IL261174A - Method for preparing an allograft material, product obtained, and uses thereof - Google Patents

Description

METHOD FOR PREPARING AN ALLOGRAFT MATERIAL, PRODUCT OBTAINED, AND USES THEREOF The present invention relates to a method for preparing an allograft material forming a sterile, lyophilized and virally inactivated membrane obtained from mammalian placenta and consisting of at least one membrane layer, this allograft material being suitable for use as a surgical graft. The at least one membrane layer is defined by an amniotic membrane, a chorionic membrane, a spongy tissue membrane or a chorioamniotic membrane obtained from mammalian placenta. The allograft material obtained according to the method is decellularized, virally inactivated and sterilized, and may be vacuum-stored for a period of up to five years at ambient temperature. 152 METHOD FOR PREPARING AN ALLOGRAFT MATERIAL, PRODUCT OBTAINED, AND USES THEREOF

[0001] The present invention relates, in general, to the production of membranes obtained from mammalian placenta and, more particularly, to a method for preparing an allograft material forming a sterile, lyophilized and virally inactivated membrane obtained from mammalian placental tissue and consisting of at least one membrane layer, this allograft material being suitable for use as a surgical graft.

[0002] The chorioamniotic membrane which separates the fetus from the maternal endometrium in mammals comprises the amniotic membrane, or amnios, and the chorionic membrane, or chorion. These two membranes are connected by a spongy tissue membrane, also referred to as spongy layer, consisting, inter alia, of collagen and proteoglycans, the spongy tissue membrane comprising protein bridges attached on both sides to the amnios, on the one hand, and to the chorion, on the other hand.

[0003] The amniotic membrane is the innermost layer of the chorioamniotic membrane. Its role is to protect the fetus and to maintain the amniotic fluid around the fetus. This amniotic membrane is a very fine, transparent tissue comprising several layers, namely an epithelial layer, a basal membrane, a compact layer, and a fibroblastic layer.

[0004] The chorionic membrane is the outermost layer of the chorioamniotic membrane, namely the layer in direct contact with the wall of the maternal uterus (the decidua). This chorionic membrane is a thick and fibrous tissue also comprising several layers, namely a reticular layer, a basal membrane, and a trophoblastic layer.

[0005] The amniotic membrane, obtained from the placenta after a delivery, is a tissue which has been used for more than a hundred years in the treatment of burns and wounds. Indeed, as early as 1910, Davies was using fetal membranes both on burns and on ulcerated tissues. Trelford and Trelford-Sauder report that, in 1935, authors published clinical applications of the amniotic membrane in vaginoplasty, conjunctival reconstruction, treatment of burns or wounds, treatments relating to intra-abdominal adhesion. It was in 1940 that De Roetth for the first time used a fetal membrane in reconstructive ophthalmology for treating conjunctival alterations of patients. Trelford et al. also report that, in 1952, Douglas used the amnios for treating extensive wounds. For the first time, it was indicated that the stromal layer of the amniotic membrane, comprising the compact layer and the fibroblastic layer, enabled stronger adhesion of the graft and thus its effectiveness. In 1972, the work of Trelford et al. confirmed this fact. Gindraux et al. report that, starting in 1972, and especially since its rediscovery in 1995, other authors confirmed all the previously presented clinical applications and also reported new indications such as the genitourinary tract,3 the stomach, the larynx, the oral cavity, the head and the neck, in clinical trials or in case reports.

[0006] Non-vascularized and not innervated, the amniotic membrane is rich in collagen and in various growth factors and has properties that participate in the cicatrization process.

[0007] With the recent progress in tissue engineering, it became necessary to use matrixes as support for the adhesion and for the proliferation of the cells and tissues, said matrices used as a scaffolding for this proliferation being derived from mammalian tissues. The amniotic membrane and the chorionic membrane are considered as an important source of support matrix in this field.

[0008] In the context of tissue graft activity, the amniotic membrane can be used as graft tissue which can be sutured or glued with a biological glue. The graft thus implanted serves as support matrix which is colonized by the cells of the patient during the cicatrization. The most common uses of the amniotic membrane as a graft in surgery are, for example, ophthalmologic surgery, and in particular repair of the cornea, gastric surgery for the repair of ulcers, reconstructive surgery of the ear, in particular reconstruction of the tympanus, surgery for cutaneous lesions repair, in particular for repair of ulcerative disorders, nerve, ligament and tendon repair surgery.

