MXPA98009863A - Increase of colag construction resistance - Google Patents

Abstract

A method to improve the tensile strength of collagen strands or constructs of wet collagen strands for their implantation to replace or repair oral tissues, where the resistance of the strands is improved by plasticizing the strands with a plasticizing agent, imparting a load traction to the strands of collagen to strain the collagen strand, allow the stress in the strand to decrease by the methods of conditioning stress relaxation or plastic deformation and, finally, remove the plasticizing agent. It is intended that prosthetic devices comprising collagen strands with improved strength characteristics repair stressed tissues, such as ligaments and tendons.

Description

INCREASE OF RESISTANCE OF COLLAGEN CONSTRUCTS Field of the Invention The present invention is in the field of implantable medical devices designed as tissue and is directed to prosthetic devices made of collagen strands, which are used to replace or repair tissues or organs. It is intended that such prosthetic devices repair load bearing tissues such as ligaments and tendons, for example. This invention describes a method to improve the ultimate tensile stress (UTS) of collagen strands and collagen strand constructs by the use of relaxation efforts.

BACKGROUND OF THE INVENTION One of the most important attributes of living organisms is their ability to self-repair. Several mechanisms have evolved to achieve this, including wound healing, compensatory growth and epimorphic regeneration (J. Gross, Regeneration versus repair, pp. 20-39 (1992), In: LK Cohen, RF Diegelman and J. Lindblad ( eds.), Wound Healing: Biochemical and Clinical Aspects, WB Saunders, Philadelphia). Although all tissues and organs (with the possible exception of teeth) are capable of some degree of repair, mammals unfortunately have lost the ability to completely regenerate severely damaged body parts. (G. Gross, supra, (1992)). In an attempt to overcome this deficiency, numerous synthetic devices have been developed, with the intention that the implants are biologically inert, and also work during the life of the recipient. Experience with synthetic devices, however, has shown that biological inertness is not only seemingly impossible, but that the interaction between the biomaterial and the surrounding living tissue can really contribute to the long-term success of the implant. (J. Kohn, Med. Dev. Technol., 1: 34-38 (1990)). The science of tissue design has begun to exploit this biological reality. The biomaterials used to produce biorremodelables grafts are the focus of this study area. Numerous researchers have evaluated two families of materials: the biological components of the extracellular matrix (ECM), such as collagen and proteoglycan, and synthetic, non-biological materials. Materials derived from biological products are advantageous since they contain properties that facilitate cell function and binding, while synthetic materials may not interact with cells in the desired form (R. Langer, Science, 260: 920-926 (1993)) . Researchers are also attempting to alter synthetic materials by coupling peptide sequences recognized by cell adhesion proteins, such as integrins (JA Hubbell, Ann NY Acad. Sci., 665: 253-258 (1992); Lin, HB , et al., Biomaterials; 13: 905-914 (1992)). The original theory that the extracellular matrix is simply an inert support material in or on which the cells reside, has recently been considered as false (Hay, ED, et al., Cellular Biology of the Extracellular Matrix, 2nd edition ( 1991), Plenum Press, New York, Nathan, CJ Cell Biol, 113: 981-986 (1991)). The cells continue to interact with the main components of the extracellular matrix, which continues to serve the functions of adhesive, biomaterial, filter, receptor, signal and text (Nathan, C, supra (1991); Trelstad, RL, Textbook of Rheumatology, pp. 35-57 (1993) 4th edition, B. Saunders, Philadelphia). Therefore, it seems reasonable to assume that the complex interactions between the cells and the extracellular matrix are such that the implants derived from biological materials will continue to provide stimuli to guide the remodeling, while the synthetic ones will not, unless they are modified to such a degree that they become essentially identical to the natural molecules that they intend to imitate. Although biological in origin, the extensive chemical modification of collagen tends to become "alien". To improve the long-term functioning of implanted collagen devices, it is important to retain many of the properties of natural collagen tissue. In this "tissue design" method, the prosthesis is designed not as a permanent implant, but as a support structure or pattern for regeneration or remodeling. The principles of tissue technology incorporate an iso orfo tissue replacement requirement, where the biodegradation of the implant matrix occurs at approximately the same functional rate of tissue replacement by the host, so that a functional analogy of original tissue. (Yannas, I. V. (1995) Patterns of Regeneration, pp, 1619-1635, In: Bronzino, J. D. (ed.), The Manual of Biomedical Engineering, CRC Press, Inc., Boca Raton, Florida). Although Type I collagen has been used as a biomaterial for more than 50 years, such implants have generally not exploited the body's ability to repair an implant. On the contrary, the implants were intended to be permanent, and the manufacturing procedures used to produce those devices used collagen extracted with enzymes, partially degraded, stabilizing the collagen by crosslinking it using glutaraldehyde or chromium salts (Chvapil, M., Industrial uses of collagen, in DAD Parry and LK Creamer (eds.), Fibrous proteins: scientific, industrial and medical aspects, (1979) Academic Press, London; Chvapil, M., Int Rev Connect Tiss Res, 6: 1 (1972), Stenzel, KH, et al., Ann Rev Biophys Bioeng, 3: 231-253 (1974)), or mounted on collagen in non-polymer structures such as films and sponges (Burke, JF, supra) (1981); Chvapil, M., supra (1979); Chvapil, M. (1973), supra, Rubin, A., J Sci Chem., A3: 113-118 (1969); Stenzel, K. H., supra (1974); Yannas, I.V., Science, 215: 174-176 (1982); Yannas, I.V., Proc Nati Acad Sci USA, 86: 933-937 (1989), Heimbach, D., Ann Surg, 208: 313-320 (1988)). Since prostheses that must function under significant loads require significant mechanical resistance, such as ligament and tendon replacements and in the repair of hernias, alternative biomaterials are being sought. An important area for tissue design technology is the development of a material to permanently replace a damaged ligament or tendon. The tendon or ligament most commonly replaced is the anterior cruciate ligament (ACL) of the knee, due to damage commonly attributed to athletic injuries such as football or skiing. Once the ACL is broken, healing does not occur by itself, as it can be in other knee ligaments (for example, the middle collateral ligament) mainly because the position of the broken ligament ends is impossible, due to to the elastic contraction. Patellar tendon autografts are the current standard for the care of anterior cruciate ligament (ACL) replacement (Markolf KL et al., Transcript ORS 20: 644, 1995). Autografts are not regulated medical devices and are not subject to any of the manufacturing and marketing regulations that govern biomaterials. The patellar tendon is readily available for use by the surgeon and, being an autograft material, immune rejection is not a concern. Typically, one third of the width of the patellar tendon is removed from the same knee joint and placed in the anterior cruciform position; the fixation is usually done by piercing precisely the femur and the tibia, placing the graft between, and fixing the ends in the bone cement inside the perforated holes. Although functional as a support structure for new tissue growth and biological fixation, the collection procedure causes additional trauma to the ACL patient. In addition, the patellar tendon weakens during the remodeling process and can therefore be damaged. Finally, the failure rate of patellar tendon autografts is high because they are revascularized slowly, and often lengthened to the point where knee loads are no longer supported. After the failure of a patellar tendon autograft, surgeons consider a prosthetic device for the replacement of the ACL. Advances in the science of materials have produced grafts, which are very strong and durable, and still surpass the natural resistance of the ACL. The main materials considered for ACL replacement grafts are synthetic polymers, carbon fibers and collagen. Allogeneic materials (ie, cadaverous grafts) avoid the trauma of collection, but may carry risks of transmission of viral pathogens. The synthetic graft material, usually polyethylene terephthalate (Dacron, manufactured by both Howmedica and Stryker) or polytetrafluoroethylene (Teflon, made by Gore-Tex), and carbon fiber grafts (DuPont) can be twice as strong as the tissue of the native ACL, and can last up to 1 x 107 cycles without failure.

