MXPA96002719A - Preparation of bone for transplantac - Google Patents

Abstract

The present invention relates to a method for preparing bone for transplantation, the bone contains an internal matrix comprising the removable material, the method comprising: (a) contacting the bone or matrix with a solution comprising a detergent or a decontamination agent; (b) contacting the bone with an atmosphere having a pressure that is less than ambient pressure; and (c) contacting the bone or matrix with a solution comprising a detergent or an agent of decontamination

Description

"PREPARATION OF BONE FOR TRANSPLANTATION" The present application is a continuation in part of the North American Application Serial Number 5 07 / 815,394, filed by December 31, 1991, the entire description, claims and figures of which are hereby incorporated by reference in their entirety. . 0 FIELD OF THE INVENTION The present invention relates to methods of bone processing for transplantation. More particularly, the invention is directed to the provision 5 of decontaminated bone, the transplantation of which substantially minimizes the exposure of the transplant to , • _ contaminating pathogens or immunogenic material.

DEVELOPMENTS GIVEN TO KNOW 0 The acquisition and processing of human bone for transplantation is a complicated task that requires the coordinated efforts of several groups including the donor's family, the hospital staff, the local acquisition group, the specimen processing laboratory of blood, the bone processing laboratory, the transplant patient, and the transplant team. A primary consideration is minimizing the risk of transferring potentially harmful diseases to tissue recipients. In fact, the provision of safe bone tissue for transplantation provides a very special challenge as an immunogenic material and also microorganisms and viruses that are found deep within the internal matrix of the bone samples. In this regard, blood samples can be analyzed in the processing laboratory for a variety of known infectious agents including, for example, Human Immunodeficiency Virus (HIV-1) Human Immunodeficiency Virus (HIV-2 Human T cell lymphotropic virus (HTLV-1) Hepatitis B Hepatitis C Cytomegalic (CMV) virus Treponema pallidum (syphilis) With regard to the serious clinical consequences resulting from the transplantation of contaminated bone, see for example from Kakaiya et al., "Tissue transplant-transmitted infections, "Transfusion 31 (3), 1277-284, 1991; Shutkin," Homologous-serum hepatitis following use of refrigerated bone-bank bones, report of a case ", Journal of Bone and Joint Surgery, 16-A (1), 160 -162, 1954. The transmission of human immunodeficiency virus (HIV) through bone as well as bone marrow has been reported by "Transmission of HIV through bone transplantation case report and public health recommendat ions "Novbid. Mortal. Weekly Rep. , 37, 597-599, 1988; Furlini et al., "Antibody response to human immunodeficiency virus after infected bone marrow transplanb", Eur. J. Clin. Microbiol. Infect. Dis. 7 (5) 554-665, 1988. HIV has been cultured from fresh bone as well as chilled bone and freeze-dried bone. Buck et al., "Human immunodeficiency virus cultured from bone Implications for transplantation", Clin. Ortho , 251, 249-253, 1990. In addition, the protection of bone processing laboratory technicians is of great concern due to the serious potential for transmission of HIV and hepatitis B. An additional and very important consideration with respect to the design of bone processing methodologies is to avoid or minimize the immune response (including rejection of the transplant) in the recipient patient to donor macromolecules remaining in the transplanted bone, such as collagens, and superficial antigens of the major histocompatibility complex cell or other glycoproteins. See, for example, Friedlander and Horowitz, Orthopedics, 15 (10), 1171-1175 (1992), and Man in et al., Id., 1147-1154. Consequently, there is a great need for processing method. of bone that decreases the risk of the recipient's immune response or disease transmission associated with the use of and preparation and acquisition of transplantable bone. In this regard it is also important to recognize that even the current state of the art methodology is used to select donadox, recent infections in a specific donor may not be detected, thus underlining the importance of improved cleaning and decontamination treatments that offer prophylactic protection against potential infectious agents or as yet undetected. The combination of donor selection treatments and antibiotics traditionally used during bone processing reduces but does not limit to an acceptable level the risk of transmission of known virus contaminants and a variety of bacteria. See, for example, N.L. Scarborough, Orthopedics, 15 (10), 1161-1167 (1992) and T.I. Malinin "Acquisition and Banking of Bone Allografts", in Bone Grafts and Bone Substitutes, Habal and Reedi, editors, Chapter 19, pages 206 to 22c >;, .B. Saunders Company, Philadelphia, PA (1992). As mentioned above, the methods currently available do not offer prophylactic protection of viruses, selection of batteries and fungi, which are the common flora in humans or in a hospital environment. Even though the sensitivity and specificity of screening tests for these pathogens are high, screening tests are not fail-proof, and may be false-negative, for example, of low antibody levels (e.g., recent infection). or immunodeficiency) or even a technical error. In addition, screening tests may be useful only to identify known infection agents. In addition, the traditionally used antibacterial antibiotic cocktails mentioned above that are currently used do not actually kill or kill all types of bacteria. For example, a solution of polymyxin / bacitracin (50,000 units of bacitracin / 500,000 units of polymyxin B) does not inactivate the Proteus species. In addition, traditional antibiotic cocktails have no significant effect on viruses or fungi. There are also significant limitations in the extent to which decontamination agents have been successfully used to penetrate and decontaminate the bone matrix. See Prolo and Oklund, "Sterilization of Bone by Chemicals," Osteochondral Allografts-Biology, Banking and Clinical Applications, by Friedlaender et al., Editors, Chapter 22, pages 233-238, Litte, Bro n and Company, Boston MA ( 1983). The bone matrix contains potentially removable materials, e.g., marrow, cells and lipid that prevent access of decontamination agents deep into the bone matrix where as mentioned above there may be present infectious agents or immunogenic macromolecules. Certain of the difficulties encountered in extracting the removable materials from the bone matrix are described, but are not resolved in accordance with British Patent Specification Number 964,545, published in 1964. Specification Number '545 describes a procedure to use a fatty solvent (for example, a chloroform / methanol mixture) to clean the bone. There are considerable time periods involved that are incompatible with the preferred bone bank procedures such as rapidly matching a donor bone piece of the appropriate size to a recipient. An additional disadvantage that manifests itself as inherent in this methodology is that it seems to be restricted to a series of specific steps that must be carried out in a specific order. If this is not done, the proteins of the immunogenic donor are said to remain in the bone due to denaturation in situ caused by the fatty solvent. These and other difficulties associated with the provision of decontaminated bone appropriate for transplantation are resolved in accordance with the practice of the invention.