[0009] A detailed history and a procedure for collection, storage by freezing, and use of an amniotic membrane as ophthalmological surgery graft are described in the patent US 6,152,142. In this document, the technique described comprises a step of removal of the chorion before the placement of the amnios in a preservation solution before freezing.

[00010] Also known is the use of the complete chorioamniotic membrane as tissue graft, without the amnios and the chorion having been separated. The most common uses of the complete chorioamniotic membrane as surgical graft are, for example, in ligament surgery, surgery involving fascia, and treatment of cutaneous lesions and ulcers.

[00011] Also known are applications in intra-articular injection of crushed chorioamniotic membrane, which is reported to present properties associated with the limitation of arthrosis.

[00012] The cryopreserved amniotic membranes also have demonstrated their anti-inflammatory, anti-fibrosis and anti-angiogenetic properties, as well as their ability to promote the epithelialization. The amniotic membranes treated in order to be decellularized, so that only the extracellular matrixes remain, are thus tissues that are known for their physiological advantages in the context of tissue grafts.

[00013] However, problems of sanitary security and tissue preservation exist for these products when used as graft.4

[00014] Since the amniotic, chorionic, spongy tissue membranes or the chorioamniotic membranes are biological tissues, there is a high risk of transmission of an infection from a donor to a receiver, or of contamination during the collection of the tissue at the time of a delivery, during tissue preparation treatments, or during the transport of this tissue.

[00015] For example, the patent US 8,709,494 discloses grafts obtained by separate methods for preparing an amniotic membrane and a chorionic membrane from a complete chorioamniotic membrane before reassembling said membranes by lamination before dehydration. More particularly, it should be noted that the method described in this document comprises a step of separating the amniotic and chorionic membranes for cleaning them, the membrane retained for the treatment being heated, after cleaning, in a bag for dehydrating it at a temperature between 35 °C and 50 °C. For collecting grafts that are free of any pathogenic contamination, the grafts are tested and are harvested and treated in a sterile medium; the method described does not comprise a disinfection step. This selection leads to a very high rate of graft rejection.

[00016] In fact, an effective method for disinfection and for preservation of these tissues is vital for the safety of the patients.

[00017] Although the method by cryopreservation is effective, it has the disadvantage of requiring the installation of freezers for clinical use, which are expensive and of large size. This method also involves the need to ensure the preservation of the cold chain between the site of amniotic membrane preparation and the site of use, namely the surgical operation room or the bedside table of the patient.

In addition, the use of cyropreservatives according to this technique involves the need of having to remove the cryopreservatives from the tissues before use. Indeed, these cryopreservatives are known for their toxicity, as in the case of DMSO, for example, or for the structural modifications that they induce in tissues, as in the case of glycerol, for example.

[00018] Alternative methods have been developed in order to avoid this technical constraint, for example, treatments comprising a step in which the grafts are put in the presence of antibiotics and/or antimycotics.

[00019] For example, in 2004, Nakamura proposed, in Investigative Ophthalmology & Visual Science, January 2004, Vol. 45, No. 1, a method for disinfection, sterilization and lyophilization of an amniotic membrane intended for the reconstruction of an ocular surface. The step of disinfection of the amniotic membrane is carried out by incubation in phosphate buffered saline (PBS) containing antibiotics and antimycotics. The decellularization of the amniotic membrane is carried out by incubation in ethylenediaminetetraacetic acid (EDTA). The decellularized amniotic5 membrane is then lyophilized under a vacuum; the product thus obtained is packaged and can be stored at ambient temperature. A last step of gamma irradiation at 25 kGy ensures sterilization of the resulting final product.

[00020] In the patent application CN 104 083 803, a method for treating the amnios is described, comprising in particular a first step of disinfection carried out by treating the membrane with an agent which is a solution of sodium hydroxide (0.1 to 0.5 M), hydrogen peroxide (1 to 3 g/L), acetate peroxide (0.1 to 0.5 g/L) or ethanol (60% to 90%); followed by a second step in a buffer solution comprising a mixture of antibiotics. The amnios is then dried under a vacuum before being packaged.

