrix 2). From these results it is evident that the optimized silk matrices showed comparable values ​​to native ACL, for which they were reported to have an average tensile strength (UTS) of about 2100 N, rigidity ~ 250 N / nm, yield point ~ 2100 N and 33 % elongation at break Woo, SL-Y, et al., The Tensile Properties of Human Anterior Cruciate Ligament (ACL) and ACL Graft Tissue in Knee Ligaments: Structure, Function, Injury and Repair, 279-289, ed. D. Daniel et al., Raven Press 1990).

[0178] Regression analysis of fatigue data of a fiber construct, shown in Fig. 3B for Matrix 1 and in Fig. 3D for Matrix 2, when extrapolated to physiological load levels (400 N) predicts the number of cycles to damage in vivo, indicating the lifetime of the fiber construct for 3, 3 million cycles for Matrix 1 and longer than 10 million cycles for Matrix 2. The spiral pattern of the fiber construct utilizing washed silk fibers results in a fiber construct with physiologically equivalent structural properties, confirming its usefulness as scaffolding for ligament tissue engineering.

[0179] In another example involving cell isolation and culture, bone marrow stromal cells (BMSCs), pluripotent cells capable of differentiating into osteogenic, chondrogenic, tendonogenic, adipogenic and myogenic lines were selected, as the creation of appropriate conditions may direct their differentiation to the desired fibroblast cell line (Markolf et al., J. Bone Joint Surg. 71A: 887-893, 1989; Caplan et al., Mesenchymal stem cells and tissue repair, The Anterior Cruciate Ligament: Current and Future Concepts, Ed. DW Jackson et al., Raven Press, Ltd, New York, 1993; Young et al., J. Orthopedic Res. 16: 406-413, 1998).

[0180] Human BMSC was isolated from the bone marrow from the iliac plate from compatible at least 25 years old donors obtained from a commercial supplier (Cambrex, Walkersville, MD). Twenty-two milliliters of human bone marrow were aseptically aspirated into a 25 ml syringe containing three milliliters of heparinized (1000 units per milliliter) physiological saline. The heparinised bone marrow solution was sent to the laboratory overnight for isolation of bone marrow stromal cells and culture. Upon arrival from the supplier, twenty-five milliliter aspirates were resuspended in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 0.1 mM endogenous amino acids, 100 U / ml penicillin, 100 mg / L streptomycin ( P / S), and 1 ng / ml, basic fibroblast growth factor (bFGF) (Life Technologies, Rockville, MD) and seeded 8-10 microliters of aspirate / cm² for tissue culture bottles. Fresh medium was added to bone marrow aspirants twice a week for up to nine days of culture. BMSCs were selected based on their ability to adhere to tissue culture plastics; non-adherent hematopoietic cells were removed during media exchange after 9-12 days of culture. The medium was then changed twice a week. When primary BMSC were close to confluence (12-14 days), they were detached using 0.25% trypsin / 1 mM EDTA and

EP 2 374 919 was re-seeded at 5x10³ cells / cm². The first passage (P1) hBMSC was trypsinized and frozen in 8% DMSO / 10% FBS / DMEM for future use.

[0181] Frozen P1 hBMSC was thawed, plated at 5x10³ cells / cm² (P2), trypsinized when they were near confluence and used to inoculate the fiber construct. Sterilized (ethylene oxide) silk matrices (in particular, single strands of Dies 1 and 2, bundles of 30 parallel extracted silk fibers and spiral collagen fiber ropes) were inoculated with cells in non-standard seeding chambers (1 ml total volume) processed into Teflon blocks to minimize the amount of cell medium and increase cell-fiber contact. Inoculated matrices, after a 4-hour incubation period with cell suspension (3.3x10⁶ BMSC / ml) was transferred to a Petri dish containing the appropriate amount of cell culture medium for the duration of the experiments.

[0182] To determine the rate of silk fibroin degradation, tensile strength (UTS) was measured as a function of culture time under physiological growth conditions, i.e. in cell culture medium. Groups of 30 parallel 3 cm long silk fibers were extracted, seeded with hBMSC and grown on fibroin for 21 days at 37 ° C and 5% CO2. Non-vaccinated control groups were grown in parallel. UTS silk fibroin was determined as a function of culture time for vaccinated and non-vaccinated groups.

[0183] The reaction of bone marrow stromal cells to a silk fiber construct was also investigated.

[0184] BMSCs readily attached and increased on silk and collagen matrices after 1 day in culture (see Figs. 7A-C and Fig. 16A) and formed cell elongations to bridge adjacent fibers. As shown in Fig. 7D and Fig. 16B, a uniform cell sheet covering the construct was observed on days 14 and 21 of culture, respectively. MTT analysis confirmed complete coverage of the fiber construct by inoculated BMSC after 14 days of culture (see Figs. 8A-B). Quantification of total DNA of cells cultured on Matrix 1 (see Fig. 9A) and Matrix 2 (see Fig. 9B) confirmed that BMSC proliferated and increased in silk construct culture with the highest amount of DNA measured after 21 and 14 days, respectively.

