WO2008097882A1

WO2008097882A1 - Treatment of soft tissue to increase porosity

Info

Publication number: WO2008097882A1
Application number: PCT/US2008/052882
Authority: WO
Inventors: Manh-Dan Ngo; Richard Seeman; Alison Lee
Original assignee: Manh-Dan Ngo; Richard Seeman; Alison Lee
Priority date: 2007-02-02
Filing date: 2008-02-04
Publication date: 2008-08-14
Also published as: CA2677308A1; WO2008097884A2; WO2008097885A3; EP2114136A2; CA2677229A1; EP2114135A2; WO2008097884A3; WO2008097885A2; CA2677305A1; EP2117462A1

Abstract

The present invention relates to a process for treating a decellularized soft tissue such as dermis by freezing the same at a plurality of decreasing temperatures at atmosphere or higher to cause the formation of ice crystals having a size greater than 2.0μ and lyophilizing the soft tissue under vacuum to remove the water to less than 6%forming a porous matrix.

Description

tty. oc et o. -

TREATMENT OF SOFT TISSUE TO INCREASE POROSITY

RELATED APPLICATIONS

This application claims the benefit of priority of U.S. Provisional Application Serial Nos. 60/899,021, filed February 2, 2007; 60/899,020, filed

February 2, 2007; 60/899,018, filed February 2, 2007; and 60/924,249, filed May

4, 2007, the specifications of each of which are hereby incorporated by reference in their entirety.

FIELD OF INVENTION

The present invention is generally directed toward methods of treatment of allograft soft tissue taken from a human donor to increase porosity of the tissue.

BACKGROUND OF THE INVENTION Techniques for restoring structure and function to damaged tissue are used routinely in the area of reconstructive surgery. Tissue transplantation is another way of restoring function by replacing or rebuilding the damaged tissue. Tissue when lyophilized becomes hard and is difficult to rehydrate so that it is flexible for surgical use. In an attempt to solve this problem, physical puncturing and slitting of the implant has been utilized which can reduce the implant's structural integrity and introduce contamination into the implant.

The advantages of retaining an acellular matrix, composed primarily of a collagenous component, have been explored by Klaus and Duhamel (WO 84/0488)) for the production of sterile body implants. In this method, a variety of tissues were extracted sequentially with non-ionic and ionic detergents to yield structures essentially free of cellular membranes, nucleic acids, lipids and cytoplasmic components. The treatment consists of sequential extractions with a non-denaturing detergent and a denaturing detergent to form an acellular matrix of collagen. A soft tissue process is shown in U.S. Patent Number 6,734,018, which relates to a process for preparing an acellular soft tissue graft for implantation into a mammalian system. The process extracts a soft tissue sample with an extracting solution including one or more nonionic detergents and one or more endonucleases, to produce extracted tissue and treats the extracted tissue with a tty. oc et o. - treating solution including one or more anionic detergents, to produce a treated tissue. The treated tissue is washed with a decontaminating solution including one or more decontaminating agents to produce the acellular soft tissue graft; and acellular soft tissue graft is then stored in a storage solution comprising one or more decontaminating agents.

A number of patents are directed towards punching holes in soft tissue implants or cutting slits in soft tissue implants. U. S. Patent Number 6,197,036 pertains to a natural or synthetic material having a plurality of apertures cut therein. The natural material can be fibrous tissue, fascia including the fascia, dura, pericardium, striated muscle or part of the vaginal wall. Tissue from allogenic sources may be freeze dried to produce the immune response to the material. Patches made from this material include a plurality of apertures formed in the center portion. Another patent U.S. Patent Number 6,755,781 relates to a sheet having one or more cuts disposed upon its length or apertures disposed through the full thickness of the sheet. A material used is chemically processed acellular human dermis which is freeze-dried for storage. U.S. Patent Number 5,997,575 pertains to a submucosal tissue graft construct in the form of perforated laminate.