SUMMARY OF THE PRESENT INVENTION The present invention relates to the discovery that the manipulation of the atmospheric pressure to which the internal matrix of the bone is subjected during cleaning and / or decontamination thereof is particularly effective for the provision of a bone suitable for transplantation. Accordingly, a method is provided for preparing the bone for transplantation, the bone containing the internal matrix itself comprising removable material, the method comprising the step of contacting the bone with an atmosphere at less than ambient pressure. In a preferred aspect, the method comprises a further step of contacting the bone or matrix thereof with a solution comprising a decontamination agent or a detergent. Representative of the clinical devices that can be treated with decontaminated bone produced in accordance with the practice of the invention are knee and hip surgery of femoral heads, proximal tibias and distant femurs. In accordance with the practice of present development, it has also been determined that the lipid in matter to the bone interferes considerably with the cleaning and decontamination thereof. An important aspect of the invention therefore provides a method for treating the internal matrix of the bone containing a predetermined amount of renewable material, the matrix also containing a predetermined amount of the lipid that essentially immobilizes the removable material, the method comprising the step of contacting the matrix with an atmosphere at less than ambient pressure for an effective period of time to reduce the lipid content to less than the predetermined amount thereof. A further aspect of the invention provides a method for treating the bone containing the internal matrix itself comprising a predetermined amount of removable material having considerable affinity for the bone, the method comprising the step of contacting the matrix and an atmosphere at less than the ambient pressure and then maintaining the atmosphere in contact therewith for an effective time to reduce the amount of the removable material to less than the predetermined value thereof. In addition, the invention provides a method wherein the bone is subjected to an elevated temperature before, during or after contacting the atmosphere at less than ambient pressure. These and other particular features and advantages of the present invention are described in accordance with the detailed description of the invention which will be given directly below.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram of a femur showing the site of perforated holes, suitable to facilitate access to the removable material in the bone. Figure 2 is a diagram of a femoral head showing the site of appropriate perforated holes to facilitate access of the removable material into the bone.

Figure 3 is a diagram of a femoral head showing the site of appropriate punched holes to facilitate access to the removable material in the bone. Figure 4 is a diagram of a right femur showing the site of the appropriate punched holes to facilitate access to the removable material in the bone. Figure 5 is a photograph showing the improved bone produced in accordance with the practice of the present invention.

DETAILED DESCRIPTION OF THE ORIGINAL INVENTION According to the invention, a simple, safe and effective method for treating the bone and making it suitable for transplantation is provided which comprises: a) contacting the bone with an effective global decontamination agent to inactivate bacteria, fungi, viruses and parasites; b) clean the bone; and c) decontaminating the terminal between the clean bone by contacting it with an effective global decontamination agent to inactivate bacteria, fungi, viruses and parasites.

