WO1996014095A1

WO1996014095A1 - Synthetic collagen orthopaedic structures such as grafts, tendons and other structures

Info

Publication number: WO1996014095A1
Application number: PCT/US1995/014308
Authority: WO
Inventors: Frederick H. Silver; Yasushi Pedro Kato
Original assignee: The University Of Medicine And Dentistry Of New Jersey
Priority date: 1994-11-07
Filing date: 1995-11-07
Publication date: 1996-05-17
Also published as: AU4280896A; CA2204753A1; EP0793512A1

Abstract

An improved biodegradable and biocompatible reconstituted monofilament collagen fiber having increased strength and elasticity is disclosed. The method of manufacture of the fiber, comprising a water bath step following a dehydration step, is also disclosed. Several embodiments of the collagen fiber are disclosed, including grafts, prosthetic devices, bundles of the fibers where the fibers of the bundle are tensioned and fail substantially in unison, and methods thereof. The fibers of the invention, or the embodiments of the fibers, may be coated or embedded in a polymer to protect the fibers and to bind them together. Additionally, proteoglycans may be incorporated into the fibers to enhance the ultimate tensile strength of the fibers.

Description

SYNTHETIC COLLAGEN ORTHOPAEDIC STRUCTURES SUCH AS GRAFTS, TENDONS AND OTHER STRUCTURES

This is a continuation in part of pending application Ser. No. 07/990,967, filed Dec. 15, 1992 which is a divisional application of application Ser. No. 07/297,115, filed Jan. 13, 1989, now U.S. Patent No. 5,171,273. The patent application and its parent (now the issued patent and referred to collectively herein as the "parent applications") are incorporated herein by reference.

BACKGROUND OF THE INVENTION

This invention relates to high strength reconstituted collagen fibers which are particularly well suited as grafts for orthopaedic, dermal, cardiovascular, and dental implants, prosthesis and other applications particularly in living subjects, like animals, especially human subjects.

From the description of the prior art it is evident that a serious and urgent need exists for high strength fibrous materials suitable for use as a graft that is long lasting and has biocompatibility with a host and is biodegradable. The fibers and grafts of the invention overcome many of the prior art difficulties and problems and have a combination of advantageous properties generally absent in the prior art. The collagenous fibers and grafts of the invention can be manufactured without sacrifice of the host's tissue. The graft of the invention quickly incorporates repair tissue, a necessary characteristic in the design of biomaterials that enhance the deposition of repair tissue in skin, tendon and the cardiovascular system. Although high-strength oriented and unoriented collagenous materials are reported in the literature no report is known of collagen fibers of small diameter that can be processed into woven and non-woven textile prostheses which have the necessary properties that simulate or exceed those of the natural body part. The collagen fibers of the invention are especially well suited for the repair of soft tissue injuries.

In addition, attempts at arranging fibers in parallel which retain the strength of the individual fibers when bundled together for use as prosthetic tendons and ligaments or other suitable devices for the repair of tissue have not been successful. Up to 70 to 80% of the predicted strength of the bundle is lost when the fibers are bundled in parallel.

This phenomenon has been documented in fields distinct from that of collagen implants. In rope making, for example, fibers are bundled and braided in order to retain as much of the individual strength of the individual fiber as possible. This approach is, of course, unfeasible, however, in animal bodies, where tendons and ligaments should be oriented in parallel if they are to simulate the natural body. EMBODIMENTS OF THE INVENTION

In this description of the invention, the following terms have the following meaning.- "Autograft" means transferring a tissue or organ by grafting into a new position in the body of the same individual. "Implant" means a graft which is woven into the and secured in the surrounding tissue.

"Graft" means anything inserted into something else, or contacted upon something else so as to become an integral or associated part of the latter and it includes materials and substances which are either added to an already intact structure or serve as a replacement substitute or repair to a damaged or incomplete structure. Thus a "graft" is intended to be given the broadest possible meaning and encompasses a prothesis, implant or any body part substitute for any mammal (animal or human) .

As used in this application, the terms "the same",

"uniform", "parallel", "simultaneously" and "coated" include

"substantially the same", "substantiallyuniform", "substantially parallel", "substantially simultaneously" and "substantially coated".

"Wet" when referring to fibers or embodiments of the invention means more than 15% water content. "Dry" means 15% or less water conten . The invention provides a high strength collagen fiber for grafts, as well as a method for the manufacture of the high strength collagen fibers.

In a particular important embodiment, the invention further provides a bundle of monofilament collagen fibers arranged in parallel in which the fibers are tensioned by stretching so that the bundled fibers retain a high percentage of their individual strength and so that the fibers fail simultaneously. The invention further provides a method for the production of monofilament collagen fibers arranged or organized in parallel in which the fibers are tensioned by stretching so that the bundled fibers retain a high percentage of their individual strength and so that the fibers fail simultaneously. The invention further provides a prosthetic device, such as a tendon or a ligament device, made from a multiplicity of the tensioned fibers arranged in parallel or from a multiplicity of the bundles arranged in parallel.

The invention also provides collagen devices like fibers and grafts for numerous applications, particularly where high tensile strength and biocompatibility are essential. The invention also provides collagen proteoglycan fibrous grafts which have even greater tensile strength than the non- proteoglycan grafts of the invention. The invention further provides a method for making improved collagen proteoglycan fibers for use in such grafts. The invention provides further implants in which the collagen grafts are woven and secured into the surrounding tissue. The surrounding tissue then invades the graft material. The graft is revascularized and eventually replaced by the host's tissues.

The invention further provides for grafts with physical properties that can be manipulated or processed into a variety of shapes, thicknesses, stiffnesses in woven or non-woven forms.

Other embodiments provided by the invention will become apparent from the description which follows.

SUMMARY OF THE INVENTION

Several major new embodiments of the invention are described herein.