It can thus be seen that previous processes require extensive physical treatment of the soft tissue with a multitude of process steps in an attempt to obtain an acellular soft tissue specimen which has increased flexibility.

SUMMARY OF THE INVENTION

The present invention is directed toward a process for use in the preparation of acellular (essentially lacking in living cells and/or non-living cells), soft-tissue implants such as dermis which have been lyophilized and must be rehydrated to obtain flexibility. The decellularized soft tissue implants produced typically provide long-term durability, flexibility, and ability to hold quantities of fluid and be easily used by the surgeon when used in clinical applications. In certain embodiments, the present invention provides a process for treating soft tissue to increase the porosity of the tissue. The process comprises the following:

(1) obtaining acellular dermis; tty. oc e o. -

(2) freezing the dermis at 2O⁰C for about 15 minutes or for a time period sufficient to stabilize the temperature of the dermis not under vacuum;

(3) freezing the dermis at a second lower temperature of -20°C for about 80 to 120 minutes preferably 100 minutes not under vacuum; (4) freezing the dermis at a third lower temperature until the dermis is frozen solid or at -40°C for 60 minutes not under vacuum;

(5) placing the acellular dermis under vacuum at a temperature of - 40°C for 30 minutes;

(6) drying the acellular dermis under vacuum at -10°C to +1O⁰C, preferably -5°C for 300 to 500 minutes preferably 400 minutes; and

(7) drying the acellular dermis under vacuum at 25°C to 35⁰C preferably 25⁰C for 100 to 400 minutes preferably 240 minutes.

It is thus an object of the invention to provide acellular porous disinfected soft tissue for implantation into a human being. It is another object of the invention to provide acellular disinfected or decontaminated soft tissue which is packaged for usage as an implant by a surgeon.

It is still another object of the invention to provide acellular soft tissue which can be stored for long periods of time for later use by a surgeon for implantation into a human being. It is yet another object of the invention to provide acellular dermis which can be quickly hydrated for use by a surgeon.

These and other objects, advantages, and novel features of the present invention will become apparent when considered with the teachings contained in the detailed disclosure along with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic flow chart showing a treatment process of the soft tissue;

DESCRIPTION OF THE INVENTION

In certain embodiments, the present invention provides a method for the preparation of soft tissue from human donors which is decellularized and then treated to increase the tissue porosity. y. oc e o. -

For the purpose of this application, epidermis is the outer most layer of the skin and dermis is the layer of skin lying immediately under the epidermis. The term skin may refer to either epidermis, dermis or subcutaneous layers or all of the same, depending on the usage. As used herein, the term "decontamination" refers to a process or treatment that renders a medical device, instrument, or environmental surface safe to handle. For example, according to OSHA, decontamination is "the use of physical or chemical means to remove, inactivate, or destroy bloodborne pathogens on a surface or item to the point where they are no longer capable of transmitting infectious particles and the surface or item is rendered safe for handling, use, or disposal" [29 CFR 1910.1030].

The term "disinfection" refers to the destruction of pathogenic and other kinds of microorganisms by physical or chemical means. Disinfection is generally less lethal than sterilization, because it destroys most recognized pathogenic microorganisms, but not necessarily all microbial forms, such as bacterial spores.

An "acellular soft tissue" is a tissue-derived biomatrix structure that is made from any of a wide range of soft tissues by removing all, or substantially all, viable cells and all detectable subcellular components and/or debris generated by killing cells. As used herein, an acellular soft tissue lacking substantially all viable cells is one in which the concentration of viable cells is less than about 1 % (e.g., less than 0.1 %, 0.01 %, 0.001 %, 0.0001 %, 0.00001 %, or 0.000001 %) of that in the tissue or organ from which the acellular soft tissue was made.

The methods and related compositions described herein relate to treating soft tissue, and in particular embodiments, treating soft tissue to increase its porosity. The novel methods described herein can be applied to any number of suitable tissue types, including dermis, fascia pericardia, dura, tendons, ligaments, and muscle.

