ES2392725A1 - Method for the processing of the alogenic bone tissue and recipient to carry out it (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Method for the processing of allogeneic bone tissue, and container to carry it out, which comprises the following four successive application procedures: - individualized washing of each bone allograft in an airtight container and immersed in physiological saline at 60ºC for 10 minutes followed by 1 minute of vigorous manual agitation. - individualized centrifugation of each allograft, at 2,000 revolutions per minute for 15 minutes for 1500 g. In a specific pvc container. - individualized pasteurization of each allograft for 90 minutes in sterile, hermetic container with physiological saline preheated to 60º c. - final microbiological control, weighing the pasteurization serum through paper filter with pore size of 0.45 μm, and cultivating said filter in culture medium for aerobic, anaerobic and fungal microorganisms. (Machine-translation by Google Translate, not legally binding)

Description

Method for the processing of allogeneic bone tissue and container to carry it out.

OBJECT OF THE INVENTION

The invention, as stated in the present specification, refers to a method for the processing of allogeneic bone tissue and a container for carrying it out.

More particularly, the object of the invention is focused on the development of a new processing methodology for bone allografts, which meets the characteristics of simplicity, effectiveness, effectiveness and efficiency, allowing allografts of higher biological quality to be obtained than those obtained with other methods. processing, assuming, therefore, an improved alternative to what is already known in this field. Furthermore, the invention comprises a second aspect related to a specific container adapted to carry out one of the process steps.

BACKGROUND OF THE INVENTION

As it is known, we call any biological material composed of bone tissue, tendon or combination of both, extracted from its original site to be used in a place other than this.

Osteotendinous allografts are carriers of cellular and humoral elements capable of producing a host immune reaction against graft in the recipient.

The cellular content of the graft (bone tissue cells, tissue stromal cells, hematopoietic precursors, vessels and nerves) undergoes ischemic necrosis phenomena from the moment of their extraction (autografts) or from the time of donor death (allografts); The duration of ischemia and its cold or hot character determines irreversible changes inside the graft in a double way: on the one hand there is deterioration of the organic matrix and inactivation of osteoinductive humoral factors (BMPs, cytokines of the family of TGF type, related to the collagen content of the matrix), on the other hand necrosis phenomena favor graft contamination.

Both phenomena should be avoided to guarantee the quality of the grafts. In addition, hydrolysis and oxidation reactions such as lipid peroxidation can occur.

All these phenomena highly harmful to the quality and even graft survival need the presence of "free water" (15, 33) inside the tissue. In fact, the immobilization of free water is one of the main objectives that must be met by the preservation of osteotendinous allografts, which can be achieved through several routes:

-

 Freezing: Below –80º there is no free water in the tissue stroma (H2O molecules are immobilized in the form of ice crystals), in addition to the effect of cell destruction caused by rapid freezing. Freezing (20, 26, 28, 29, 34, 41) is, therefore, an excellent means of preserving osteotendinous grafts.

-

 Lyophilization (17): Application of a freeze-dehydration procedure (sublimation of water content from solid ice particles to water vapor). Dehydration from the solid ice phase also prevents protein denaturation that can occur during the isolated freezing process.

-

 The third method to immobilize free water is immersion in fluids with a very high concentration of solutes. The salting of certain foods as a method of conservation for prolonged periods achieved precisely the immobilization of free water, preventing or delaying the phenomena of putrefaction. In the case of tissues for clinical use, glycerol is sometimes used at a certain concentration, especially for the skin and to obtain demineralized bone matrix (27, 33).

The incorporation of osteotendinous allografts is achieved through osteoconduction and osteoinduction mechanisms. Allografts are not direct osteoformers of bone tissue. Therefore, everything that improves those two mechanisms will be beneficial for the final result.

The allogeneic bone tissue is composed of the bone matrix, the own bone tissue cells (osteoblasts / osteocytes and osteoclasts), hematopoietic tissue, fatty bone marrow, vessels and nerves, all of them included in the three-dimensional network of calcium salts that constitutes the support Physical bone tissue.

In view of the optimal biological properties of an osteotendinous allograft, the

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preservation of the bone matrix, both its organic and inorganic fraction. The remaining tissue components lack beneficial properties and could, or should, be eliminated.