[00021] The use of antibiotics for preparing grafts to be implanted has numerous disadvantages, in particular the disadvantage of the risk of inducing resistances or of using antibiotics whose spectrum is not consistent with the microorganisms present in the treated grafts.

[00022] Treatment methods without antibiotics are also described for soft tissues or more resistant tissues such as bone.

[00023] Described in the patent application US 2013/0247517 is a method for treating an amnios which is intended to be used as an anti-adhesion cicatrization support in the treatment of wounds. The method described relates to a chemical treatment with 1% glutaraldehyde. A decontamination of the material is considered, consisting in dipping the amnios in an ethanol solution or in a hydrogen peroxide solution or in both, without further specification or monitoring of the decontamination.

[00024] Other products and methods for treating soft tissues have also been described in the patent literature. For example, the patent US 6,024,735 describes compositions and a method for cleaning soft tissues attached to bone and obtained from cadavers. The compositions in question comprise a detergent having functions based on lauryl ether and another detergent having functions based on alkylphenol oxyethylate and are marketed under the name Allowash™.

[00025] The patent US 6,482,584 describes a method for cleaning a porous implant by cyclic perfusion for the inactivation of pathogens, as well as the resulting cleaned and sterilized product consisting, for example, of ligaments attached to bone and intended to form allografts. The treatment method uses hydrogen peroxide in the form of an aqueous solution, inter alia, and requires the alternation of positive and negative pressures within the washing chamber in which the implant is placed.

[00026] The patent application FR 2 949 042 in the name of the applicant describes a method for treating cartilaginous tissue which is intended to be used as support in bioengineering, or as a graft. This method comprises in particular a step of washing with ethanol (70 to 100%), a GAG extraction step, a disinfection step6 (including in particular with alcohol or hydrogen peroxide (5 to 30%)), a lyophilization step, and a sterilization step by gamma irradiation.

[00027] In the document DePaula C.A. et al. "Effects of Hydrogen Peroxide Cleaning Procedures on Bone Graft Osteoinductivity and Mechanical Properties," Cell Tissue Bank, 2005; 6(4):287-98, the effects of the cleaning of bone tissues by hydrogen peroxides on osteoinductivity and mechanical properties of said tissues are described. The method comprises a first step of chemical treatment with 3% hydrogen peroxide, and a second step with 70% denatured alcohol.

[00028] The above-mentioned treatment conditions applied without antibiotics are suitable for relatively resistant tissues such as bone and even ligament, but when applied to a membrane layer obtained from placenta for durations and at the concentrations described as having sufficient efficacy to guarantee decontamination, they would risk causing the complete destruction of the membrane layer or at the very least the loss of all the biological and physiological functionalities of interest, including, inter alia, the different sublayers, the growth factors, the components of the extracellular matrix, such as elastin, and laminin.

[00029] Indeed, it is known from Mbithi J. N. et al., Appl. Environ. Microbiol. 1990, Vol. 12, No. 1, 147-179 that non-enveloped viruses such as the HAV virus are not destroyed by 3 or 6% hydrogen peroxide solutions.

[00030] In the same way, the lowering of polioviruses reported in the study Reckitt and Colmann, 1997: 1-12 are limited to a 3 log decrease in spite of a treatment at H2O2 concentrations of 7.5% for a duration of 6 hours.

[00031] In addition, the structure in layers and sublayers of the amniotic, chorionic, spongy tissue membranes, or the chorioamniotic membranes, is detrimental to the efficacy of the treatments and there is a risk of destroying said structure to the detriment of the efficacy of said membranes.

[00032] Thus, one aim of the present invention is to propose a method for preparing an allograft material forming a sterile, lyophilized, virally inactivated membrane obtained from mammalian placenta and consisting of at least one membrane layer, the allograft material being suitable for use as surgical graft, and according to which the treatment conditions do not induce, or in any case, minimize losses or destruction of at least one membrane layer or of the sublayers constituting said membrane layer.

[00033] The method according to the invention comprises at least two steps of viral inactivation and at least one step of lyophilization, according to which the first step of viral inactivation is carried out by treating said at least one membrane layer7 with a first viral activation agent selected from the family of the alcohols; and a second step of viral inactivation is carried out after said first step of viral inactivation, by treating said at least one membrane layer with a second viral inactivation agent selected from the family of the peroxides.

