WO1996034910A1

WO1996034910A1 - Cross-linking of proteins, including collagen, using high osmolality storage medium

Info

Publication number: WO1996034910A1
Application number: PCT/US1996/006285
Authority: WO
Inventors: Mark A. Moore; David T. Cheung; Gerald L. MECHANIC
Original assignee: The University Of North Carolina At Chapel Hill; Carbomedics, Inc.; MECHANIC, Laurie
Priority date: 1995-05-04
Filing date: 1996-05-02
Publication date: 1996-11-07
Also published as: AU5789796A

Abstract

A storage medium for use in a method of cross-linking collagenous tissue samples is disclosed. The medium is high osmolality aqueous solution of a salt and a sugar that is buffered to maintain pH. The high osmolality of the medium serves both to preserve the native state of the collagen comprising the sample and to suppress the growth of naturally occurring and opportunistic microbial agents in and/or on the tissue sample.

Description

CROSS-LINKING OF PROTEINS, INCLUDING COLLAGEN, USING HIGH OSMOLALITY STORAGE MEDIUM

The present invention relates to an aqueous solution for storing and preserving proteinaceous, and particularly coUagenous, tissue samples for cross-linking. In more detail, the present invention relates to a high osmotic pressure (compared to physiologic) solution which is used as a storage medium for proteinaceous tissue samples such as tissue samples including collagen in a process for cross-linking the protein in the tissue, for instance, in the cross-linking of collagen with glutaraldehyde or in accordance with the method described in U.S. Patent No. 5, 147,514.

Photooxidative cross-linking of the collagen fibrils of tissue samples such as bovine pericardium in accordance with the method of U.S. Patent No. 5,147,514 results in a product having physical and chemical properties which make that product useful as a biomaterial for use as an artificial tendon, heart valve leaflet, pericardial patch, and in many other applications. The product is produced by harvesting a sample of such tissue, incubating the sample in an aqueous media solution of a photooxidative catalyst buffered to about physiological pH (6.8-8.6) for sufficient time to equilibrate the concentrations of media solution, collagen fibrils, and photooxidative catalyst, and then irradiating with light to cross-link the collagen fibrils. It has been discovered that it was advantageous to "precondition" the tissue sample by incubation in a media solution which did not include the catalyst before transfer to the solution including the catalyst for irradiation. When preconditioned in this manner, the resulting product shows decreased susceptibility to proteolytic degradation. It has also been discovered that a first media solution of high osmolality gives desirable results when used in that process and when used before glutaraldehyde and other processes known in the art for cross-linking coUagenous tissue samples. The solution is also advantageously used as a storage medium for the coUagenous tissue sample. Storage in the medium substitutes for both the steps of immersion in cold saline with frequent rinses and changes of fresh saline and the preconditioning of the tissue sample before equilibrium in the second, catalyst-containing media solution as described in the above-referenced U.S. Patent No. 5,147,514. Further, the storage medium both maintains collagen fibrillar structure and neutralizes harmful agents.

It has long been standard practice, for instance, in histological laboratories, to freeze dry tissue samples for storage for long periods of time. Standard practice for storage of samples for relatively short periods of time usually involves incubation in one of several known saline solutions, buffered to physiological pH, at low temperature. However, both methods jeopardize the maintenance of the native structure of structural proteins such as collagen. Maintaining collagen fibrillar structure is of particular concern in light of experimental data indicating that the method described in U.S. Patent No. 5,147,514 cross-links the collagen fibrils in their true, native state, e.g., as intact collagen fibrils, and that this capability of that method appears to be responsible for the excellent mechanical properties of the resulting product and the ability of the product to resist in vivo degradation. It is, therefore, an object of the present invention to provide an improved process of photooxidative cross-linking of coUagenous tissue utilizing the storage media of the present invention. However, it is apparent from the results obtained when used in that process that the media solution of the present invention is useful in other cross-linking processes, such as glutaraldehyde cross-linking, known in the art and for preservation of any proteinaceous material or tissue, and perhaps even more broadly, as a storage medium for many different types of tissues, biomaterials, and/or extracts or solutions of same and/or their component parts or molecules.