However, laboratory tests can not simulate the internal environment of the knee joint. Although these grafts begin to be strong enough to withstand the load required, those materials are not biologically inert, and are subject to fatigue and abrasion. The problems of chronic inflammation and abrasion persist and worsen over time, until a mechanical failure occurs, and it is necessary to operate again. ACL grafts made from reconstituted collagen fiber have been reported. U.S. Patent No. 5, 171,273 discloses a collagen graft comprising synthetic collagen fibers embedded in a loose matrix of non-crosslinked collagen. The starting material for the fibers was dissociated insoluble collagen from bovine skin (dermis). The dissociated material is a suspension of fragments of fibrillar bovine collagen type I, native in band, which is believed to contain small amounts of other tissue proteins. U.S. Patent No. 5,263,984 discloses a prosthetic ligament comprising filaments formed from fibrils or short pieces of native polymeric connective tissues, such as collagen. The collagenous starting material in this case was also insoluble.

They have been proposed fibers reconstituted collagen arranged in bundles (Dunn, FH, et al, Am J Sports Med 20:.. 507, 1992; Cavallaro, JF et al, Biotech Bioeng 43: 781, 1994) or braids (Cavallaro, JF et al., supra., Chvapil, M., et al., J. Biomed Material Res 27: 313, 1993) as replacements of the ACL with the properties of tissue technology. Studies of promising implants in models with smaller animals, such as the rabbit (Dunn, FH et al., Supra) and the dog (Cavallaro, JF et al., Supra), have not been successful in larger models, as in the goat (Chvapil, M., et al., supra), perhaps due to the relatively low cooperative force of the composite structure, despite the high final tensile stress (UTS) of the individual fibers. This loss of strength is due to tensions, lengths and non-uniform orientations between the fibers in the construct (Zurek W., et al., Textile Res J 57 (8): 439, 1987). It is desirable to have a prosthetic device prepared from a biomaterial, such as collagen, that approximates the strength of the synthetic materials. A continuing goal of the researchers is to develop implantable prostheses, which can be used successfully to replace or repair mammalian tissues.

