Title:  Light energized tissue adhesive conformal patch

United States Patent:  6,773,699

Issued:  August 10, 2004

Inventors:  Soltz; Barbara A. (Spring Valley, NY); DeVore; Dale P. (Chelmsford, MA); DeVore; Braden P. (Westerly, RI); Soltz; Robert (Spring Valley, NY); Soltz; Michael A. (Pleasanton, CA)

Assignee:  Tissue Adhesive Technologies, Inc. (Spring Valley, NY)

Appl. No.:  973385

Filed:  October 9, 2001

Abstract

Consistent with the present invention, tissue adhesive compositions and an associated laser exposure system are provided for bonding or sealing biological tissues. The compositions are comprised of chemically derivatized soluble collagen which is formulated to concentrations ranging from 300 mg/ml (30%) to 80 mg/ml (80%) collagen protein. In particular, Type I collagen, for example, is first prepared by extraction from bovine or porcine hide and purified. The collagen preparations are then chemically derivatized with sulfhydryl reagents to improve cohesive strength and with secondary derivatizing agents, such as carboxyl groups, to improve the adhesive strength of the solder to the tissue. The compositions are then formed into viscous solutions, gels or solid films which are used to encapsulate structural components such as a cojoinal network or mesh. The resultant patch which when exposed to energy generated from an infrared laser, for example, undergo thermally induced phase transitions. Solid or semi-solid protein compositions become less viscous enabling the high concentration protein to penetrate the interstices of treated biological tissue or to fill voids in tissue. As thermal energy is released into the surrounding environment, the protein compositions again become solid or semi-solid, adhering to the treated tissue or tissue space and are reinforced by the embedded cojoinal network or mesh.

SUMMARY OF THE INVENTION

Consistent with the present invention, a suitable solder and associated laser welding system are provided which avoid the shortcomings of conventional systems described above. For example, the biological solders and sealants consistent with the present invention are biodegradable and do not require chormophores or dyes to promote adhesion. Further, consistent with the present invention, the laser system provides accurate temperature control to eliminate peripheral tissue damage, damage to stay sutures (if required), and large area exposure to reduce treatment time. Further, the use of a feedback controller reduces required surgeon skill. The laser system can be comprised of inexpensive off-the-shelf components and has been designed to be compact, nonintrusive in a surgical setting, inexpensive to manufacture and user friendly.

It is believed that the laser energy disrupts the three-dimensional structure of collagen fibers found in tissues, promoting tissue crosslinking and improving cohesive strength. The films or gels are easy to apply and fix to the tissue surface.

Further consistent with the present invention, a method is provided for preparing suitable protein compositions for use with the laser system. The compositions are comprised of chemically derivatized soluble collagen, which is formulated to concentrations ranging from 300 mg/ml (30%) to 800 mg/ml (80%) collagen protein. In particular, Type I collagen, for example, is first prepared by extraction from animal hide, skin or connective tissue and purified. The collagen preparations are then chemically derivatized with sulfhydryl reagents to improve cohesive strength and with secondary derivatizing agents, such as carboxyl groups, to improve the adhesive strength of the solder to the tissue. The compositions are then formed into liquid, gels or solid films which when exposed to energy generated from an infrared laser, for example, undergo thermally induced phase transitions. Solid or semi-solid protein compositions become less viscous enabling the high concentration protein to penetrate the interstices of treated biological tissue or to fill voids in tissue. As thermal energy is released into the surrounding environment, the protein compositions again become solid or semi-solid, adhering to the treated tissue or tissue space.

In accordance with an additional feature of the present invention, minute quantities of any derivative of commercially available, medical grade cyanoacrylate can be applied to fix and appose tissue edges. Next a layer of the high concentration collagen can be applied adjacent to and on the surface of the tissue to be bonded. This top layer is typically exposed to an infrared laser to melt, denature, and mix the two components to promote both chemical and mechanical bonds to the tissue as well as enhancing intrinsic strength of the composite solder. Infrared laser exposure increases temperature at the bonding site. Feedback control of the laser can be used to adjust solder temperature for optimizing the bonding mechanism to the tissue while preventing thermal damage to adjacent healthy tissue.

It is believed that high concentration collagen compositions described in this invention have an increased volume of linkages to improve the strength performance of the solder while modulating the thermal melt temperatures and solder viscosities. Laser exposure parameters were chosen to rapidly flow the solder and promote physical contact of the solder with the tissue. These parameters include choice of an operating wavelength in the infrared range, power density, pulsed or cw mode, exposure times and tissue temperature.

DETAILED DESCRIPTION OF INVENTION

The family of proteins known as collagens are ubiquitous in nature and predominant components of all connective tissues. Type I collagen is the most abundant of the twenty characterized collagens and is commonly used as a biomaterial for medical devices. Collagen-based devices include hemostats, bulking agents, eye shields, punctal plugs, adhesives, drug delivery systems, and others. Collagen is well known to be a biocompatible and safe biomaterial.