From the point of view of osteoinduction phenomena, the most important thing is the preservation of the bone matrix, composed of collagen fibers and other proteins (cell adhesion proteins, osteocalcin, BMPs, etc.).

Thanks to the combination of the two phases of the bone matrix, organic and inorganic, the bone tissue has its characteristic mechanical properties, combining two apparently contradictory properties such as stiffness and elasticity.

Collagen fibers flexibility and resistance to bending, torsion and tension. The mineralized matrix confers resistance to compression and the hardness necessary to support the body as a whole.

From the point of view of osteoconduction phenomena, the important thing is to keep the mineralized bone matrix intact, that is, the physical structure of the bone tissue.

There is evidence that the elimination of certain components of the organic matrix of allografts improves their biological properties:

-

 The elimination of lipid content, abundant material especially in metaphysodiaphyseal areas, improves the incorporation of allografts. Experimental animal studies comparing the biological behavior of allografts with and without lipid removal, showed that those subjected to lipid content removal (4, 17, 26, 39, 40, 44) by chloroform-methanol showed:

-

 Better incorporation

-

 Histomorphometry: Increased formation and bone resorption.

-

Scintigraphy at 3 weeks of the implant: isotope uptake by the graft (higher rate of bone remodeling).

-

 Conservation of mechanical properties.

-

 The presence of fat inside the allografts can make certain clinical applications difficult: the impact of spongy tissue fragments for cavity filling, during hip replacement surgery, is facilitated if the fragments have been washed to remove the fat content . Otherwise, since fat is an incompressible liquid, mechanical stressing efforts will be less effective (7) and some tendency to slip due to the lubricating effect of the grease itself will persist.

-

The elimination of free protein content decreases the risk of transmission of

diseases carried by prions (5,8, 10, 11, 13, 18, 19, 25, 31, 38, 42, 43)

.

-

 The reduction of cellular content decreases the immunogenic capacity of allografts and reduces the possibility of intracellular pathogen transmission: unprocessed bone tissue can be a vehicle for the transmission of pathogens, including HIV, HVC and HTLV. We must also take into account that the tests we have are capable of detecting the pathogens known today, but at any time, new ones, intra or extracellular, that cannot be detected until the knowledge of the disease transmitted and transmitted develop reliable screening tests, something similar to what happened with HIV during the first years of the 80s (9).

On the other hand, the application of low heat doses (up to 60º C and up to 10 hours) destroys bacteria and pathogenic viruses without altering the mechanical properties or clinical effectiveness (3, 6, 12, 16, 21, 24, 30, 32 , 35-37).

The comprehensive review of the literature on current trends in the processing of osteotendinous allografts, in particular the work of Lomas (26), Galea (17) and Yates (44), allowed us to glimpse a new design in this field of activity where , as mentioned above, there are some basic mandatory standards (legal regulations), but also a wide range of possibilities for biological and microbiological improvement of allografts for clinical use.

Taking into account the current scientific evidence indicating the desirability of eliminating certain components of the organic matrix, it seems equally evident the need to develop the necessary methodology to achieve such biological improvement without the added manipulation resulting in an increase in pollution. The achievement of this balance is not easy, but scientific research in this field should advance in that line:

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1 - Eliminate as far as possible the lipid content inside the allografts.

2 - Eliminate as much as possible the free protein content inside the allografts.

3 - Remove the cellular content inside the grafts.

4 - Keep the protein fraction with osteoinductive abilities.

5 - Preserve the three-dimensional mineral structure of bone tissue.

6 - Get, at the end of the procedure, a tissue free of microorganisms transmissible to the recipient.

In practice it is difficult or impossible to establish standards regarding the tissue and cellular content of a bone segment, given the variability that exists not only between different individuals but also between different bone fragments of the same individual.

On the other hand, the physical structure of each segment has a lot to do with the ease or difficulty of the process: spongy bone allografts with a more open structure, such as vertebral bodies, facilitate the elimination of humoral and cellular content quickly and easily. , very different from other bones, also spongy but much denser, like the one that makes up a femoral condyle.