[00034] Another aim of the present invention is also to propose a method for preparing an injectable material obtained from the sterile, lyophilized, virally inactivated spongy tissue membrane obtained from mammalian placenta.

[00035] The product obtained according to the method is also decellularized, sterilized and packaged while preserving its properties of surgical interest in order to fulfill its function as graft or as injectable material. The technical advantages of this method in comparison to the existing methods are: • ensuring a virally inactivating and disinfecting chemical treatment of the tissue at depth in order to ensure the safety of the graft or of the injectable material.

This treatment thus makes it possible not to have to separate the amniotic membrane from the chorionic membrane and as a result to preserve the chorioamniotic structure and the protein bridges, for example, collagen bridges, linking these two membranes; • providing a disinfecting chemical treatment of the tissue, so that the obligation of asepsis of the tissues or the empirical use of antibiotics are not necessary as in the case of a method by cryopreservation, this chemical treatment also having the technical advantage of allowing the use of placental tissues obtained by vaginal delivery, in spite of the fact that they are known to be associated with microbial contamination when obtained by vaginal delivery, • providing a simple lyophilization method which is such that the amniotic or chorionic or chorioamniotic membrane has a perfectly flat presentation after treatment according to the method, that is to say without curling of the membrane onto itself and without exhibiting a gondola-shaped appearance.

Indeed, the preparation methods known from the prior art which use a chemical treatment followed by a lyophilization step tend to cause the membrane to assume a gondola-shape at its edges during this step.

[00036] Definitions used in the present application.

[00037] Certain expressions or words may be used in the present application in order to indicate technical elements, facts, properties or features. Their meaning is given below:

[00038] - "membrane layer" is understood to mean one of the constitutive tissue sheets of the placenta. In the present invention, it refers to the amniotic membrane, the chorionic membrane, the spongy tissue membrane, or the chorioamniotic8 membrane, each of these sheets possibly comprising or not comprising the constitutive physiological substructures of the amnios and of the chorion.

[00039] - "amniotic membrane" or "amnios" is understood to mean the tissue envelope which develops around the embryo and then around the fetus during pregnancy in mammals. Its role is to protect the developing organism by maintaining the amniotic fluid around it. It is attached to the second membrane namely the chorion. The amniotic membrane comprises the following physiological sublayers: the epithelial cell layer, the basal membrane, the compact layer, the fibroblastic layer, the spongy layer.

[00040] - "chorionic membrane" or "chorion" is understood to mean the tissue envelope located between the amnios and the uterine wall which develops around the embryo and then the fetus in mammals during pregnancy. The chorionic membrane comprises the following physiological sublayers: the reticular layer, the basal membrane, the trophoblastic cell layer.

[00041] - "chorioamniotic membrane" is understood to mean the amnios and the chorion together, which have not been separated naturally or artificially and are connected by protein bridges.

[00042] - "spongy tissue membrane" or "spongy layer" is understood to mean a layer located between the amnios and the chorion.

[00043] - "viral inactivation" is understood to mean a technique which makes it possible to reduce, considerably or completely, and definitively, the capability of action of the viruses. Said viruses, defined as inactivated, lose their pathogenic and replication capabilities due to a 4 log decrease in their population during residual titrations after one or two independent chemical steps, performed on enveloped or non-enveloped DNA or RNA viruses.

[00044] - "lyophilization" is understood to mean a technique the purpose of which is to dry a product that has been deep frozen by sublimation beforehand. More precisely, the liquid to be removed from the product is transformed into ice by freezing in a first step; then the ice is sublimated, by primary desiccation under a vacuum; finally, by a secondary desiccation, the water molecules on the surface of the product are extracted by desorption.

[00045] - "not to damage the structural, functional and biological integrity of the membrane layer" is understood to mean that the product obtained according to the method exhibits sufficient preservation of the biological molecules ensuring the mechanical structure of the tissue, such as collagen of various types, the proteoglycans, elastin, and laminin, as well as biologically active molecules such as, for example, the hormones, enzymes, growth factors, in order to ensure a subsequent use of the product. Thus, according to these parameters, the product obtained9 according to the method exhibits biological properties that are similar or quasi-similar to the initial tissue before treatment. In the present invention, these molecules are, for example, laminin and elastin; as well as the growth factors of the types EGF (Epidermal Growth Factor), TGF (Transforming Growth Factor) and KGF (Keratinocyte Growth Factor).