[0185] Extracted silk fiber fibroin control groups, whether inoculated or uninoculated with BMSC, maintain their mechanical integrity as a function of culture duration for 21 days (see Fig. 10).

[0186] RT-PCR analysis of BMSC seeded on Mat 2 strings showed that both collagen I and III were upregulated for 14 days in culture (Fig. 14). Type II collagen and bone sialoprotein (as indicators of specific differentiation of cartilage and bone, respectively) were not detectable or slightly expressed during culture. Using real-time quantitative RT-PCR on day 14, a collagen I to collagen III transcript ratio of normalized to GAPDH of 8.9: 1 was obtained (see Fig. 17). The high ratio of collagen I to collagen III indicates that the reaction is not characteristic of wound healing or scar tissue formation (as seen with high levels of type III collagen), but rather characteristic of a ligament; the relative ratio of collagen I to collagen III in native ACL is ~ 6.6: 1 (. Amiel et al., Knee Ligaments: Structure, Function, Injury, and Repair, 1990).

[0187] In addition, research is underway to provide an overview of the effect of targeted multidimensional mechanical stimulation on ligament formation from bone marrow stromal cells in a bioreactor system. In the bioreactor, independent, but at the same time cyclic, multidimensional deformations (e.g. translation, rotation) can be applied to the developing ligaments. After 7 to 14 days of static rest period (time after inoculation), the rate of translational and rotational deformations and linear deformation were kept constant for 1 to 4 weeks. Translational strain (3.3% -10%, 1-3 mm) and rotational strain (25%, 90 °) at 0.0167 Hz (one complete load and stress cycle) were applied simultaneously to silk-based matrices inoculated with BMSC

EP 2 374 919 per minute); a set of bioreactors with inoculated matrices without mechanical loading, but otherwise identical, served as controls. The ligaments are exposed to permanent cyclic deformations during the days of the experiment.

[0188] After the culturing period, ligament samples, both mechanically strained and control (static) were characterized in terms of: (1) general histomorphological (visual) appearance, (2) cell distribution (histological image processing and coloration of MTT sections), ( 3) cell morphology and orientation (histological analysis), and (4) production of specific tissue markers (RT-PCR, immunostaining).

[0189] Mechanical stimulation significantly affects the morphology and alignment of BMSC and the newly developed extracellular fiber construct, cell distribution along the fiber construct, and up-regulation of the ligament-specific differentiation cascade; BMSCs line up the long axis of the fiber, adopt spheroid-like ligament / tendon fibroblast morphology, and up-regulate ligament / tendon specific markers. It is expected that the newly formed extracellular fiber construct (i.e., the composition of proteins produced by cells) will line up along the load line as well as the longitudinal axis of the fiber construct. Targeted mechanical stimulation is expected to enhance ligament development and in vitro bioreactor formation resulting from BMSC inoculation on a new silk fiber-based construct. The longitudinal orientation of the cells and the newly obtained fiber construct is similar to ligament fibroblasts found in ACL in vivo (Woods et al. Amer. J. Sports Med. 19: 48-55, 1991). In addition, mechanical stimulation maintains the correct expression ratio between type I collagen transcripts and type III collagen transcripts (e.g., above 7: 1) indicating the presence of newly formed ligament tissue as opposed to scar tissue formation. The above results indicate that the mechanical apparatus and bioreactor system provide suitable conditions (e.g. multidimensional deformation) for in vitro formation of ligaments by tissue engineering, starting from the bone marrow stromal cells and a new silk fiber based construct.

[0190] The culture conditions used in these preliminary experiments can be further developed to more accurately reflect the physiological environment of the ligament (e.g., by increasing the amount of different types of mechanical forces) to obtain in vitro functional equivalents of native ACL for potential clinical use. These methods are not limited to ACL production by bioengineering. By applying the appropriate size and diversity of forces experienced in vivo, the methods of the present disclosure can produce ex vivo any type of ligament in the body as well as other types of tissue.

