Prosthetic ligaments.

IS 2 174 847 T3

DESCRIPTION

Prosthetic ligaments.

The present invention is in the field of implantable medical devices and prosthetics. More specifically, this invention is directed to useful devices such as prosthetic ligaments and in vivo base structures for the regeneration of ligament tissue, and to methods for their manufacture and use.

Ligaments connect one bone to another, usually where bones form joints in human and animal species. The ligaments act in the joint as a mechanism for maintaining joint stability, for guiding joint movement, and for resistance to joint relaxation forces. Without ligaments, the human and animal species would be unable to maintain the erect form. Ligament injuries result in either a normal physiological process of repair, which can lead to the resumption of normal joint mechanics, or inadequate repair with loss of joint stability, abnormal joint movements, and occasionally painful arthritis as a result of abnormal wear and tear on the joint surface. In general, ligaments that are outside the joints, and washed by rich vascular supply, have a good chance of healing normally after injury. The ligaments within the joints, called intra-articular ligaments, are generally bathed in synovial fluid, have relatively poor blood supply, and heal with difficulty.

In the prior art, treatment of injured ligaments has generally been either by attempts to protect the ligament from further deforming stress and thereby allow a normal physiological repair process to occur or by attempting surgical repair with sutures, replacement, or excisián (Johnson, RJ et al. (1992) J. Bone Joint Surg. 74-A: 140-151; Arnold et al. (1979) Am. J. Sports Med. 7: 305; McDaniel et al. (1983 ) Clin Orthop. 172: 158; Rovere et al. (1983) Am. J. Sports Med. 104: 205). With non-operative and operative repair, healing and regeneration of the ligamentous tissue can occur. Generally, if the ligament is located intra-articularly, the repaired tissue is usually inferior to the original tissue and sometimes inadequate to resist the normal forces of the joint. In view of the insufficiency of many primary repairs, several previous attempts have been made to replace ligamentous tissue with natural and artificial materials. Unfortunately, this has also led to significant problems related to these replacement materials.

In particular, the replacement of ligaments in the prior technique has been by autografts (Friedman et al. (1985) Clin. Orthop. 196: 9), allografts (Webster (1983) Clin Orthop. 181: 238), xenografts (McMaster ( 1988) in "Prosthetic Ligament Reconstruction of the Knee" (Friedman and Ferkel, (eds.),

WB Saunders, Philadelphia, pp. 96-100), or using synthetic materials (Woods (1985) Orthop. Clin. North Am. 16: 227). Autograft, or replacement of the injured ligament with one's own tissue, is still the preferred modality (Amiel et al. (1986) Am. J. Sports Med. 14: 449-462; Warren et al. (1990) AAOS 57th Annual Meeting, Anaheim, CA p. 84). Autograft reduces the risk of disease transmission between donor and recipient, immunological complications, and complications from foreign body reactions. However, the weakening of the part of the body from which the replacement tissue is collected , the extensive surgical procedures both with the collection of the donor tissue and with the replacement of the injured tissue, and the inadequate mechanical strength of the replaced tissues They have promoted the search for alternative repair methods.

Allograft, or replacement of the injured ligament with tissue from another person (either preserved living tissue or chemically processed tissue) has been performed (Noyes et al. (1990) J. Bone Joint Surg. 72-A: 1125-1136 ; Shine et al. (1990) Am. J. Sports Med. 18: 457-465; Webster (1983) Clin. Orthop. 181: 238; Bright et al. (1981) J. Pediatr. Orthop. 1:13) . This approach has been only partially successful in the long term due primarily to the immune response of the host to the graft and to failures in the preservation and sterilization processes (Jackson et al. (1990) Am. J. Sports. Med. 08: 1 -10; Minami et al. (1982) Hand 14: 111). Furthermore, the risk of disease transmission is of particular importance in the allograft (Prewett et al. (1991) Orthop. Res. Soc. 16: 456).

Xenograft, or replacement of the injured ligament with tissue from animal images, has been attempted. However, due to improper processing of the material leading to the presence of toxic and immunological substances in the graft, this method has had minimal success (Teitge (1988) in The Crucial Ligament, (Feagin, ed.) New York, Churchill-Livingston, pp. 529-534).

