Title:  Solvent dehydrated microbially-derived cellulose for in vivo implantation

United States Patent:  6,599,518

Issued:  July 29, 2003

Inventors:  Oster; Gerry Ann (Hatfield, PA); Lentz; Kevin (Perkasie, PA); Koehler; Kevin (Keyport, NJ); Hoon; Russell (Furlong, PA); Serafica; Gonzalo (Troy, NY); Mormino; Richard (Round Lake, NY)

Assignee:  Xylos Corporation (Langhorne, PA)

Appl. No.:  989297

Filed:  November 21, 2001

Abstract

A solvent dehydrated microbially-derived cellulose material is described for use as an implantable material in general and plastic surgery.

SUMMARY OF THE INVENTION

The materials of the present invention comprise an implantable form of solvent dehydrated microbially-derived cellulose, particularly cellulose produced from cultures of Acetobacter xylinum propagated in a nutrient media and incubated under controlled conditions. The cellulose film or pellicle is produced via A. xylinum propagation inclusive of incubation under controlled conditions. The pellicle is chemically treated with sodium hydroxide to destroy pyrogens and viable microorganisms then the pellicle is bleached with hydrogen peroxide to whiten the cellulose. Following compression of each pellicle, the material is treated with a water-miscible organic solvent such as acetone for several cycles to optimize dehydration. The material is then again compressed, undergoes a final drying step, is cut and packaged, and gamma sterilized.

In one aspect of the invention, there is provided a method for producing solvent dehydrated cellulose from microbially derived cellulose. The method comprises the steps of propagating cellulose-producing microbes in a nutrient media under controlled conditions followed chemically treating the microbially-derived cellulose with sodium hydroxide to depyrogenate the material and destroy viable organisms, followed by bleaching the microbially-derived cellulose with hydrogen peroxide to whiten the cellulose prior to further processing.

In another aspect of the invention, the cellulose is dehydrated (water is removed) by processing the cellulose with a water-miscible organic solvent selected from the group consisting of methanol, ethanol, propanol, isopropanol, acetone and mixtures thereof.

In a further aspect of the invention the solvent dehydrated microbially-derived cellulose is used as an implantable medical material for plastic and general surgery. The solvent dehydrated microbially-derived cellulose is useful in general and plastic surgery because it can be cut into desirable sizes and shapes to meet surgical requirements.

A further aspect of the invention relates to a kit comprising microbially-derived cellulose and a package comprising a sealed waterproof pouch, optionally placed within a secondary waterproof pouch, and gamma sterilized.

DETAILED DESCRIPTION OF THE INVENTION

In preparing the solvent dehydrated microbially-derived cellulose (SDMC) of the present invention, the cellulose was synthesized by a bacteria, preferably the bacteria Acetobacter xylinum (wild type), and was recovered from inoculation flasks and propagated via continued inoculation and incubation for linear growth in subsequent flasks and carboys of optimized media to attain the desired volume of microbially derived cellulose. The media is comprised of nutrients such as sucrose, ammonium sulfate, sodium phosphate, magnesium sulfate, citric acid, acetic acid and trace elements resulting in a growth media having a pH of about 4.0 to about 4.4. The sterilized media is inoculated from propagation cultures of A. xylinum and filled into bioreactor trays at the appropriate volume to yield the a final cellulose to water ratio of about 90% to 95% water to about 5% to 10% cellulose. The bioreactor trays are sealed and incubated in a controlled environment at 30^oC.+2^o until growth of a pellicle of microbially-derived cellulose is complete. The pellicles are removed from the bioreactor trays and are chemically treated to remove bacterial by-products and residual media. A caustic solution, preferably sodium hydroxide at a preferable concentration of about 0.1M to 4M, is used to remove viable organisms and pyrogens (endotoxins) produced by bacteria from the pellicle. The treated pellicles are then rinsed with filtered water to reduce microbial contamination (bioburden). The chemically processed cellulose films (pellicles) are then exposed to a "bleaching" process to attain a "whitening" effect on the material. A typical bleaching solution of hydrogen peroxide is in the range of about 0.25% to about 3% and is prepared from concentrated hydrogen peroxide and filtered water.