Table 1. Tensile strength and stiffness (N / mm for a given sample of 3 cm) as a function of sericin extraction from 10-fiber silk yarn from silkworms having 0 twists per inch (i.e. in parallel and (i) temperature and (II) time. Samples the repetitions were processed two years after

<td>Yarn</td><td>number the fiber</td><td>temp</td><td>Time</td><td>UTS (N)</td><td>Std. Stand.</td><td>Stiffness (N / mm)</td><td>Std. Stand.</td><td>UTS / fiber (N)</td><td>Stiffness / fiber (N / mm)</td>

<td> 10(0)</td><td> 10</td><td>RT</td><td>60 min</td><td> 10,74</td><td> 0,83</td><td> 6,77</td><td> 0,65</td><td> 1,07</td><td> 0,68</td>

<td> 10(0)</td><td> 10</td><td>RT</td><td>60 min (repeating)</td><td> 10,83</td><td> 0,28</td><td> 6,36</td><td> 0,14</td><td> 1,08</td><td> 0,64</td>

<td> 10(0)</td><td> 10</td><td>33C</td><td>60 min</td><td> 10,44</td><td> 0,17</td><td> 6,68</td><td> 0,55</td><td> 1,04</td><td> 0,67</td>

<td> 10(0)</td><td> 10</td><td>37C</td><td>60 min</td><td> 9,60</td><td> 0,84</td><td> 6,09</td><td> 0,59</td><td> 0,96</td><td> 0,61</td>

<td> 10(0)</td><td> 10</td><td>37C</td><td>60 min (repeating)</td><td> 9,54</td><td> 0,74</td><td> 5,81</td><td> 0,67</td><td> 0,95</td><td> 0,58</td>

<td> 10(0)</td><td> 10</td><td>90C</td><td>15 min</td><td> 9,22</td><td> 0,55</td><td> 4,87</td><td> 0,62</td><td> 0,92</td><td> 0,49</td>

<td> 10(0)</td><td> 10</td><td>90C</td><td>30 minutes</td><td> 8,29</td><td> 0,19</td><td> 4,91</td><td> 0,33</td><td> 0,83</td><td> 0,49</td>

<td> 10(0)</td><td> 10</td><td>90C</td><td>60 min</td><td> 8,60</td><td> 0,61</td><td> 4,04</td><td> 0,87</td><td> 0,86</td><td> 0,40</td>

<td> 10(0)</td><td> 10</td><td>90C</td><td>60 min (repeating)</td><td> 8,65</td><td> 0,67</td><td> 4,55</td><td> 0,69</td><td> 0,87</td><td> 0,46</td>

<td> 10(0)</td><td> 10</td><td>94C</td><td>60 min</td><td> 7,92</td><td> 0,51</td><td> 2,42</td><td> 0,33</td><td> 0,79</td><td> 0,24</td>

EP 2 374 919

<td>9 (12s) χ 3 (9z)</td><td> 27</td><td colspan="2">Non-extracted</td><td> 24,50</td><td> 0,38</td><td> 8,00</td><td> 0,49</td><td> 0,91</td><td> 0,30</td>

<td>9 (12s) χ 3 (9z)</td><td> 27</td><td>90C</td><td>60 min</td><td> 21,88</td><td> 0,18</td><td> 7,38</td><td> 0,34</td><td> 0,81</td><td> 0,27</td>

<td>9 (6s) χ 3 (3z)</td><td> 27</td><td colspan="2">Non-extracted</td><td> 24,94</td><td> 0,57</td><td> 9,51</td><td> 0,57</td><td> 0,92</td><td> 0,35</td>

<td>9 (6s) χ 3 (3z)</td><td> 27</td><td>90C</td><td>60 min</td><td> 21,36</td><td> 0,40</td><td> 7,95</td><td> 1,00</td><td> 0,79</td><td> 0,29</td>

<td>9 (12s) χ 3 (6z)</td><td> 27</td><td colspan="2">Non-extracted</td><td> 24,69</td><td> 0,65</td><td> 9,08</td><td> 0,56</td><td> 0,91</td><td> 0,34</td>

<td>9 (12s) χ 3 (6z)</td><td> 27</td><td>90C</td><td>60 min</td><td> 21,80</td><td> 0,47</td><td> 7,48</td><td> 0,97</td><td> 0,81</td><td> 0,28</td>

Table 2. Weight loss as a function of sericin extraction. The standard deviation + / -0.43% for n = 5, reflects the highest accuracy that can be obtained when confirming the removal of sericin, i.e., 0.87 or 1% error will always be appropriate for the methods used, and a weight loss of around 24% indicates for constructs essentially free of sericin .__

<td>Yarn</td><td>Non-extracted and dried (mg)</td><td>extracted and dried (mg)</td><td>% weight loss</td>

<td>9 (12) x 3 (6)</td><td> 57,6</td><td> 43,6</td><td> 24,31</td>

<td>9 (12) x 3 (6)</td><td> 58,3</td><td> 43,9</td><td> 24,70</td>

<td>9 (12) x 3 (6)</td><td> 57,0</td><td> 42,9</td><td> 24,74</td>

<td>9 (12) x 3 (6)</td><td> 57,2</td><td> 42,7</td><td> 25,35</td>

<td>average</td><td> 57,53</td><td> 43,28</td><td> 24,77</td>

<td>tilt. stand.</td><td> 0,57</td><td> 0,57</td><td> 0,43</td>

Table 3. Illustrates the change in mass as a function of the second sericin extraction. Correlated with Fig. 1E 1G, less than 3% weight loss is likely to indicate fibroin weight loss due to mechanical damage during the 2nd extraction.