BRIEF DESCRIPTION OF THE INVENTION The invention provides a method for improving the tensile strength of collagen threads and constructs made from collagen threads which includes laminating a strand or strands of collagen construct with a plasticizing agent; imparting a tensile load to the collagen strand or construct to deform the collagen strand, and then allow the strain in the strand to decrease by stress relaxation or plastic deformation. Additionally, the method may include crosslinking the strand along with a crosslinking agent. Collagen strands and constructs comprising collagen strands with improved tensile strength properties are useful for implantation as prosthetic devices. It is intended that prosthetic devices comprising collagen strands with improved strength characteristics, repair load-bearing tissues, such as ligaments and tendons.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides methods for increasing the strength of biocompatible prosthetic devices comprising collagen threads useful as implants for repairing tendons, ligaments or other damaged structures for hernia repair, replacement of blood vessels, support prolapsed , and reconstruction of the chest wall after trauma or resection of a tumor. In a preferred embodiment, the ligament replaced is the anterior cruciate ligament (ACL) at the knee joint of mammals. The devices of the present invention comprise an array of collagen strands formed from a solution of collagen molecules that mimic the chemical and organizational structure of natural collagen. In a preferred embodiment, the device comprises collagen strands, arranged in a bundle, in which the collagen strands have been conditioned by stress relaxation to improve the tensile strength of the device. Once implanted, the device provides a support structure for infiltration and population by the connective tissue cells of the host, which will eventually supplement or replace the device with natural tissue, thereby allowing it to perform its natural functions. Collagen strands comprising collagen can be prepared by any of the numerous methods known in the art for the formation of collagen strands. In accordance with the present invention, acid extraction is preferably used instead of extraction with enzymes to produce the collagen solution. Extraction with enzyme (pepsin) removes telopeptide regions from the ends of collagen molecules; Such collagen preparations produce weaker strands than the preparations extracted with acid. Similarly, collagen extracted with pepsin has been shown to produce contracted fibroblast collagen reticles, which are twenty times weaker than the reticles produced from collagen extracted with acid (Bell, E., INSERM, 177: 13-28 (1989)). Collagen solubilized with acid can be prepared using techniques and sources known to those skilled in the art. Sources of collagen include skin and tendons. A preferred collagen composition for use herein is obtained from a novel source, the common bovine digital extensor tendon, and by a novel extraction method, both described in US Pat. No. 5,106,949, the description of which is incorporated here as a reference Although monomers and mixtures of monomers and polymers of higher collagen can be used, for example, dimers up to and including fibrils, in the practice of the present invention; for many applications monomers are preferred.

The collagen solutions for use in the present invention are generally in a concentration of about 2 to 10 mg / mL, preferably about 4 to 6 mg / mL, and more preferably about 4.5 to 5.5 mg. / mL and at a pH of about 2 to 4. A preferred solvent for the collagen is acetic acid diluted to about 0.05 to about 0.1% volume / volume. Other conventional solvents for collagen may be used, as long as the solvents are compatible with the formation of the strand and the desired properties of the collagen strand. These collagen solutions may also contain optional components known to those skilled in the art to modify or regulate the interaction between the host and the implant; components such as neutral and charged polymers, including but not limited to polyvinyl alcohol, polyethylene glycol, hyaluronic acid, growth factors and other components of the extracellular matrix such as proteoglycans. The formation of collagen strands using type I collagen solubilized in acid is described in U.S. Patent No. 5,378,469, incorporated herein by reference. A preferred method for making collagen strands for use in the present invention comprises: (a) extruding a solution comprising collagen in a dehydrating agent, the dehydrating agent having an osmotic pressure higher than that of the collagen solution and a pH of about 5 to 9; and, (b) keeping the dehydrating agent under conditions to allow the formation of the collagen strand. In another preferred method for making collagen strands, the method further comprises rinsing the formed strand to remove the dehydrating agent to provide additional flexibility. This optional step is particularly useful in applications where the collagen strand is to be woven in the form of a knitted or woven fabric in the form of a fabric. A preferred rinse agent is purified water. Another preferred rinse agent comprises a phosphate buffered saline solution (PBS) having a phosphate concentration of about 0.001 to about 0.2 M and a sodium chloride (NaCl) concentration of about 0.05 to about 0.1 M. When solutions are used damped, the pH of the rinse bath is maintained above a pH of about 5 to prevent overhydration of the strand. A preferred pH range is from about 6 to about 8.