In a broad sense, therefore, an object of the present invention is to provide a storage medium for preserving biological specimens, tissues, extracts, biomolecules, and/or isolates which both maintains the native state of the sample and helps protect the sample from damage or degradation caused by harmful agents found in the sample or opportunistic, invasive agents.

Another object is to provide a media solution for storage of tissue samples for longer periods of time than previously possible, e.g., for several weeks, at room temperature. ^•

These, and other, objects are met by providing a medium for storage of proteinaceous tissue samples comprising an aqueous solution of a salt in a weight: volume (W:V) ratio higher than about 11.7% and a sugar in a concentration of from about 30 to about 80% (W:V). Also provided is an improved method for cross- linking a coUagenous tissue sample in which the improvement comprises maintaining the ionic interaction between the collagen fibrils comprising the sample by immersing the sample in a high osmolality aqueous solution of a salt and a sugar, the salt being a salt capable of penetrating the sample to inhibit hydration of the collagen molecules comprising the fibril, the sugar functioning to maintain the high osmolality of the

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^SBrøπWESHffr(^βWif26) solution as salt concentration in the solution decreases as the salt penetrates the tissue sample.

The media solution of the present invention not only helps to preserve the protein in the sample but also suppresses propagation of any microbial agents which are present. The solution is further advantageous in that it causes precipitation (inactivation) of protease and other hydrolytic enzymes, rendering them inactive and preventing degradation of the proteins present in the sample, and specifically, preventing enzymatic degradation of the collagen.

Once removed from the animal, coUagenous tissue becomes hydrated and thickens. This thickening is believed to result from partial unwinding of the collagen fibrils, making the fibrils more susceptible to enzymatic degradation. The interaction of native helical collagen molecules inside the collagen fibrils must be kept intact to maintain the stability of the fibrils. It has been discovered that this interaction can be kept intact by increasing the ionic strength of the solution in which the sample is stored to the point that the hydrophobic interaction between collagen molecules is maximized. This increase is accomplished by using a high salt concentration in the media solution of the present invention.

However, as the salt in the solution penetrates the tissue sample, and depending upon diffusion time across the thickness of the tissue and the thickness of the tissue, a concentration gradient is set up between tissue and solution. As the salt concentration in the tissue increases to about one molar at physiological pH, the ionic interaction between collagen molecules in the fibril is interrupted by interaction between the collagen molecules and the salt, resulting in the unwinding of the fibril and subsequent solubilization of the fibril. The high concentration of sugar in the media solution of the present invention therefore maintains the osmolality of the solution and causes the short term aggregation of the collagen fibrils.

The osmolality (represented by the Greek letter μ) of the storage medium of the present invention is higher than the osmolality of 3.0 M NaCl solution, e.g., 4500 mosm, and in a particularly advantageous embodiment, the osmolality is higher than about 6000 mosm. The upper limit of the osmolality is imposed by the practicalities of handling the solution, e.g., the increasing viscosity that results from high solute content. Osmolality is also limited by the ability of the solvent to hold solute, e.g., the point at which it is saturated. Both sugar and salt contribute to the osmolality of the medium (as compared to the ionic strength of the solution, which results from inclusion of the salt, which dissociates in water) and the relative contributions of salt and sugar to the osmolality of the solution are not as important to the function of the solution as total osmolality. Maintaining the high osmolality of the solution while effective salt concentration decreases by penetration into the tissue appears to mitigate collagen denaturation by maintaining the hydrophobic interaction of the collagen fibrils in the same manner as the salt functions to maintain collagen aggregation.