<td>Yarn</td><td>Mass after 1x extraction, dried (mg)</td><td>Mass after 2x extraction, dried (mg)</td><td>% weight loss</td>

<td>9 (12) x 3 (6)</td><td> 42,5</td><td> 41,7</td><td> 1,88</td>

<td>9 (12) x 3 (6)</td><td> 43,1</td><td> 42</td><td> 2,55</td>

<td>9 (12) x 3 (6)</td><td> 43,1</td><td> 42,1</td><td> 2,32</td>

<td>9 (12) x 3 (6)</td><td> 42,5</td><td> 41,7</td><td> 1,88</td>

<td>9 (12) x 3 (6)</td><td> 42,6</td><td> 42,4</td><td> 0,47</td>

<td>9 (12) x (6)</td><td> 43,7</td><td> 42,4</td><td> 2,97</td>

<td>9 (12) x 3 (6)</td><td> 43,4</td><td> 42,9</td><td> 1,15</td>

<td>9 (12) x 3 (6)</td><td> 43,7</td><td> 43,1</td><td> 1,37</td>

<td>9 (12) x 3 (6)</td><td> 44</td><td> 43,2</td><td> 1,82</td>

<td>average</td><td> 43,18</td><td> 42,39</td><td> 1,82</td>

<td>tilt. stand.</td><td> 0,56</td><td> 0,57</td><td> 0,76</td>

EP 2 374 919

Table 4

<td>Geometry</td><td>Method layering</td><td>Conditions</td><td>number levels nawarstw a dowry</td><td>Whole number the fiber</td><td>Average value UTS (N)</td><td>UTS bias stand. (N)</td><td>Average% exp.</td><td>% exp. tilt. stand.</td><td>Wed. stiffness (N / mm)</td><td>Stiffness (N / mm) tilt. stand.</td><td>UTS on fiber</td><td>Fiber stiffness</td>

<td>1 (0) x 3 (10)</td><td>multiple flossing</td><td>extracted</td><td> 2</td><td> 3</td><td> 1,98</td><td> 0,05</td><td> 10,42</td><td> 1,63</td><td> 2,17</td><td> 0,51</td><td> 0,66</td><td> 0,72</td>

<td>1 (0) x 4 (10)</td><td>multiple flossing</td><td>extracted</td><td> 2</td><td> 4</td><td> 2,86</td><td> 0,14</td><td> 11,98</td><td> 1,54</td><td> 2,08</td><td> 0,31</td><td> 0,72</td><td> 0,52</td>

<td>3 (0) x 3 (3)</td><td>multiple flossing</td><td>extracted</td><td> 2</td><td> 9</td><td> 6,72</td><td> 0,17</td><td> 12,30</td><td> 0,72</td><td> 4,54</td><td> 0,16</td><td> 0,75</td><td> 0,50</td>

<td>1 (0) x 3 (10) x 3 (9)</td><td>multiple flossing</td><td>extracted</td><td> 3</td><td> 9</td><td> 6,86</td><td> 0,23</td><td> 13,11</td><td> 1,45</td><td> 4,06</td><td> 0,36</td><td> 0,76</td><td> 0,45</td>

<td>2 (0) x 6 (11</td><td>multiple flossing</td><td></td><td> 2</td><td> 12</td><td> 7,97</td><td> 0,26</td><td> 10,05</td><td> 0,91</td><td></td><td></td><td> 0,66</td><td></td>

<td>4 (6) x 3 (3)</td><td>turning</td><td>unextracted</td><td> 2</td><td> 12</td><td> 10,17</td><td> 0,18</td><td> 19,86</td><td> 1,16</td><td></td><td></td><td> 0,85</td><td></td>

<td>1 (0) x 3 (10) x 4 (9)</td><td>multiple flossing</td><td>extracted</td><td> 3</td><td> 12</td><td> 9,29</td><td> 0,19</td><td> 14,07</td><td> 0,98</td><td> 5,10</td><td> 0,31</td><td> 0,77</td><td> 0,43</td>

<td>1 (0) x 4 (11) x 3 (11)</td><td>turning</td><td>extracted</td><td> 3</td><td> 12</td><td> 9,70</td><td> 0,14</td><td> 12,56</td><td> 1,03</td><td> 7,60</td><td> 0,33</td><td> 0,81</td><td> 0,63</td>