The properties of the strands and fibers of collagen can be evaluated in a similar way to other strands and fibers used in the textile industry. Textile fibers are generally measured as the mass of the strand by length, or denier (mass in grams per 9000 meters in length). Typically from about 40 to 80, the denier may vary from about 15 to about 300, altering the rate of infusion of the collagen to the dehydration bath, the rate of flow of the dehydration bath, and / or the size (of the orifice) of the needle. The tenacity of a strand is measured as grams of tensile force per denier. For example, if a strand with a denier of 50 has a tensile force of 220 grams, the tenacity is 220/50 = 4.40 grams per denier (gpd). The final load is the maximum load of a strand or thread construct, just before its breaking, usually measured in grams. The final tensile stress (UTS) is calculated by dividing the final load by the cross-sectional area and is measured in Newtons per square millimeter, which are also known as mega pascals (MPA), or in pounds per square inch (psi) . Grams per denier (gpd) can also be used to express the UTS. The constructs can be formed from collagen strands by techniques for processing fibers known to those skilled in the art., for example, by knitted fabric or mesh fabric. Most techniques for handling fibers, both natural fibers, for example, cotton, silk, etc., and synthetic fibers, for example, nylon, cellulose acetate, etc., could be useful in the processing of the strands provided herein. , including the techniques used to produce three-dimensional textiles. See, for example, Mohamed, American Scientist. 78, (1990) 530-541. Three-dimensional collagen constructs and methods for making them are described in US Patent Application Serial No. 08 / 215,760, the disclosure of which is incorporated herein by reference. Collagen strands have been used to form twisted, folded or folded knit constructions, and knitted fabrics as described in US Patent Application Serial No. 08 / 216,527. The collagen strands can be woven using techniques known to those skilled in the art to produce a knitted construct. A knitted tube comprising two pleated yarn, the braiding of a strand of crosslinked collagen and a strand of non-crosslinked collagen has been used in the preparation of a blood vessel construct also described in US Patent Application No. of Series 08 / 216,527.