One such embodiment is an improved biodegradable and biocompatible reconstituted monofilament collagen fiber which has increased strength and elasticity of the fiber when compared to fibers made by other than the method of the invention. The fibers may be made from soluble or insoluble collagen and may be crosslinked or not crosslinked. The fibers may be stretched from 2.5% to 100% of their length to increase the strength of the fibers. Generally, the fibers are stretched until the strength of the fibers reaches a maximum level. The stretching may be performed in a single stretching application or may be performed by repeated cycles of stretching, drying, wetting, and stretching as deemed appropriate under the circumstances. The fibers may be left uncoated or may be coated with a suitable material, preferably biocompatible and biodegradable, to protect the fiber from the environment and from trauma during handling. The coating is applied as a low weight fraction of the fibers, generally less than 10% of the weight of the fibers. The fibers may or may not be embedded in a matrix compound of a suitable material, preferably biocompatible and biodegradable, to strengthen the fibers by binding the fibers together in a fiber/matrix composite. The matrix is applied in a high weight fraction of the fibers, generally more than 10% of the weight of the fibers. Proteoglycans may or may not be incorporated into the interfibrillar spaces to increase the tensile strength of the fibers.

A method for the production of the improved biodegradable and biocompatible collagen fibers is another embodiment of the invention.

Another embodiment is a bundle of physically associated collagen fibers arranged in parallel, or substantially parallel, in which the fibers are tensioned by stretching so that the fibers each have the same, or substantially same, strength and fail in, or substantially in, unison at the same load. This results in a stronger bundle of fibers than could otherwise be achieved if the fibers had different strengths and consequently broke individually at different times. The bundles of the invention retain a high percentage, generally more than about 50%, of the combined strength of the individual fibers. In one preferred embodiment, in a bundle comprising 10 fibers, the load to failure of the bundle was about 60% of the calculated cumulative load to failure of the individual fibers. The fibers of the bundle may or may not be coated or embedded in a matrix as described for the monofilament fibers. Proteoglycans may or may not be incorporated in the bundle as described for the monofilament fibers. A process for making bundles of fibers in which the fibers are tensioned and fail in unison or substantially in unison at the same or substantially the same load comprises the following. Fibers made in accordance with the invention, as disclosed in the parent applications, are attached at each end to a tensioning device which stretches the fibers from about 2.5 to about 100.0%, preferably from about 5 to about 7.5% of their original length. The stretching results in an increase in strength of the fibers and, very importantly, results in uniform strength of the fibers. Fibers other than those made by the process of the invention can likewise to treated in the same manner. Another embodiment of the invention is a prosthetic device for a graft, such as a tendon or ligament device, which comprises the tensioned fibers of the invention, aligned in parallel, either bundled or unbundled. The tendon or ligament prosthetic device has increased strength, compared with tendon or ligament devices comprising fibers made by means other than that of the invention, as the individual fibers of the device are stronger and fail in unison.

Other embodiments will become apparent as described further herein.

The invention, as disclosed in the parent applications, has several embodiments. In one of its embodiments the invention provides a high strength synthetic collagen graft constituted of high strength reconstituted crosslinked collagen fibers embedded into a loose uncrosslinked collagen matrix.

The grafts of the invention are biologically compatible with a host. They simulate the morphological and biomechanical characteristics of the host's natural tissue.

In one embodiment of the invention high molecular weight chondroitin sulfate proteoglycan is added during the latter stages of collagen fiber synthesis to be incorporated into interfibrillar spaces and as a result enhances the ultimate tensile strength of the collagen fibers formed.

A further embodiment of the invention is a process for the manufacture of the reconstituted collagen fibers.

The fibers of the invention are prepared from soluble or insoluble collagen. In a preferred embodiment, the collagen is in solution or dispersion in an acid solution. The solution or dispersion is extruded through polyethylene tubing into a fiber formation buffer ("FFB") . The fibers are then successively bathed in alcohol, to dehydrate the fibers, and then again in water, following which the fibers are dried and wound on a tensioning spool. Prior to drying, as the wet fibers are collected and tensioned on the spool, the fibers may be stretched, up to about 100% of their original length. It was a surprising finding that for production of optimum strength in the manufactured fibers a water bath following the dehydration bath was so highly desirable. Methods of forming collagen fibers without this water bath yield fibers of inferior strength. Other embodiments of the invention will become apparent to one of average skill in the art to which the invention pertains.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure l shows a diagrammatic example of an automated method for production of continuous collagen fibers. Figure 2 is a diagram outlining the various embodiments of the fibers of the invention.

Figure 3 shows ultimate tensile strength of collagen fibers which were stretched by varying percentages of the original lengths of the fibers. Fibers stretched 5.0% had an increase in tensile strength of about 50%. Fibers stretched 7.5% had an increase in tensile strength of about 57%.

Figure 4 shows load at failure of collagen fibers which were stretched by varying percentages of the original lengths of the fibers. The most dramatic increase in strength occurred at 5.0% stretch, which resulted in an increase in strength of about 160%. Fibers stretched 7.5% of their length almost doubled their strength. Fibers stretched by more than 7.5% or less than 5.0% had smaller strength gain, although these fibers were still stronger than control fibers. Figure 5 shows strain at failure of collagen fibers which were stretched by varying percentages of the original lengths of the fibers. All stretched fibers had a higher amount of strain at maximum (point of breakage) . Fibers stretched 5.0% had the highest strain at maximum. Figure 6 shows modulus (stress/strain) at low levels of applied stress of collagen fibers which were stretched by varying percentages of the original lengths of the fibers.

Figure 7 shows modulus (stress/strain) at high levels of applied stress of collagen fibers which were stretched by varying percentages of the original lengths of the fibers.

DETAILED DESCRIPTION OF THE INVENTION The method for production of the high strength collagen fibers of the invention is as follows:

Collagen fibers are placed in or passed through a fiber formation buffer ("FFB") . The collagen precursor fibers which are placed in the FFB may be soluble fibers in solution or insoluble collagen in a dispersion in a dilute acid solution. Before being placed in the FFB, the precursor collagen in the acid solution may be extruded through tubing with the desired diameter to yield fibers of the desired thickness.