The invention also provides a method for cleaning the bone that can be used in step (b) of the method described above and comprises contacting the bone with a detergent under conditions of high pressure washing at elevated temperatures. For purposes of this disclosure, the term "bone" is used in the most general sense and includes all types of human or aniaml bone tissue, including whole bones, bone pieces, bone blocks, and fixed connective tissues such as ligaments and tendons, as well as preparations of ground bone and preparation of ground demineralized bone. Initial or primary decontamination is achieved by contacting the bone with an effective global decontamination agent to inactivate bacteria, viruses, fungi and parasites. The contact time must be sufficient to effectively inactivate the infectious agents. Preferably, the bone is soaked in the overall decontamination solution for at least two or more minutes, preferably 10 or more minutes and more preferably at least one or more hours. During this primary decontamination soak the bone can be removed from the solution for debridement of the larger external tissue and fat and then returned to the solution for further soaking. The global decontamination agent must be effective to inactivate bacteria, viruses, fungi and parasites. The preferred decontamination agents are the iodophors. Useful iodophors include polyvinyl-iodine pyrrolidone preparations (PVP-I or povidone iodine) which are commercially available from Purdue-Frederick Company, ISP (formerly GAF) and BASF. PVP-I preparations useful in the practice of the invention include those of molecular weight less than 20,000 such as PVP-Iodine 17/12 from BASF. It has been determined that the preferred PVP-I preparations are those of molecular weight less than 100,000, and particularly having a median molecular weight of about 35,000, or a k-value of about 26 to 32, eg, PVP-Iodine 30 / 06 of BASF. Alternatively, the appropriate iodophor solutions can be prepared by mixing together a solution of the complexing agent (polyvinyl pyrrolidone in the case of PVP-I) having the desired molecular weight and molecular iodine in sufficient quantities to provide the desired concentration of available iodine . For example, an available iodine concentration of about 1 weight percent can be obtained by dissolving 90 grams of PVP in water and then with stirring adding 10 grams of iodine, and finally adding enough water to bring the total volume to 1 liter. Other PVP ratios with respect to iodine can be used to obtain a PVP-I solution that provides the desired available iodine concentration. Appropriate available iodine concentrations are from .03 percent to 1 percent by weight of iodine with respect to the solution, preferably from 0.1 percent to 0.5 percent. Other decontamination agents that have been found to inactivate a broad range of infectious agents (including bacteria, fungi, parasites and viruses) are hydrogen peroxide, ethanol, ozone, ethylene oxide, irradiation and use of the foregoing in combinations, and with PVP-I. The solution of the global decontamination agent must be of an effective concentration to inactivate bacteria, viruses, fungi and parasites. The ioopher concentration of a primary decontamination solution is preferably within the range of 0.5 percent to 10 percent and more preferably 1 percent to 5 percent by weight, with the available iodine concentration of 0.05 percent to 1 percent, preferably from 0.1 percent to 0.5 percent by weight. The concentration of PVP-I is preferably within the range of 0.5 percent to 10 percent and especially preferably 1 percent to 5 percent by weight of PVP-I, with an available iodine concentration of 0.05 percent to 1 percent, preferably from 0.1 percent to 0.5 percent by weight. The primary decontamination solution may include a detergent, preferably at a concentration of 0.1 percent to 5 percent of the solution, more preferably from 1 percent to 3 percent, and especially preferably 0.1 percent to 1 percent. percent in weight. Cationic, amphoteric and nonionic anionic detergents are suitable. Preferred detergents are nonionic detergents such as polyethoxyethylene ethers (for example those sold under the registered trade name Triton >); R) by Rhom Haas by Union Carbide) or the fatty acid esters of polyoxyethylene sorbitan (Tween series sold by ICI and Sigma among others). More preferably, the detergent is octylphenoxypolyethoxyethanol (Triton X-10 (R) from Rhom &Haas). Of the polyoxyethylene sorbitans, polyoxyethylene sorbitan monooleate is especially preferred. (20) (Tween (R) 80). Advantageously, primary decontamination is carried out by soaking the bone in a solution of 0. 1 percent to 5 percent PVP-I and 1 percent (by weight) of Triton X-10? (R) for at least two or more minutes, preferably 10 or more minutes and more preferably so less one or more hours. It has recently been determined that a highly advantageous primary decontamination procedure involves soaking the bone in a solution of 1 to about 5 percent PVP-I, without detergent, for at least about 1 to 2 hours. Preferably, due to the ratio of free iodine to the iodine complexed to the different concentrations of PVP-I, it has been determined that the especially preferred modification of this step involves first soaking the bone in 5 percent PVP-I for about 1/2 to 1 1/2 hours followed by debridement and further soaking in 1 percent PVP-I for approximately 1/2 to 1 1/2 hours. As in Example 1, it shows the preliminary decontamination step of the invention is more effective than the antibiotic cocktail of the prior art. PVP-I is the preferred decontamination agent due to its rapid action, the broad spectrum of infectious agents it can inactivate (viruses as well as batteries, fungi and parasites) and its relatively low toxicity to human tissue. In addition, PVP-I has been found to inactivate HIV. The preliminary decontamination step not only protects the bone recipient by significantly reducing the risk of infection from the bone, but also protects the laboratory technicians. The primary decontamination solution that either contains PVP-I or PVP-I and a detergent further makes the bone easier to clean by initially loosening or softening soft tissues, lipids and blood products. Cleaning after primary decontamination can be carried out by conventional methods but preferably by contacting the bone with a detergent in such a manner as to remove the fat, marrow and other debris. The detergent lyses the cells (e.g., blood cells) dissolves the fat, and solubilizes the proteins comprising the bone marrow. The cleaning process may include agitation and / or elevated temperatures. Vigorous stirring is especially preferred and can be effected by a rotary stirrer. This wash produces bone that has marrow and fat cells and insignificant wastes and therefore adds an additional margin of safety for the transplant recipient by removing cells that can house the agent? infectious and / or molecules that can cause an immune response. The preferred and especially preferred suitable detergents are the same as those described above for the primary decontamination step. The detergent concentration is recommended to be from about 0.1 percent to 5 percent, more preferably from 1 percent to 3 percent and especially preferably from about 0.1 percent to 1 percent by weight. Although the iodophor can be added to the solution containing the detergent in a concentration of 0.1 percent to 10 percent, it has been determined that it is preferred to omit the addition thereof. Advantageously, the bone is cleaned with a detergent solution under conditions of high pressure washing and elevated temperatures. The high pressure washing conditions provide sufficient force to drive the cleaning solution into the internal matrix of the bone. These high pressure washing conditions include, for example, vigorous stirring, such as a rotating paint can stirrer, or high pressure washing such as with a high pressure (or speed) jet stream. Suitable paint can agitators include those manufactured by Red Devil, preferably model number 0-5400-OM (615 revolutions per minute and .25 horsepower). The pressure of the liquid jet stream is preferably from 7.03 to 210.90 kilograms per square centimeter and more preferably from 35.15 to 103.45 kilograms per square centimeter. Especially preferred, the liquid jet stream is sterile and includes the detergent. The cleaning is significantly accelerated and is more complete if carried out at temperatures within the range of 20 ° to 80 ° C, and preferably at an elevated temperature of 37 ° C to 80 ° C, particularly preferably of about 50 ° C. C at 65 ° C. High-pressure washing effectively loosens the marrow and progressively removes trash within the spongy bone matrix. After this high-pressure washing procedure, the bone is considerably cleaner and whiter than the bone processed by normal methods (see Figure 5). To speed up cleaning, the solution can be changed, for example by transferring the bone from a fresh solution during the cleaning operation. Preferably the solution is changed at least twice. After cleaning, the detergent can finally be removed by repeated washing with sterile water. A biologically acceptable alcohol such as ethanol can also be used to remove the detergent. If alcohol is used, it should be removed by rinsing with sterile water. If blocks of bone are to be cleaned with fixed connective tissue, the connective tissue (tendons, ligaments, meniscus, for example) should be covered with a sterile coating such as a plastic wrap or a sterile wrap during the cleaning procedure so that it is avoid contact with the detergent. The bone can also be cleaned and decontaminated by exposing it to hydrogen peroxide, which also has bactericidal properties. After washing with the detergent, the bone is transferred to a hydrogen peroxide solution of 0.5 percent to 10 percent, preferably 3 percent, for a sufficient period of time to allow additional bleaching and removal of the fat from the body. trace Agitation may be applied. The incubation time is appropriately 5 to 120 minutes, preferably 5 to 60 minutes, and especially preferably 15 to 30 minutes. After the treatment, residual peroxide is removed by extensive washing with sterile water. After cleaning the bone it is finally decontaminated before packing. This terminal decontamination is effected by contacting the bone with a global decontamination agent for at least about 2 or more minutes, preferably at least about 10 or more minutes, and especially preferably 30 to 60 minutes or more. A preferred global decontamination agent is 1 percent (weight / volume) polyvinylpyrrolidone iodine.