For this reason, the objective of the present invention is to develop a new method of processing for bone allografts, looking for it to be sufficiently efficient and quick to be applied immediately, as well as sufficiently safe from the microbiological point of view to ensure non-transmission. of disease In short, a method that meets the following requirements:

-

 Safe from the point of view of the transmission of infectious or other diseases.

-

 Effective from the biological point of view.

-

 Reproducible.

-

Superior to other known methods.

-

 Efficient from the point of view of the clinical cost / benefit ratio.

On the other hand and as a reference to the state of the art, it should be noted that the applicant is unaware of the existence of any other method or procedure of similar application that has similar characteristics to those presented here, being the details that characterize it conveniently set out in the final claims that accompany the present specification.

EXPLANATION OF THE INVENTION

Thus, the objective of the invention is to develop a processing methodology for allogeneic osteotendinous tissue, which allows the elimination of the blood material (blood and bone marrow), protein and lipid from the osteotendinous tissue in the greatest possible amount. This must be achieved without involving prolonging and / or complicating the technique to the point of making it unfeasible for clinical practice, either because of its excessive prolongation over time, or because the proposed changes increase the risk of microbiological contamination of allografts.

The elimination of these elements, considered harmful to the biological behavior of the grafts, can be carried out by various procedures, from the application of chemical solvents to the use of mechanical mobilization systems of cells and lipid and protein components.

After reviewing the different known lines of research, the use of physical methods of cleaning the grafts is chosen, based on the experiences of the Anglo-Saxon researchers, and consists essentially of applying the following procedures to the osteotendinous tissue, in the sequence that is indicates:

one.

 Washing under vigorous agitation in an orbital incubator at 60º C.

2.

 Centrifugation

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3. Pasteurization. This methodology significantly simplifies the procedure designed by the Anglo-Saxon authors and shortens it over time, so that it is perfectly applicable to the real working conditions of the tissue banks in our environment. In addition, it does not require sophisticated technological means or, therefore, specific training by personnel already working in allogeneic tissue processing units, so it could be applied to the practice almost immediately. Specifically, the method for the processing of allogeneic bone tissue set forth below, object of the present invention as the original method, responds to the interest of obtaining bone grafts of higher biological quality than those obtained with other processing methods, eliminating inside, the greatest possible amount of lipids, soluble proteins and cells, achieving a better and faster incorporation after implantation in the recipient patient, together with a lower immune reaction of the latter against the graft and a lower probability of transmission of diseases transmitted by Bone allografts. For this, the proposed method consists of four procedures or successive application phases consisting of: First.- Washing of each allograft individually, in an airtight container and submerged in physiological serum at 60 ° C in temperature, under sterility conditions. The duration of this first procedure is 10 minutes, and will be followed by vigorous manual agitation for 1 minute in the same airtight wash container. Second.- Centrifugation of each bone allograft individually, maintaining sterility conditions, at 2,000 revolutions per minute (1,500 g) in a laboratory centrifuge (HERAEUS CRYOFUGE 8,000), for 15 minutes. It should be noted that the aforementioned centrifugation is performed inside a container designed and constructed specifically for this procedure in autoclave-resistant PVC, with a grid in its lower part to allow the exit of the materials to be removed and keep them separated from the allograft during centrifugation. . Said container will have the measures adapted to each centrifuge, preferably being

following interior and exterior measurements:

EXTERNAL MEASURES.-

Height: 120 mm.

Width: 99 mm.

Closing thread: 6 turns with 5 mm thread pitch.

INTERNAL MEASURES.-

Depth: 97 millimeters (with grid placed: 82 millimeters).

Width: 76 mm.

GRID MEASURES.-

Height: 15 mm

Width: 74.5 mm.

Hole diameter: 6 mm.

Third.- Pasteurization of each bone allograft individually. This procedure

Its purpose is the destruction by heat of bacteria, fungi and pathogenic viruses. Each allograft is introduced into a sterile hermetic container with an individual identification code for each allograft, adding preheated physiological serum at 60 degrees Celsius until the bone graft is completely covered. The containers, in groups of four, are introduced into the laboratory thermo-agitator (SELECT

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UNITRONIC 320 OR), with temperature control and stirring speed, the procedure being programmed at 60 degrees Celsius for 90 minutes at an orbital stirring speed of 40 cycles per minute.