The FFB may be an aqueous buffer which may contain neutral salts or it may be composed solely of distilled water. In a preferred embodiment, the FFB comprises NaCl, TES (N- Tris(hydroxymethyl) methyl-2-aminoethane sulphonic acid), and sodium phosphate dibasic. Chemically similar or equivalent compounds may also be used as well as other collagen fiber formation buffers well known in the art. The temperature of the fiber formation buffer should be sufficiently high to allow fiber formation to occur but should not be so high as to disturb the fiber formation process. Generally temperatures between about 4° to about 4i°C have been found to be suitable. The pH of the FFB should be higher than the isoelectric point. A pH about 7.5 has been found to be acceptable.

The fibers remain in the FFB for a period of time sufficient to allow the fibers to become strong enough to support their own weight when lifted from the FFB. The necessary time will vary depending on the temperature of the FFB, with lower temperatures requiring a longer immersion in the buffer. At higher temperatures, an immersion of 15 minutes may be sufficient whereas at lower temperatures, an immersion of 8 hours may be necessary at low FFB temperatures.

After the fiber formation step, the water is removed from the collagen. Water removal can be achieved by immersing the fibers in a dehydrating solvent, such as an alcohol for a period of time sufficient to remove of the water from the fibers, or may be achieved by air drying. Depending on the dehydration method used, the length of time of for this step will vary. Using an alcohol, times from 30 minutes to several hours may be employed. Any alcohol may be used which effects water removal from the collagen. That is, the solubility parameter of water in the alcohol used must be high. Examples of alcohols are suitable for the dehydration step include lower alkanols (up to 6 carbon atoms), like methyl, ethyl, and isopropyl alcohols. Thereafter, the fibers are placed in a water bath for a time sufficient to remove the excess solvent and other chemicals and to allow the fibers to align in or near parallel during the subsequent drying process. The length of time required varies inversely with the temperature of the water bath. Generally, water temperatures between about 4° and about 41°C have been found to be suitable, with longer immersion times being required at lower water temperatures. The water is preferably ion free water which may be distilled water. Following the water bath, the fibers are dried. The drying may be by air drying at room temperature or an external heat source, such as a «at lamp may be used. in a preferred embodiment, the drying is performed with the fibers under tension. Drying should preferably continue until the fibers retain from 0% to about 30% moisture by weight. Preferably, the fibers are dried to retain about 15% moisture by weight.

Following drying, the fibers are collected. In a preferred embodiment, the fibers are collected onto a spool under tension. The collagen fibers may be formed using a manual method such as the method described above or by means of a continuous automated process, such as described in Kato and Silver, Formation of Continuous Collagen Fibres: Evaluation of Biocompatibility andMechanical Properties, Biomaterials, 11:169- 175 (1990) which is incorporated herein by reference. An automated method for production of continuous collagen fibers may be performed as follows: A collagen solution or dispersion is extruded through a continually flowing solution of FFB with the aid of a syringe pump (Sage Instruments, Cambridge MA) containing the collagen dispersion. To prevent pulsatile flow during the process of aggregation and to, therefore, prevent tensile stress on the freshly extruded collagen fibers, a micro gear pump (Cole Palmer, Chicago, IL) is used. A conveyor belt mechanism is used to carry the fibers through the various processes. The fiber formation buffer is recycled and reheated to the desired temperature by a pump system. The extruded collagen flows down a tubing conveyed under the flow of the fiber formation buffer and is collected on a conveyor belt. The belt carries the collagen fiber via a pulley mechanism designed to immerse the belt in solution through an alcohol bath, followed by a water bath. The fiber is then picked up by a spool a distance from the conveyor belt, drying under tension with a heat lamp during the time of travel between the conveyor belt and the spool. A diagrammatic representation of an automatic process to produce continuous collagen fibers is shown in Figure 1.

Following the formation of the fibers, the fibers may be treated in various ways to produce the various embodiments of the invention. An outline of the treatment of the fibers is illustrated in Figure 2.

SUBSTITUTE SHEET (RULE 2β The collagen fibers, made by a manual or automatic process, may then be crosslinked using known crosslinking techniques. U.S. Patent No. 4,703,108 to Silver, et al. which discloses numerous crosslinking techniques is incorporated herein by reference.

Further details as to suitable methods of crosslinking are also given herein in Examples I and II.

The bundles of the invention are comprised of a multiplicity of suitable monofilament collagen fibers bundled together in parallel alignment. A method of making suitable collagen fibers is detailed herein, but other suitable methods can be used to make collagen fibers to be bundled in accordance with the invention.

Monofilament collagen fibers are placed side to side in parallel alignment to form a bundle. The bundle may be placed in a fiber formation buffer solution or may be left dry. The wet bundle is stretched. The bundle is then air dried at which time the monofilaments will have become associated to form a single bundle. The term "associated" in the context of the bundles means that the fibers are in contact with each other substantially throughout the entire lengths of the fibers and that the contact between the fibers is self-sustaining. If desired, the fibers of the stretched bundle may be crosslinked.

A bundle of collagen fibers of the invention comprises a multiplicity of fibers generally over 5 and may include several hundred fibers. For most applications, bundles comprising 7 to 15 fibers are suitable. For certain applications bundles comprising from about 2 fibers to about 100 fibers seem more suitable; for other applications, bundles comprising 100 fibers to about 10,000 fibers may be better suited. The number of fibers in what is termed a "bundle" is suited to the particular application or use selected.

Any suitable FFB may be used to wet the fibers for bundling in accordance with the method of the invention. Any of the FFBs described above to make the collagen fibers may be used in the bundling .process.