[00046] - "alcohols" is understood to mean any organic compound one of the carbons of which is bound to a hydroxyl group (-OH).

[00047] - "peroxides" is understood to mean any chemical compound containing a functional group of general formula R-O-O-R' (two adjacent oxygen atoms bound to one another), where R and R' are hydrogen atoms or any alkyl radicals.

[00048] The physiological tissue intended to be treated according to the method described in the present invention is an amniotic membrane, a chorionic membrane, a spongy tissue membrane or a chorioamniotic membrane still associated with the placenta. The membrane is obtained after delivery from a donor who has been properly informed and has given her consent in compliance with the requirements of the European directives and the Law on Bioethics. This delivery can have been a cesarean delivery or a vaginal delivery. Due to the sanitary requirement relating to the donation of tissues and cells of human origin, a socio-clinical qualification of the donor and a biological qualification upstream of the donor are ensured systematically. This qualification requires screening for HIV virus, hepatitis B, C, HTLV and the treponema pallidum bacterium responsible for syphilis. The placental tissue is advantageously placed in a sterile box, frozen and transported at a temperature of -20 °C, then stored at -80 °C. The placental tissue can also be transported, at the time of the collection, at +4 °C in order to be prepared. If the clinical data of the donor are in compliance with the requirements relating to tissue donation, and if the blood analysis for screening for bacteria and viruses is negative, the eligible tissue can undergo the preparation procedure optionally after thawing.

[00049] The physiological tissue of interest is separated from the placenta, and a cleaning and a quality control are carried out. The physiological tissue can be an amniotic membrane naturally separated from the chorionic membrane during the delivery or artificially separated by a human mechanical intervention; or a chorionic membrane naturally separated from the amniotic membrane during the delivery or artificially separated by a human intervention; or also a whole chorioamniotic membrane, without the separation between the amniotic membrane and the chorionic membrane having been performed. The spongy tissue membrane, which is naturally present between the amniotic membrane and the chorionic membrane, can have been removed or not.10

[00050] If the placental tissue was not frozen initially, the cutting and separation of the different membranes from the placenta can be carried out after the transport at +4 °C; the membranes obtained can be stored at -80 °C.

[00051] The placenta which has not been separated from its physiological tissues can be frozen for storage at a temperature of -80 °C for a duration of up to 2 years if the physiological tissue was not treated immediately.

[00052] The method according to the invention is characterized by a succession of non-interchangeable steps applied to the physiological tissue which is a membrane layer which may or may not have been cut beforehand, as the case may be.

[00053] According to another embodiment, the spongy tissue membrane is extracted by pressure from the amniotic membrane so as to preserve only the layer of interest. Alternatively, this spongy tissue membrane can also be collected and preserved in a bottle under a vacuum.

[00054]

[00055] In order to guarantee the harmlessness of the finished product as an allograft material, a first phase of chemical treatment is carried out. Indeed, the finished product must be free of microbiological agents such as bacteria, viruses, parasites.

[00056] The membrane layer in the frozen state can advantageously be washed in purified water or kept in a bath of purified water. This step ensures both the thawing of the tissue, if necessary, up to ambient temperature as well as lysis of the cells present in the tissue by the effect of osmotic pressure.

[00057] The first step of virally inactivating chemical treatment is to subject the membrane layer to a washing or to keep it in a bath consisting of a first virally inactivating agent, namely ethanol. A washing of the membrane layer with purified water or keeping it in a bath of purified water can advantageously be carried out after this step.

[00058] According to an embodiment, the first viral inactivation agent is ethanol with an alcohol content between 50% and 80%, and preferably at 70% v/v.

[00059] According to another embodiment, the first step of viral inactivation is carried out by treating the membrane layer with ethanol at 70% v/v for approximately 60 minutes.