Various synthetic polymers have been manufactured for ligament replacement, such as polypropylene.

ES 2 174 847 T3 pylene, polyethylene terephthalate, carbon, polytetrafluoroethylene (Claes et al. (1991) Orthop. Res. Soc.

16: 598). Ligament replacement devices are intended to function as permanent implants, and thus are subjected to continuous intra-articular wear. However, none of the present synthetic polymeric ligament devices have functioned successfully as a substitute for ligaments. Such devices have failed due to ligament rupture, reduction of joint particles with resulting synovitis, abrasion of opposing surfaces in the joint, extensive joint debridement and removal of the ligament required by infection, persistent effusion , and bone tunnel widening (Woods et al. (1991) Am. J. Sports Med. 19: 48-55; Woods (1985) Orthop. Clin. North Am. 16: 227): Thus, artificial materials are generally of insufficient durability or of insufficient mechanical compatibility to withstand repetitive loading on the joint and have never been shown to restore normal joint mechanics.

The concept of a resorbable mold or base structure for tissue repair and regeneration has received rigorous attention in recent years. Repair of tissues such as the skin, nerves, and meniscus has been attempted using both natural and synthetic resorbable polymers. For example, Yannas et al. (US Patent No. 4,060,081) prepared endodermal implants from glycosaminoglycans and natural collagen. Nyiles et al. (Trans. Am. Soc. Artif. Intern. Organs (1983) 29: 307-312) reported the use of synthetic resorbable polyesters for applications in the regeneration of peripheral nerves. Li (US Patent No.^° 4,963,146) used a porous, semipermeable, resorbable collagen conduit as the base structure for nerve regeneration.

WO 91/16867 by Regen Corporation describes an intervertebral proteosic disc made of fibers by molding and WO 90/09769 also by Regen Corporation describes a prosthetic meniscus made of fibers by molding.

However, even with the aforementioned technologies, which have been applied to the reconstruction of anatomical structures, a successful structure has not yet been developed as a prosthetic ligament and built from fully resorbable materials, or their anaologs. Consequently, what is needed is a prosthetic ligament that includes a base structure composed of biocompatible materials that is soft, strong, resorbable, and that can support the growth of the ligaments.

Accordingly, it is an objective of this invention to provide a substitute for ligaments or a prosthesis that is biomechanically capable of withstanding the normal forces of the joint and that is able to function with these loads to protect the cartilage that surrounds it.

Another objective is to provide a ligament substitute or prosthesis that is biomechanically capable of providing stability to the joint.

Another objective is even to provide a resorbable prosthesis that acted in vivo as a temporary base structure for the infiltration of the ligament with fibroblasts and the regeneration of the ligament.

A further objective is to provide a method for insertion and fixation of a ligament prosthesis.

Yet a further object is to provide a method by which such prosthetic ligaments can be fabricated.

Summary of the invention

The present invention provides a biocompatible and bioresorbable structure for implantation adjacent to and within the joints that assumes the shape and role of the ligament. This prosthetic ligament promotes and creates a base structure for tissue regeneration that has the phosphoric characteristics of the natural tissue of the ligament, with which the base structure and the fibroblasts that grow inside maintain the forces of resistance to the traction of the natural ligaments. .

The prosthetic ligament of the invention is a prosthetic ligament that contains multiple elongated filaments, essentially aligned, each of said filaments being a matrix with volume, dry and porous, formed by the extrusion of biocompatible and bioresorbable fibrils, said fibrils constituting 75 to 100 % by dry weight of said matrix and being fiber segments of a connective tissue type polymeric component, or an analogous thereof, said fibrils being crosslinked, and including said matrix from 0 to 25% by dry weight of

ES 2 174 847 T3 polysaccharide molecules, in which each of said filaments establishes a bioresorbable base structure adapted for inward growth of ligament fibroblasts, and in which said base structure and said fibroblasts that have grown inside maintain the forces of resistance to the traction of the natural ligaments.