When a cartilage or connective tissue is present, the decontamination and cleaning solutions preferably contain sodium chloride, or another biologically acceptable salt, in an amount sufficient to prevent the PVP-I from concentrating in the cartilage or connective tissue. It is preferably used 0.01 to 0.75 M NaCl, and more preferably 0.15 M NaCl. The global decontamination agent used for terminal decontamination can be removed from the bone by washing with sterile water, or a thin layer to further protect the bone against infectious agents. The bone coated in this way can be lyophilized. More preferably, a layer of PVP-I is allowed to form. The PVP-I solution that adheres to the bone imparts a rich golden amber color, which can serve as an indicator that the bone has been treated. If desired, the amber-colored bone can be directly lyophilized and packaged and stored at room temperature, preferably in amber-colored bottles. Although various methods for lyophilizing tissues are known in the art, a process that has been found to be suitable for lyophilizing bone is freeze drying for about 10 to 168 hours preferably about 20 to 28 hours. The residual PVP-I on lyophilized bones continues to offer protection until it is removed by washing or by body fluids after implantation. Also, the bone can be coated with other appropriate global decontamination agents, PVP-I, or mixtures thereof. Alternatively, the residual global decontamination agent can be removed by rinsing with sterile water or inactivated by chemical reaction. The color of the original whitish bone may be restored in this way. The iodophors can be chemically inactivated by adding a reducing agent such as sodium ascorbate or thiosulfate to the soaking solution after the required soaking time has elapsed. The reducing solution must be of a sufficient molarity and quantity to inactivate the remaining molecular iodine. For example, 50 to 100 microliters of a 1 M sodium ascorbate solution should be sufficient to inactivate 10 milliliters of 1 percent PVP-I. This treatment returns to the solution again from a dark brown to a light color and returns the bone to its natural color. After the terminal decontamination step, the bone can be lyophilized or cryopreserved or frozen fresh for storage.

It should be appreciated by those skilled in the art that bone treated in the manner disclosed herein is appropriate for all therapeutic uses for which bone is required, eg, bone transplants, hip surgery and surgery. knee.

DETAILED DESCRIPTION OF THE PRESENT INVENTION Introduction It is recognized that there is a need for technologies capable of providing bone for transplantation that effectively minimizes the serious risks of ineffectiveness and immunogenicity associated with current transplant procedures. Unfortunately, bone preparation for transplant purposes has shown that it is a very difficult problem due to the presence of bone structure (the internal matrix) which is recognized to substantially prevent the penetration into the bone of effective amounts of useful substances in the bone. the cleaning and decontamination of it. Bone is a specialized form of connective tissue that owes its hardness to the deposition of a mineral substance in a soft organic matrix. The internal matrix of the bone is understood as a technique that refers to the materials found within the bone, whether it is cortical (compact) or spongy (trabecular) type that are typically found in the structure and including, for example, bone fluids, extravascular and vascular fluids, matrix of calcified bone, bone marrow (including red or fatty marrow) and cells thereof, osteogenic cells, extracellular and intracellular lipids and erythrocytes. These materials (except usually the calcified bone matrix) are recognized in the art to be removable materials, that is, they are preferably removed from the bone during cleaning and / or decontamination thereof even when it is recognized that these materials have affinity typically a considerable affinity for the bone and the spaces defined in it occupy. For purposes of the invention, the removable matrix material is said to have considerable affinity for a bone sample when a water-based cleaning solution or decontamination solution in contact with it can not dislodge a considerable portion of the material when The solution is provided for about 1 to 5 minutes as a stream at room temperature from a source tapped from the normal household key.

As provided in the original embodiment of the invention, the aforementioned materials of the internal matrix interfere considerably with the cleaning and decontamination of the bone. Manifesting widely, the developments of the present embodiment of the invention provide the important discovery that the removal of these matrix materials, either directly or indirectly, from the bone intended for transplantation is greatly facilitated by placing the bone sample in an environment where it is contacted with (or placed in) an atmosphere of less than ambient pressure. In accordance with the practice of the invention, it is understood as the ambient pressure, approximately 1.0 atmosphere of gas pressure. It has also been discovered in accordance with the practice of the invention that the lipid, whether found as an extracellular or intracellular lipid in the matrix, tends to immobilize the removable components of the matrix considerably (eg, marrow, cells, macromolecules). arithigenic, or garbage of any of them, and also the lipid itself) preventing or limiting the removal of these components and by cleaning and decontamination procedures such as, for example, those described in the original embodiment herein. Without wishing to be bound by any theory, it is believed that aqueous cleansing or decontamination solutions effectively penetrate the internal bone matrix due to the presence of packed medullary cells and a hydrophobic lipid barrier. This effect is considerable since it is known, for example, that fat cells are an important component of the bone matrix, the yellow bone marrow being composed almost entirely of fat cells (see for example by Y Tanaka and VR Goodman "Electron Microscopy of Human Blood Cells, "Chapter 7 on page 380, Harper and Row, New York, NY 1972. Accordingly, a preferred aspect of the present embodiment of the invention involves treating the internal matrix of the bone containing a predetermined amount of removable material. , the matrix also contains a predetermined amount of lipid that immobilizes the removable material considerably, the method comprises the step of contacting the matrix with an atmosphere less than the ambient pressure for an effective period of time to reduce the lipid content to less of the predetermined amount of the same. An amount of lipid manifests itself as immobilizing considerably the removable material of the bone matrix when a water-based cleaning or decontamination solution can not dislodge the material when the solution is provided in contact with the bone sample for about 1 to 5 minutes such as a stream at room temperature from a pressure source of the domestic tap approximately normal. For the purposes of the invention, "lipid" includes all substances recognized as such by the technique including for example triglycerides, free fatty acids, cholesterol and esters thereof, and lipids "polar" such as lecithin and sphingomyelin.