Many salts are suitable for use in the storage medium of the present invention, but those which will function as described above to penetrate a tissue sample to inhibit hydration of the proteinaceous material in its native configuration are specifically contemplated. Due to their low cost, high solubility, and ready availability, sodium chloride and potassium chloride are preferred for use in the storage medium of the present invention and the examples set out below refer to those salts. However, many other salts are known in the art and readily available, including, for instance, the ammonium, sodium, calcium, magnesium, manganese, and potassium salts of halides, nitrites, nitrates, phosphites, phosphates, sulfites, sulfates, and alkanoic acids such as propionates, acetates, and formates, and specifically, the aforementioned sodium and potassium chloride, magnesium chloride, and sodium nitrite. All such salts, as well as many not listed here but which are recognized in the art to function in substantially the same way to achieve substantially the same result as those which are listed, are contemplated by the present invention.

As a general guideline, the desired contribution to the osmolality of the storage medium of the present invention attributable to the salt component is achieved by inclusion of, in the case of NaCl, about 2.0 to about 5.0 M NaCl in the medium (e.g., from about 116.8 to about 284.0 g per kilogram (e.g., one liter) final volume of water, or about 11.7 to about 28.4% (W:V) concentration), the preferred range being about 2.25 - 4.0 M. For salts such as calcium chloride, the desired contribution to the osmolality of the medium is achieved by inclusion of from about 1.3 to about 3.4 M CaCl2 in the medium, and so on.

Similarly, many sugars are used as the second component of the storage medium of the present invention. Again because of its low cost, high solubility, and ready availability, most of the examples set out below use sucrose as the sugar in the storage medium. However, other sugars and sugar derivatives such as glucose, fructose, mannose, galactose and other monosaccharides, disaccharides such as maltose, cellobiose, and lactose, trisaccharides, or polysaccharides such as amylose or amylopectin. as well as sugar derivatives such as sorbitol (derived from glucose by reduction of the aldehyde group), mannitol, etc., glycosides such as methyl glucoside (derived from glucose by acid-catalyzed reaction of methanol with glucose), or proteoglycans also function in substantially the same way to maintain the proteinaceous components of a tissue sample in their native state, and all such sugars are contemplated as falling within the scope of the present invention.

In the case of sucrose, the desired contribution to the osmolality of the storage medium of the present invention can generally be obtained by using a concentration of from about 30 to about 85% W:V (e.g., from about 0.3 to about 0.85 kg per kg, e.g., one liter, final volume of water) sucrose, the preferred range being from about 30 to about

80% W:V concentration, e.g., about 0.85 to about 2.35 M sucrose. In other words, the contribution of the sugar to the total osmolality of the storage medium of the present invention ranges from about 3400 mosm upwardly to the saturation point of the solvent.

In the case of certain salts which are not as soluble as others, the desired contribution to the osmolality of the media is obtained by increasing the amount of sugar in the solution. By compensating for solubility in this manner, satisfactory results are obtained even with salts such as potassium chloride, potassium iodide, sodium citrate, sodium acetate, and sodium sulfate. These salts are listed here because it was discovered that, at least when the media of the present invention is made by the following method, they were not completely solublized. This method is but one way to prepare the media- is described here to illustrate the teaching set out above as to the upper limits on the concentration of the salt imposed by the practicalities of dissolving one or both of the components of the solution, and was an attempt to prepare the solution of the present invention using various salts and a standard of 67 g of sucrose per 100 ml final volume. Because sugar occupies a large volume, the first step in the method was to attempt to dissolve 0.3 moles of various salts in 40 ml phosphate buffered saline (PBS) by overnight stirring at room temperature. If soluble, then the 67 g sucrose was added (again by stirring overnight at room temperature) and final volume was adjusted 100 ml with PBS to prepare the 3 M salt and 67% W:V sucrose solution that was desired. The combinations of salts and sucrose were as follows:

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SUBSTΓΓUTE SHEET (RULE 26) SALT WEIGHT ADD 40 ml SOLUBLE? pH ADD 67 g SOLUBLE? PBS SUCROSE