<td>1 (0) x 4 (10) x 3 (9)</td><td>multiple flossing</td><td>extracted</td><td> 3</td><td> 12</td><td> 8,78</td><td> 0,17</td><td> 14,25</td><td> 1,09</td><td> 5,10</td><td> 0,32</td><td> 0,73</td><td> 0,43</td>

<td>15 (Textured)</td><td>texturing</td><td>not extracted, dry</td><td> 1</td><td> 15</td><td> 10,62</td><td> 0,68</td><td> 10,76</td><td> 1,70</td><td> 4,75</td><td> 0,30</td><td> 0,71</td><td> 0,316</td>

<td> 30(0)</td><td>parallel</td><td>extracted, wet</td><td> 1</td><td> 30</td><td> 20,24</td><td> 1,46</td><td> 26,32</td><td> 3,51</td><td> 1,14</td><td> 0,15</td><td> 0,67</td><td> 0,038</td>

<td> 30(0)</td><td>parallel</td><td>incubated 21 days, wet</td><td> 1</td><td> 30</td><td> 19,73</td><td> 2,10</td><td> 20,70</td><td> 6,03</td><td></td><td></td><td> 0,66</td><td></td>

<td> 30(0)</td><td>parallel</td><td>Inoculated with cells 21 days, wet</td><td> 1</td><td> 30</td><td> 20,53</td><td> 1,02</td><td> 29,68</td><td> 7,08</td><td></td><td></td><td> 0,68</td><td></td>

<td>2 fibers (tumble in 8</td><td>weave</td><td>extracted, dry</td><td> 2</td><td> 16</td><td> 10,93</td><td> 0,13</td><td></td><td></td><td> 6,96</td><td> 1,14</td><td> 0,68</td><td> 0,435</td>

<td>4 the fiber / drum in 8</td><td>weave</td><td>extracted, dry</td><td> 2</td><td> 32</td><td> 24,60</td><td> 0,22</td><td></td><td></td><td> 12,39</td><td> 0,53</td><td> 0,77</td><td> 0,387</td>

<td>4 (6) x 3 (3) in 4 drums</td><td>weave</td><td>extracted, dry</td><td> 3</td><td> 48</td><td> 37,67</td><td> 0,18</td><td> 22,38</td><td> 0,98</td><td></td><td></td><td> 0,78</td><td></td>

<td>15 (0) x 3 (12)</td><td>multiple flossing</td><td>dry</td><td> 2</td><td> 45</td><td> 27,39</td><td> 0,62</td><td> 31,68</td><td> 1,35</td><td> 4,63</td><td> 0,49</td><td> 0,61</td><td> 0,102889</td>

EP 2 374 919

<td>15 (0) x 3 (12) x 3 (10)</td><td>multiple flossing</td><td>unextracted, 1 day after manufacture</td><td> 3</td><td> 135</td><td> 73,61</td><td> 6,00</td><td> 33,72</td><td> 5,67</td><td> 12,33</td><td> 1,53</td><td> 0,55</td><td> 0,091333</td>

<td>15 (0) x 3 (12) x 3 (10)</td><td>multiple flossing</td><td>unextracted, 2 days after manufacture</td><td> 3</td><td> 135</td><td> 72,30</td><td> 5,68</td><td> 31,18</td><td> 4,35</td><td></td><td></td><td> 0,54</td><td></td>

<td>15 (0) x 3 (12) x 3 (10)</td><td>multiple flossing</td><td>unextracted, 3 days after manufacture</td><td> 3</td><td> 135</td><td> 70,74</td><td> 2,97</td><td> 29,50</td><td> 4,47</td><td></td><td></td><td> 0,52</td><td></td>

<td>15 (0) x 3 (12) x 3 (10)</td><td>multiple flossing</td><td>non-extracted, 4 days after manufacture</td><td> 3</td><td> 135</td><td> 75,90</td><td> 1,57</td><td> 34,57</td><td> 4,12</td><td></td><td></td><td> 0,56</td><td></td>

<td>15 (0) x 3 (12) x 3 (10)</td><td>multiple flossing</td><td>unextracted, 5 days after manufacture</td><td> 3</td><td> 135</td><td> 71,91</td><td> 5,71</td><td> 36,72</td><td> 3,75</td><td></td><td></td><td> 0,53</td><td></td>

<td>15 (0) x 3 (12) x 3 (10)</td><td>multiple flossing</td><td>non-extracted, 6 days after manufacture</td><td> 3</td><td> 135</td><td> 74,57</td><td> 1,45</td><td> 37,67</td><td> 4,27</td><td></td><td></td><td> 0,55</td><td></td>