Preparation of the Transplantable Bone The appropriate bone for transplantation was prepared using a process comprising the step of contacting the bone with an atmosphere at less than ambient pressure and at least one additional step either before or after contact, comprising also contact the bone with a solution consisting of a detergent or a decontamination agent. Preferably, at least one additional step is carried out after contact with a low pressure atmosphere in order to take advantage of the opening of channels towards the matrix that is caused by the removal of lipid. The original embodiment of the invention defines a large number of additional cleaning or decontamination steps including those carried out at elevated temperature that can be combined in a sequence to define a specific cleaning or decontamination procedure. Preferred decontamination agents include, as above, ethyl alcohol, hydrogen peroxide, chlorhexidine, hypochlorite and iodophors such as PVP-I (which have a molecular weight of less than approximately 100,000). In this connection, the term "elevated temperature" refers to a temperature of about 37 ° C or higher, preferably from about 37 ° C to about 80 ° C and, in general, particularly preferably from about 50 ° C to approximately 65 ° C, even though optimum temperatures can be determined for individual samples. The cleaning and decontamination steps can be carried out in accordance with the practice of the original and present embodiments of the invention at less than 37 ° C. Preferred and representative examples of the present embodiment are given in Examples 6 to 9 which will be given below. The term "bone", as used in accordance with the practice of the present development of the invention, is used in the most general sense and includes all types of human or animal bone tissue including whole bones, pieces of bone, blocks of bone with fixed connective tissues such as ligaments and tendons, the sample being capable of restoring the natural integrity of the bone in a patient, and of supporting the weight at a transplant site, typically having these pieces of bone at least one dimension of approximately 10 millimeters or greater. Bone samples that are representative of those useful in the practice of the invention include the following products obtainable from Cryolife Orthopedics, Inc., of Marietta, GA (typically sold under the VIP trademark, Viral Inactivation Process): a block spongy; spongy cubes; condyl, whole; cortical strips; pivot, bicortical "cloward", pivot, bicortical crock; pivot; "tricortical" crock "of the ilium, pivot," crock "protero-tricortical: femur, distant, head of the femur, fibula, entire, head of the humerus, ilium, entire, plate of the ilium, he-jaw, pelvis, entire, rib , medium, rotating bracelet, and whole cube Excluded from the definition of bone in the present are the preparations of ground bone, the ground demineralized bone preparations and the bone cuttings and all are excluded categories that have in common the following particularities : (1) they are not bone structures that carry weight since they can not sustain the weight in a therapeutic context, for example being less than about 5 millimeters thick of the cortical bone of a tibia or femur in a patient; ) due to the very high surface area or volume ratio of cuttings or a sample of ground bone, the lipid thereof does not participate significantly in the immobilization of the removable material therein. unless the ambient value is within the practice of the invention even though it has generally been found (see Examples 6 to 10 below) that pressures of about 0.7 atmosphere are generally required or less for a useful effect, with pressures from about 0.3 atmosphere to about 0.1 atmosphere being especially preferred. It will be noted that large pieces of bone (for example the head of the femoral, the distant femur and the proximal tibia) that have large spongy regions are best treated at pressures of about 0.2 to 0.13 atmosphere. Optimal atmospheric pressures for use with specific bone pieces can be determined for specific applications and may depend on the specific sequence and the combination of other cleaning or decontamination steps that have preceded one or more of the contact steps of low atmospheric pressure. The optimum temperature for contacting the bone with a pressure lower than the ambient is usually approximately 20 ° C to 60 ° C. Similarly, the optimal times to maintain the pressure unless the ambient pressure and usually fall within the range of 30 to 60 minutes that can be determined for each application by monitoring the progress of blood and lipid extraction (see Example 10). In general, the use of a gas pressure lower than the ambient pressure for less than two minutes will be ineffective and use for more than five hours will not confer any additional benefit. In relation to the practice of the invention, however, and with respect to bringing to the optimum the conditions of the low atmospheric pressure useful for it, it has been determined that the use of too low a pressure is generally ineffective. The use of too low pressure causes a freezing effect not unlike that seen in the lyophilization equipment, ie, the removable bone materials leave the bone quickly first but then an additional cleaning or decontamination fails due to the freezing in situ of the materials including the lipid. The precise low pressure at which a fault occurs varies as a function of numerous parameters including freezing temperature of the lipids, blood and marrow, and the surface area of the bone with an atmosphere of 0.001 being representative of an unacceptable pressure. The scale of unacceptable low pressures can be determined for any specific combination of the bone sample (s) and the equipment. It has also been discovered that direct or indirect contact of an atmosphere at less than 1.0 atmosphere of pressure with the internal matrix of a bone sample is facilitated by drilling one or more holes of sufficient depth of the sample (see, for example, Examples 7 to 10 and Figures 1 to 4 which are presented below). In connection with this aspect of the invention, the following serves as a guideline for the practice of the invention: (1) where possible, the hole or holes should be drilled towards a region of the bone sample bearing minimal weight as It will be recognized in the technique. For example, see Figures 1 to 4 in the case of a femur. (2) Preferably, the holes are between 5 and 100 millimeters in depth depending on the nature of the bone sample and with about 10 to about 30 millimeters in depth preferred for many human bone samples (see Examples 7 to 10) ), including the human femur. Preferably the holes are of a diameter between 0.5 and about 3 millimeters in diameter, even when the number, size, depth and placement of the holes are subject to the wide discretion of the physician. The following representative examples serve to illustrate and describe more particularly the original and present embodiments of the invention.