[00060] The second step of the virally inactivating chemical treatment is to subject the membrane layer to a washing or to keep it in a bath consisting of a second viral inactivation agent, namely hydrogen peroxide.

[00061] As presented above, it is known that the treatment with hydrogen peroxide solution is effective on non-enveloped viruses only at concentrations above %.11

[00062] Moreover, it has been shown by the applicant that the treatment of the membranes with hydrogen peroxide solutions at concentrations above 10% for durations necessary for inactivating non-enveloped viruses destroys said membranes.

[00063] The second viral inactivation agent is hydrogen peroxide in a form selected from an aqueous solution and a gas.

[00064] According to an embodiment, the second viral inactivation agent is hydrogen peroxide in the form of an aqueous solution at a concentration between 3% and 30% w/v.

[00065] Surprisingly, it was shown that dividing the second step of viral inactivation of said at least one membrane layer into two substeps of treatment with hydrogen peroxide at different concentrations and for different durations made it possible to achieve an unexpected efficacy in particular on the non-enveloped viruses.

[00066] According to an embodiment, the method is characterized in that the second step of viral inactivation of said at least one membrane layer comprises two substeps of treatment with the second viral inactivation agent selected from the family of the peroxides.

[00067] A method for preparing an allograft material forming a sterile, lyophilized, and virally inactivated membrane obtained from mammalian placenta and consisting of at least one membrane layer, said method comprising at least two steps of viral inactivation and at least one step of lyophilization, and wherein: • the first step of viral inactivation is carried out by treating said at least one membrane layer with a first viral inactivation agent selected from the family of the alcohols; • a second step of viral inactivation is carried out after said first step of viral inactivation, by treating said at least one membrane layer with a second viral inactivation agent selected from the family of the peroxides, wherein the second step of viral inactivation of said at least one membrane layer comprises two substeps of treatment with the second viral inactivation agent selected from the family of the peroxides.

[00068] According to an embodiment, the second step of viral inactivation of said at least one membrane layer comprises two substeps of treatment with hydrogen peroxide: • a first substep of treatment being carried out with a solution of hydrogen peroxide at a concentration of more than 10% w/v for less than 20 minutes; and • a second step of treatment being carried out with a solution of hydrogen peroxide at a concentration of less than 5% w/v for a duration of more than 50 minutes.12

[00069] According to another embodiment, the second step of viral inactivation of said at least one membrane layer comprises two substeps of treatment with hydrogen peroxide: • a first substep of treatment being carried out with a solution of hydrogen peroxide at 30% w/v for approximately 15 minutes; and • a second substep of treatment being carried out with a solution of hydrogen peroxide at 3% w/v for approximately 60 minutes.

[00070] At the end of this second step of viral inactivation, it is advantageous to proceed with a step of neutralization by subjecting the membrane layer to a washing or by keeping it in a bath consisting of at least one alkaline buffer.

[00071] According to a preferred embodiment, two baths take place in an alkaline buffer containing diluted sodium hydroxide whose pH is adjusted to 8.5.

[00072] The next step of chemical treatment of the membrane layer is advantageously to subject the membrane layer to a washing or to keep it in a bath consisting of at least one buffer solution ensuring a physiological reequilibration of the membrane layer.

[00073] According to a preferred embodiment, two baths take place in a PBS solution.

[00074] According to a preferred embodiment, the steps of thawing, viral inactivation, washing, pH adjustment and buffering are carried out at ambient temperature.

[00075] According to this first phase of chemical treatment, the membrane layer is subjected to a second treatment phase, namely lyophilization. This second phase makes it possible both to ensure the quality of the storage under a vacuum of the product obtained according to the method and to ensure an additional step of disinfection and decellularization of the membrane layer after the chemical treatment.

[00076] According to a preferred embodiment, the lyophilization method is carried out between two substantially flat supports.

[00077] According to an even more preferred embodiment, the method for lyophilization of the membrane layer is carried out by sandwiching the membrane layer between two substantially flat supports, which are permeable to gas, including steam, and whose surface in contact with the membrane layer is substantially hydrophobic, preventing the known adhesion of the cellulose products to cellular residues of the epithelial layer of the amnios.