Each filament is a dry, porous bulky matrix of biocompatible and bioresorbable fibrils, at least some of which are cross-linked. "Fibrils" as used herein are segments or short fragments of fibers of connective tissue-type natural polymeric components, such as those obtained from human and animal tissues, plants, insects or their analogues. Preferable connective tissue-type components include collagen, elastin, reticulin, cellulose, alginic acid, and chitosan, with type I cholegen being most preferred.

The fibrils in the filament are connected by intramolecular and / or interfibrillary crosslinking bonds. In one aspect of the invention, these crosslinking bonds are formed by a chemical crosslinking reagent such as one selected from the group consisting of glutaraldehyde, formaldehyde, carbodiimides, hexamethylene diisocyanate, bisimidates, glyoxal, polyglycerol-polyglycidyl-adipyl chloride, and adipyl chloride. A preferred chemical reagent is formaldehyde.

In some embodiments of the invention, the fibrils are randomly oriented throughout the matrix. In other forms, these fibrils are oriented predominantly along the axis of the filament.

A preferred prosthetic ligament also includes polysaccharide molecules dispersed between the fibrils. In various forms of the invention, these polysaccharides participate directly in the formation of covalent crosslinking with fibrils, or mechanically interact with fibrils in the form of enmeshment or through an interlocking mechanism, forming stable fibril-polysaccharide complexes. As such, these polysaccharide molecules provide lubrication, hydrophilicity, and strength to the prosthetic ligament. Preferably, these polysaccharide molecules have a molecular weight greater than 1000, and are selected from the group consisting of chondroitin-4-sulfate, chondroitin-6-sulfate, kerataen sulfate, dermaten sulfate, heparan sulfate, heparin, hyalureonic acid. , alginic acid, quitoséan. These polysaccharides can be uniformly dispersed throughout the prosthetic ligament as individual molecules, or they can be present in varying amounts in different regions of the structure.

The matrix includes about 75 to 100% natural and / or synthetic fibers and about 0 to 25% polysaccharide molecules by dry weight, the proportions of which can be constant throughout the structure or can be variable.

In a preferred embodiment of the invention, the prosthetic ligament is primarily a mixture of two densities of filaments, with the most porous, or "low density" filament having a density of about 0.05 to 0.4 g of matrix. / cm³ , and more preferably, about 0.07 to 0.3 g matrix / cm³, (where “g of matrix / cm³"Is a unit that indicates the number of grams in a cubic centimeter of the matrix) and the less porous, or" high density "filament has a density in the range of about 1.0 to about 1.3 g of matrix / cm³.

In one embodiment, the prosthetic ligament is in the form of a filamentous braid with a loop at one end and a straight bundle of filaments at the other end to aid insertion and fixation in the bone. In another, multiple filaments twist around each other.

In yet another embodiment of the invention, the prosthetic ligament further contains a mesh or membrane composed of a biocompatible, bioresorbable material that is attached to portions of the outer surface of the matrix. The mesh or membrane helps the implantation1 of the prosthetic ligament in the joint by providing a temporary anchoring mechanism.

This invention also encompasses a method for manufacturing a prosthetic ligament of the type described above. Usually the method includes the following stages. A plurality of essentially pure fibers are provided from a polymeric connective tissue component and cut into multiple segments or fibrils that are shorter than the fibers. The cutting can be carried out by mechanical disintegration, for example. The fibrils are then aggregated into a plurality of elongated filaments and contacted with a crosslinking reagent for a time sufficient to crosslink at least a portion of the fibrils within the filaments. The filaments are then aligned in a mutually adjacent relationship to form the prosthetic ligament. Polysaccharide molecules are

ES 2 174 847 T3 can add to fibrils prior to the aggregation step to form prosthetic ligaments containing polysaccharides.

According to the invention, the aggregation step is carried out by creating a dispersion of fibrils; causing the dispersion to acquire a filamentous shape; and drying it to form a filament. The forming step includes extruding the dispersion from a syringe into a coacervation bath of a high concentration neutral salt. To produce the low-density filaments, portions of the coacervated fibrils are freeze-dried, and other portions are air-dried to obtain the high-density filaments. Both high-density and low-density filaments are crosslinked to increase the strength and in vivo stability of the protein device.