The tensioning of the fibers is carried out by any suitable means, such as a stretching device which may be a screw driven frame to which parallel oriented supports, which may be tongue depressors, are clamped at each end using screws. The ends of collagen fibers to be stretched are attached to the supports by any suitable means so that the fibers do not slip when stretched. The supports are separated, thus stretching the fibers, by turning a screw. The amount of stretching can be adjusted by number of turns of the screw. Thus, the stretching device acts like a rack to stretch the fibers. The stretching device is manufactured from means (mechanical or otherwise) which accomplish the desired stretching function.

The fibers are stretched or tensioned to a degree that, when compared to unstretched fibers, the stretched fibers have increased strength. The stretching, or tensioning, of collagen fibers, whether in bundles or as monofilaments, results in an increase in tensile strength of the fibers, especially when fibers are stretched from about 2.5 to about 10.0% of their original lengths. It has been found, in accordance with the invention, that the increase in strength of stretched fibers is optimal when fibers are stretched from 5.0 to 7.5% of their original length. See Figure 3. Stretched fibers have higher load at failure and strain at failure than unstretched fibers, the increase being most pronounced with stretching from 5.0 to 7.5% of the fibers' original lengths. See Figures 4 and 5. Additionally, stretched fibers are less stiff than unstretched fibers, especially with higher degrees of applied stress. See Figures 6 and 7.

When implanted in animal bodies, collagen implants often lose up to 75% of the initial tensile strength within four weeks of implantation. It is therefore essential to maximize the initial collagen fiber tensile strength to compensate for this rapid loss of tensile strength of implants, especially in orthopedic applications such as tendon or ligament replacement. The monofilament and bundled fibers of the invention, having higher tensile strength than fibers previously known in the art, are especially well suited for implants requiring long lasting strength of fibers. As an example, bundles of fibers made in accordance with the invention were found to retain a high percentage, approximately 60%, of the predicted strength computed by multiplying the load at failure of individual fibers times the number of fibers in the bundle. This compares favorably with other known methods of bundling fibers which result in a loss of up to 70 to 80% of predicted strength.

If desired, the bundle may be coated with a suitable protective material. Individual unbundled fibers may also be coated. The bundle may be embedded in a matrix. Individual unbundled fibers may also be embedded in a matrix. Preferably the coating or matrix material is a biodegradable and biocompatible polymer, which may be a synthetic or natural polymer, and may be water or non-water soluble. Suitable polymers include water soluble polymers derived from natural sources, such as alginates, pectins, gelatins, and polysaccharides, and synthetic polymers such as polylactic acid, polyglycolic acid, polyurethane, and copolymers thereof.

The coating is performed by covering the bundles or the unbundled fibers with a liquid coating material. Preferably the coating is performed by dipping the stretched bundles into, or running the fibers through, the liquid coating material, or spraying the coating material onto the bundles or fibers until the bundles or fibers are coated. Generally, less than a 10% weight fraction of coating material is applied to the fibers. After coating, the bundles may be dried and crosslinked.

Embedding of the bundled or unbundled fibers in a matrix is performed in substantially the same way as the coating. However, a higher weight fraction of matrix compound is used than is used in coating so that the matrix compound becomes embedded between the fibers of the bundle. When applying a matrix to unbundled fibers, the matrix is applied as a coating.

Alternatively, or in addition to coating, the collagen fibers may be embedded in a biodegradable and biocompatible polymer matrix to bind the fibers together. The procedure for producing the matrix/fibers composite may be identical to the process for coating except that a higher weight portion of the matrix is used, greater than 10% of the fibers.

The tendon or ligament prosthetic devices for grafts of the invention are constructed as follows. The device may be made in various ways, as illustrated in Figure 2. In one suitable method, individual fibers made in accordance with the invention are placed in parallel alignment. The fibers are made into bundles and the ends of each bundle is secured. A multiplicity of bundles, each consisting of a multiplicity of fibers, for example about 10 fibers per bundle although as few as two to five fibers and as many as several hundred fibers may be used per bundle, are joined in parallel to form a tendon or ligament device. Generally, between 7 and 15 fibers per bundle will are used to form the prosthetic device of the invention, in a second method, the parallel fibers are wrapped with a braided bundle of fibers. The parallel fibers are secured at each end to form the final device. In a third method, fibers are made into bundles which are then wetted, stretched, and dried. The stretched bundle may be crosslinked. The bundles are arranged in parallel and secured at the ends to form the final prosthetic device. In a fourth method, the parallel bundles are wrapped with a bundle of fibers which may be braided. The ends of the parallel bundles are secured to form the prosthetic device. The graft of the invention has numerous applications which can assume different physical embodiments or different geometrical shapes.

The synthetic collagen graft material of the invention is useful as a mesh, sheet, film, tube, circular casing, filament, fiber or as a woven or non-woven fabric.

The graft material comprise collagen fibers with a dry diameter in the range of about 20-60 microns. The diameter of the wet collagen fibers may be much greater, often 1.5 to more than 2 times as thick. The collagen fibers used in the grafts of the invention have tensile strengths in the range of about 30 to about 91 MPa. It is a noteworthy aspect of the invention that the fibers of the invention can have ultimate tensile strengths exceeding that of autograft materials or naturally occurring tendon fibers in laboratory animals. The fibers of the invention also have improved strength compared to fibers made by means other than the method of the invention. The collagen materials of the invention can have an index of refraction in the range of about 1.4 to about 1.7, generally about 1.6. The graft material is biodegradable with the host's naturally produced repair tissue supplanting the graft material. Furthermore, the graft is biologically, morphologically and biomechanically compatible with surrounding tissue of the subject treated.

