WO2002028417A1

WO2002028417A1 - Methods for treating joints using viscoelastic collagen solutions

Info

Publication number: WO2002028417A1
Application number: PCT/US2001/031062
Authority: WO
Inventors: Dale P. Devore; Peter D. Ciarametaro
Original assignee: Collagenesis, Inc.
Priority date: 2000-10-03
Filing date: 2001-10-03
Publication date: 2002-04-11
Also published as: AU2001294983A1

Abstract

The present invention features a method of treating joint tissue that has been damaged by disease or trauma. The treatment involves administering a soluble viscoelastic collagen solution into the joint space, to protect the joint tissue, to increase the viscosity of the synovial fluid, to lubricate and reduce friction at the articular surfaces, to reduce inflammation, to reduce further cartilage degeneration, and to increase cartilage repair in the joint.

Description

METHODS FOR TREATING JOINTS USING NISCOELASTIC COLLAGEN SOLUTIONS

Background of the Invention This invention relates to a method of treating joints damaged by disease or injury. Damage to joint tissue, caused by either traumatic injury or various acute or chronic diseases, often involves the cartilage of the joint and may also involve the synovium (the epithelial lining of the joint). The cartilage of the joint is produced by chondrocytes and forms the articular surface as well as a layer between the bone and the articular surface. At the articular surface, the cartilage is a hyaline cartilage that plays the dual role of absorbing shock and lubricating the apposing surfaces of the movable joint. The articular cartilage is smooth, white, semirigid, and generally not thicker than 6 mm in humans. It is a hyperhydrated structure normally composed of 70-80% water. The water molecules may be bound by hydrogen bonding to other principal components of the cartilage, such as proteoglycans or collagen. The proteoglycans are elastic macromolecules that form the intercellular matrix of hyaline cartilage and the nucleus pulposus of the intervertebral disks. Proteoglycans resemble a bottle brush, with a linear protein forming the core. Linked to the core and radiating outwards are numerous glycosaminoglycan chains that extend stiffly into the matrix space because of repelling negative charges. The result is the creation of a molecular sieve.

The collagen in joint cartilage is principally Type II. The Type II fibers run up to the surface of the articulation from the deeper layers of cartilage, bend to run parallel along the surface, and then bend again to run back to the deeper layers. Any cracks which appear in the cartilage (fibrillations), run in the plane of the collagen fibers. Accordingly, superficial cracks run parallel to the articular surface, but, as they run deeper, the cracks turn to a perpendicular orientation.

All the internal aspects of a joint, with the exception of the articular cartilage, are lined by the synovium. The recesses and internal aspects of the capsule, as well as areas around the intra-articular structures, such as the menisci and ligaments, show synovial lining. The synovium controls a number of functions, including (1) diffusion in and out of the joint; (2) ingestion of debris; (3) lubrication of the joint by secretion of hyaluronate and glycoproteins into the synovial fluid; and (4) secretion of immunoglobulins and lysosomal enzymes.

Joint damage may be initiated with an abnormal force on the joint cartilage, which can occur, for example, as a result of joint trauma or osteoarthritis. This abnormal force increases the unit load on the chondrocytes, killing the cells and reducing the formation of new cartilage. As a result, the existing cartilage degrades and surface cracks form, leading to weakened and sometimes abnormally shaped cartilagenous structures. Eventually, pieces of cartilage break off from the articular surface and lodge in the synovium, inducing an inflammatory response. This inflammatory response causes the infiltration of inflammatory cells and fibrotic cells

(plasma cells, fibroblasts, macrophages, leukocytes, and polymorphonuclear cells), pain, swelling, reduced joint mobility, and the formation of scar tissue. It can, under conditions not yet fully understood, induce further degradation of the cartilage by causing the release of lysosomes from both the synovial cells and the chondrocytes.

The inflammation response, in addition to playing a role in mediating joint damage caused by an abnormal force on the joint, can also be one of the initial steps in the damage to a joint, especially with inflammatory diseases such as rheumatoid arthritis. Inflammation in this case is also associated with hyperplasia of the synovium, which forms a pannus (cloak) that fills the recesses of the joint and covers the articular cartilage. This produces additional degradation of the cartilage due to impaired contact of the cartilage with the nutritional synovial fluid, increased chondrocyte death, and the release of collagenases by the hyperplastic synovial cells and the chondrocytes.