In a preferred aspect of the invention, the crosslinking step is carried out using chemical crosslinking reagents that form interfibrillar and intermolecular crosslinking bonds. In other aspects of the invention, a further crosslinking step is carried out by subjecting the chemically crosslinked matrix to a heat and vacuum dehydrothermal crosslinking process.

The invention will be described below in connection with certain illustrated embodiments.

Brief description of the drawings

The aforementioned and other objectives of this invention, its various characteristics, as well as the invention itself, can be more fully understood from the following description, when read in conjunction with the accompanying drawings in which:

Figs. 1A and 1B are diagrammatic representations of a human knee joint showing normal positioning of the articular ligaments (Fig. 1A), and an injury to an articular ligament (Fig. 2B);

Figs. 2A-2E are perspective views of exemplary prosthetic ligaments in accordance with the present invention;

Fig. 3 is a perspective view of a section of the prosthetic ligament of Fig. 2A;

Fig. 4 is a cut perspective view of low density filaments of the present invention; and Fig. 5 is a sectional view of high density filaments of the present invention.

Description of the invention

Ligament fibroblasts have the ability to regenerate ligament tissue if the correct physical and chemical environment is given in which to do so (Frank et al. (1983) J. Orthoped. Res. 1: 179-188). Additionally, ligament fibroblasts can migrate to a defective area filled with a fibrin clot and form apparently ligament-like tissue (Franck et al. (1983) J. Sports Med. 11: 379-389). When a suitable matrix base structure is present within a defective area of ​​the ligament, such ligament tissue can be formed. Complete regeneration of an injured ligament in an otherwise healthy joint can provide normal joint movement and stability, thereby preventing arthritic changes.

It has been discovered that a prosthetic ligament made from short fiber segments of biocompatible and bioresorbable polymeric connective tissue components can be surgically implanted in the knee, shoulder, or other joint to provide normal joint movement and stability. This prosthetic ligament also acts as a base structure to regenerate the ligament tissue whose inward growth is stimulated by the physical characteristics of the implanted device.

Fig. 1A shows a diagrammatic representation of the normal positioning of a natural anterior cruciate ligament 10 in the human knee joint 100. This ligament connects the femur 12 with the tibia 14 between the lateral meniscus 16 and the median meniscus 19. The ligament The prosthetic of the present invention can be used to replace or enhance the function of such a native ligament. Fig. 1B shows a typical lesion of the articular ligament 50 that could be repaired by the prosthetic ligament of the invention.

IS 2 174 847 T3

An example of prosthetic ligament 10 is shown in Fig. 2A. Prosthetic ligament 10 is generally a plurality of essentially aligned elongated filaments 12 extending along a central axis 11. In this embodiment, the filaments 12 have been brought together in a braid or interlacing around axis 11. The clamp or tube 13 holds the filaments 12 together. In an alternative embodiment shown in Fig. 2C, the filaments 12 have been twisted together, forming a rope-like structure. Other types of structures that include filaments aligned in a mutually adjacent relationship are also useful (Fig. 2B, 2D, and 2E).

The prosthetic ligament can be manufactured from any biocompatible, bioresorbable fibers of a natural, synthetic or biosynthetic connective tissue polymer component or a connective tissue-like plant component. Examples of such materials include collagen, reticulin, elastin, cellulose, alginic acid, and chitosan. The following procedure can be used to prepare type I choleagene from bovine Achilles tendon.

The tendin is first cleaned of the fasciae and foreign tissues and it is crumbled. The shredded tendon is extracted in 1M NaCl, pH 7.0, to separate a small portion of the collagen molecules that are of recent synthesis and that have not yet been incorporated into the stable fibrils, as well as glycoproteins and proteoglycans that are associated with collagen through non-covalent interactions. Other salts such as potassium chloride and the like can be used as substitutes for sodium chloride.

The lipids that are associated with cell membranes or collagen tissues are separated by first extracting them with detergents such as Triton X-100 (Sigma Chemical Co., San Luis, MO), followed by extraction with ether-ethanol mixtures. The concentration of Triton X-100 is usually between about 2% and 4%, but is preferably about 3%. The preferred ether-ethanol mixture is usually in a ratio of about 1: 1 (volume / volume). The extraction period is usually between around 8 hours and around 96 hours, but is preferably between around 24 and 48 hours.