EXAMPLE 1 Step 1: Primary Decontamination Human bone was harvested, cultured and found to be contaminated with a variety of bacteria and fungi including: The species Staphylococcus (negative coagulase) The species Enterococcus Candida albicans Acinetobacter anitratus Klebsiella pneumoniae. The ilium was soaked in a 5 percent solution of PVP-I (polyvinyl pyrrolidone-iodine, C15 complex of GAF). The bulky outer tissue and fat were removed, the bone was returned to 5 percent PVP-I for a total time of one (1) hour and the thickness was tested (in five duplicates) to determine residual contamination. The following table shows a comparison of the present method with incubations in a saline solution, the positive control and the bacitracin / polymyxin cocktail.

Total Microorganism / bone solution Pre-treatment 8,700 Post-treatment Saline solution 11,000 Antibiotic cocktail 4,300 5% PVP-I 330 Results indicate that 5 percent PVP-I is superior to antibiotic treatment to reduce the number of infectious organisms . Step 2: Cleaning The bone was transferred to a bottle with the screw top containing 1 percent (by volume) of octylphenoxypolyethoxyethanol, (Triton X-100-R) at 37 ° C and stirred vigorously in a can shaker Of paint (Model Number 0-5400-OM manufactured by Red Devil) during minutes. After transferring the bone to a clean solution of Triton X-10 (R) in 1 hot percent, the bone was incubated overnight (approximately 15 to 18 hours at 37 ° C to 42 ° C). and stirred vigorously for 10 minutes. The bone was transferred to 1 percent fresh Triton X-10 (R) and again vigorously stirred for 10 minutes. Any remaining marrow was removed by washing with sterile water. Then, the bone was placed in 3 percent hydrogen peroxide, stirred for 10 minutes, and incubated for a total time of 60 minutes. The clean bone was thoroughly washed with sterile water washing and rinsing repeatedly until there was no evidence of the detergent foam.

Step 3: Terminal Decontamination The clean bone was placed in 1 percent PVP-I at room temperature, stirred vigorously for 10 minutes and incubated for a total time of 30 minutes and removed from the solution.

Step 4: Storage If desired, PVP-I can be allowed to dry on the bone giving the bone a rich golden color and additional protection against infectious agents. The coated bone can then be lyophilized. Also, if desired, the bone can be coated with PVP allowing the PVP to dry on the bone.

EXAMPLE 2 Step 1. Primary Decontamination A human knee en bloc was harvested medianate the local procurement agency was packed and embarked on wet ice to a bone processing laboratory. In the processing laboratory, the knee was placed in 1 percent to 5 percent PVP-I, 0.15M sodium chloride for 10 to 60 minutes.

Step 2: Preparation and Cleaning of the Tissue The following knee tissues were removed with adjacent bone blocks: - patella tendon - posterior cruciate ligament - anterior cruciate ligament - menisci The pieces were trimmed to remove excess tissue and fat. The ligament or tendon was wrapped in a sterile coating (eg, a plastic wrap or sterile wrap) while the bone blocks were cleaned by washing with hot 1% Triton X-100 (R) (from 40 °). C at 65 ° C followed by complete rinsing with sterile water or a saline solution.

Step 3. Terminal sterilization The tissues were placed in 1 percent PVP-I, 0.15 M sodium chloride, shaking lightly for 1 hour at room temperature and rinsing thoroughly with sterile water or saline. Each piece was cryopreserved, packed and stored in liquid nitrogen.

EXAMPLE 2 (Supplement) It has been determined that with respect to the use of Triton X-10 (R) in Step 2 of Example 2, that 0.1 percent is a preferred concentration thereof.

EXAMPLE 3 Step 1. Primary decontamination Human diaphyseal bones were harvested by the local procurement agency, packaged and shipped on wet ice to the bone processing laboratory. The processing laboratory placed the bones in 5 percent PVP-I, 1 percent Triton X-10? (R) for 10 to 60 minutes.

Step 2. Preparation and Cleaning of the Tissue The bones were debrided to remove excess tissue and fat, placed in 1 percent PVP-I 1 percent Triton X-10 ™ (R). Then, the bones were further cleaned by washing and incubating in 1 percent warm Triton-X-100 (R) (40 ° C to 65 ° C) followed by thorough rinsing with sterile water. The bones were ground into chips in a bone mill, rinsed with sterile water and lyophilized. The chips were milled to a finer size in a Tekmar mill.

Step 3. Demineralization The bone powder was demineralized with 0.6N cold hydrochloric acid and rinsed with sterile water.

Step 4. Terminal Sterilization The demineralized powder was placed in 1 percent PVP-I for 1 hour, rinsed thoroughly with sterile water. The powder was transferred into small bottles, lyophilized, packed and stored at room temperature.

EXAMPLE 4 Terminal sterilization with inactivation of PVP-I.

A bone, which was treated in a manner similar to the bone of Example 1, was placed in 20 milliliters of 1 percent PVP-I and incubated for 1 hour. After incubation, 0.132 milliliter of sodium ascorbate 0.91 was added. The solution almost immediately became crystal clear and after 10 minutes, the bone returned to its natural whitish color.