<td>13 (0) x 3 (11) x 3 (10) x 3 (0)</td><td>multiple flossing</td><td>not extracted, dry</td><td> 4</td><td> 351</td><td> 189,01</td><td> 14,00</td><td> 45,87</td><td> 3,72</td><td></td><td></td><td> 0,54</td><td></td>

<td>13 (0) x 3 (11) x 3 (10) x 3 (0)</td><td>multiple flossing</td><td>non-extracted, subjected to 30x initial stress, dry</td><td> 4</td><td> 351</td><td> 170,12</td><td> 7,37</td><td> 39,95</td><td> 1,37</td><td></td><td></td><td> 0,48</td><td></td>

EP 2 374 919

Table 5. Comparison of UTS and stiffness between mechanical testing conditions for freeze (2 hours incubation in PBS at 37 ° C) and dry. N = 5. The results illustrate an approximately 17% decrease in the UTS value as a function of wet testing

<td>Yarn</td><td>number the fiber</td><td>Conditions test yarn</td><td>UTS (N)</td><td>UTS tilt. stand.</td><td>Stiffness (N / mm)</td><td>Stiffness tilt. stand.</td><td>UTS / fiber (N)</td><td>Stiffness / fiber (N / mm)</td>

<td>9 (12s) x 3 (9z)</td><td> 27</td><td>extracted dry</td><td> 21,88</td><td> 0,18</td><td> 7,38</td><td> 0,34</td><td> 0,81</td><td> 0,27</td>

<td>9 (12s) x 3 (9z)</td><td> 27</td><td>wet extracted</td><td> 18,52</td><td> 0,25</td><td> 2,56</td><td> 0,31</td><td> 0,69</td><td> 0,09</td>

<td>9 (6s) x 3 (3z)</td><td> 27</td><td>extracted dry</td><td> 21,36</td><td> 0,40</td><td> 7,95</td><td> 1,00</td><td> 0,79</td><td> 0,29</td>

<td>9 (6s) x 3 (3z)</td><td> 27</td><td>wet extracted</td><td> 17,94</td><td> 0,30</td><td> 2,40</td><td> 0,28</td><td> 0,66</td><td> 0,09</td>

<td>9 (12s) x 3 (6z)</td><td> 27</td><td>extracted dry</td><td> 21,80</td><td> 0,47</td><td> 7,48</td><td> 0,97</td><td> 0,81</td><td> 0,28</td>

<td>9 (12s) x 3 (6z)</td><td> 27</td><td>wet extracted</td><td> 18,74</td><td> 0,22</td><td> 2,57</td><td> 0,11</td><td> 0,69</td><td> 0,10</td>

<td>12 (0) x 3 (10s)</td><td> 36</td><td>extracted dry</td><td> 30,73</td><td> 0,46</td><td> 16,24</td><td> 0,66</td><td> 0,85</td><td> 0,45</td>

<td>12 (0) x 3 (10s)</td><td> 36</td><td>wet extracted</td><td> 25,93</td><td> 0,29</td><td> 6,68</td><td> 0,70</td><td> 0,72</td><td> 0,19</td>

<td>4 (0) x 3 (10s) x 3 (9z)</td><td> 36</td><td>extracted dry</td><td> 30,07</td><td> 0,35</td><td> 15,49</td><td> 1,06</td><td> 0,84</td><td> 0,43</td>

<td>4 (0) x 3 (10s) x 3 (9z)</td><td> 36</td><td>wet extracted</td><td> 22,55</td><td> 0,66</td><td> 7,63</td><td> 1,00</td><td> 0,63</td><td> 0,21</td>

Table 6. Impact of TPI on UTS and Stiffness. N = 5

<td colspan="2">Yarn</td><td>TPI</td><td>UTS (N)</td><td>tilt. stand.</td><td>Stiffness (N / mm)</td><td>tilt. stand.</td><td>UTS / fiber (N)</td><td>Stiffness / fiber (N / mm)</td>

<td> 12(0) 3(2)</td><td>x</td><td> 2</td><td> 23,27</td><td> 0,28</td><td> 6,86</td><td> 0,60</td><td> 0,65</td><td> 0,19</td>

<td> 12(0) 3(4)</td><td>x</td><td> 4</td><td> 24,69</td><td> 0,31</td><td> 7,61</td><td> 1,17</td><td> 0,69</td><td> 0,21</td>

<td> 12(0) 3(6)</td><td>x</td><td> 6</td><td> 25,44</td><td> 0,42</td><td> 6,51</td><td> 1,35</td><td> 0,71</td><td> 0,18</td>

<td> 12(0) 3(8)</td><td>x</td><td> 8</td><td> 25,21</td><td> 0,23</td><td> 5,80</td><td> 0,67</td><td> 0,70</td><td> 0,16</td>