While chondrocytes are normally capable of replacing damaged collagen and proteoglycan matrix material, the process is fairly slow, and significant time is required for proper healing. In addition, the healing process in the joint is often hampered by the inflammatory response which causes chondrocyte death and further cartilage degradation. The inflammatory response also affects the synovial fluid. For example, because the synovial fluid in damaged joints becomes filled with inflammatory cells, this fluid cannot properly sustain and lubricate the cartilage. The fluid also has increased protein content, increased turbidity, and, perhaps most significantly, decreased viscosity. The impaired function of the synovial fluid increases the friction of the joint and further stimulates the inflammatory response. As a result, the response to joint injury can trigger a continuous cycle of inflammation, impaired joint function, and cartilage degradation. Given that inflammation produces such counterproductive effects, for example, the infiltration of inflammatory and fibrotic cells, a local increase in temperature, swelling, pain, reduced joint mobility, hyperplasia, the formation of scar tissue, and further degeneration of the cartilage of the joint, there is a clear need for treatments which reduce or prevent this process. Such a treatment would produce rapid improvement of joint function by reducing swelling, pain, and immobility, and also mediate long term positive results by reducing further cartilage degeneration and enhancing chondrocyte-mediated repair of damaged cartilage. One common mode of treatment to reduce joint inflammation involves the use of drugs such as corticosteroids. However, these drugs must be used with caution due to possible adverse side effects. In one particular example, corticosteroids can sometimes cause steroid arthropathy and contribute to the degeneration of articular cartilage in addition to producing an acute reduction in inflammation.

An alternative treatment involves the intra-articular injection of hyaluronate into the joint space to increase the viscosity of the synovial fluid and lubricate the apposing articular surfaces of the joint. As previously mentioned, hyaluronate is produced by the synovium, and it is naturally found in the synovial fluid. It is a high molecular weight molecule with highly viscous or viscoelastic characteristics that provide its lubricating properties. Hyaluronate treatment, however, also has shortcomings. Due to its relatively short half-life in the joint of less than 24 hours (Lindenhayn et al., Eur. J. Clin. Chem. Clin. Biochem. 35: 355-363, 1997), multiple injections are generally required to produce an effect that lasts for more than a few days.

Summary of the Invention The present invention provides a novel treatment for joint tissue damaged by disease or trauma. This treatment involves the injection of a viscoelastic collagen solution into the joint tissue to increase the viscosity of the synovial fluid, to lubricate and reduce friction at the articular surfaces, to reduce inflammation, to reduce further cartilage degeneration, and to increase cartilage repair in the joint. The viscoelastic collagen solution preferably remains stable at the site of injection for at least seven days.

The invention also features methods for preparing viscoelastic collagen solutions that are long lasting without the use of use of bi- or multi-functional agents, which tend to be less biocompatible than mono-functional agents. The method involves enhancing stability by using controlled mono- derivatization only, which allows for a greater degree of control over acylation. The mono-functional agent should be present at a concentration between 1-10% of collagen, preferably between 1-5%. The degree of derivatization is less than 50%, preferably less than 25%.

By "damaged joint" is meant a joint in which the structure or function of the joint cartilage, synovial tissue, or synovial fluid has been impaired.

By "soluble viscoelastic collagen solution" is meant a purified collagen solution, prepared by the method disclosed in U.S. Pat. No. 5,631,243 ("the '243 patent"), wherein the collagen molecules are not crosslinked to each other by bi- or multi-functional groups, and wherein the collagen solution is soluble at physiological pH and has a kinetic viscosity sufficient to increase the viscosity of the synovial fluid when between 0.5 to 3.0 cc of collagen solution with a collagen concentration of between 0.5-10% (wt/mL) is injected into the intra-articular space of a joint. The kinetic viscosity of the solution preferably ranges between 1,000-800,000 cps, more preferably between 50,000-400,000 cps, and most preferably between 100,000-250,000 cps. By "trauma" is meant an abnormal force event on a joint. The force can be acute, for example, as associated with a fall or a sprain. Alternatively, the force can be chronic, for example, as associated with repetitive motion.

By "disease" is meant an inflammatory or degenerative condition with a genetic and/or environmental pathological basis that causes joint damage.

By "mono-functional agent" is meant an acylating agent that has only one reactive moiety to react with a deprotonated amine of a collagen molecule. "Bi- functional agent" means an acylating agent that has two reactive moieties to react with at least two depronated amines either on a single collagen molecule or two adjacent collagen molecules. When the two deprotonated amines are on different collagen molecules the bi-functional agent crosslinks the two collagen molecules.