Consistent with the present invention, collagen is isolated and purified from various tissue sources including dermis, tendon and ligaments. While collagen is currently prepared from human dermis, most commercial products are prepared using collagen from animal sources. Once a pure form of collagen is prepared, it can be treated in several ways to provide a suitable base protein for use as an adhesive or sealant. These treatments include chemical derivitazation to add functional or reactive groups as well as reduce the pKa of the solder to a more acidic value, and gelatinizing the collagen to create a viscous liquid, gel or solid film having a relatively high protein concentration.

The exemplary solder formulations described herein are suitable for infrared laser activation and include chemically derivatized bovine Type I collagen formed into a liquid, gel or solid film. Collagen was chosen due to its long history as a safe, biocompatible biomaterial and due to its ability to be chemically functionalized into a base formulation with unique cohesive and adhesive characteristics.

1. Exemplary Preparation of Pure Type I Collagen Solutions

Pure, Type I collagen was prepared from calf corium. Corium was cut into small pieces approximately 1 cm², soaked in reagent alcohol and extensively washed with sterile, deionized water. The washed pieces were then swollen in acetic acid and digested using porcine mucosal pepsin (Sigma Chemical Company, 1:60,000 crystallized and lyophilized). Following 1 or more pepsin treatments, residual pepsin was removed by dialysis and Type I collagen isolated by differential salt precipitation. Purified Type I collagen was dialyzed against acetic acid and filtered through 0.45.mu. and 0.22.mu. filters. Solder formulations prepared from calf collagen were not successful unless the solutions were extensively dialyzed to remove residual salt (NaCl) and filtered through a 0.22.mu. filter. It is believed that 0.22.mu. filtration is required to produce monomolecular collagen for subsequent chemical derivatization. Details of pure, soluble Type I bovine collagen are described in U.S. Pat. Nos. 4,713,446; 5,104,957; 5,219,895; 5,476,515; 5,631,243, each of which is incorporated by reference herein. An exemplary collagen purification process is provided below.

a. Skin Preparation:

1) Remove about 25 grams of bovine skin (split calf hide) and cut into small pieces using a scalpel and blade. Wash the skin with sterile, deionized water.

2) Cut the skin into small pieces using a scalpel blade. Weigh pieces and place in a 4 liter beaker containing 3 liters of 0.5M acetic acid. (This is 28.6 ml of concentrated acetic acid per liter or a total of about 86 ml. Add the 86 ml of acetic acid to 3 liters of water).

3) Attach stirrer and stir for 18 hours.

4) Add 2% pepsin to the container. First weigh out 0.5 grams of pepsin and dissolve in 50 ml of deionized water. Then add to the beaker.

5) Continue stirring for 12 hrs. If all of the skin pieces have not dispersed into the acetic acid, add another aliquot of 1% pepsin or 0.25 gram of pepsin powder. Stir for 12 hrs.

6) Filter the suspension through cheesecloth to remove pieces of skin that have not dispersed into the acetic acid. (remove a small sample for analysis-40 ml in a 50 ml centrifuge tube and place in refrigerator.

7) Add solid NaCl (salt) to 0.8 M. This is 140 grams. Add while stirring the collagen solution. Stir for at least two hours to precipitate collagen. Allow the precipitate to settle for 12 hrs.

8) Siphon-off the clear liquid leaving the collagen precipitate. Then centrifuge the precipitate in centrifuge bottles and collect all the precipitate. Weigh and redissolve in 3 liters of 0.5M acetic acid. (see above for details in preparing the acetic acid solution). Stir for 12 hrs.

9) Take sample of solution, 40 ml in a 50 ml centrifuge tube and cool to 5 C.

10) Again add solid NaCl to 0.8M. Same as above. Stir for at least 2 hrs or until the precipitate is dissolved. Allow precipitate to settle for 12 hrs and centrifuge to collect the pellet. Weigh and redissolve in 0.1 M acetic acid (add 12 ml of acetic acid to 2 liters of deionized water).

11) Stir for 12 hrs to redissolve the collagen. Then dialyze the collagen solution using Spectrapore dialysis tubing, >30,000 molecular weight cut-off. This step will remove dissolved pepsin. Then dialyze against 0.1M acetic acid.

12) Dialyzed volumes will then be filtered through 0.45.mu. and then 0.2.mu. filters and stored in 1 liter or 4 liter bottles in the refrigerator. This is the collagen stock solution for making collagen-based solder formulations.