<td> 12(0) 3(10)</td><td>x</td><td> 10</td><td> 25,94</td><td> 0,24</td><td> 6,45</td><td> 0,77</td><td> 0,72</td><td> 0,18</td>

<td> 12(0) 3(12)</td><td>x</td><td> 12</td><td> 25,87</td><td> 0,19</td><td> 6,01</td><td> 0,69</td><td> 0,72</td><td> 0,17</td>

<td> 12(0) 3(14)</td><td>x</td><td> 14</td><td> 22,21</td><td> 0,58</td><td> 5,63</td><td> 0,71</td><td> 0,62</td><td> 0,16</td>

Table 7. Additional tpi data to confirm that it can be used up to 30tpi without causing damage to the yarn, which would result in a drastic decrease in UTS and stiffness; note, all matrices were twisted (N = 5 per group).

<td>Yarn</td><td>number the fiber</td><td>UTS (N)</td><td>tilt. stand. (N)</td><td>Stiffness (N / mm)</td><td>tilt. stand.</td><td>UTS / fiber (N)</td><td>Stiffness / fiber (N / mm)</td><td>Conditions</td>

<td>1 (30) x 6 (20) x 3 (4.5)</td><td> 18</td><td> 10,92</td><td> 0,44</td><td> 1,21</td><td> 0,02</td><td> 0,61</td><td> 0,07</td><td>not extracted, wet</td>

<td>1 (30) x 6 (20) x 3 (10)</td><td> 18</td><td> 11,48</td><td> 0,37</td><td> 1,25</td><td> 0,06</td><td> 0,64</td><td> 0,07</td><td>not extracted, wet</td>

<td>1 (30) x 6 (6)</td><td> 6</td><td> 3,83</td><td> 0,24</td><td> 0,37</td><td> 0,04</td><td> 0,64</td><td> 0,06</td><td>not extracted, wet</td>

<td> 15(20)</td><td> 15</td><td> 13,19</td><td> 0,27</td><td> 6,03</td><td> 0,67</td><td> 0,88</td><td> 0,40</td><td>extracted, dry</td>

EP 2 374 919

Table 8. Influence of yarn on hierarchy and mechanical properties (i.e. number of levels and number of fibers per level can significantly affect yarn and material results).

<td>Geometry</td><td>Conditions</td><td>number levels layering</td><td>Whole number the fiber</td><td>UTS (N)</td><td>UTS tilt. stand. (N)</td><td>% ed average</td><td>% exp. tilt. stand.</td><td>Stiffness average (N / mm)</td><td>Stiffness deflection stand. (N / mm)</td><td>UTS for fiber</td><td>Fiber stiffness</td>

<td>1 (0) x 3 (10)</td><td>extracted</td><td> 2</td><td> 3</td><td> 1,98</td><td> 0,05</td><td> 10,42</td><td> 1,63</td><td> 2,17</td><td> 0,51</td><td> 0,66</td><td> 0,72</td>

<td>1 (0) x 3 (10) x 3 (9)</td><td>extracted</td><td> 3</td><td> 9</td><td> 6,86</td><td> 0,23</td><td> 13,11</td><td> 1,45</td><td> 4,06</td><td> 0,36</td><td> 0,76</td><td> 0,45</td>

<td>1 (0) x 3 (10) x 4 (9)</td><td>extracted</td><td> 3</td><td> 12</td><td> 9,29</td><td> 0,19</td><td> 14,07</td><td> 0,98</td><td> 5,10</td><td> 0,31</td><td> 0,77</td><td> 0,43</td>

<td>1 (0) x 4 (10)</td><td>extracted</td><td> 2</td><td> 4</td><td> 2,86</td><td> 0,14</td><td> 11,98</td><td> 1,54</td><td> 2,08</td><td> 0,31</td><td> 0,72</td><td> 0,62</td>

<td>1 (0) x 4 (10) x 3 (9)</td><td>extracted</td><td> 3</td><td> 12</td><td> 8,78</td><td> 0,17</td><td> 14,25</td><td> 1,09</td><td> 5,10</td><td> 0,32</td><td> 0,73</td><td> 0,43</td>

<td>15 (0) x 3 (12)</td><td>not extracted, dry</td><td> 2</td><td> 45</td><td> 27,39</td><td> 0,62</td><td> 31,68</td><td> 1,35</td><td> 4,63</td><td> 0,49</td><td> 0,61</td><td> 0,10</td>

<td>15 (0) x 3 (12) x 3 (10)</td><td>not extracted, dry</td><td> 3</td><td> 135</td><td> 73,61</td><td> 6,00</td><td> 33,72</td><td> 5,67</td><td> 12,33</td><td> 1,53</td><td> 0,55</td><td> 0,09</td>

EP 2 374 919

<td colspan="2">Table 9. Effect of m extracted matrix</td><td colspan="7">surface modification (RGD and ETO gas sterilization) for the mechanical properties of silk life; PBS was used as a negative control during modification treatments.</td>