By "multi-functional agent" is meant an acylating agent that has two or more reactive moieties to react with two or more deprotonated amines on a collagen molecule or two or more adjacent collagen molecules.

The present invention can be used to treat a variety of joint-related diseases in animals and humans, including primary or secondary osteoarthritis, chondromalacia, rheumatoid arthritis, congenital hip displasia, spondyloarthropathy, acute yersinia arthritis, pyrophosphate arthritis, gout arthritis (arthritis urica), septic arthritis, arthritis of traumatic etiology, juvenile arthritis, or lupus.

The present invention has advantages over other treatments for joint damage. For example, the injection of the viscoelastic collagen provides all the therapeutic benefits of hyaluronate, but has an increased half-life (i.e., it generally remains stable in the joint for at least 5-7 days) that extends the therapeutic effect of the viscoelastic collagen and, therefore, reduces the need for multiple injections.

Other features and advantages of the invention will be apparent from the following detailed description and from the claims. Brief Description of the Drawings Figure 1 is a light micrograph of synovial membrane from a rabbit joint treated with a viscoelastic collagen following induction of synovitis. Figure 2 is a light micrograph of synovial membrane from a non- treated rabbit joint following induction of synovitis.

Detailed Description As described in more detail below, the present invention is based on the discovery that the injection of a viscoelastic collagen solution into a damaged joint can be used as a treatment to reduce symptoms of inflammation, to reduce further joint damage due to cartilage degeneration, to reduce pain, and to promote joint healing. Importantly, the viscoelastic collagen maintains its effectiveness on a relatively long term basis, particularly in comparison to hyaluronate injections. In initial studies, the viscoelastic collagen solutions injected into damaged joints produce therapeutic effects for at least 7 days.

The injection of a viscoelastic collagen solution into a joint space increases the viscosity of the synovial fluid and exerts its therapeutic effect by lubricating the surfaces of the joint and reducing the amount of cellular infiltration into the joint tissue. Due to the reduced inflammation response in the joint, the viscoelastic collagen solution mediates reduced pain, temperature, swelling, and immobility in the joint. In addition, the treatment reduces joint tissue hyperplasia, scar tissue formation, the rate of chondrocyte death, and further degeneration of the joint cartilage.

The present invention is effective in the treatment of joints damaged by an acute or chronic trauma or by degenerative or inflammatory diseases, such as primary and secondary osteoarthritis, chondromalacia, rheumatoid arthritis, congenital hip displasia, spondyloarthropathies, such as ankylosing spondylitis and psoriatic arthritis, acute yersinia arthritis, pyrophosphate arthritis, gout arthritis (arthritis urica), septic arthritis, various forms of arthritis of traumatic etiology, juvenile arthritis, and lupus.

Viscoelastic collagen can also be' used in animals to treat degenerative joint disease (DJD) characterized by synovitis, degradation of articular cartilage, and loss of synovial membrane lubrication. For example, viscoelastic solutions have been used as a therapeutic agent to treat damaged carpal and fetlock joints in race horses and to treat hip and shoulder joints in dogs with displasia.

Viscoelastic Collagen Preparations and Injections The purified viscoelastic collagen solutions used for the joint treatment of the present invention have viscoelastic properties that increase the viscosity of the synovial fluid. It is preferred that the kinetic viscosities of the viscoelastic solutions fall between 1 ,000-800,000 cps. It is more preferred that the viscosity ranges between 50,000-400,000 cps; most preferably the viscosity ranges between 100,000-250,000 cps (measured at 0.5 m using a Brookfield viscometer (Brookfield Instruments, Stoughton, MA)). The viscoelastic collagen suitable for use in the present invention is soluble at physiologic pH and is prepared by the methods described in U.S. Pat. No. 5,631,243 ("the '243 collagens"). The viscoelastic collagens of the present invention are distinct from other viscoelastic collagens, such as the collagens disclosed in U.S. Patent No. 4,713,446 ("the '446 patent"), because they are not crosslinked to each other by means of bi- or multi-functional groups. In a preferred embodiment, a long lasting viscoelastic collagen solution is prepared by controlling the degree of modification, using one or more mono-functional agents only. The '243 collagens are longer lasting if derivatization is carefully controlled to minimize derivatization (preferably less than 50% derivatization). The amount of mono-functional agent should be less than 10% of the total collagen weight. The '446 collagens, even with crosslinking, are not as long lasting as the '243 collagens with controlled mono-derivation only.