Acellular soft tissue can be obtained from human sources, such as tissue from elective surgery or from a cadaver, or may be obtained from non-human sources, such as non-human primates (e.g., monkeys, baboon, chimpanzees), pigs, cows, horses, goats, sheep, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, or mice. In further embodiments, where the soft tissue is from a non-human source, the non-human source is a genetically engineered non-human animal, e.g., tty. oc et o. - one that has been genetically engineered to lack an immunogenic epitope of collagen-containing material, such as a terminal α-galactose moiety.

In particular embodiments, a process of the invention uses allograft human skin which has been previously taken from a human donor. Typically, the soft tissue being processed is full thickness skin which includes epidermis, dermis and subcutaneous layers.

The skin which has been previously obtained from the human donor is generally frozen after recovery. The frozen skin is then taken from the freezer, removed from its packaging (e.g., Kapak bag), if any, and the skin thawed in a basin filled with sterile purified water. Prior to processing, tissue is typically inspected for damage (holes or tears) and distinctive features (moles, warts, tattoos) which are removed using a scalpel. Tissue is inspected for hair and the same is removed using any one of a number of techniques including chemical removal and physical removal. A visual inspection is performed to ensure the skin tissue has uniform thickness. Thickness may be recorded using a thickness gauge. To identify the orientation (dermal or epidermal side) of tissue such as skin, the skin is typically positioned such that the epidermis faces the processor and an incision is cut into the upper left corner of each piece of tissue to indicate the epidermal side. In processing, the epidermal layer is removed and the dermis is typically decellularized using, for example, Sodium Chloride (NaCl) solution at a concentration of 0.1 - 1OM, preferably about IM with a pH ranging from 5.0 - 9.0, preferably 6.8 - 7.2, and is agitated, for example, at a speed of 65 rpm on an orbital shaker for 1-96 hours, preferably 12 hours to a maximum of 48 hours. After 12 hours, the container holding the skin is generally checked to ascertain if the epidermal layers have been sloughed off. If not, the container may be checked every 2 hours until the epidermis has sloughed off. The dermis is then typically removed and placed on, for example, a cutting board with the epidermal side up, and any remaining epidermal layer are picked off and discarded as well as any remaining hairs. In some embodiments, the remaining dermis pieces are replaced in the tissue flasks, filled with sterile water and agitated on the orbital shaker for about 15 minutes. In some embodiments, the sterile water is refreshed and the rinse procedure is repeated one more time for a total of two rinses. y. oc e o. -

In further embodiments, once the final rinse (e.g., sodium chloride soak or sterile water rinse) is complete, the dermis pieces may be trimmed into shaped pieces, preferably rectangular, by removing all of the rough edges of each piece with a scalpel. The trimmed dermis pieces are then typically immersed in a detergent such as Triton X-IOO solution having a concentration of 0.01 - 10.0%, preferably about 0.1% with a pH ranging from 4.5 - 8.5, preferably 6.2 — 7.0 and agitated on the orbital shaker for 1 - 96 hours, preferably 24 hours to 48 hours. In further embodiments, the dermis is then placed in tissue flasks filled with sterile water, and agitated on the orbital shaker at 65 rpm for 15 minutes. The sterile water is typically refreshed and the rinse procedure is generally repeated a minimum of 7 more times for a total of 8 water rinses. In some embodiments, a residual detergent test may be performed on the rinsate after the 6^th water rinse to ensure the detergent has been adequately removed.