Additional purification can be achieved by extracting the tendoen under aecid and basic conditions. Both acidic and basic extraction weaken non-covalent intermolecular interactions, thus facilitating the release of covalently linked glycoproteins, glycosaminoglycans (GAGs), and other molecules other than collagen.

Extraction of the tendoen under alkaline conditions is achieved by treating the tendoen with Ca (OH) 2, NaOH, or similar substances, at a pH of approximately 13 for a period of 8 to 96 hours in the presence of a salt that stabilizes the structure such as (NH4) 2SO4, or Na2SO4 to minimize the potential risk of denaturing choleagene. Alkaline treatment cleaves non-covalently bound glycoproteins and GAGs from choleagene matrices. The alkali also separates the residual lipids by saponification.

The acid extraction must be carried out at a pH below 3 in the presence of a salt that stabilizes the structure. Acids such as acetic acid, hydrochloric acid, or the like can be used. Like alkaline extraction, acid extraction removes glycoproteins and non-covalently bound GAGs.

The portions of the molecule that do not form a triple helix (telopeptides) are involved in the formation of intermolecular crosslinks. They are weak antigens and are susceptible to being attacked by proteases, such as pepsin and trypsin. Prolonged digestion with such proteases dissociates the fibrils into individual molecules. However, if the digestion process is properly controlled such that telopeptides are maximally separated without complete dissociation, the immunogenicity of the fibrils can be further reduced without significantly compromising mechanical strength. For example, to isolate molecular choleagene, pepsin digestion of skin or tendons is usually carried out with an enzyme: choleagene ratio of about 1:10 (w / w) for about 24 to 96 hours at a temperature below of the environment. In comparison, fibrils can be obtained by limited digestion with pepsin carried out with a ratio of about 1: 100 (enzyme: collagen in w / w) for about 24 to 96 hours at 4 ° C.

Type I choleagene fibrils obtained according to this method are used to manufacture the prosthetic ligament of the present invention. However, it should be understood that collagen obtained from other sources, such as biosynthetically produced collagen or its analogs, can also be used in the manufacture of the prosthetic ligament. These fibers can be arranged in an essentially longitudinal arrangement with the density of the fibers being essentially uniform throughout the matrix.

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The following general procedure can be used to fabricate a prosthetic ligament.

The purified connective tissue fibers are swollen in phosphate buffered saline at pH 7.4. The swollen fibers are then subjected to a homogenization step to further break down the fibers into smaller fibrils without denaturing the protein. Low shear rate homogenization is preferred, such as with a Silverson Homogenizer fitted with a disintegration head. The homogenized connective tissue is then filtered first through a 40 mesh then through a 100 mesh stainless steel screen to remove large fibers that have not been homogenized. Uniformly dispersed connective tissue fibrils can now be used for the production of high-density and low-density filaments.

One method of forming a filament includes an extrusion process. Briefly, a dispersion of collagen or other fibrils (2% to 4% w / v) is fed into a reservoir that is attached to a device driven by a piston on one side and a needle on the other side such as a pump device. syringe. The dispersion is pushed out through the needle by means of a continuous and constant thrust of the piston to ensure a continuous and constant flow of dispersion. The wet filaments can be extruded into a coacervation bath of buffer acetate solution, pH 4.7, in the presence of 1% NaCl. Alternatively, the wet filaments can be extruded into a bath of saline solution containing 5% to 20% NaCl.

Another method of forming a filament includes placing or adding the dispersion into a mold that is filamentous in shape. The fibrils of the dispersion can be oriented in the mold randomly or uniformly, depending on the desired application. Uniform orientation in the mold can be achieved for example by painting the dispersion into the mold directionally with a bristle brush.

The prosthetic ligament contains low-density porous filaments, and may also contain high-density filaments to add strength. A typical prosthetic ligament consists of between about 10% and about 50% low density filaments that have a density of between about 0.05 and about 0.4 g / cm³, and preferably between about 0.07 and about 0.3 g / cm³, and between about 50% and about 90% of high density filaments having a density of between about 1.0 and about 1.3 g / cm³.