In a preferred embodiment, a bundle of multiple filaments is formed by winding the strands around a device comprising at least two stitches, such as legs firmly mounted to a frame, to produce a closed ring. When the closed ring is removed from the legs, the opposite sides of the ring are joined, so that the bodies of the strands are parallel to each other, to form two rings at either end of the bundle. The rings at either end of the bundle are then secured and either or both rings can then be optionally cut to form a bundle of individual collagen strand segments that are almost about the same length. A ring or rings at either end of the construct can be used to fix the construct or bundle when implanted or grafted into a host or patient. To form a bundle of 500 folds by this method, the strand is wrapped around a device 250 times. The bundles can also be formed by assembling and aligning the ends of segments of individual strands that are comparatively of approximately the same length and then securing the ends to form a bundle construct. Other techniques and sources known to those skilled in the art can also be used to form a bundle of strands. A beam can be used to form a multi-filament construct of three or more bundles of strands, a helix of two or more bundles, or a single braided beam or a non-braided bundle. At least one ring may also be formed at one end of the bundle to provide means for holding the device when it is implanted in a host or patient. The theoretical strength of identical collagen fibers in a beam, in parallel, exceeds that of any twisted or braided construct containing the same number of fibers, due to the fact that any twisted or braided construct contains elements not really aligned with the axis of the construct In practice, however, twisted and twisted constructs often have the capacity to withstand much higher traction loads than parallel beams, because it is impossible to keep the lengths and tensions of the fibers across the bundle absolutely uniform. One method to combat the practical loss of resistance in a parallel bundle of collagen fibers is to influence the important properties of collagen: it is easily plasticized, and the imposed stresses easily relax over time. The viscoelasticity of the collagenous tissues was investigated by Fung, which defines stress relaxation, also called simply "relaxation", as a process when a tissue is loaded to a constant finite deformation and the length remains constant, the corresponding stresses induced in the tissue diminish with time. (Fung, Y. C, (1981) Biomechanics: Mechanical Properties of Living Fabrics, pp. 211 Springer-Verlag New York Inc., New York). In this way, by plasticizing and then lengthening or extending a parallel beam of uncrosslinked collagen fibers, all the fibers are brought to the same length. Next, if the beam subjected to stress is allowed to relax, the effect is that all the fibers are brought to approximately the same degree of tension. Alternatively, after plasticizing, the construct may be allowed to undergo plastic deformation (i.e., under a tensile load), thereby achieving the same result. At the microstructural level, these stress relieving and plastic deformation conditioning treatments cause the collagen molecules within the fiber to align with the fiber axis, in fact, for the same reason, stretching the fiber is a method well known for improving tensile properties (Zurek, supra). Those conditioning treatments have added the benefit of aligning the beam elements together into a more coherent unit, increasing the packing density of the fibers. The coherence of the construct allows sharing the load between the adjacent collagen strands to contribute to the resistance of the construct. With consistency, packing density (also called "fiber volume") is increased, and more strands can be made to fit into a confined space such as a joint or a bone tunnel when implanted as a ligament prosthesis. factors combine to significantly improve the wet strength of a bundled collagen fiber construct In the preferred embodiment, a single collagen strand or bundle of collagen strands of between 2 and 1000 folds is produced by the methods of formation of beams known in the art Each strand in the bundle is preferably of approximately the same length as compared to the others in the bundle The strands may, however, be of different denier and composition. orientation of the strands in the beam can be such that the core of the beam is composed of thinner or smaller denier threads and the peripheral threads are thicker or denier threads. The composition of any of the internal or external strands in a bundle may have cytokines or growth factors coated on, or incorporated within, the strands to improve or regulate cell compatibility or biofemodelation. The beam can also be modified so that the ends of the beam that are implanted-within the bone are treated with a bone morphogenic protein or cytokines that increase bone formation. The beam is then placed inside a device that can pull the beam from the ends in opposite directions to impose an effort along the length of the beam. The initial value of the tensile strength for non-reticulated, dry beams is obtained by pulling the bundles beyond the breaking point, noting the peak load. The method for improving the tensile strength of a collagen strand or bundle comprising collagen strands comprises fixing the ends of a bundle of strands in a device or means for pulling the bundle at the ends in opposite directions. A preferred device for pulling the beam at the ends is a mechanical test system such as the Mini-Bionix 858 mechanical test system (MTS Systems Corp., Eden Prairie, MN). Once the ends of the beam are fixed by the clamps of the mechanical test system, the beam is then plasticized. The plasticizing agents are preferably water or aqueous solutions or buffers such as phosphate buffered saline (PBS). Buffer solutions with lower pH have been used to plasticize the collagen strands at a faster rate than at a higher pH. Plasticizers such as glycerol or other hygroscopic agents known in the art may also be used, but to preserve strength after the conditioning treatment, the plasticizer must also be removed. Once plasticized, the bundles are lengthened by the mechanical test system to impart a total stress, preferably between about 20 to 200%, more preferably between about 50 and 100%. The elongation step can be performed incrementally or in a single elongation step to impart an effort of about 5 to 10% and is allowed to relax to decrease the effort in the construct between about 0.5 and zero grams per end, before an elongation additional. Alternatively, (as in plastic deformation) a tensile load can be applied and maintained while increasing the length. The effort in the beam bundle construct is preferably left to relax or dissipate at a load of approximately zero. The less effort remaining after relaxation, the greater the effect of conditioning on the increase in resistance. The stress relaxed or conditioned by plastic deformation construct is preferably crosslinked after the conditioning treatment. Crosslinking provides resistance, stability and some durability to the collagen strands and constructs that comprise collagen. The crosslinking is effected by any of the numerous methods known to those skilled in the art, including lyophilization, ultraviolet (UV) radiation, or contacting the construct with a chemical crosslinking agent. Various types of chemical crosslinking agents are known in the art, such as acyl azide, hexamethylene diisocyanate, bisimidates, glyoxal, polyglycerol polyglycidyl ether, adipyl chloride, ribose and other sugars, carbodiimides such as cyanamide or l-ethyl-3- (3-diimethylamino-propyl) carbodiimide hydrochloride (EDC), aldehydes such as glutaraldehyde or formaldehyde, and oxidizing agents may be used. Preferred crosslinking agents are those that produce a biocompatible material capable of being remodeled by host cells. A preferred crosslinking agent is EDC. The carbodiimides activate the carboxyl groups on the collagen molecule, which then forms synthetic peptide bonds with the adjacent amino groups, releasing a urea. The crosslinking solution containing EDC and water may also contain acetone. In a preferred embodiment, sulfo-N-hydroxysuccinimide is added to the crosslinking agent (Staros, 1982). However, crosslinking agents are not necessarily limited to those examples, since other crosslinking agents and methods known to those skilled in the art may be used. The increase of the final tensile strength (UTS) of the bundles of collagen strands conditioned by stress relaxation on the bundles of collagen strands not conditioned by stress relaxation, is due to the improved alignment and reduction of the area of total cross section (ie, thinning of the fiber) caused by the elongation treatment. The increased UTS of bundles of collagen strands conditioned by relaxation of stress on similar denier controls, seems to be due to the improved distribution of the load, when the length, tension and orientation become more uniform within the beam. The collagen strands with higher strength of the invention are preferably sterilized before being implanted or grafted to a patient or host. Sterilization can be effected by the use of gamma radiation with 2.5 Mrad typically, ethylene oxide or by chemical sterilization. A preferred method of chemical sterilization of the stress-relaxed collagen construct is by contacting the construct with diluted peracetic acid solution with a neutral pH or a high salt concentration. Methods for sterilizing collagen are described in U.S. Patent No. 5,460,962, the disclosure of which is incorporated herein by reference. However, sterilants and sterilization methods are not limited to those examples, since other sterilants and methods for sterilizing collagen known to those skilled in the art may alternatively be used. Collagen fibers can also be coated with agents such as pharmaceuticals; growth factors; hormones; other components of the extracellular matrix; or genetic material. The coating of the agent can be carried out by immersion or chemical bonding. The coatings can be selected so as to affect the bioremolability of the construct by promoting or regulating the internal growth of host cells. In addition to the implant in a host or patient, cells can be grown on the fibers since collagen is a natural substrate to which the cells bind. The following examples are provided to better explain the practice of the present invention, and should not be construed in any way, as limiting the scope of the present invention. Those skilled in the art will recognize that various modifications may be made to the methods described herein, without departing from the spirit and scope of the present invention.