The method for obtaining collagen generally is not critical. The collagen may be solubilized by any standard extraction method, such as acid or salt extraction (see, e.g., U.S. Pat. Nos. 4,713,446 and 5,492,135). The preferred acylating reaction for the solubilization of collagen is described in U.S. Pat. Nos. 4,851,513 and 4,713,446. To be soluble at physiologic pH, the collagen is preferably reacted with an acylating agent having two or more carboxylic acids or carboxylic acid derivatives. To be soluble at physiological pH, the collagen is preferably reacted with acylating agents that reduce the pKa of the collagen to acid pH. Conversely, collagen may be esterified to increase the pKb to alkaline pH (esterification is performed by reacting protein with acidified alcohol). Both will result in a preparation being soluble at physiological pH. The collagen may be prepared from the recipient's own connective tissue. Methods for the extraction, dispersion, and solubilization of collagen from human tissues are described in U.S. Pat. No. 4,969,912. The collagens may be derived from human dermis. Alternatively, the collagen can be prepared from a nonhuman crude collagen source, such as bovine hide, as described in U.S. Patent Nos. 5,476,515 and 5,861,486. The collagen may also be prepared using recombinent or transgenic techniques or secreted from cultured cells.

It is preferred that the chemicals used during collagen preparation be free of inflammatory contaminants (e.g., pyrogens) and that the collagen preparations be sterilized (e.g., with filter sterilization).

In one embodiment of the invention, the stability of mono-derivatized collagen is enhanced by controlling the degree of derivatization. Derivatization is maintained at less than 50%, preferably less than 25%. Derivatization causes destabilization of collagen preparations resulting in an increased rate of dissolution. Conversely, too little derivation results in fibrous dispersion. Thus, the degree of derivatization must be sufficient to reduce the pKa to a level wherein collagen molecules do not interact to form fibril units but not to a level so high that the collagen preparation undergoes rapid dissolution.

Preferably, derivatization is conducted by adding a mono-functional acylation agent, such as glutaric anhydride, at a concentration from 1-10% of collagen weight, preferably from 1-5%. The optimal pH for acylation reactions is between 8.5 and 10.5, preferably between 8.5 and 9.5. In this range, free amines become deprotonated and available for reaction. At concentrations greater than about 2% it is preferable to maintain the pH of the solution at about 9.0 during mixing, preferably using a NaOH solution. Other mono-functional acylation modifiers may be used, including those described in U.S. Patent No. 4,713,446 and WO 99/39238. Preferred modifiers are those resulting in pendent moieties with known physiological catabolism, such as glutarates and succinates which are part of intermediary metabolism.

After derivatization, the pH of the collagen solution is reduced to its pK_a, usually between about 4.0-5.0, preferably between about 4.2 and 4.8, to precipitate the derivatized collagen (proteins precipitate at the pK_^ (negative log of the dissociation constant for

«-COOH group)). It may be necessary to optimize the pKa for precipitation of derivatized collagen. The pKa depends on the degree of derivatization — the more derivatization, the lower the pKa. The precipitate can be recovered by centrifugation. Prior to injection, the purified collagen is stabilized in buffer at physiological pH. In a preferred embodiment, the buffer is a phosphate buffer with about 2% glycerol as the osmolality enhancer to bring the osmolality to about 305-325 mOsm (preferably about 315 mOsm). Other physiological buffers can be used, and supplemented with, for example, either glycerol or a physiologic concentration of NaCl (e.g., 0.9%, 0.15M) to bring the solution to isotonicity or physiological osmolality of about 315 mOsm. Examples of other suitable buffers include, but are not limited to, Tris-HCl and HEPES. Viscoelastic collagen solutions prepared by the methods described in the '243 patent are stable in vivo (i.e., following intra-articular injection). The concentration of derivatized collagen is preferably between 0.5-10% (wt/mL), more preferably between 1.5-5.0%, and most preferably between 2.0-4.0%. The preferred volume of injectate is from about 0.5 to 3.0 cc, most preferably about 2.0 cc. Following injection into the intra-articular space, the viscoelastic collagen generally is stable and effective for at least three weeks. The collagen of the invention can be further modified to reduce degradation by collagenases and noncollagenase neutral proteases.

Although crosslinking using bi- or multi-functional agents is not necessary to provide long lasting collagens, crosslinkers may be used to provide additional stability. The total concentration of bi-functional and mono-functional modifiers should not exceed 10% total collagen. If bi- functional agents are used, the concentration should be less than 2% total collagen and the mono-functional agent should not exceed 3% total collagen concentration.