To form low density filaments, the wet filaments are removed from the coacervation bath and freeze dried. These frozen and dry filaments are highly porous and do not have a high tensile strength. Porosity and strength are adjusted by stretching the filaments and then subjecting the filaments to a crosslinking agent. Steam crosslinking with formaldehyde is preferred. Alternatively, the drawn filaments can be cross-linked by dehydrothermal heat and vacuum treatment by methods well known in the art.

High density filaments are obtained as follows. Filaments are extruded from a dispersion (2 to 4% w / v) as described above. However, after the wet filaments have been collected, they are air dried under a hood to obtain collagen filaments of a given diameter. The air-dried filaments are then subjected to crosslinking either in a solution phase using a crosslinking agent well known in the art such as glutaraldehyde, formaldehyde, and the like, or in a vapor phase also known in the art such as exposing formaldehyde steam filaments.

The filaments are then manipulated into a desired shape such as a multifilament bundle 20 which can be used to form a multifilament braid or three or more high-density bundles 20 and / or low-density filaments 12 (Fig. 2A), or two or more more beams (Fig. 2B), or a beam with a single turn (Fig. 2C) or a beam without twists (Fig. 2D). Fig. 2E shows an embodiment 20 similar to that shown in Fig. 2D where loops 22 are formed at each end to be used to secure the prosthetic ligament in vivo.

The following procedure can be used to prepare a device with a braided filament weave, as shown in Fig. 2A. High density and low density filaments are longitudinally combined in a ratio of 2: 1 (w / w) into small bundles, which have about 100 to 300 high density filaments and 50 to 150 low density filaments. Three bundles are first braided in the central area and then folded in the braided region to form a curved area and a loop 22. The six bundles are then braided. The end of the ligament is sealed by inserting it into a 13 mesh tube of a resorbable polymer such as a polylactate mesh tube. The ligament

ES 2 174 847 T3 braided in this way can be easily stretched from 10 to 15% without straining the collagen molecules.

The cross-linked device maintains a sufficient degree of hydrophilicity and elasticity that simulates the properties of the natural ligament, that is, the ability to withstand mechanical stress and stabilize the joint. In addition, the framework provides an ideal environment for cell infiltration, extracellular matrix synthesis, and deposition resulting in regeneration of natural ligament tissue.

Polysaccharides may be dispersed throughout the fibrils of the filaments. They can act as lubricants and / or intermolecular crosslinking bonds between fibrils. The useful crosslinking bonds by polysaccharides are typically composed of at least one of the group of molecules consisting of chondroitin-4-sulfate, chondroitin-6-sulfate, kerataan sulfate, dermatan sulfate, heparan sulfate, heparin sulfate, alginic acid , chitosan and hyaluraonic acid. The polysaccharide dispersion is preferably uniform throughout the fibril dispersion and may be in the range of between about 1 and 25% (w / w) for example.

Intermolecular crosslinking bonds can also be established by means of a dehydrothermal process (heat and vacuum) which is well known in the art. This procedure can be carried out as an additional step after chemical crosslinking to achieve added strength.

The cross-linked device has a relatively high thermal stability between about 55 and 85 ° C, preferably between about 65 and 75 ° C, to have sufficient in vivo stability. This can be achieved by manipulating the crosslinking conditions, which include reagent concentration, temperature, pH, and time.

Following the processes described above and in the examples set forth below, a prosthetic ligament can be constructed with the shapes shown in Fig. 2 having the characteristics listed below in TABLE 1.

TABLE 1

1. Physical characteristics

to. Density (g / cm³) low density filaments 0.05 - 0.4 high density filaments 1.0 - 1.3

b. Initial tensile strength (Newtons) 300 - 600 after growth into tissue

c. Length (cm)

two. Constituents

to. Type I or type I + type II collagen fibers

b. Polysaccharides

1000 - 3000 15 - 17 (% by weight) 75 - 100 (% by weight) 0 - 25

The following non-limiting examples describe in vivo manufacturing and testing methods of the prosthetic ligament of the invention.