EXAMPLE 5 This example compares the results obtained by the cleaning method with high pressure / high temperature detergent with those obtained by normal methods. After cleaning, the femoral heads were separated to better show the degree of cleanliness. The head of the femoral shown on the right (Figure 5) was incubated at 60 ° C in 1 percent (by volume) of Tween 80 for 2 days with periodic agitation of 10 minutes using a paint can shaker (Model number 0- 5400-OM manufactured by Red Devil). The head of the femoral was then washed with lukewarm water, incubated in 3 percent hydrogen peroxide for 20 minutes, and then washed again with warm water to remove the hydrogen peroxide.

The head of the femoral that was shown on the left was cleaned according to normal methods. It was washed with water at 60 ° C for 15 minutes; it was incubated in 3 percent hydrogen peroxide for 15 to 20 minutes, washed again with water at 60 ° C to remove the hydrogen peroxide; it was incubated in 70 percent ethanol for 1 hour; and then washed with water at 60 ° C to remove the ethanol. Figure 5 is a photograph of the bones cleaned in this manner.

EXAMPLE 6 Treatment of a Human Femur With Atmosphere At Least of Ambient Pressure The femur of a human donor was debrided and cut about 15 centimeters from the far end. The distant part was placed in a vacuum desiccator in a vertical position in such a way that the head of the fer was in the upper part and the arrow was in the lower position. A vacuum (approximately 635 millimeters of mercury resulting in a chamber pressure of 0.15 atmosphere) was applied for 1 hour using a commercial vacuum pump (Gast, model DOA-P104-AA). The temperature of the bone remained from 20 percent to 25 percent during the procedure. Approximately 37 milliliters of the lipid was collected. The bone was then washed and placed in an aqueous solution of 0.1 percent Triton X-10 ™ (R 'at 60 ° C, and then stirred vigorously for 10 minutes on a shaker (a commercial paint shaker, Red Devil model 5400-CM) .Incubation was continued at 60 ° C for approximately 3 hours.The bone was then rinsed with sterile water.

EXAMPLE 7 Use of an Atmosphere of Less than Ambient Pressure to Facilitate Displacement of Removable Material from the Internal Matrix of Multiple Bone Samples The human bone was debrided and cut into pieces including a proximal femur, a proximal tibia, spongy pivots and ilium wedges. The bone samples were then incubated in 5 percent of an iodopher (PVP-I, as mentioned above, product of BASF 30/06) for 50 minutes, followed by transfer and incubation, in 1 percent of the aforementioned iodophor for between 30 and 60 minutes. Then holes (1 millimeter by 10 millimeters cylindrical) were drilled in the femur and tibia pieces as shown in Figures 1 to 4 (excluding pivots). All the pieces were then placed in a vacuum desiccator in such a way that the bone samples were placed therein above a recipient container for the lipids. Vacuum was applied for one hour using a Gast pump model DOA-P104-AA resulting in a chamber pressure of 635 millimeters of mercury (0.16 atmosphere) with the bone being maintained at a temperature of about 23 ° C. Approximately 37 milliliters of lipid and 7.5 milliliters of blood were collected.

EXAMPLE 8 Use of an Atmosphere of Less than Ambient Pressure to Facilitate the Displacement of Removable Material from an Ilio Wedge A wedge of ilium prepared according to the procedure of Example 7 (approximately 3.81 centimeters in length, and 1.90 millimeters on the transverse side) was preheated for 1 hour in a 0.1 percent solution of Triton X-100 (R) in water at 60 ° C, with occasional slight agitation and then transferred to the 50 milliliter capacity collection tube. Vacuum (620 millimeters of mercury) was applied for 80 minutes leading to a chamber pressure of 0.15 atmosphere (with bone temperature remaining at 23 ° C). Approximately 0.8 milliliter of lipid was collected over a period of 30 minutes.

EXAMPLE 9 Treatment of Bone Samples of the Human Femur For this procedure, samples from the right and left distant femurs of a human donor were cut approximately 13.97 centimeters inward from the distant ends thereof. The femur sample on the right side was treated by a process known in the art as described above using a polymyxin / bacitracin solution while the sample from the left femur was treated by a viral inactivation process as described in FIG. directly described below (see also Example 1). Accordingly, the sample on the left side of the bone was soaked in a 5 percent solution of iodophor (5 percent PVP-I, product of BASF 30/06), for approximately 1 hour, and then transferred to a volume of 1/2 liter of a fresh iodophor solution (1% PVP-I, product of BASF 30/06) for about 1 hour, after which it was debridged. Then, approximately 5 holes (1 to 2 millimeters wide and 10 to 20 millimeters deep) were punched through both the cortical regions and also the regions of cancellous bone as shown in Figure 4. After rinsing with warm water and dried, the bone was placed in a vacuum desiccator and a vacuum of approximately 26 millimeters of mercury was applied using a Gast Model DAA-V175-E8 pump for 30 minutes leading to the presence through the bone of a pressure of 0.132 atmosphere . The bone temperature of approximately 23 ° C to 35 ° C or slightly warmer. Approximately 30 milliliters of lipid was removed by the vacuum treatment. After washing with warm water for 5 minutes, the bone was placed in a 0.1 percent surfactant solution and stirred as in Example 8, for 10 minutes. The fresh surfactant solution was then added and the process repeated until the solution was quite clear. The bone was then incubated with an additional volume of the surfactant solution for three hours at 60 ° C. After a rinse with water, the bone was transferred for 5 to 15 minutes to an aqueous solution of 3 percent peroxide, and then rinsed with water. The clean bone was then transferred to a 1 percent iodophor solution (PVP-I) for one hour, rinsed with water, after which the container was refilled with water., A reducing agent, thiosulfate Sodium was added to convert any residual iodine into iodide and the bone was rinsed thoroughly with water for storage. In order to compare the transplantable bone of the invention with that which was available from the traditional methods, the right femur sample was treated as mentioned, already according to the process recognized in the polymyxin / bacitracin technique. Therefore, the right femur sample was debrided and placed in a container of warm water. The container was manually stirred, the water containing lipid was discarded and fresh hot water was added. The process was repeated until the amount of lipid in the water was minimal (approximately 0.5 hour). The bone resulting from the traditional process retained characteristics that demonstrate the lowest level of clinical utility, for example a reddish brown color due to residual blood and oily residue due to residual liquid.