In a controlled environment, the pellicles are compressed to the desired thickness. It is the thickness of the compressed film that achieves the final desired density of the microbially-derived cellulose. The original fill volume as well as the compression steps are integral to the present invention to attain the desired density that affects the strength, integrity, and function of the cellulose. Further processing of the present invention continues with the use of a water-miscible organic solvent selected from the group consisting of methanol, ethanol, propanol, isopropanol, acetone and mixtures thereof to dehydrate the cellulose. Without being bound to any one theory, it is believed that soaking the compressed films in a water-miscible organic solvent cross-links the cellulose fibers, thereby yielding a product having increased tensile strength, reduced elongation (stretch) and increased suture retention when used as an implantable medical device for various surgical procedures. Depending on the desired level of dehydration, the solvent treated films are exposed to one or more applications of the organic solvent then the films are subsequently compressed to the desired thickness in a controlled environment. The solvent is removed by either air-drying at ambient temperature or oven-drying at about 30^oC. under controlled conditions. Dried samples are tested on a residual moisture balance as confirmation of the desired residual moisture of less than about 15%.

In a controlled environment, the films can be cut to various shapes and sizes that those skilled in the art will understand. It is possible for each unit to be packaged in a waterproof double-pouch system and sterilized by exposure to gamma irradiation at a dose level as high as 35 kGy, but preferably a lower dose would be used. The gamma dose is determined by the bioburden level of the non-sterile material as described in ISO 11137 Sterilization of Health Care Products--Requirements for validation and routine control--Radiation Sterilization.

The waterproof packaging is comprised of waterproof inner and outer chevron peelable pouches. The material is a polyester/LDPE/foil blend sealed to silica coated polyester, suitable for sterilization, by, for example gamma irradiation.

The inventive microbially-derived cellulose can be used in tissue augmentation which involves implantation of the subject microbially-derived cellulose material for general as well as plastic surgery applications. Examples of general and plastic surgical uses include but are not limited to, general soft tissue augmentation, pelvic floor reconstruction, bladder neck suspension, hernia repair, inguinal hernia patch and duraplasty.

Another use of the present inventive cellulose material involves their application in sutures. Suture retention is critical for implantable medical articles to secure and maintain position during surgery, healing and function. The surgeon must rely on the ability of the implantable material to not only accept suture without tearing during needle insertion, but to also retain the suture without tearing away from the sutured edge of the implant.

The ability of the present inventive microbially-derived cellulose to be used in surgical procedures requires that the material is safe and effective for its intended purpose and achieves sufficient biocompatibility.

The ability of the present invention to withstand depyrogenation and sterilization processes is necessary toward producing an implantable medical device for general and plastic surgery. Often, biomedical polymers have lower thermal and chemical stability than other materials such as metals, ceramics and synthetics; therefore, they are more difficult to sterilize using conventional methods. For any material used as an implantable medical device, it must be free from endotoxins (non-pyrogenic), microorganisms and other possible contaminants that will interfere with the healing process and cause harm to the recipient.

The present invention undergoes depyrogenation by using a heated caustic solution (0.1M to 4M sodium hydroxide) known to destroy endotoxins that may be present due to bacteria or cross-contamination from materials exposed to pyrogens. The material is then gamma irradiated at doses sufficient to destroy microorganism contamination by pre-determined sterility assurance levels based on bioburden levels (the amount of microorganisms typically present on the non-sterile material.) Samples were gamma irradiated at a dose of about 35 kGy. It can be concluded that the material can be depyrogenated with a strong alkaline sodium hydroxide solution at an elevated temperature and that it can withstand gamma sterilization without any significant affect to mechanical properties.

Medical devices intended for implant must meet vorious criteria to comply with either U.S. Food and Drug Administration (FDA) regulations or the International Organization for Standardization (ISO) requirements in order to be deemed fit for their intended use. Cytotoxicity studies are considered relevant to prove that the implant device is safe/biocompatible with human tissue. In vitro biocompatibility studies, based on the International Organization for Standardization 10993: Biological Evaluation of Medical Devices, Part 5: Tests for Cytotoxicity: in vitro Methods guidelines, were conducted on the present invention to determine the potential for cytotoxicity.

The mechanical properties of the microbially-derived cellulose relates to tensile strength, % elongation and suture retention. The material is considered multidirectional as well as possessing the properties of a linear polymer whereas the polymer chains tend to line up in the direction of draw; therefore no regard was made for the direction of the cutting.

Claim 1 of 17 Claims

We claim:

1. A method for preparing an implantable material for medical and surgical applications comprising the steps of:

a) providing a microbially-derived cellulose;

b) treating said microbially-derived cellulose to render said cellulose non-pyrogenic;

c) dehydrating said microbially-derived cellulose in a continuous manner by applying a water-miscible organic solvent selected from the group consisting of methanol, ethanol, propanol, isopropanol, acetone and mixtures thereof to said microbially-derived cellulose; and

d) subsequently removing said solvent.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.