[00078] According to an even more preferred embodiment, the method for lyophilization of the membrane layer is carried out by sandwiching the membrane layer between two substantially flat supports made of cellulose paper or cellulose derivative.13

[00079] The virally inactivated membrane layer is thus subjected to a lyophilization under the following conditions: • freezing in two steps: - the first freezing step being carried out at an acclimation temperature selected so as not to damage the structural, functional and biological integrity of the membrane layer, - the second freezing step being carried out at the final freezing temperature which is lower than the acclimation temperature; • a lyophilization in two main steps, referred to as primary step and secondary step: - the primary lyophilization step being carried out by application of a vacuum at approximately 200 microbar and of a rising temperature profile; - the secondary lyophilization step being carried out by application of a vacuum at approximately 50 microbar and of a falling temperature profile.

[00080] In an embodiment, the acclimation temperature is between -5 and -20 °C, and the final freezing temperature is between -40 and -60 °C.

[00081] The rising temperature profile is advantageously a profile according to which the lyophilization temperature is set initially at a low initial temperature and then increased to a final primary lyophilization temperature, in one or more intermediate rising temperature steps. The falling temperature profile is advantageously a profile according to which the lyophilization temperature is initially set at a temperature higher than the final temperature of the primary lyophilization step, and is then lowered to a final secondary lyophilization temperature which is higher than the initial temperature of the primary lyophilization step.

[00082] These lyophilization conditions make it possible to obtain a product, namely a virally inactivated and lyophilized membrane layer, which is characterized in particular in that it has a residual water content at ambient temperature of less than %.

[00083] The product obtained, namely a virally inactivated and lyophilized membrane layer, has a perfectly flat presentation. It can advantageously be cut according to pre-defined parameters in order to meet the needs of the practitioner performing the grafting of the product obtained to a patient.

[00084] The product obtained, namely a virally inactivated and lyophilized membrane product, has a perfectly flat presentation and can be stored in such a form in a sterile packaging under sterile conditions.

[00085] According to another embodiment, the product obtained is a virally inactivated and lyophilized spongy tissue membrane.14

[00086] The virally inactivated and lyophilized membrane layer, which is packaged, is subjected to a last sterilization step, namely a gamma irradiation.

[00087] According to a preferred embodiment, this sterilization step is carried out by exposure of the membrane layer to gamma rays at 25-32 kGray.

[00088] The product obtained according to the method is thus a sterile, lyophilized, virally inactivated allograft material consisting of at least one membrane layer selected from the amniotic membrane, the chorionic membrane, and the chorioamniotic membrane in which the amnios and the chorion are connected to one another by protein bridges.

[00089] The product obtained according to the method can also be a sterile, lyophilized, virally inactivated injectable material consisting of at least one membrane layer, namely the spongy tissue membrane.

[00090] The product obtained according to the method can be used as an allograft material selected from the group consisting of ligament graft, fascia lata graft, tendon graft, dura mater graft, sub-mucosal membrane graft, nerve sheath graft, ligament graft, cartilage graft, rotator cuff graft, conjunctival tissue graft, wound and/or ulcer treatment agents, and suture improvement agents.

[00091] Some uses that can be considered for the product obtained according to the method comprise in particular the repair of corneal ulcers, the repair of tendons and/or ligaments, ligament replacement, nerve repair by sheathing, cartilage repair, replacement of the dura mater, replacement of the submucosal membrane, reinforcement of the rotator cuff, treatment of conjunctival and/or corneal ulcers, treatment of cutaneous ulcers and/or wounds, and cicatrization and suture improvement.

[00092] Some uses that can be considered for the product thus obtained according to the method from the spongy tissue membrane are, for example, the treatment by intra-articular injection of the prearthritic or arthritic condition, and ligament, meniscus or cartilage repair.

[00093] Another use that can be considered for the product according to the invention is, for example, a tissue engineering support matrix for culturing mesenchymatous or fibroblast cells.

[00094] The invention will be understood better and completed by helpful reference to the figures which are included purely for illustration and a summary of which is given below:

[00095] Brief Description of the Figures.

[00096] Figure 1: Synthetic representation of the steps of the method for preparing the allograft material.15

[00097] Figure 2a: Photograph by optical microscopy of a section of an untreated chorioamniotic membrane stained with hematoxylin-eosin. The enlargement used is 40x. (1) denotes the amniotic membrane. (2) denotes the spongy tissue membrane. (3) denotes the chorion.