EXAMPLES Example 1: Manufacture of Reconstructed Collagen Strands. Collagen strands were prepared according to U.S. Patent No. 5,378,469, the disclosure of which is incorporated herein by reference. The manufacture of the collagen strands is briefly described below. A. Materials and Equipment 1. Collagen: Collagen extracted with acid, such as the one prepared in US Pat. No. ,106,949, it was stored at 4 ° C in 0.05% acetic acid at a concentration of 5.0 mg / mL and degassed before use. 2. Syringe of 140 cc (Becton Dickinson) 3. Needle of stainless steel of blunt tip, caliber 18 (Popper &Sons, Inc.), with front tube and bridge of silicone. . Syringe Pump 5. PVC dehydration channel 5.5 meters (18 feet) in length and 5.08 centimeters (2 inches) in diameter, with Masterflex Pump and neoprene tube. 6. Dehydration agent: Prepared by mixing 1200 g of MW polyethylene glycol (Molecular Weight) of 8000 (PEG-8000), 20 g of monobasic sodium phosphate (monohydrate) and 71.6 g of dibasic phosphate (anhydrous) in approximately 4000 mL of water . The pH was then adjusted to approximately 7.6-7.8 with INN NaOH and water was added to a final volume of 6000 L. 7. A rinsing channel of 1.83 meters (6 feet) and 5.08 centimeters (2 inches) in diameter, of PVC 8. Rinsing agent: purified water. 9. Drying cabinet with pulleys and hot air bellows. 10. Coil and motor of catchment at wind level. B. Extrusion Approximately 5000 mL of dehydrating agent was poured into a dewatering channel and the recirculation pump was started. The speed of the dehydration agent was maintained at about 5 cm / sec. to produce a laminar flow of the agent along the length of the dehydration channel. Approximately 400 L of rinsing agent was added to the rinsing channel. A needle was placed in the dehydration agent at approximately 30.5 centimeters (12 inches) from the upstream end. The cylinder of the collagen syringe was attached to the pump of the syringe, the pump was set at an infusion rate of approximately 2.5 mL / min., And the infusion pump was started. When sufficient clearance was generated in the dewatering channel, the strand was manually transferred through the rinsing channel and placed on the first pulley in the dehydration cabinet. The strand typically remains approximately 3 minutes in the rinsing channel. The heating bellows was then ignited at approximately 35/40 ° C. Gradually, as the strand dried, the collagen strand was carefully placed on the pulley in a zigzag fashion. The free end of the formed strand was wound onto the pick-up coil. The pickup coil was placed so that the strand emerged dry on contact, from the cabinet. Continuous strands of up to several hundred meters have been produced.

Example 2: Coftipation of Beams A __ »__ di < Opened by Relaxation of Efforts with the Beams of Several Folds Not Aconified by Relaxation of Efforts Reconstituted collagen strands were produced, as described in Example 1. Beads of strands of 10, 50 and 200 folds were made by winding strands of collagen around two legs firmly mounted to a frame. At the points where the strand wound around the legs, the strands were secured with tape to form a ring. In the tape, the ends of the ring were cut to form a bundle of strands, where each strand was about the same length as the others. The strains of the individual strands, when compared through the beam, varied. A mechanical Mini-Bionix 858 test system (MTS Systems Corp., Eden Prairie, MN) was used to test the bundles of collagen strands. The bundles were fixedly held in vertically opposite jaws, where the upper jaw pulled up and down the jaw in a fixed lower position. The initial values of the tensile strength for non-crosslinked beams, dried, were obtained by pulling the bundles beyond the breaking point. Beams were broken gradually, one strand at a time, in many places along the measured length. To generate a load-elongation curve, the beams were mounted on the fasteners of the mechanical test system, and were saturated with phosphate buffered saline (PBS). The beams were gradually lengthened to a total effort of about 50% of the initial, and allowed to relax to a zero load. The beams were then rinsed in purified water and allowed to dry. All beams were crosslinked with 2.5% glutaraldehyde, rinsed with water, and dried in air. The mechanical test was carried out in bundles plasticized with PBS at an effort speed of approximately 50% / minute; the peak load was noted. Similar denier control constructs were manufactured, as described above to compare the constructs of the same denier with the constructs conditioned by resultant stresses. Stress-conditioned samples (SC) were compared with non-stress-conditioned samples (NSC) and similar denier (DMC) controls of a post-conditioning size. The data were analyzed using the Student's t-test, with a level of significance of p > 0.05 (the insignificance was designated as?, Ns "), p <0.05, p <0.01, and p <0.001 The results are shown in Table 1.