The features and other details of the invention will now be more particularly described and pointed out in the examples. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. The principle features of this invention can be employed in various embodiments without departing from the scope of the invention. Example 1 : Therapeutic Viscoelastic Collagen Solution Injections in an Animal Model of Joint Disease.

To determine the effects of a viscoelastic collagen solution injection into a damaged joint, a rabbit model of antigen-induced synovitis was used. As shown below, the viscoelastic collagen solution reduced cellular infiltration and inflammation in the joint. These experiments demonstrated that this treatment is effective for use in reducing symptoms of inflammation, in reducing further degeneration of the joint cartilagenous tissue, and in promoting the healing of joint tissue damaged by trauma, degenerative disease, or inflammatory disease.

The Collagen Formulation

To carry out these experiments, purified, monomolecular collagen was prepared from bovine hide using standard techniques. The collagen was chemically derivatized to render a physiologically soluble solution, according to the methods described in U.S. Pat. No. 5,631,243. The purified solution was filter sterilized (0.22 μm), and subsequent steps were conducted under aseptic conditions. The collagen was precipitated at pH 4.5 and washed in deionized water. The final pellet was dissolved in about 20 mg/ml (2%) phosphate buffered glycerol at pH 7.2. This solution was viscoelastic, exhibiting a viscosity of approximately 100,000 cps at 0.5 φm.

Experimental Protocol

To test the viscoelastic collagen solution, a total of 33 New Zealand White rabbits (2-3 kg each) were divided into four groups. Groups 2, 3, and 4 consisted often rabbits each. To "sensitize" the animals in groups 2, 3, and 4, each was injected in the hindpaw with 2 mg BSA (4X crystallized) in 1 ml Freund's Complete Adjuvant. To induce damage of joint tissue, eight out of the ten animals in groups 2, 3, and 4 were then "challenged" by injecting 1 mg BSA in 1 ml sterile saline into the joint space of both knees 21 or 28 days following sensitization. The two remaining animals in each of groups 2, 3, and 4 were "non-challenged." All animals in groups 2, 3, and 4 then received "treatment" of a viscoelastic collagen injection in one treated knee. The injection included 0.5 cc of collagen solution with a concentration of 2.0% (20 mg/ML) collagen in phosphate buffer supplemented with 2% glycerol at a pH of about 7.2. The other, untreated knee received a control injection of sterile saline. The schedule of challenges and treatments are shown in Table 1. Prior to all injections, the animals were anesthetized with Fentanyl IV injected in the marginal ear vein. Group 1 consisted of 3 rabbits that received no sensitization, no challenge, and no collagen treatment. Table 1. Protocol

S = BSA sensitization; NS = no BSA sensitization; C = BSA challenge; NC = no BSA challenge; T = viscoelastic collagen treatment; NT = no viscoelastic collagen treatment.

At 1, 7, and 14 days after the final BSA challenge, all knee joints were evaluated for warmth, swelling, and range of motion. Histopathological evaluation was conducted on joint tissue 21 days after final BSA challenge.

Joint tissue was dissected, preserved in 10% formalin, sectioned, and stained with H & E (hematoxylin & eosin). Specimens were examined for cellular infiltration as an index of inflammation and were assigned a score of 0 to 4, where 0 = normal infiltration and 4 = heavy infiltration.

Results The inflammation scores (cellular infiltration) for the animals in each treatment group are shown in Tables 2-4. All Group 1 animals and all non- challenged control animals had cellular infiltration scores of 0.

Table 2. Group 2: Treated before BSA challenge

Table 4. Challenge - Challenge - Treat

As shown in Tables 2-4, treatment with viscoelastic collagen increased the number of animals exhibiting normal ("0") inflammation scores, decreased the number of animals exhibiting the more severe inflammation scores ("2-4"), and reduced the mean inflammation score for each group. For example, in Group 3, five of the eight animals treated with viscoelastic collagen had inflammation scores of 0, whereas only two nontreated animals had a 0 score. Similarly, only two of the treated animals had an inflammation score of 2-4, whereas five of the non-treated animals had a score of 2 or greater. Treatment in Group 3 also reduced the mean inflammation score from 1.8 to 0.6.

These results demonstrate that viscoelastic collagen injections are effective at reducing inflammation in joint tissue and maintain their effect for at least 3 weeks.