Example 1

Preparation of Purified Type I Collagen

A) Fabric

Bovine, swine, or sheep Achilles tendon is obtained from USDA-approved slaughterhouses. The preferred age of the animals is between 12 and 18 months. The tissue is kept cold during the purification process except where specified to minimize bacterial contamination and tissue degradation.

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B) Mechanical Disintegration

The carefully chosen adherent tendon tissues are first mechanically removed by scraping. The tendons are then shredded or cut into small pieces and washed in excess amounts (10 volumes) of cold water to remove residual blood proteones and water-soluble materials.

C) Extraction with salt

The washed tendons are extracted in ten volumes of 5% NaCl, 0.1M phosphate buffer, pH 7.4 for 24 hours to remove salt soluble materials. The salted tendons are repeatedly washed in approximately 10 volumes of water to remove the salt.

D) Removal of lopids

The material is extracted into 3% Triton X-100 for 24 hours. The detergent is removed by extensive washing with water. The material is then extracted in 3 to 4 volumes of ether-ethanol (1: 1 vol / vol) for 24 (+/- 2) hours to minimize even more the lipid content. The material from which the lopids have been extracted is washed extensively to remove the ether and ethanol.

E) Extraction with acid and base

The material is subjected to acidic and basal extractions to separate materials other than collagen. The alkaline extraction is carried out with 3 to 4 volumes of 0.5M NaOH at pH 13 to 13.8 at room temperature in the presence of 1.0M Na2SO4 for 24 (+/- 2) hours with gentle stirring. After alkaline extraction, the pH is neutralized with HCl. The pH is then adjusted to 2.5 by adding concentrated lactic acid to a final concentration of 0.2 M. The acid extraction was continued for 24 (+ / 2) hours with stirring.

F) Limited proteolytic digestion

The swollen or acidic tendon is then subjected to a pepsin-limited proteolytic digestion (enzyme: collagen = 1: 100 in w / w) for 24 (+/- 2) hours to 4^°C. Pepsin and telopeptides are separated by dioalysis.

The swollen fibrillar material is then coacervated by adjusting the pH to its isotoonic point with NaOH or 1M HCl or adjusting the ionic strength to 0.7 with NaCl. The coacervated collagen fibrils are collected by filtration, and the filtrate is washed extensively with cold distilled water. Highly purified type I collagen can be stored at -20 to -40^°C until use. Alternatively, the purified fibril dispersion can be freeze dried and stored at room temperature as dry fibrils.

Example 2

Low density filament preparation

15 grams of purified collagen fibrils are swollen in 500 ml of phosphate buffered saline (PBS) to obtain a 3% (w / v) collagen dispersion. The swollen collagen is homogenized with a Silverson Homogenizer using a disintegration head for two minutes. The homogenized collagen is then filtered first through a 40 mesh stainless steel mesh followed by vacuum filtration through a 100 mesh stainless steel mesh. The filtered and dispersed collagen is deaerated in vacuo. The uniform collagen dispersion is fed into a syringe pump which extrudes the collagen through a modified 12 gauge needle at a rate of 1 ml per minute. The extruded collagen filaments are collected in an acetate buffer coacervation tank at pH

4.7 in the presence of 1% NaCl at 35^°C. The coacervated filaments are then placed on freeze drying trays and frozen at -40 ° C followed by drying at -10 ° C under vacuum at 150 µm Hg for 24 hours. Final drying is done at 20^°C for six hours before removing the filaments from the freeze drying trays. The dried collagen filaments after freezing are placed in a crosslinking chamber and subjected to crosslinking under formaldehyde vapor generated from a 2% formaldehyde solution for 90 minutes at 95% humidity at room temperature. The cross-linked collagen filaments are stored at room temperature in a clean plastic bag until later use.