Magnesium 61.0 g chloride V YES 3.15 YES

Potassium 22.4 g chloride V NO NA NA NA

Potassium 49.8 g Iodide V NO NA NA NA

Manganese 45.3 g YES 0.54 NO sulfate

Sodium citrate 88.2 g NO NA NA NA

Sodium acetate 24.6 g V NO NA NA NA

Sodium 20.7 g YES 5.75 NO phosphate 21.3 g (mono- and dibasic-, equimolar)

Sodium nitrite 20.7 g YES 6.52 V YES

Sodium sulfite 42.6 g NO NA NA NA

The weights of each salt added were calculated for a total of 0.3 moles of salt to be added for each solution and take into account the total molecular weight of the salt as provided in the anhydrous or hydrated form (NA = not applicable).

In a second set of experiments, the media was prepared using a standard of 17.5 g sodium chloride along with 67 g of various sugars per 100 ml final volume. Because the sugar occupies a large portion of the volume, the first step was to dissolve the 17.5 g of sodium chloride in 40 ml PBS then 67 g of the particular sugar was added. The final volume was adjusted to 100 ml with PBS to prepare the 3 M salt and 67% W:V sucrose solution that was desired.

SALT WEIGHT ADD 40 ml SOLUBLE? SUGAR TO ADD 67 g SOLUBLE? PBS ADD SUGAR

Sodium 17.5 g chloride V YES Amylose NO

Sodium 17.5 g chloride V YES Glucose V NO

Sodium 17.5g chloride V YES Lactose V No

Sodium 17.5g chloride V YES Fructose V YES

Sodium 17.5 g chloride V YES Sucrose V YES

The fructose was not completely soluble in the total volume of 100 ml. Therefore, an additional 33 ml PBS was added to solublize all of the components. Thus, this solution was 50% fructose and 2.25 M NaCl. It will be understood by those skilled in the art who have the benefit of this disclosure that those solutions which were not able to be prepared these methods may be prepared by other methods known in the art and that even a solution such as the sodium chloride-fructose solution, in which additional PBS was added to solublize the fructose, gave satisfactory results (as set out below) when used in accordance with the present invention.

Although the examples set out above disclose a number of high osmolality solutions which are used to advantage in the method of the present invention, there are manv other such solutions which may also be utilized to advantage, including solutions w ich are comprised of the very salts and/or sugars listed above as being insoluble by the method described in those examples. Those skilled in the art who have the benefit of this disclosure will recognize that additional solute can be solublized by using, for instance, elevated temperature, more efficient and more vigorous stirring and/or agitation, and by other methods known in the art. It will also be recognized that some salts cause a decrease in pH when dissolved

(note the pH of the above-described solutions including manganese sulfate and magnesium chloride). Further, this lowering of pH can have detrimental effect(s) on the coUagenous tissue to be cross-linked. Reference is made, for instance, to Example 4, infra, wherein it is reported that a solution made in accordance with the present invention and comprised of magnesium chloride and sucrose had a pH of 3.59. When that tissue was evaluated by heat shrink test, there was no sharp decrease in tissue length as a function of temperature, indicating that the tissue was likely damaged by this low pH. 1-ven so. magnesium chloride/sucrose solutions are used to advantage in connection with the present invention by, for instance, neutralizing the acidity of the solution by adding sufficient magnesium hydroxide to raise the pH or by using a stronger buffer. Such adjustments in pH are known to those skilled in the art and the resulting solution gives satisfactory results when used in the method of the present invention.

The storage medium is buffered to physiological pH with any of a number of commonly used buffers such as phosphate buffered saline (PBS). Other suitable buffers include those containing potassium or sodium phosphate, or potassium or sodium chloride, such as a Good's buffer, e.g., HEPES, TES, or BES (Research Organics, Inc.), preferably at concentrations of from about 0.2 to about 1.0 M. However, the molar concentration of the buffer is not as important as the concentration of the other two components of the storage medium of the present invention. Almost any concentration of buffer components which maintains pH between about 3.5 and about 10, and preferably at about physiological pH, functions effectively in the storage medium of the present invention. The term "physiological pH" refers to nominal hydrogen ion concentration in vivo; those skilled in the art will recognize, and the term is specifically intended to encompass, a pH range of from about 6.8 to about 8.6 as may be encountered, depending upon the system, in normal living systems.