Example 2: Preparation of Stable Viscoelastic Collagen Solution Using Controlled Acylation and no Crosslinking.

Purified collagen was prepared from bovine hide using procedures described in U.S. Patent Nos. 5,476,515 and 5,861,486, and from human dermis. Collagen solution with a concentration of 3mg/mL (0.3%) was passed through 0.45 μm filters and placed in a 4L reaction vessel with a stirrer. The pH was adjusted to 9.0 using ION NaOH and IN NaOH. Glutaric anhydride was added to the solution at 2% of the total collagen weight. The pH was not maintained at 9.0 pH and was carefully monitored until stable. Generally, the pH drop was less than two pH units. , After 15 minutes, the clear solution was refiltered through 0.45 μm filters to remove aggregates and particulates. The pH was then reduced to 4.2-5.0 to precipitate the derivatized collagen. The pellet was recovered by centrifugation at 9,000 φm and the pellet was washed 2 times using sterile water at pH 4.2-5.0. The pellet was then redissolved at about 20mg/mL (total collagen) in

0.005M sodium phosphate buffer, pH 7.2-7.6, containing 2% glycerol to provide a final osmolality of 305 milliosmoles (315 mOsm is optimal). The thick, viscous material was then filtered through a 5μm filter to remove any gelatinous aggregates. Measurements of pH, collagen concentration, osmolality, and viscosity were made as described in U.S. Patent No. 5,631,243.

Other Embodiments Although the present invention has been described with reference to preferred embodiments, one skilled in the art can easily ascertain its essential characteristics and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed in the scope of the present invention.

All publications and patent applications mentioned in this specification are herein incoφorated by reference. What is claimed is:

Claims

1. A method of treating a damaged joint, said method comprising administering a soluble viscoelastic collagen solution into the joint space.

2. The method of claim 1, wherein said viscoelastic collagen solution has a kinetic viscosity between 50,000 - 400,000 cps.

3. The method of claim 2, wherein said viscoelastic collagen solution has a kinetic viscosity between 100,000 - 250,000 cps

4. The method of claim 1, wherein said joint is damaged by trauma.

5. The method of claim 1 , wherein said joint is damaged by disease.

6. The method of claim 4, wherein said disease comprises primary or secondary osteoarthritis, chondromalacia, rheumatoid arthritis, congenital hip displasia, spondyloarthropathy, acute yersinia arthritis, pyrophosphate arthritis, gout arthritis (arthritis urica), septic arthritis, arthritis of traumatic etiology, juvenile arthritis, or lupus.

7. The method of claim 5, wherein said spondyloarthropathy is ankylosing spondylitis or psoriatic arthritis.

8. The method of claim 1, wherein said collagen solution is administered in an amount sufficient to reduce inflammation.

9. The method of claim 8, wherein said inflammation comprises an increase in joint inflammation, cellular infiltration, pain, temperature, swelling, immobility, hypeφlasia, scar tissue formation, chondrocyte death, lysosomal release or further degeneration of the joint cartilage.

10. The method of claim 1, wherein said collagen solution is administered in an amount sufficient to reduce joint pain in a patient.

11. The method of claim 1 , wherein said collagen remains stable at the site of injection for at least seven days.

12. The method of claim 1, wherein said solution has a collagen concentration between 0.5-10% (wt/mL) .

13. The method of claim 1, wherein the degree of derivatization of the viscoelastic collagen of said solution is less than 50%.

14. The method of claim 13, wherein the degree of derivatization is less than 25%.

15. A method of preparing a viscoelastic collagen solution comprising: a) obtaining a solution of purified collagen; b) adding an acylation agent to said collagen solution, said acylation agent consisting essentially of one or more mono-functional acylation agents, at a concentration between 1-10% of collagen weight, to produce derivatized collagen, wherein the degree of derivatization is less than 50%; c) precipitating said derivatized collagen; and d) dissolving said precipitated derivatized collagen in a physiologically acceptable buffer solution.

16. The method of claim 15, wherein said concentration is between 1- 5% of collagen weight.

17. The method of claim 15, wherein said mono-functional acylation agent is selected from the group consisting of glutarates and succinates.

18. The method of claim 15, wherein said mono-functional acylation agent comprises glutaric anhydride.

19. The method of claim 15, wherein said derivatization is less than 25%.

20. A collagen solution prepared by the method of claim 15.

21. A collagen solution prepared by the method of claim 19.