In certain embodiments, the acellular dermis is disinfected in a solution containing a disinfection solution of e.g., 35% peracetic acid, ethano, e.g.,1 95% (undenatured), propylene glycol and sterile water. The disinfection mixture is generally stirred, e.g., with magnetic stir bar for at least 15 minutes or until homogenous. The dermis is then typically soaked under vaccum and agitated at 65 rpm for 30 minutes to 12 hours, preferably 4 hours, at 4°C to 40°C preferably 20°C - 25°C. In particular embodiments, the disinfection solution is formed with peracetic acid (v/v) 0.05% - 5.0%, preferably 0.5% - 0.7%; propylene glycol (v/v) 20% - 60%, preferably 37.5%; ethanol (undenatured) (v/v) 10% - 50%, preferably 23% to 26% and sterile water 30% to 40%, preferably 35%. The disinfection solution typically has a pH ranging from 2.0 - 5.0, preferably 3.2 - 3.8. In further embodiments, the disinfected dermis is subjected to a rinse series with sterile water followed with agitation at 65 rpm under vacuum; two 5-minute rinses, followed by two 10-minute rinses, followed by two 15-minute rinses for a total of 6 rinses or until the peracetic acid has been adequately remove. In some embodiments, after the last rinse, a residual test may be performed on the rinsate to ensure that the peracetic acid has been adequately removed with less than 1 ppm remaining on the tissue.

In further embodiments, the disinfected dermis tissue is cut to a final size and the tissue is then placed in a freezing and lyophilization cycle, which is thought to increase porosity of the dermis tissue. In certain embodiments, the tty. oc et o. - acellular dermis is conditioned (e.g., equilibrated) for 15 minutes at about 20°C or for a time period sufficient to stabilize the temperature of the dermis with a temperature ramp up from ambient with no vacuum. In further embodiments, the acellular dermis is next frozen at about -20°C for 80 minutes to 120 minutes, preferably 100 minutes with no vacuum and in the third step, the tissue is frozen at about -40°C for 45 minutes to 75 minutes or until the dermis is frozen solid, preferably 1 hour (60 minutes) with no vacuum.

It is thought that this stepped freezing which is initially without vacuum causes small solid ice particles to form within the dermis creating cavities which make the dermis more flexible when hydrated for use in surgery or other medical operations. Under conventional lyophilization techniques, ice particles do not form in the size necessary to increase dermis porosity. In embodiments of the present invention, however, after a three step freezing treatment with the dermis not being under vacuum thereby allowing large ice particles to form within the dermis, the dermis is placed under vacuum, e.g., about 400 to about 800m Torr, e.g., about 600m Torr at -4O⁰C for an additional 30 minutes so that the soft tissue is placed under vacuum after which it is subjected to a drying phase at about -10°C to +10°C, preferably -5°C for 300 to 500 minutes, preferably 400 minutes under vacuum at, e.g., 600m Torr. In further embodiments, the frozen lyophilized dermis is then dried, e.g., at 25° C to 35⁰C, preferably 25⁰C for 100 to 400 minutes, preferably 240 minutes under vacuum at, e.g., about 400 to about 800mTorr, e.g., 600m Torr until the residual moisture of the lyophilized dermis is less than 6% and the dermis has a porosity throughout its body formed by the cavities left by the removal of the solid frozen ice. In certain embodiments, the pore cavity size left by the water being removed from the ice particles formed in the dermis during the initial three freezing stages ranges from about 2.0μ to about 200μ and allows the dermis to soak up more saline or other moisturizing compound than if it was conventionally lyophilized and dried so that the same is flexible upon hydration in the operating room. In further embodiments, the finished dermis implant is placed, e.g., in foil and a Tyvek® pouch. While the present description has used dermis as an example of soft tissue, it is to be understood that the applicable process applies to all soft tissue and not just dermis. Atty. oc et o. 4 45-2 3

While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. One skilled in the art will appreciate that numerous changes and modifications can be made to the invention, and that such changes and modifications can be made without departing from the spirit and scope of the invention. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Each patent, patent application, and publication cited or described in the present application is hereby incorporated by reference in its entirety as if each individual patent, patent application, or publication was specifically and individually indicated to be incorporated by reference.