The following non-limiting examples describe the invention in further detail.

Example 1

A rectangular illumination cell was constructed from clear plastic with an outer jacket of the same material and tubes communicating with the inner chamber for circulation of media and dye. A frame, comprised of narrow strips of plastic including spaced holes therealong for suturing tissue samples thereto, was constructed in a size fitting into the inner chamber of the cell. After suturing a piece of bovine pericardium to that frame and inserting the frame into the inner chamber, a media comprised of 2.8 M potassium chloride, μ = 0.164, potassium phosphate buffer, pH 7.4, including 50% W:V sucrose, was circulated through the inner chamber of the illumination cell. After soaking in this high osmotic pressure media, the tissue was incubated in media including 0.02 M sodium phosphate buffer, pH 7.4, containing 0.01% (wt/vol) methylene green and illuminated for 24 and 48 hours by two 150 watt flood lamps at a distance of about 4.5 cm while holding temperature at between - 2°C and 6°C.

After irradiation, small pieces of tissue from each sample were digested with pepsin ( 1% pepsin solution in 3% acetic acid at 4°C for 24 hrs.) or bacterial collagenase ( 1% collagenase solution in 0.15 TES buffer, pH 7.5, in 0.01 M CaCl2 at 37°C for

6÷hrs.). The following ratios of hyp/mg of tissue in the enzyme columns clearly demonstrate the cross-linking of the tissue samples (the control samples were not illuminated).

Time of Irradiation (hrs.) Pepsin Collagenase

0 (Control #1) 26 314

(Control #2) 31 314

24 (Sample #1) 0 410

(Sample #2) 0 290

48 0 303

Additional tissue samples were further stabilized (without apparent change in tactile properties, e.g.. tissue texture and suppleness) by reduction of the newly formed iminium

SUBSΠTUTE SHEET (RULE 26) bonds by immersion in a solution of NaBH4 for one hour as demonstrated by the following hyp/mg ratios:

Time of Irradiation fhrs Pepsin Collagenase

0 (Control #1) 26 314

(Control #2) 7 208

24 (Sample #1) 0 180

(Sample #2) 0 170

48 0 170

Example 2

Soluble BAPN rat type I collagen in 0.5 M HAc was divided into six 4 ml samples and each sample placed in a dialysis bag with 300 mg NaCl (no salt was added to sample 5 and 6). Samples were dialyzed into the high osmotic strength buffer described in Example 1 (samples 5 and 6 were dialyzed into PBS, pH 7.4) and 2 ml of

0.2% methylene blue. Samples 2 and 3 were transferred to buffer including 0.1% methylene blue in PBS, sample 4 was transferred to PBS including 0.1% methylene blue, and samples 5 and 6 remained in PBS. Sample 2 was exposed to a 150 watt white floodlight located about 7 inches from the surface of the fluid while holding temperature between about 8 and 12°C for eight hours, samples 3 and 4 were exposed for 24 hours under the same conditions, and samples 5 and 6 were exposed for two hours under the same conditions. All samples were then dialyzed back into HAc until the solutions were no longer blue and then analyzed by SDS-PAGE. The samples exposed for 24 hours were more cross-linked than those exposed for eight hours, and all samples were more cross-linked than samples 5 and 6.

Example 3

Bovine pericardial tissues were harvested from a local abattoir and stored in plastic ZIP LOCK® bags on ice until cleaned. Residual fact was removed from each tissue and the tissues maintained on wet ice with minimal fluid contact until immersed in the appropriate storage solution (each buffered with 0.13 M sodium chloride-phosphate buffered saline).