In another embodiment of the invention, proteoglycans are associated with the collagen fibers of the invention. For that purpose the extruded fibers are immersed in a fiber formation buffer containing the proteoglycan. The fibers are Soaked for a sufficient time at an appropriate temperature to cause the proteoglycan to be incorporated into the fibrous structure. For instance, the fibers can be soaked for 60 minutes at 37°C. The fibers are then rinsed with appropriate liquids to remove excess proteoglycan and dried. Soaking temperature can be in the range from about 15°C to 50 or 60°C with either longer or shorter soaking periods, as may be desirable. Specifically this embodiment of the invention may be prepared as follows. Proteoglycans in a concentration between 0.01 and 0.02 g/100 ml were added to the fiber formation buffer and stirred. A 1% w/v collagen dispersion was placed in a syringe to which polyethylene tubing of internal diameter 0.58 mm was attached. Fibers were extruded into a fiber formation buffer. The fiber formation buffer is composed of l35mM NaCl, 30mM TES (N-Tris(hydroxymethyl) methyl -2- aminomethane sulfonic acid) and 30mM sodium phosphate dibasic. The final pH is adjusted to about the neutral range such in the range of about 6.5 to 7.5. Chemically similar or equivalent compounds may also be used as well as other collagen fiber formation buffers well 22

known in the art. The extruded fibers were left in the tray containing fiber formation buffer for 60 minutes. The buffer was maintained at 37°C. The buffer was removed and replaced by isopropanol. The fibers were soaked in isopropanol overnight and were then soaked in distilled water for 15 minutes. The fibers were then removed from the distilled water and air dried under tension. The extruded collagen fibers were then crosslinked by exposure to glutaraldehyde. Fibers which were formed in the presence of high molecular weight proteoglycan were found to have significantly increased ultimate tensile strengths compared to low molecular weight, chondroitin sulfate, glycosaminoglycans or controls. Furthermore collagen fibers formed in the presence of high molecular weight proteoglycans exhibit higher tensile strength than collagen fibers that are crosslinked. Further details are given in Example III below.

The high molecular weight proteoglycans which are generally preferred in the invention are large proteoglycans with a core protein with a molecular weight greater than about 100,000 and glycosaminoglycans chain with a molecular weight greater than about 5,000.

These proteoglycan generally have a molecular weight of the range of about 1,000,000 to about 3,000,000 typically about 1,200,000. Other proteoglycans desirable for use in the invention include large proteoglycans from tendon with chondroitin sulfate chains of average molecular weight of 17,000 and a core protein molecular weight of 200,000. It is not unlikely that other proteoglycans will also be useful in the invention providing they impart the desirable properties to the collagen fibers, in particular the desired tensile strength.

Once the high strength collagen fibers which constitute the graft material of the invention are formed in accordance with the various embodiments of the invention, the fibers are collected. The collected fibers are shaped, pressed or formed into sheets, tubes and numerous other shapes of varying dimensions and thickness as desired for the particular application. The fibers can be processed into woven materials. They can be packed with various pharmacologically active agents. These structures then can be directly used as the graft, prosthesis or implant of the invention depending on the need and how the particular structure has been prepared. The fibrous graft can be woven or secured to surrounding tissue as an implant or graft or topically applied and topically secured.

One skilled in the art will shape the structure to the desired application.

The following examples are exemplary of the various embodiments of the present invention discussed hereinabove. They are not to be construed as limiting but as illustrative of the process and products of the present invention.

Whenever "means" or "steps" are disclosed in the specification, equivalent "means or "steps" which perform the same function may be used by one skilled in the art. EXAMPLE I Collagen fibers were prepared from a 1% (w/v) dispersion of insoluble type I collagen derived from bovine corium in dilute HC1, pH 2.0. This collagen dispersion was extruded through polyethylene tubing with an inner diameter of 0.28mm into a 37°C bath of aqueous sodium phosphate fiber formation buffer as described elsewhere. After immersion of 45 minutes, the fibers were placed in isopropanol for at least four hours. They were then rinsed in distilled water for 15 minutes and allowed to air dry under tension overnight. Fibers were placed in a sealed desiccator containing 10 ml of a 25% (w/v) glutaraldehyde solution at room temperature and allowed to vapor crosslink for 24 hours. Alternatively, collagen fibers were placed in an oven at 110°C in a vacuum of between 50 and 100 m torr for 72 hours. These fibers were then placed in a sealed desiccator containing 20 g of cyanamid in 5 ml of distilled water for 24 hours.

Prostheses containing 200 to 250 individual crosslinked collagen fibers were coated with a 1% (w/v) collagen dispersion in HC1, pH 2.0, air dried overnight and then extensively washed in distilled water. One ml Alcide^® activator and one ml Alcide^® base were added to 10 ml of distilled water and after 10 minutes diluted with 24 ml of phosphate buffer solution. Each implant was immersed in this cold sterilant for at least four hours, and then soaked in one liter of sterile physiological saline prior to implantation. EXAMPLE II Reconstituted Collagen Fibers Insoluble collagen type I from fresh, uncured corium was obtained from Devro, Inc. (Somerville, NJ, USA) . The corium was limed, fragmented, swollen in acid, precipitated, washed with distilled water and isopropanol, lyophilized and stored at -30°C. A 1% (w/v) dispersion of type I collagen in dilute HC1, ph 2.0 was prepared by adding i.2g of lyophilized collagen to 120ml of HC1 solution in a blender (Osterizer) and mixing at a speed of 10,000 rev min^"1 for 4 min. The mixture was allowed to settle for 10 min and then remixed at 10,000 rev min^"1 for 4 min. The resulting dispersion was placed under a vacuum of 0.01 m torr at room temperature to remove any trapped air bubbles. Dispersion was then stored in disposable 30 cc syringes at 4°C. Collagen fibers were produced by extruding the collagen dispersion through polyethylene tubing with an inner diameter of 0.28 mm into a 37°C bath of aqueous fiber formation buffer composed of 135mm NaCl, 30mM TES (N-Tris(hydroxymethyl) methyl - 2- aminomethane sulfonic acid) and 30mM sodium phosphate dibasic. The final bath pH was adjusted to 7.5 by adding 5.On NaOH drop- wise. Fibers were allowed to remain in the buffer for 45 min, and then placed in 500 ml of isopropyl alcohol for at least 4 hours. The fibers were immersed in distilled water for 15-20 min and air dried under tension. Collagen fibers were crosslinked using glutaraldehyde or by a combination of severe dehydration and treatment with cyanamid. Glutaraldehyde crosslinking was accomplished by placing air-dried collagen fibers in a sealed desiccator containing 10 ml of a 25% (w/v) aqueous glutaraldehyde solution in a petri dish. The fibers were placed on a shelf in the desiccator and were crosslinked in a glutaraldehyde vapor for 1-4 d at room temperature. Collagen fibers were also cross-linked by placing in an oven at 110°C and at vacuum of 50-100 m torr for 3 d. Subsequent to dehydrothermal crosslinking (DHT) , collagen fibers were placed on a shelf in a sealed desiccator containing a petri dish with 20 g of cyanamide in 5 ml of distilled water. Collagen fibers were crosslinked for one day in contact with cyanamide vapor.