Table 1 10 PLANS NCS SC DMC Total Size (denier) 600 400 400 Peak Load Average (g) 642 619 570 • _ > ! Table 1 (continued) 10 PLANS UTS (g / denier) 1,069 1,548 1,426% Increase in SC + 45% + 9% Significance p < 0.001 insignificant 50 PLANS NCS SC DMC Total Size (denier) 3000 2000 8000 Peak Load Average (g) 1 1663333 2405 1767 UTS (g / denier) 0.544 1.202 0.884% Increase in SC + 121% + 36% Significance p < 0.001 p < 0.001 20Q PLIEGUES NCS SC DMC Total Size (denier) 12000 8000 8000 Peak Load Average (g) 5294 6211 4537 UTS (g / denier) 0.441 0.776 0.567% Increase in SC + 76% + 37% Significance p < 0.001 p < 0.05 The increase of the UTS of the SC beams over the NSC beams seems to be due to the improved alignment and reduction in the total cross-sectional area. { that is, the thinning of the fiber) caused by the elongation treatment. The increase of the UTS of SC over the DMC seems to be due to an improved distribution of the load, when the length, tension and orientation become more uniform within the beam. The application of these findings can improve the mechanical properties of a collagenous ACL replacement, and can provide a basis for the development of other types of implants with better mechanical properties.

Example 3: Comparison of Conditioned and Non-Conditioned Collagen Strand Beams The reconstituted collagen strands were produced as described in Example 1. The strands were made by rolling 20 folds (20 hours) of collagen strands with a denier. 50 around a frame, securing the ends with tape, and cutting the tape. First, the dried samples were tested to obtain an initial value of tensile strength of the construct without preconditioning, which is actually the dry strength of the sample. The beams are gradually reprinted, one thread at a time, in many places within the measured length, this served as the value for the final load (maximum load before breaking).

The beam was then secured inside the pneumatic jaws of a test machine of a Mini-Bionix 858 mechanical test system (MTS Systems Corp., Eden Prairie, MN), and the specimen (length of approximately 50 mm) was sprayed with PBS. The beam was then rinsed by saturation with purified water, and allowed to dry at room temperature. The bundle was reticulated by saturating the construct with 2.5% glutaraldehyde in PBS, rinsed with purified water, and allowed to dry. The tape was applied in the middle part of the substance of the test sample, and it was mounted again in the MTS, again at approximately a total length of 50 mm. The sample was plasticized with PBS, then tested to determine the failure using an effort velocity of approximately 50% / minute. Unconditioned control samples were treated and tested identically, except that they were not subjected to conditioning. The results of the mechanical test were tabulated as shown in Table 2, comparing the non-conditioned controls with the conditioned samples. The cross-sectional area taken, of a single, cross-linked, wet, non-conditioned strand, was approximately 0.006 mm2.

Table 2 Unconditioned Controls Conditioned Samples Maximum Load 433 g ± 42 423 g ± 0 Total Area (20) (. 0Ó6) = 0.120mm2 (0.12) 7 (1.85) = 0.065 mm2 UTS (0.433) (9.81) /0.12 = 35 MPa (0.423) (9.81) /0.06 = 63.8 MPa Grams per end 433/20 = 21.6 g / end 423/20 = 21.2 g / end Denier (20) (50) = 1000 (20) (50 / 1.85) = 540 T Nacity in Humid 433/1000 = 0.433 g / denier 423/540 = 0.783 g / deüier Compared with unconditioned control samples, the constructs that were conditioned showed much greater tenacity, almost double. Effort-relaxed bundles also exhibited a much greater degree of packing than strands packed more closely together.

Example 4 - Large Beam Constructs Beads of collagen strands with a total of about 510 folds were conditioned as described above. The total effort with conditioning averaged approximately 26.7%, thus reducing effective denier to an average of approximately 40.4 per end (20182 total) within the beam. The controls were tested without stress conditioning, both dry and wet with PBS. Tensile tests were performed on cross-linked cbn glutaraldehyde samples after plasticizing the strands with PBS. The results are shown in Table 3. Like the other samples on materials of similar size, they often occurred breaks in the jaws; therefore, the results of the resistance had to be considered as minimum values.

Table 3 15 TYPE of Sample Maximum Cavity Deiiier Total Tenacity in Humid (grams) (denier) (grams / denier) Control 6279 g 26500 den 0.251 g / den ^? [Not moistened, 7279 g 25000 den 0.291 g den not subjected to effort] 7296 g 25000 den 0.292 Mean ± SD 6951 g ± 582 0.278 g / den ± 0.023 Table 3 (continued) Sample Type Carg Maximum Denier Total Tenacity in Wet (grams) (denier) (grams / denier) Control N / A N / A N / A [Moistened with PBS, 7905 g 25000 den 0.316 g / den not subjected to stress] 8325 g 25000 den 0.333 Mean ± DE 8116 ± 298 0.325 g / den ± 0.012 [p < 0.01] Conditioned samples 10620 g 22069 den 0.481 g / den [Moistened with PBS] 8150 g 19290 den 0.422 g / den 9030 g 19186 den 0.471 g / den Mean ± DE 9267 g ± 1251 0.45 & g den ± 0.032 (p <0.001) The wet toughness values of those large beams showed an increase of approximately 17.9% on average after moistening and drying with common PBS. This increase is probably due to improved cohesion within the beam after wetting. Compared with the wet controls, the beam stress conditioning increased the peak load only approximately 14.2%, but increased with wet tenacity by approximately 40.9%, a much higher margin; compared to the dry controls, the conditioning caused a significant increase in peak load (approximately 33.3%) and an even greater increase in wet toughness (approximately 64.7%). The increase in tenacity was due both to the decrease of the total denier after conditioning and to the increase in the peak load. Stress relief conditioning has been shown to increase the wet toughness of collagen strand bundles with a content of up to 500 folds. This increase is attributable to two factors: (1) a decrease in the total denier of the construct resulting from the conditioning effort; (2) an increase in peak load. Although the above invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be obvious to one skilled in the art that certain changes and modifications may be practiced within the scope of the appended claims. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (20)