Fourth and last.- Final microbiological control. After the pasteurization phase, the microbiological control that completes the process is carried out.

To do this, under a laminar flow hood to maintain sterility conditions at all times, the serum in which the pasteurization was carried out is passed through a paper filter with a pore size of 0.45 μm, directly cultivating the filter in culture medium for aerobic, anaerobic and fungal microorganisms.

Given the foregoing, it is found that the new method proposed by the invention meets the characteristics of effectiveness, efficiency and simplicity of application, significantly improving the traditional methods for processing this kind of human tissues.

The new methodology has a high effectiveness for the reduction of the humoral and cellular content of the grafts, improving their capacity for integration by the recipient and reducing the probability of transmission of diseases carried by intracellular pathogens.

On the other hand, the application and use of the new processing methodology is simple and fast, and can easily be incorporated by any osteotendinous tissue bank. Its application times are perfectly compatible with the usual working hours of this type of units.

The washing / stirring procedures (10 minutes at 60 ° C) and centrifugation (1500 g for 15 minutes), very effective for the elimination of the humoral and cellular content of the allografts, have low antiseptic effectiveness. However, the pasteurization phase (90 minutes at 60 ° C), included as the final procedure of the new methodology, provides, according to our studies, 100% bactericidal and fungicidal efficacy.

The pasteurization procedure as a decontaminating method, associated with the final filtering as a method of detection of contaminated grafts, configures the new processing methodology as a process of high microbiological safety.

It should be mentioned that a comparative study of results between the traditional processing methodology and the new methodology presented here has been carried out. This study has been carried out under the real conditions of the daily practice of a tissue bank, with a sufficiently large sample (n = 693). Under these conditions, the contamination rate observed for grafts processed with the new methodology has been significantly lower than that of the traditional methodology: 2.08% versus 7.84% of the latter.

Finally, it should be mentioned that the cost / effectiveness of the new methodology is significantly better than that of the traditional methodology. The new methodology would allow savings greater than

108,000 euros for a period of 10 years, at the expense of a significant reduction in the rates of allograft contamination.

This represents a greater number of grafts available for clinical use, which would more than compensate for the investment in equipment necessary for its implementation, as well as the increase in costs derived from the greater complexity of the procedure.

It is therefore found that the described method for the processing of allogeneic bone tissue represents, an innovation of structural and constitutive characteristics unknown until now for this purpose, reasons that together with its practical utility, provide it with sufficient basis to obtain the privilege of exclusivity that is requested.

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 Lomas RJ, Cruse-Sawyer JE, Simpson C, Ingham E, Bojar R, Kearney JN. Assessment of the biological properties of human split skin allografts disinfected with peracetic acid and preserved in glycerol. Burns 2003;

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 Paulos L et al. Comparative material properties of allograft tissues for ligament replacement: effects of type, age,

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sterilization and preservation. Trans Orthop Res Soc 1987; 12: 129.

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32

 Pruss A, Seibold M, Benedix F, Frommelt L, Von Garrel T, Gürtler L et al. Validation of the ‘Marburg bone bank system’ for thermodisinfection of allogenic femoral head transplants using selected bacteria, fungi, and spores. Biol 2003; 31: 287-94.

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 Ross A, Kearney JN. The measurement of water activity in allogeneic skin grafts preserved using high concentration glycerol or propylene glycol. Cell Tissue Bank 2004; 5 (1): 37–44.

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 Salai M, Brosh T, Keller N, Perelman M, Dudkiewitz I. The effects of prolonged cryopreservation on the biomechanical properties of bone allografts: a microbiological, histological and mechanical study. Cell Tissue Bank 2000; 1 (1): 69-73.

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 Shimizu K, Masumi S, Yano H, Fukunaga T, Ikebe S, Shin S. Revascularization and new bone formation in heattreated bone grafts. Arch Orthop Trauma Surg 1999; 119 (1-2): 57-61.

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 Shin S, Yano H, Fukunaga T, Ikebe S, Shimizu K, Kaku N et al. Biomechanical properties of heat-treated bone grafts. Arch Orthop Trauma Surg 2005; 125 (1): 1-5.