Claims

tty. oc et o. 5-2 3What is claimed is:

1. A method for the treatment of soft tissue to provide porosity for the same for implantation into a human comprising:

(a) conditioning acellular soft tissue for about 15 minutes at 20°C without vacuum;

(b) freezing acellular soft tissue at about -20°C for 2.5 to 3 hours without vacuum;

( c) freezing acellular soft tissue at about -4O⁰C for about 1 hour without vacuum; (d) drying the frozen acellular soft tissue in a range of about -

5⁰C to about 25°C for a period of time to reduce the residual moisture of the soft tissue and

(e) packaging the soft tissue in a sealed package.

2. A method for the treatment of soft tissue to provide porosity for the same for implantation into a human comprising:

(a) freezing acellular soft tissue for less than 0.5 hour at about - 20⁰C without vacuum;

(b) freezing acellular soft tissue at about -2O⁰C for about 2.5 to 3 hours without vacuum; ( c) freezing acellular soft tissue at about -40⁰C for about 1 hour without vacuum;

(d) drying the frozen acellular soft tissue at about -5⁰C for a period of time to reduce the residual moisture of the soft tissue and

(e) packaging the soft tissue in a sealed package.

3. The method as claimed in claim 2 wherein said soft tissue is dermis.

4. The method as claimed in claim 2 wherein said vacuum is 600m Torr.

5. The method as claimed in claim 2 wherein said drying in (d) runs from about 360 minutes to about 460 minutes.

6. The method as claimed in claim 2 wherein (a) runs for a time period of about 15 minutes.

7. The method as claimed in claim 2, further comprising freezing the frozen acellular soft tissue at about -40⁰C under vacuum for about 30 minutes. tty. oc et o. -

8. The method as claimed in claim 7, further comprising drying the frozen acellular soft tissue above 0° C for about 100 to about 400 minutes under vacuum to reduce the residual moisture content of the soft tissue to less than 6%.

9. The method as claimed in claim 7 wherein said acellular soft tissue after treatment has a pore size ranging from 2.0μ to 200μ.

10. The method for the treatment of soft tissue to increase the porosity of the soft tissue comprising:

(a) freezing the soft tissue at a series of reduced temperatures without the application of vacuum to create solid ice particles in the soft tissue having a size greater than 2.0μ within the soft tissue; and

(b) drying the soft tissue under vacuum to reduce the residual moisture of the soft tissue to less than 6% leaving the tissue with a plurality of cavities greater than 2.0μ.

11. The method as claimed in claim 10 wherein said soft tissue is dermis.

12. The method as claimed in claim 10 further comprising freezing the frozen acellular soft tissue at -40°C under vacuum for about 30 minutes.

13. The method as claimed in claim 11 wherein said dermis after treatment has a pore cavity size ranging from 2.0μ to 200μ.

14. A method for the treatment of dermis to increase the porosity of the dermis comprising:

(a) freezing the dermis at a series of reduced temperatures without the application of vacuum to create ice particles in the dermis having a size greater than 2.0μ within the soft tissue; and (b) drying the dermis under vacuum to reduce the residual moisture of the dermis to less than 6% leaving the dermis with a plurality of cavities having a size substantially the same as the ice particles formed during the freezing step (a).

15. The method as claimed in claim 14 wherein said cavities range from 2.0μ to 200μ.

16. The method as claimed in claim 14 further comprising freezing the frozen acellular soft tissue at about -40°C under vacuum for about 30 minutes.

17. A method for the treatment of dermis tissue to prepare the same for implantation into a human comprising the steps of: tty. oc et o. 45-2 3

(a) conditioning acellular dermis tissue for less than 0.5 hour at about 20°C without vacuum;

(b) freezing acellular dermis tissue at about -20°C for 2.5 to 3 hours without vacuum; ( c) freezing acellular dermis tissue at -40⁰C for about 1 hour without vacuum;

(d) freezing the frozen acellular dermis tissue at -4O⁰C under vacuum for about 30 minutes;

(e) drying the frozen acellular dermis tissue for 5 to 10 hours at about -5°C under vacuum;

(f) drying the acellular dermis at 25⁰C for a period of time to reduce the residual moisture of the soft tissue to less than 6%; and

(g) packaging the dermis tissue in a sealed package.