EXAMPLE III Extraction of Soluble Type I Collagen

Acid soluble type I collagen was extracted from tail tendons of young rats. The tendons were stripped from the tails and dissolved in 0.01 M HC1 at 4°C followed by centrifugation for 30 min. at 30,000 X g. The supernatant was sequentially filtered through 0.8. , 0.65, and 0.45 μm Millipore filters. The collagen preparation was analyzed by SDS polyacrylamide gel electrophoresis and amino acid analysis.

Purification of insoluble Type I Collagen The raw material (bovine corium) was prepared from fresh uncured bovine hide which was obtained from Devro, Inc. (Somerville, N.J.). The hides were split into two components, the grain layer (papillary dermis) and the corium (reticular dermis) . Fresh corium was frozen and stored at -20°C until it was used. One liter of the frozen raw material was defrosted at room temperature and placed in an 18 liter Nalgene processing tank (Consolidated Plastics, Twinsburg, Ohio) , equipped with air and water lines. Distilled water was added until the total volume of the processing mixture reached 14 liters. Air at a pressure of 6 psi was introduced into the tank for 5 minutes, to create a homogeneous mixture. This mixture was then left to sediment for 20 minutes. After complete sedimentation occurred, the liquid phase was drained and fresh distilled water was added until the total volume reached 14 liters. This procedure was repeated three times.

Eight liters of 99.8% of isopropyl alcohol

(Mallinkroft, Inc., Paris, Kentucky) was added to the solid phase; the sediment was mixed using air in a tank placed on a gyrotory shaker (New Brunswick Scientific Co., New Brunswick, N.J.) for 12 hours at a speed of 34 rev/min.

The liquid phase was then removed using a Becton siphon pump (Consolidated Plastic, Twinsburg, Ohio) and 8 liters of 99.8% isopropyl alcohol was mixed with the solid phase. The mixture was then placed on the shaker for another 12 hours. After removal of the liquid phase, the material was washed with 2 liters of distilled water, poured into plastic trays and placed in a freezer until frozen solid.

The frozen material was then placed in the cold trap of a freeze dryer (Freeze Mobile 12, Virtis, Inc., Gardner, N.Y.) at -65°C. A vacuum of 10 microns was then applied for 48 to 96 hours. The vacuum was then released and material removed. The freeze dried collagen was removed from the trays and stored in air tight bags.

Preparation of Insoluble Type I Collagen for Fiber Formation A IN solution of HC1 was slowly added to 120.0 ml of distilled water until the pH was 2.0. A 1.2 g sample of insoluble type I collagen, extracted by the procedure described above, was then put into a blender (Osterizer Model, Oster Corporation, Milwaukee, Wisconsin) with the HC1 (pH 2.0). This 1% w/v collagen HC1 dispersion was blended at high speed (10,000 rpm) for 3 minutes. The dispersion was then emptied from the blender into a 600 ml sidearm flask. A vacuum (Vacuum Pump, Model 150, Precision Scientific Company, Chicago, 111.) of 100 microns was applied at room temperature until the air bubbles were removed from the dispersion. This procedure required approximately 15 minutes. The vacuum was removed and the dispersion was ready for making fibers. Glycosaminoglycans and Proteoglycans Dermatan sulfate (chondroitin sulfate B from porcine skin) , chondroitin sulfate (type A from whale cartilage) , glycosaminoglycans (GAG) and dextran sulfate (Dexs) were obtained from Sigma Chemical Company (St. Louis, Mo.). Chondroitin sulfate proteoglycan (CS-PG) and dermatan sulfate proteoglycan (DS-PG) from hypertrophic scar tissue and high molecular weight proteoglycan from articular cartilage (PGi) were prepared and characterized as previously described.

FIBRIL ASSEMBLY STUDIES Turbidity-Time Studies Lyophilized soluble type I collagen was dissolved at l mg/ml in HC1, pH 2.0, stirred at 4°C for 24 hours, dialyzed against HC1, pH 2.0, centrifuged at 1600 g for 60 minutes and the supernatant was then filtered through a 0.65 μm Millipore filter. This collagen stock solution was stored at 4°C for periods of up to one week.

Fibril formation was initiated by mixing 0.9 ml of a collagen solution with 0.1 ml of buffer on ice to give a final composition of 30 M n-tris [hydroxymethyl]methyl-2- aminoethanesulfonic acid (TES) , 30 mM phosphate and NaCl to a final ionic strength of 0.225 at pH 7.3. Cuvettes were filled with sample, sealed and transferred to a water-jacketed sample compartment of a Gilford Model 250 spectrophotometer. The compartment was maintained at the desired experimental temperature and the absorbent was recorded as function of time. Absorbent was defined as the natural logarithm of the ratio of the incident light and the scattered light intensities. Absorbent at 131 nm was converted to turbidity by multiplying by 2.303.