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 Thoren K, Aspenberg P Increased bone ingrowth distance into lipid-extracted bank bone at 6 weeks. A titanium chamber study in allogeneic and syngeneic rats. Arch Orthop Trauma Surg 1995; 114 (3): 167-71.

40

 Thoren K, Aspenberg P, Thorngren KG. Lipid extracted bank bone: bone conductive and mechanical properties. Clin Orthop Rel Res 1995; 311: 232–46.

41.

 Van P, Thevelein J. Determinants of freeze tolerance in microorganisms, physiological importance and biotechnological applications. Adv Appl Microbioly 2003; 53: 129.

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Claims (8)

1.- METHOD FOR THE PROCESSING OF ALLOGENIC BONE TISSUE, characterized in that it comprises four procedures or successive application phases consisting of:

-

 Washing of each allograft individually, in an airtight container and submerged in physiological serum at 60 ° C temperature, in sterile conditions, with a duration of 10 minutes, and followed by vigorous manual agitation for 1 minute in the same airtight washing container .

-

 Centrifugation of each bone allograft individually, maintaining sterility conditions, at 2,000 revolutions per minute (1,500 g) in a laboratory centrifuge for 15 minutes.

-

 Pasteurization of each bone allograft individually, with the purpose of destroying fungi and pathogenic bacteria by heat, for which it is introduced into a sterile sealed container, with an individual identification code for each allograft, adding preheated physiological serum at 60 degrees Celsius until the bone graft is completely covered, shaking for 90 minutes.

-

 Final microbiological control, for which, under laminar flow hood with sterile conditions, the serum in which the pasteurization was carried out is passed through a paper filter with a pore size of 0.45 μm, directly cultivating the filter in culture medium for aerobic, anaerobic and fungal microorganisms.

2. METHOD FOR THE PROCESSING OF THE ALLOGEN BONE TISSUE, according to claim 1, characterized in that in the pasteurization phase, the containers are introduced in groups of four in a laboratory thermo-agitator with temperature control and stirring speed, the procedure being programmed at 60 degrees Celsius and at an orbital agitation speed of 40 cycles per minute. 3.- CONTAINER for carrying out a method for the processing of allogeneic bone tissue as described in claim 1 or 2, specifically for carrying out the centrifugation step comprised in said method, characterized in that it consists of a vessel designed and constructed specifically for this procedure in autoclave-resistant PVC, with a grid in its lower part to allow the exit of the materials to be removed and keep them separated from the allograft during centrifugation. 4.- CONTAINER, according to claim 3, characterized in that said container has the following dimensions: EXTERNAL MEASURES.- Height: 120 mm. Width: 99 mm. Closing thread: 6 turns with 5 mm thread pitch. INTERIOR MEASURES.- Depth: 97 millimeters (with grid placed: 82 millimeters). Width: 76 mm. GRID MEASURES.- Height: 15 mm. Width: 74.5 mm. Hole diameter: 6 mm. SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201031896 SPAIN Date of submission of the application: 21.12.2010 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl. See Additional Sheet RELEVANT DOCUMENTS

Category

@ Documents cited Claims Affected

X

GARCÍA RODRÍGUEZ, LA. Improved processing project for osteotendinous allografts. Development and validation of a new processing methodology for allogeneic osteotendinous tissue. Doctoral thesis [on line], [recovered on]. Recovered from the internet: <URL: http://ruc.udc.es/dspace/handle/2183/7325>; <URL: https://www.educacion.gob.es/teseo/createpdf?origen=3&idFicha=295853>, especially pages 77-80, 87, 89, 94-98, 102, 215, 239. 1-4