Solution Salt Sucrose (W:N>

1 2.5 M 50%

2 3.0 M 60%

3 4.0 M 67%

4 2.5 M 67% Solution Salt Sucrose fW:N>

5 4.0 M 50%

6 PBS control (pH 7.4, 300 mosm)

Tissues preserved in solutions 1-5 were stored at room temperature; solution 6 was presterilized by filtering through 0.2 micron filters. Tissue preserved in solution 6 was stored at 4°C. Samples were removed from each solution at time periods of 1, 7, 28, and 56 days, washed free of the storage solution with freshly prepared, sterile PBS for 2 hrs., and aerobic bioburden was measured using the method described at U.S. Pharmacopeia XXII, The U.S.P. Convention, Inc.; Rockville, MD, pp. 1481-2 (1990). The results are set out below as average total recoverable aerobic bioburden, in CFU's per sample, for two specimens: Preservation Time (Days)

Solution 0 1 7 28 56

1 3643 760 1700 8130 22.5

2 2400 363 68 43 2958

3 7455 308 220 80 65,425

4 2665 245 100 365 19,700

5 4378 3375 313 9355 74,200

6 1610 9175 12,053 10,983 7,785,000 These data suggest that solutions 1-5 had a significant effect on the bioburden levels of the tissue. The bioburden level of the tissue stored in PBS (solution 6) increased dramatically after one day to an approximately constant level for 28 days whereas the microbial levels of tissue stored in solutions 1-5 all decreased after one day of storage. After one week of storage, the bioburden levels were significantly lower than at harvest (T = 0). At 28 days, the bioburden levels or solutions 1 and 5 (50% sucrose) were comparable to the levels of tissue stored in PBS (solution 6), but tissues maintained microbial levels of less than 500 CFU/sample. At 56 days, the microbial levels for all solutions except solution 1 were significantly increased compared to the 28 day specimens, but these levels were still 100-fold less than that of PBS-stored tissue. Example 4

Bovine pericardial tissue was received on ice from the abattor on the day after harvest. Separate bioburden analysis of the tissue received in this shipment indicated: aerobic count = 3.6 x 10^ cfu (colony forming units)/g tissue aerobic spore formers < 10 cfu/g fungal count = 30 cfu/g. Twenty-five pieces approximately 3/4" x 1" were cut from a sac and five each were placed in 50 ml each of the following solutions:

1. phosphate buffered saline (PBS), pH 7.3 - 7.4

2. sucrose, magnesium chloride media

3. sucrose, sodium nitrite media

4. fructose, sodium chloride media 5. sucrose, sodium chloride media.

Solutions 2-5 were made by dissolving 0.3 moles salt in 40 ml PBS, adding 67 g sugar, and adjusting final volume to 100 ml with PBS to give a 3 M salt, 67% W:V sugar solution in accordance with the present invention. The fructose was not completely soluble in the total volume of 100 ml. An additional 33 ml of PBS was added to give a solution that was 50% fructose and 2.25 M NaCl. The salt added was calculated as the weight of the salt as provided in the anhydrous or hydrated form, for a total of 0.3 moles of salt for each solution.

After samples were stored in solution at room temperature for 12 days, one piece of tissue and 10 ml of each solution were analyzed for bioburden and two pieces of tissue were removed from each sample container and placed in a 50 ml volume of 0.5% glutaraldehyde in PBS, maintained for two days, split lengthwise and analyzed by shrinkage temperature analysis. Shrinkage temperature is a measure of thermal stability and is known to rise upon fixation by glutaraldehyde (CA. Pereira, et al., "Effect of alternative crosslinking methods on the low strain rate viscoelastic properties of bovine pericardial bioprosthetic material," 24 J. Biomed. Mater. Res. 345-361 (1990)). To perform this test, a piece of tissue was mounted on two extensometers, placed in a water bath, and the temperature was slowly raised. The length of the tissue was monitored as a function of temperature. When the tissue reached its shrinkage temperature there was a very rapid decrease in length. The temperature at the maximal rate of tissue shortening was taken as the shrinkage temperature. Values reported are an average of two separate measurements. The data are summarized below. Solution Tissue bioburden* Liquid Shrink temperature- Shrink temperature- (NOTE: pH of Aerobic count bioburden# tissue from solutions tissue from solutions were (Cfil/g) Aerobic count (°C) glutaraldehyde taken upon (cfu ml) (°C) completion of experiment)