<td>Yarn</td><td>number the fiber</td><td>Modification surface / sterilization</td><td>UTS (N)</td><td>tilt. stand.</td><td>Stiffness (N / mm)</td><td>tilt. stand.</td><td>UTS / fiber (N)</td><td>Stiffness /fiber (N / mm)</td>

<td>12 (0) x 3 (10s)</td><td> 36</td><td>Non-treated</td><td> 25,94</td><td> 0,24</td><td> 6,45</td><td> 0,77</td><td> 0,72</td><td> 0,18</td>

<td>12 (0) x 3 (10s)</td><td> 36</td><td>RGD</td><td> 23,82</td><td> 2,10</td><td> 3,79</td><td> 2,06</td><td> 0,66</td><td> 0,11</td>

<td>12 (0) x 3 (10s) x3 (9Z)</td><td> 108</td><td>Non-treated</td><td> 48,89</td><td> 4,84</td><td> 9,22</td><td> 0,84</td><td> 0,45</td><td> 0,09</td>

<td>12 (0) x 3 (10s) x3 (9Z)</td><td> 108</td><td>RGD</td><td> 55,28</td><td> 3,28</td><td> 8,17</td><td> 0,81</td><td> 0,51</td><td> 0,08</td>

<td>4 (11s) x 3 (11z) x 3 (10s)</td><td> 36</td><td>ECA</td><td> 18,72</td><td> 0,45</td><td> 5,52</td><td> 0,42</td><td> 0,52</td><td> 0,15</td>

<td>4 (11s) x 3 (11z) x 3 (10s)</td><td> 36</td><td>RGD + ETO</td><td> 19,30</td><td> 0,62</td><td> 4,67</td><td> 0,3</td><td> 0,54</td><td> 0,13</td>

Table 10

<td></td><td>UTS (N)</td><td>Stiffness (N / mm)</td><td>The yield point. (N)</td><td>Elongation (%)</td>

<td>Silk matrix 1</td><td> 2337+/-72</td><td> 354+/-26</td><td> 1262+/-36</td><td> 38,6+/-2,4</td>

<td>Silk matrix 2</td><td> 3407+/-63</td><td> 580+/-40</td><td> 1647+/-214</td><td> 29+/-4</td>

<td>Human ACL</td><td> 2160+/-157</td><td> 242+/-28</td><td> -1200</td><td> -26-32%</td>

Mechanical properties of two different cords for 3 cm long cords compared to the 5 properties of human ACL.

Table 11

<td>Twist level (number of turns / cm)</td><td>Matrix 1</td><td>Matrix 2</td><td>Matrix 3</td><td>Matrix 4</td><td>Matrix 5</td><td>Matrix 6</td><td>Matrix 7</td>

<td>number of fibers per group</td><td> 30 (0)</td><td> 15 (0)</td><td> 1300 (0)</td><td> 180 (0)</td><td> 20 (0)</td><td> 10 (0)</td><td> 15 (0)</td>

<td>number of groups per bundle</td><td> 6 (3)</td><td> 3 (3)</td><td> 3 (2)</td><td> 3 (3,5)</td><td> 6 (3)</td><td> 6 (3)</td><td> 3 (3)</td>

<td>number of beams per strand</td><td> 3 (3)</td><td> 6 (2,5)</td><td> 1 (0)</td><td> 3 (2)</td><td> 3 (2)</td><td> 3 (2,5)</td><td> 3 (2,5)</td>

<td>number of strands per string</td><td> 6 (0)</td><td> 3 (2,0)</td><td> -</td><td> 2 (0)</td><td> 3 (1)</td><td> 3 (2)</td><td> 3 (2)</td>

<td>number of strings per ACL</td><td> -</td><td> 6 (0)</td><td> -</td><td> -</td><td> 3 (0)</td><td> 6 (0)</td><td> 12 (0)</td>

<td>UTS (N)</td><td> 2337</td><td> 3407</td><td> 2780</td><td> 2300</td><td> 2500</td><td> 2300</td><td> 3400</td>

<td>Stiffness (N / mm)</td><td> 354</td><td> 580</td><td> 300</td><td> 350</td><td> 550</td><td> 500</td><td> 550</td>

<td colspan="8">Examples of several geometric hierarchies that may result in appropriate mechanical properties for ACL replacement. Note: Matrix 1 and 2 were developed as shown in the examples, Matrix 3 would provide a single-beam prosthesis, Matrix 4 would provide a 2-strand denture, Matrix 5 would provide a 3-string prosthesis, Matrix 6 would be another 6-string prosthesis, and the Matrix 7 provides a prosthesis consisting of 12 strings.</td>