REI-VINDICATIONS '' i r '

1. A method for increasing the strength of at least one strand of collagen, characterized in that it comprises: (a) plasticizing a collagen strand with a plasticizing agent; (b) imparting a tensile load to the collagen strand by lengthening the collagen strand to impart stress on the strand; (c) allow the stress in the strand to decrease by stress relaxation or plastic deformation; and (d) removing the plasticizing agent from the collagen strand.

2. The method according to claim 1, characterized in that it further comprises the step of: (e) crosslinking the strand with a crosslinking agent.

3. The method according to claim 1, characterized in that the plasticizing agent is water or aqueous buffer.

4. The method according to claim 1, characterized in that the tensile load is imparted by lengthening the strand at an effort of between about 20 to 200%.

5. The method according to claim 1, characterized in that the tensile load is imparted by lengthening the strand at an effort of between about 50 to 100%.

6. The method according to claim 1, characterized in that the elongation is carried out at repeated increments. The method according to claim 1, characterized in that the increments are at an effort of approximately 5 to 10%. 8. The method according to claim 1, characterized in that the load decreases by stress relaxation or plastic deformation to less than 0.5 grams per end. The method according to claim 2, characterized in that the crosslinking agent is selected from the group consisting of lyophilization, ultraviolet radiation (UV), acyl-azide, hexamethylene diisocyanate, bisimidates, glyoxal, polyglycerol polyglycyl ether, adipyl chloride , ribose and other sugars, carbodiimides, and aldheidos. The method according to claim 9, characterized in that the cross-linking agent is l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). The method according to claim 1, characterized in that the collagen strand contains a plurality of collagen strands, which form a bundle of multiple folds. The method according to claim 11, characterized in that the multiple-pleated bundle is between 2 and 1000 folds. The method according to claim 11, characterized in that the multiple-pleated bundle is between 20 and 500 folds. A method for increasing the strength of a multi-pleated bundle of collagen fibers, characterized in that it comprises: (a) plasticizing a multi-pleated bundle of collagen strands with water, a buffered aqueous solution, or glycerol; (b) imparting a tensile load to the multiple pleated bundle by elongating the bundle to impart an effort on the multiple pleated bundle; (c) allowing the effort of the multiple-fold bundle to decrease by stress ratio or plastic deformation; and, (d) removing the plasticizing agent from the multiple-pleated bundle. 15. The method according to claim 14, characterized in that the tensile load is imparted by elongation of the strand at an effort of between about 20 to 200%. The method according to claim 14, characterized in that the tensile load is imparted by the elongation of the strand at an effort of between about 50 to 100%. 1

7. The method according to claim 14, characterized in that the elongation is carried out in repeated increments. 1

8. The method according to claim 17, characterized in that the tensile load of the increasing elongation is at an effort of about 5% to about 10%. 1

9. The method according to claim 14, characterized in that it further comprises the step of: (e) crosslinking the multiple-pleated bundle with a cross-linking agent. The method according to claim 19, characterized in that it further comprises the step of: (f) sterilizing the crosslinked multiple-fold bundle. 2) The method according to claim 20, characterized in that it further comprises the step of: (g) coating the multiple-pleated bundle with pharmaceuticals, growth factors, hormones, components of the extracellular matrix, or genetic material. 22. The method according to claim 19, characterized in that the cross-linking agent is l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). 23. The method according to claim 14, characterized in that the multiple-pleated bundle is between 2 and 1000 folds. 24. The method according to claim 14, characterized in that the multiple-pleated bundle is between 20 and 500 folds. 25. A method for increasing the strength of a multi-pleated beam of collagen strands, characterized in that it comprises: (a) plasticizing a multiple-pleated bundle of collagen strands of between 2 and 1000 folds with a plasticizing agent selected from the group consists of water, a buffered aqueous solution, or glycerol; (b) imparting a tensile load to the multiple-pleated bundle by elongating the bundle to impart an effort on the multiple-pleated bundle of about 5% to about 10%; (c) allow the effort in the multiple-fold bundle to decrease by stress relaxation or by plastic deformation to less than 0.5 gram; (d) repeating the elongation in repeated increments; (e) removing the plasticizing agent from the multiple-pleated bundle; (f) crosslinking the multiple folds bundle with a crosslinking agent; and (g) sterilizing the crosslinked beam, 26. An implantable prosthetic device made by the method according to any of claims 1-25.