PBS 1.2 x 10⁵ 2.6 x 10⁶ 67.1 83.7

Sucrose. < 10 2 not measurablef not measurablef

Magnesium chloride. pH 3.59

Sucrose. Sodium 20 1 65.6 77.2 nitrite, pH 7.38

Fructose, Sodium < 10 0 66.3 82.5 chloride. DH 5.73

Sucrose. Sodium < 10 2 66.0 83.5 chloride, pH 6.01

* Aerobic spore formers and fungal counts were < 10 cfu/g for all 5 tissue samples.

# Aerobic spore formers and fungal counts were 0 cfu/ml for all 5 solutions. f A shrinkage temperature could not be evaluated for tissue which had been stored in the sucrose/magnesium chloride solution, with or without subsequent glutaraldehyde treatment. Although the tissue did shrink, there was no sharp decrease in tissue length as a function of temperature, indicating that the tissue was likely damaged.

§ Pericardial tissue which was freshly received was used as a control and gave a shrinkage temperature of 65.4°C.

Claims

What is claimed is: 1. In a method for cross-linking a proteinaceous tissue sample, the improvement comprising maintaining the ionic interaction between the protein molecules comprising the sample by immersing the sample in an aqueous solution of a salt in a weight to volume ratio of higher than about 11.7% and a concentration of about 30 to about 80% (W/V) of a sugar, the salt being selected form the group of salts which are capable of penetrating the sample, the sugar functioning to maintain the high osmolality of the solution even as the salt concentration in the solution decreases as the salt penetrates the sample.

2. The method of claim 1 wherein said salt is either sodium chloride or potassium chloride.

3. The method of claim 1 wherein said sugar is either sucrose or fructose.

4. The method of any of claims 1-3 additionally comprising maintaining the osmolality of the solution at higher than about 4500 mosm.

5. The method of claim 1 wherein said salt is a halide salt.

6. In a method for cross-linking a coUagenous tissue sample, the improvement comprising maintaining an osmolality of higher than about 4500 mosm by immersing the tissue sample in a storage medium before cross-linking, the storage medium comprising water, about 30 to about 80% (W:V) sucrose, and a halide salt in a concentration of higher than about 11.7% (W: V).

7. The method of claim 6 wherein said halide salt is either sodium chloride or potassium chloride.

8. The method of claim 6 wherein said storage medium additionally comprises a buffer.

9. An improved method for cross-linking proteinaceous material substantially as described herein.

10. A storage medium for preserving proteinaceous tissue comprising an aqueous solution of a salt in a weight to volume ratio of higher than about 11.7% and a concentration of about 30 to about 80% (W: V) of a sugar, the salt being a salt which is capable of penetrating the proteinaceous tissue to maintain the ionic interaction of the protein molecules comprising the tissue to maintain the molecules in their native configuration, the sugar functioning to maintain the high osmolality as salt concentration decreases as a result of penetration of the tissue by the salt.

11. The storage medium of claim 10 wherein said salt is sodium chloride or potassium chloride.

12. The storage medium of claim 10 wherein said sugar is sucrose.

13. The storage medium of any of claims 10-12 wherein the osmolality of the solution is higher than about 4500 mosm.

14. The storage medium of any of claims 10-13 wherein said salt is a halide salt.

15. The storage medium of any of claims 10-14 additionally comprising a buffer.

16. A storage medium for proteinaceous tissue samples substantially as described herein.