Collagen concentrations between 0.20 and 0.45 mg/ml and proteoglycan concentration between 0.001 and 0.2 g/lOOm were evaluated at temperatures from 27 to 37°C.

Extrusion of Collagen Fibers

An aqueous fiber formation buffer composed of 135 mM NaCl, 30 mM TES and 30 mM sodium phosphate dibasic at a final pH of 7.5 was heated to 37°C in a temperature controlled water bath. Glycosaminoglycan (concentrations between 0.001 and 0.2 g/l00 ml) or proteoglycan (concentrations between 0.01 and 0.02 g/lOOml) was added to the fiber formation buffer and stirred. A 1% w/v collagen dispersion (lg/iOOml) was placed in a syringe to which a polyethylene tubing (Clay Adams, PE-50) of internal diameter 0.58 mm was attached. A syringe pump (Sage Instruments, Model 341A) at a speed of 7 ml/minute was used to extrude the fibers into fiber formation buffer. Extruded fibers were left in the tray containing fiber formation buffer maintained at 37°C for 60 minutes. Fiber formation buffer was then emptied out from the tray using a vacuum hose and was replaced by isopropanol and left overnight. Isopropanol was removed and was replaced by distilled

SUBSTITUTESHEET(RULE26Ϊ water for 15 minutes. Fibers were then removed from the distilled water and air dried under tension.

Collagen Fiber Crosslinking Extruded collagen fibers were crosslinked by exposure to glutaraldehyde vapor for 24 hours (Glut 1) at room temperature in a sealed desiccator as described previously.

Proteoglycan concentrations present on collagen fibers were also less than 1% (data not shown) . This is another distinctive characteristic of the fibers which are particularly useful in the invention.

EXAMPLE IV Preparation of Collagen Monofilament from Soluble Collagen Collagen monofilament fibers were prepared from soluble type I collagen from fetal calf skin. Collagen fibers were prepared from 1% (w/v) solution of type I collagen in dilute HC1. The solution was extruded through polyethylene tubing with an inner diameter of 0.28 mm into a 37°C bath containing aqueous sodium phosphate fiber formation buffer. The fibers were extruded into a tray containing fiber formation buffer and then the fiber was pulled over a transfer device into a bath of isopropanol and then through a bath of distilled or demineralized water. The fiber left the last bath and was air dried using a heat lamp. The fiber was then wound on a tensioning spool. EXAMPLE V Formation of Bundles Containing Monofilaments Ten collagen monofilaments were placed side-by-side in parallel alignment to form a bundle. The bundle was glued using an epoxy adhesive onto a support beam (tongue depressor) at each end of the bundle. The assembly was then placed in phosphate buffer solution for 25 minutes. The wet bundle with support beams at each end was then attached to a stretching frame using screws to secure the beams to the stretching frame. The bundle was stretched 7 to 7.5% of its original length and allowed to air dry overnight. On drying the 10 monofilaments were associated to form a single bundle which was then removed from the two support beams. The bundle was then crosslinked for 5 days at 110°C. Bundles are made as described above using 2 monofilament fibers. Results as described in Example VII with bundles of 2 fibers are similar to results achieved using 10 fibers.

Bundles are made using about 400 monofilament fibers. Bundles are also made using about 10,000 monofilament fibers.

Results as described in Example VII with bundles of about 400 and with bundles of about 10,000 fibers are similar those achieved using 10 fibers. EXAMPLE VI Formation of Coated Bundle Collagen fibers were produced as in Example IV. Ten collagen monofilaments were placed side-by-side to form a bundle. A wet bundle of collagen fibers that was attached to support beams and stretched to 7 to 7.5% as described in Example V above was dipped in a 4% aqueous solution of sodium alginate at room temperature. The alginate coated bundle was then air dried and then crosslinked for 5 days at 110°C.

EXAMPLE VII

Mechanical Testing of Monofilament and Bundled Fibers

Collagen mono ilaments and bundles were mechanically tested while wet using an instron Model 1122 at a strain rate of 50% per minute using a 2 cm gauge length. The materials were mounted on a 2 cm gauge length frame using 2 ton epoxy (Devron Corp., Denver, CO) . Monofilaments and bundles mounted on paper frames were immersed in phosphate buffer solution (pH 7.5) for 25 minutes. The paper was cut and the samples were pulled to failure in tension. Load and strain at failure were found to be 38.0 g and 14.5% respectively for monofilament and 219 g and 10.5% for uncoated intermediate bundles.

This bundle retains 219 g of the predicted 380 g or 58% of the load to failure. The bundle breaks uniformly, has a strain at failure of about 10% and can be fashioned into a tendon/ligament device by forming either thin tapes of intermediate bundles, braiding monofilament around a group of parallel bundles, or by wrapping monofilament around groups of parallel bundles.

EXAMPLE VIII

Formation of Prosthetic Device from Unbundled Fibers

One hundred collagen monofilament fibers are placed onto spools. The fibers are then tensioned in parallel by stretching them over a pulley. An outer layer of collagen fibers is wrapped or braided around the parallel fibers. The ends of the fibers are secured to stabilize the outer layer and to preserve the parallel alignment of the fibers.

The above process is performed using 400 and using 10,000 collagen monofilament fibers. The fibers are tensioned in parallel and wrapped by an external layer of fibers which may be braided. The ends of the parallel fibers are secured to stabilize the prosthetic device.

EXAMPLE IX Formation of Prosthetic Device from Bundled Fibers

Ten stretched crosslinked bundles, each having ten collagen monofilament fibers are placed in parallel. An outer layer of collagen fibers is wrapped or braided around the parallel bundles. The ends of the bundles are secured to stabilize the outer layer and to preserve the parallel alignment of the bundles. The same process is performed using 400 collagen monofilament fibers in 40 bundles, each containing 10 fibers. The process is performed using 10,000 collagen monofilament fibers in 1000 bundles, each containing 10 fibers. The bundles are placed in parallel and wrapped by an external layer of fibers which may be braided. The ends of the parallel bundles are secured to stabilize the prosthetic device.