EXAMPLE 10 Demonstration of the Blood and Lipid Flow of a Femur as Function of Atmospheric Pressure (Less than Environment).

The effective atmospheric pressure was determined for a single piece of bone (distant human femur with perforated holes of 1 to 2 millimeters as shown in Figure 4). A manual vacuum pump was used to decrease the air pressure in increments of 63.50 millimeters of mercury (approximately 0.08 atmosphere each). The bone was maintained at each vacuum setting for 5 minutes and the number of blood and / or lipid drops produced was determined for each period of time. The results of 10 show in Table 1 that is presented below.

Vacuum% of Atmos-Volume Characterization of Femal millimeters (drops / 5 Mercury droplet minutes) 15 0 1.0 3 63.50 .92 2 Blood only 127.- 4 Blood only * - * 190.50 1 Blood only -r 3 254.- .66 5 2 lipids + 3 Blood 317. 50 Lipid only 381.- 11 Lipid only 444.50 18 508.- 28 25 571.50 38 635.- .16 82

Claims (27)

CLAIMS:

1. A method for preparing bone for transplantation, the bone contains the internal matrix itself comprising the removable material, the method comprising the step of contacting the bone with an atmosphere at less than ambient pressure.

2. A method according to claim 1, wherein the bone is kept in contact with a decontamination agent or a detergent during a period of contact of the atmosphere with the bone.

The method according to claim 1, comprising an additional step of contacting the bone or matrix thereof with a solution comprising a detergent or a decontamination agent. .

A method according to claim 3, comprising the step of contacting the bone or matrix thereof, prior to contact with the atmosphere, with a detergent or with a decontamination agent; and further comprising the step of contacting the bone or matrix thereof, subsequent to contact with the atmosphere, with a detergent or with a decontamination agent.

5. A method according to claim 3, wherein the decontamination agent has the ability to inactivate one or more species or strains of bacteria, fungi, viruses, or parasites.

6. A method according to claim 1, wherein the bone is subjected to a high pressure washing condition before or after contact with the atmosphere.

7. A method according to claim 6, wherein the high pressure washing condition comprises washing with a high speed liquid stream.

8. A method according to claim 6, wherein the condition of high pressure washing comprises vigorous stirring.

9. A method according to claim 1, wherein the bone is subjected to an elevated temperature before, during or after contact with the atmosphere.

10. A method according to claim 9, wherein the elevated temperature is between about 37 ° and about 80 ° C.

11. A method according to claim 10, wherein the elevated temperature is between about 50 ° C and about 65 ° C. - 41

12. A method according to claim 3, wherein the decontamination agent is selected from the group consisting of ethyl alcohol, hydrogen peroxide, chlorhexidine, hypochlorite and an iodophor.

13. A method according to claim 12, wherein the agent is an iodopean.

14. A method according to claim 13, wherein the iodopene is polyvinyl pyrrolidone iodide having a molecular weight of less than about 100,000.

15. A method according to claim 1, wherein the contact of the atmosphere with the matrix is facilitated by piercing one or more holes in the bone.

16. A method according to claim 15 wherein one or more holes are drilled in a region of the bone bearing the minimum weight.

17. A method according to claim 16 wherein the holes are between about 0.5 millimeter and about 3 millimeters in diameter.

18. A method according to claim 17, wherein the holes are between about 10 millimeters and about 30 millimeters deep.

19. A method according to claim 1, wherein the atmosphere has a pressure of about 0.7 atmosphere or less, and is maintained in contact with the bone for between about 2 minutes and about 5 hours.

20. A method according to claim 19, wherein the atmosphere has a pressure of about 0.3 atmosphere or less, and is kept in contact with the bone for about 30 and about 60 minutes.

21. A method according to claim 19, wherein the atmosphere has a pressure of about 0.2 atmosphere or less, and is kept in contact with the bone sample for between about 30 and about 60 minutes.

22. A method according to claim 1 which facilitates the inactivation of the virus.

23. A method for treating bone containing the internal matrix itself comprising a predetermined amount of removable material that has considerable affinity for bone, the method comprises the step of contacting the matrix with an atmosphere at less than the ambient pressure and then maintaining the atmosphere in contact therewith for an effective time to reduce the amount of the removable material to less than the predetermined value thereof.

24. A method for treating the internal bone matrix containing a predetermined amount of removable material, the matrix also contains a predetermined amount of lipid that immobilizes the removable material considerably, the method comprises the step of contacting the matrix with an atmosphere less than the ambient pressure for a period of time effective to reduce the lipid content to less than the predetermined amount thereof.

25. A method for treating bone, the bone contains a section of internal matrix having an ambient pressure that in the defined space thereof there is a predetermined amount of removable material, the method comprises subjecting the external space to and in contact with the matrix , at an atmosphere of less than ambient pressure for a period of time sufficient to reduce the amount of removable material remaining in the defined space less than the predetermined amount of the site.

26. A bone suitable for transplantation in a patient produced in accordance with the method of claim 1.

27. The bone suitable for transplantation in a patient produced in accordance with the method of claim 24.