TO

EP 1700582 A2 (ALLOGRAFT TISSUE SYSTEMS INC) 09/13/2006, the whole document 1-4

TO

US 2004219058 A1 (SHIMP LAWRENCE A ET AL.) 04/11/2004, the whole document 1-4

TO

US 5977432 A (WOLFINBARGER JR LLOYD ET AL.) 11/02/1999, the whole document 1-4

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 27.11.2012

Examiner J. Collado Martinez Page 1/6

REPORT OF THE STATE OF THE TECHNIQUE CLASSIFICATION OBJECT OF THE APPLICATION A61L2 / 04 (2006.01) B01L3 / 00 (2006.01) A61F2 / 28 (2006.01) A61L27 / 36 (2006.01) Minimum documentation sought (classification system followed by classification symbols) A61L, B01L Electronic databases consulted during the search (name of the database and, if possible, search terms used) INVENES, EPODOC, WPI, TXTUS, GOOGLE ACADEMIC Date of Written Opinion: 27.11.2012 Statement

Novelty (Art. 1 LP 11/198)

Claims Claims 1,2,4 3 IF NOT

Inventive activity (Art. 8.1 LP11 / 198)

Claims Claims 1-4 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Basis of Opinion.- This opinion has been made on the basis of the patent application as published.  1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number Publication Date

D01

GARCÍA RODRÍGUEZ, LA. Doctoral thesis [on line], [recovered on]. Recovered from the Internet: <URL: http://ruc.udc.es/dspace/handle/2183/7325>,<URL: https://www.educacion.gob.es/teseo/createpdf?origen=3&idFicha=295853 >,  

D02

EP 1700582 A2 13.09.2006

D03

US 2004219058 A1 04.11.2004

D04

US 5977432 A 02.11.1999

 2. Statement motivated according to articles 29. and 29.7 of the Regulations for the execution of Law 11/198, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The present application aims at a method of allogeneic bone tissue processing comprising the washing / agitation, centrifugation, pasteurization and final microbiological control stages of the processed material. Likewise, a container specially designed to carry out the centrifugation step of the method of the invention is the subject of the application. Document D01 discloses the development of a methodology applied to the processing of allogeneic osteotendinous tissue comprising the washing / agitation stages, centrifugation in a container specifically designed for the implementation of the method, pasteurization and microbiological control of the processed tissue (see pages 77 -80, 87, 89, 94-98, 102, 215, 239). Document D02 refers to the treatment of an osteochondral graft for later implantation. The osteochondral nucleus is subjected to a previous wash to a centrifugation stage. The centrifuged material is treated with a solution comprising antiseptic or antibiotic substances. Centrifugation is carried out in a chamber that has a graft compartment communicated with a lower receptacle where the removed material is deposited, and another compartment containing a solution to contact the graft (see the whole document). Document D03 discloses a method for the inactivation or reduction of tissue pathogenic organisms such as bone, which comprises dry centrifugation to remove tissue material and facilitate penetration of a solvent and, at least, subsequent centrifugation using a fluid that It allows inactivating or reducing tissue pathogens. Also, D03 includes a process to remove lipids and / or proteins from bone cavities by centrifuging with lipases and proteases and subsequent elimination of digested material (see the whole document). Document D04 discloses a procedure for the removal of bone marrow from portions of bone graft that combines washing / agitation with water, salts or decontaminating agents, or mixtures thereof, with centrifugation and sonication processes (see the whole document). 1.-NEW (Art. 6.1, LP 11/1986). 1.1.-Claims 1-2. The prior art document that is considered closest to the object of independent claim 1 is D01. This document aims at a method for the processing of allogeneic bone tissue comprising four successive stages:

-

Washing of each allograft individually, in an airtight container and submerged in physiological serum at 60 ° C, under sterile conditions, lasting 10 minutes, and followed by vigorous manual agitation for 1 minute in the same airtight washing container ( D01; pages 79, 102).

-

Centrifugation of each bone allograft individually, maintaining sterility conditions, at 2,000 revolutions per minute (1,500g) in a laboratory centrifuge for 15 minutes (D01; page 80).

-

Pasteurization of each bone allograft individually to destroy fungi and pathogenic bacteria, introducing the allograft in a sterile hermetic container, with physiological serum at 60 ° C until completely covering the bone graft, for 90 minutes without agitation (D01; pages 94-98).

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Final microbiological control For this, in a laminar flow hood, the serum in which the pasteurization was carried out is passed through a 0.45 μm pore size paper filter, directly cultivating the filter in media for aerobic, anaerobic and fungal microorganisms (D01; pages 87, 89, 102, 215, 239).