2. Chemical Derivatization of Pure Type I Collagen Solution

Derivatization was intended to provide functional groups to enhance both cohesive and adhesive characteristics. For cohesive functionality, thiol groups were attached by derivatization using 4-mercapto-1,8-naphthalic anhydride. Adjacent thiol groups react to form disulfide bonds between collagen molecules enhancing the cohesive properties of this solder. Immediately after derivatization with the 4-mercapto-1,8-naphthalic anhydride, monomolecular collagen is derivatized with glutaric anhydride. This reaction substitutes a COO^- for a NH_3⁺ making the composition anionic. It is believed that the net negative charge of the solder will ionically interact with positive charged proteins in tissues. This reduces the pKa of collagen to the acidic pH range resulting in a collagen preparation that remains soluble at physiological pH. The derivatization is conducted such that the resultant solder will exhibit a thermal transition at approximately 40-50^oC. when exposed to IR laser energy.

To prepare derivatized collagen, adjust 2000 ml collagen solution at 2.5-3.0 mg/ml to pH 8.5-9.5 using 10N and 1N NaOH while stirring at room temperature.

For derivatization to attach both SH^- and COO^- groups, 40 ml of an alcohol saturated solution of mercapto naphthalic anhydride (6 mg/ml) was added to the collagen and mixed for 30 minutes at 2-8^oC. and mixed for 15 minutes. Solid glutaric anhydride at 10% collagen weight (600-800 mg of glutaric anhydride) was added and mixed for another 30 minutes at room temperature. The pH was held at pH 8.5-9.5 using 1N NaOH.

At the end of 30 minutes, the solution was centrifuged at 3500 rpm for 10 minutes. The pH of the solution was adjusted to pH 6.8-7.8 as necessary. This solution was lyophilized or air-dried.

Collagen was also prepared with glutaric anhydride alone or with no derivatization. In the latter case, acid soluble collagen was precipitated at neutral pH and lyophilized or air-dried.

3. Solder Formulations

Solder formulations were prepared from chemically derivatized Type I collagen. Base compositions contained either COO^- functional groups or both SH^- (thiol) and COO^- functional groups. The degree of derivatization with SH^- functional groups was varied in attempts to modulate cohesive characteristics. Remaining free amine groups on the native collagen molecule were derivatized with COO^- groups. Thus, compositions contained approximately the same total net derivatization with SH^- and COO^- groups. The ratio of SH^- /COO^- was intentionally varied. Base preparations contained only COO^- groups. Derivatized collagen solutions were then lyophilized (or air dried and powdered). Lyophilized derivatized collagens were formulated into viscous compositions having from 30-80% collagen solids. Since collagen typically becomes saturated at less than 10% solids, novel techniques were developed to increase total collagen concentrations to 30-80%. This was accomplished by mixing lyophilized or powdered collagen preparations in 0.02M phosphate buffer at pH 6.8-7.8. Mixtures of approximately 50 mg/ml were initially prepared and exposed to thermal energy, in this case microwave energy (output power of 1100 watts, 100% power for 3-5 seconds). Microwave energy generates thermal energy causing the gelatinization of collagen. Other heating methods can be employed such as direct application of a beat source at 50 C, for example. Lyophilized or dried collagen was added to the gelatinized collagen solution and again exposed to microwave energy. This sequence continued until the desired collagen concentration was attained. The final viscous and adhesive formulation was centrifuged at 3500 rpm for 1 minute. The high concentration preparations were then cast into films by pouring hot, centrifuged gelatinized collagen in molds approximately 1 mm thick, and 4 cm square in size. Solidified gelatinized collagen films were sectioned into strips for use with the infrared laser system. An alternate method of preparing films was to gently roll the centrifuge tubes used to deaerate the thick gelatinized collagen formulations. The hot, thick formulation coated the inner surface of the centrifuge tubes in a uniform thickness. Films were removed form the tubes and sectioned into strips for use with the infrared laser system. Increased SH^- /COO^- appeared to increase thermal transition temperature and cohesive characteristics. However, cohesive strength appeared to be only minimal. Attempts to enhance adhesiveness were made by decreasing the SH^- /COO^- ratio. This typically resulted in a reduced thermal transition temperature but with little improvement in adhesive characteristics. It is postulated that a greater increase in anionicity may provide enhanced adhesiveness to tissues.

Additional solder formulations were prepared from collagen derivatized with glutaric anhydride only or from non-derivatized acid soluble collagen. In both cases, it was critical to adjust the gelatinized collagen pH to 6.8-7.8 prior to preparing solder films. Therefore, during the microwave gelatinization and prior to deaeration, the pH was adjusted to 6.8-7.8 by addition of 10N and 1N NaOH. These collagen-based solders appeared to provide the best biomechanical and adhesive characteristics.

Claim 1 of 16 Claims

What is claimed is:

1. A tissue adhesive patch, comprising:

a mesh structure, said mesh structure including a polymer, said polymer being selected from a group consisting of nylon, polyester and polycarbonate, said mesh structure being biologically compatible and non-irritating; and

a tissue adhesive including collagen, wherein the concentration of said collagen in said tissue adhesive is 300 mg/ml (30%) to 800 mg/ml (80%), said collagen being derivatized with a COO^- functional group, and being gelatinized, said mesh structure being encapsulated in said tissue adhesive.

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