IS 2 174 847 T3

Example 3

Preparation of high density filaments

15 grams of purified choleagene fibrils are swollen in 500 ml of phosphate buffered saline (PBS) to obtain 3% (w / v) of choleagene. The swollen choleagene is homogenized with a Silverson Homogenizer using a disintegration head for two minutes. The homogenized coleagene is then filtered first through a 40 mesh stainless steel mesh followed by vacuum filtration through a 100 mesh stainless steel mesh. The filtered collagen dispersion is deaerated in vacuo. The uniform collagen dispersion is fed into a syringe pump which extrudes the choleagene filaments through a modified 12 gauge needle at a rate of 1 ml per minute. The extruded humid collagen filaments are collected in an acetate buffer coacervation tank at pH 4.7 in the presence of 1% NaCl at 35 ° C. The coacervated collagen filaments are placed under a hood and air dried for 24 hours. The air-dried filaments are cross-linked in 0.1% glutaraldehyde solution in phosphate buffered saline, pH 7.4, for 24 hours at room temperature. The crosslinked filaments are extensively washed in water and stored at room temperature until later use.

Example 4

Fabrication of the ligament I device

They are longitudinally combined into small bundles to braid from 100 to 300 high-density collagen filaments and from 50 to 150 low-density collagen filaments (2: 1 ratio) obtained according to Examples 2 and 3 above. First, three bundle units each 40 cm long are braided at the midpoint. They are then folded at the midpoint to form a small loop. Then six bundle units are braided to form a mixture of collagen ligaments containing two-thirds of high-density collagen fibers interwoven with one-third of low-density porous collagen fibers. The end of the ligament is sealed by inserting it into a mesh tube of a resorbable polymer such as a polylactate mesh tube. The well-braided ligament can easily be stretched 10-15% without straining the choleagene molecules. Alternatively, the twisted bundles can be folded to have a loop at each end.

Example 5

Fabrication of the ligament II device

Same as Example 4 except that the high density choleagene filaments are replaced by polylactate monofilaments obtained from Purac America, Inc., Chicago, IL.

Example 6

Fabrication of the ligament III device

15 grams of purified collagen fibrils are swollen in 500 ml of phosphate buffered saline (PBS), containing 0.189 g of hyalureonic acid and 0.189 g of chondroitin sulfate to give a 3% (w / v) collagen dispersion. and 0.076% (w / v) of glycosaminoglycans. The rest of the procedure is the same as in Example 2. 15 grams of purified collagen fibrils are swollen in 500 ml of phosphate buffered saline containing 0.189 g of hyalureonic acid and 0.189 g of chondroitin sulfate to give a dispersion of choleagene fibrils at 3% (w / v) and 0.076 % (w / v) of glycosaminoglycans. The rest of the procedure is the same as in Example 3 except that the coleagene filaments are cross-linked in a solution of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide at pH 4.7 for 24 hours. The addition of carbodiimide is carried out every 3 to 4 hours, and the pH is adjusted to 4.7 after each addition of carbodiimide.

Example 7

Description of device insertion

A 10-mm hole is made at the anatomic insertion site of the natural cruciate ligament in the femur and tibia. A guiding suture is passed from the femuralatibia. The guiding suture is then attached to the loop at the end of the prosthetic ligament, and the ligament is pulled through the

ES 2 174 847 T3 articulation. The ligament is attached to the femur and tibia by passing a screw through the loop at the end of the ligament and into the cortical and cancellous bone. The knee is passed through a range of motion under direct visualization with open or arthroscopic techniques. All the bone that you trip over will dry out.

Example 8

In vivo and in vitro tests

In vitro tests

Each prosthetic ligament is mounted in a mechanical test gauge (MTS) with a post through each end loop of the ligament. The post is clamped to the mechanical jaws of the MTS force loading machine. The graft is pulled to rupture at a displacement rate of 15 mm / s and compared to the forces of a normal cruciate ligament (estimated to be approximately

1500 at 2500 Newton).

In vivo tests

Each prosthetic ligament is implanted in a knee joint of an animal (eg, dog, goat, monkey) and is tested six months and twelve months after implantation. After surgery, the animals are allowed to move around the cage and exercise without restrictions for six to twelve months, at which time the animal is euthanized. Each knee is left bare of extra-articular tissue. The femurs and tibiae are placed in jars and loaded into the MTS machine with the ACL aligned along the axis of the actuator. The joints are pulled to break at a displacement speed of 15 mm / s as described by McCarthy et al. (Orthopedic Research Society Meeting, Washington, DC (1991)).

The scope of the invention is indicated by the appended claims.