The comparison between D01 and claim 1 of the present application shows that there are differences in the pasteurization stage. First, D01 does not explicitly disclose the use of an individual identification code for each allograft. However, this is an irrelevant element for the appreciation of the novelty since the labeling of the samples constitutes a usual laboratory practice and would therefore be implicit in D01. On the contrary, D01 does not disclose that the physiological serum of the pasteurization stage is preheated and also explicitly excludes the agitation of the pasteurization stage, these constituting distinct differences with the method of the invention. Accordingly, it is considered that D01 does not disclose identically all the features of the method of the claim 1. Therefore, said claim 1 and its dependent claim 2 meet the requirement of novelty (art. 6.1, LP 11/1986). 1.2.-Claims 3-4. The document of the state of the art closest to the object of independent claim 3 is also D01. Said document discloses a container for carrying out the centrifugation step, this PVC container being resistant to autoclaving and with a grid in its lower part to allow the exit of the materials to be removed and keep them separated from the allograft during centrifugation (D01 ; page 80). Based on the foregoing, D01 identically anticipates all the elements of the container defined in claim 3, so that the object of said claim does not meet the requirement of novelty (art. 6.1, LP 11/1986). In opposition to the above, no explicit mention has been found in D01 of the measurements of the centrifuge containers. Therefore, the object of claim 4 is not disclosed in D01 and meets the requirement of novelty of art. 6.1 of LP 11/1986. 2.-INVENTIVE ACTIVITY (Art. 8.1, LP 11/1986). 2.1.-Claims 1-2. As already indicated, the difference between D01 and claim 1 of the present application is that in D01 the preheating of the serum in the pasteurization stage is not explicitly mentioned and in that said stage is carried out without agitation, while in the method of the invention the serum is preheated and agitation is applied during the 90 minutes of pasteurization. With regard to the preheating of the serum, it is understood that this would constitute an obvious alternative to proceed for a person skilled in the art in order to ensure that the actual treatment time at 60 ° C is as close as 90 minutes and not lower. As for agitation, it is noted that the technical problem to be solved by pasteurization is the elimination of possible pathogenic organisms from allografts. However, this problem is effectively solved by pasteurization without stirring D01 (D01; pages 240-243). Therefore, in the absence of an indication in the present application of the advantage or technical effect provided by the agitation, its inclusion in the pasteurization stage is considered to constitute a mere alternative for the person skilled in the art in view of D01 On the other hand, claim 2 defines the introduction of the containers into the thermo-agitator in groups of four for pasteurization, an orbital stirring speed of 40 cycles per minute being set. These elements are not disclosed in D01. However, D01 does contemplate the use in the pasteurization stage of a thermo-agitator like the one mentioned on page 12, lines 13-14 of the present application (D01; page 77), so that the particular placement of the containers in groups of four it is considered a simple procedural alternative that does not imply a contribution to the state of the art. As regards the setting of an orbital agitation rate of 40 cycles per minute, it has already been indicated that pasteurization with stirring is considered in itself a mere alternative to the method of pasteurization of D01 without stirring. Thus, the choice of a specific parameter of orbital velocity would, by extension, have the same consideration as a mere alternative procedure for the person skilled in the art. Consequently, the object of claims 1 and 2 lacks inventive activity in accordance with Art. 8.1 of LP 11/1986. Consequently, the object of claims 1 and 2 lacks inventive activity in accordance with Art. 8.1 of LP 11/1986. 2.2 Claim 4. Document D01 does not include the measurements of the PVC containers used in the centrifugation stage. However, it refers to a limitation of the size of the allograft samples to be processed which indicates that they cannot exceed 10 x 6 x 6 cm. These maximum allograft measurements are perfectly compatible with the capacity of the centrifugation vessels of claim 4, so that the dimensions defined in said claim are considered to be a mere choice of obvious parameters for a person skilled in the art in view of what was disclosed in D01. Therefore, claim 4 does not meet the inventive activity requirement set forth in Art. 8.1 of LP 11/1986. Documents D02-D04 reflect the state of the art and are not considered relevant for the purpose of assessing the novelty and inventive activity of the object of the present application.