Variations in technique (also "means" or "steps") of the type known in the art and understood by those of ordinary skill to be functional equivalents of those disclosed herein may be substituted as desired, for convenience or for optimization of yield, or to simplify or improve the cost- effectiveness of the overall procedure. Therefore, numerous modifications and variations of the present invention are possible which are within the scope of the appended claims.

REFERENCES

1. Wang, M.C., Pins, G.D., and Silver, F.H.,, "Collagen Fibers with Improved Strength for the Repair of Soft Tissue Injuries," Biomaterials. 15. 7:508-512, 1994.

2. Kato, Y.P. and Silver, F.H., "Formation of Continuous Collagen Fibres: Evaluation of Biocompatibility and Mechanical Properties," Biomaterials, 11. 169-175.

Claims

What is claimed is:

1. A biocompatible, biodegradable graft of improved mechanical properties which comprises crosslinked synthetic collagen fibers embedded in a collagen matrix.

2. The graft of claim l, wherein the collagen fibers are oriented in a planar array.

3. The graft of claim l, wherein the collagen matrix is crosslinked.

4. The graft of claim l, wherein the collagen matrix is not crosslinked.

5. The graft of claim l, which comprises high molecular weight proteoglycans associated with the collagen fibers.

6. A method of making a biocompatible, biodegradable graft of improved mechanical properties which comprises embedding crosslinked synthetic collagen fibers in a collagen matrix.

7. The method of claim 6, which further comprises the step of incorporating high molecular weight proteoglycans into the fibrous structure.

8. The method of claim 6, wherein the collagen matrix is crosslinked.

9. The method of claim 6, which comprises modifying the collagen fibers by exposing said fibers to an aqueous buffered medium containing high molecular weight proteoglycans, incorporating the proteoglycans into the fibrous structure, removing the excess proteoglycans and collecting the modified fibers.

10. An improved biodegradable and biocompatible reconstituted collagen monofilament fiber which has been wetted with water following dehydration and which has increased strength and elasticity as compared with a corresponding fiber which has not been wetted with water following dehydration.

11. The fiber of claim 10 which is dry.

12. The fiber of claim 11 wherein the fiber has been stretched to have a length from about 2.5% to about 100.0% longer than its unstretched length.

13. The fiber of claim 12 wherein the fiber has been stretched from about 5.0% to about 7.5% of its length.

14. The fiber of claim 11 wherein the fiber is crosslinked.

15. The fiber of claim 11 wherein the fiber is coated with a biodegradable and biocompatible polymer.

16. The fiber of claim 11 wherein the fiber is embedded in a matrix of a biodegradable and biocompatible polymer.

17. The fiber of claim 11 which comprises proteoglycans incorporated into the interfibrillar spaces of the fibers.

18. A bundle of associated collagen fibers which comprises a multiplicity of the biodegradable and biocompatible reconstituted collagen fibers of claim 11 wherein the fibers are associated and arranged substantially in parallel.

19. The bundle of claim 18 wherein the fibers have been stretched to substantially the same percentage of their length and the fibers fail at substantially the same load.

20. The bundle of claim 19 which comprises from 2 to 10,000 fibers.

21. The bundle of claim 20 which comprises from 7 to 15 fibers.

22. The bundle of claim 18 wherein the bundle is coated with biodegradable and biocompatible polymer.

23. The bundle of claim 18 wherein the fibers of the bundle are embedded in a biodegradable and biocompatible matrix.

24. The bundle of claim 18 wherein proteoglycans are incorporated into the interfibrillar spaces of the fibers.

25. A prosthetic device which comprises a multiplicity of the improved reconstituted collagen fibers of claim 10 associated and arranged substantially in parallel.

26. The prosthetic device of claim 25 wherein the fibers are arranged in bundles, each bundle containing from 2 to 10,000 fibers, wherein the fibers or the bundles have been stretched to substantially the same percentage of their length and the fibers fail at substantially the same load.

27. The prosthetic device of claim 24 wherein the fibers are coated with a biodegradable and biocompatible polymer.

28. The prosthetic device of claim 25 wherein the device is a tendon or ligament.

29. A process for making an improved biodegradable and biocompatible reconstituted collagen fiber comprising the steps of passing a collagen fiber through a fiber formation buffer to strengthen the fibers, dehydrating the fibers, wetting the fibers, drying the fibers, and collecting the fibers.

30. The process of claim 29 which further comprises, after the wetting step and prior to the drying step, stretching the fibers.

31. The process of claim 30 wherein, following the drying step, the fibers are rewet and restretched.

32. A process for making a bundle of associated biodegradable and biocompatible reconstituted collagen fibers which fibers have increased strength compared with untreated fibers and fail at substantially the same load comprising associating a multiplicity of collagen monofilament fibers side-by-side in substantially parallel alignment to make a bundle, wetting the bundle, and drying the bundle.

33. The process of claim 32 wherein the bundle comprises from 2 to about 10,000 fibers.

34. The process of claim 33 wherein the bundle comprises from about 7 to 15 fibers.

35. The process of claim 32 which further comprises the step of coating the bundle or the fibers of the bundle with a biodegradable and biocompatible polymer.

36. The process of claim 32 wherein the bundle is stretched to have a length from 2.5 to 100.0% longer than the unstretched length of the bundle.

37. The process of claim 36 wherein, following the drying step, the fibers are rewet and restretched so that the total amount of stretching and restretching is from 2.5 to 100.0% of the original length of the bundle.

38. The process of claim 36 wherein the bundle is stretched from about 5.0 to 7.5% of the original length of the bundle.

39. The process of claim 27 which further comprises the step of embedding the bundle or the fibers of the bundle in a biocompatible and biodegradable polymer matrix.

40. The process of claim 27 which further comprises the step of associating the fibers with a proteoglycan.