[00098] Figure 2b: Photograph by optical microscopy of a section of a chorioamniotic membrane treated according to the method, stained with hematoxylin- eosin. The enlargement used is 40x. (1) denotes the amniotic membrane. (2) denotes the spongy tissue membrane. (3) denotes the chorion.

[00099] Reference will be made to the figures thus identified as needed in the following detailed description of an example of an embodiment of the invention.

Example 1: Example of an embodiment of the invention in which a membrane layer is treated according to the method so as to become an allograft material.

[000100] Reference is made advantageously to Figure 1 for visualizing the important steps of the method.

[000101] A properly informed donor consenting in accordance with the requirements of the Helsinki Declaration offers a placental tissue donation obtained during a delivery. This delivery can have been a cesarean or vaginal delivery. Due to the sanitary requirement pertaining to donations of tissues and cells of human origin, systematic upstream qualification of the donor is necessary. This qualification requires screening for HIV virus, hepatitis B, C, HTLV and the treponema pallidum bacterium responsible for syphilis.

[000102] The placental tissue is recovered as early as possible in the delivery room after a delivery whether it be a vaginal or cesarean delivery. It can advantageously be placed in a sterile box, then frozen at a temperature of -20 °C.

[000103] This isolated placental tissue can be stored dry at a temperature of -80 °C for a duration of up to two years or it can be treated immediately according to the method.

[000104] In the laboratory, in a sterile room, the following procedure is applied:

[000105] The membrane layer of interest, which can be, for example, the amnios, the chorion, or the chorioamniotic membrane, is isolated from the placenta and cleaned. A photograph by optical microscopy of an as yet untreated chorioamniotic membrane which has been stained with hematoxylin-eosin is presented in Figure 2a, the different layers comprising it are indicated, namely: (1) denotes the amniotic membrane. (2) denotes the spongy tissue membrane. (3) denotes the chorion.

[000106] Alternatively, after collection, the placental tissue can be placed at approximately +4 °C. In a sterile room, the membrane layer of interest is isolated and16 cleaned. This membrane layer can be stored dry at a temperature of -80 °C for a duration of up to two years.

[000107] The membrane layer undergoes a succession of baths ensuring a chemical treatment of said membrane layer. The purpose of this treatment is a disinfection of the membrane layer of interest and quite particularly its viral inactivation. A gentle stirring at approximately 30 rpm of the liquid medium is applied during each bath in order to ensure a homogeneous penetration of the solvents into the tissues, while avoiding breaking of the protein bridges between the chorion and the amnios if the membrane layer to which the method is applied is the chorioamniotic membrane.

[000108] In a first step, the membrane layer is placed in a bath of purified water at ambient temperature for approximately 3 hours. This step ensures the thawing of the physiological tissue as well as a first step of cell lysis by osmotic pressure.

[000109] Then, the membrane layer is transferred to a decontaminating bath consisting of 70% v/v ethanol at ambient temperature for approximately 1 hour.

[000110] A washing is carried out in purified water for approximately 15 minutes at ambient temperature to remove the ethanol.

[000111] In order to ensure the second step of decontaminating treatment, the membrane layer is transferred to a bath consisting of 30% w/v hydrogen peroxide at ambient temperature for approximately 15 minutes.

[000112] Then, the membrane layer is transferred to a decontaminating bath consisting of 3% w/v hydrogen peroxide at ambient temperature for approximately 1 hour.

[000113] The chemical action applied to the membrane layer can then be neutralized in at least one bath comprising, for example, diluted sodium hydroxide at a pH of about 8.5. The at least one neutralization bath takes place at ambient temperature for approximately 15 minutes. Advantageous results were found in this neutralization step using two baths with diluted sodium hydroxide at pH 8.5.

[000114] In order to ensure reequilibration of the pH and to eliminate as well as possible the organic residues detaching from the tissue of interest, the membrane layer is transferred to at least one physiological buffer bath selected appropriately so as to ensure its physiological reequilibration. The at least one bath takes place at ambient temperature for approximately 15 minutes. Advantageous results were found in this step using two successive baths in PBS solution.

[000115] Finally, the membrane layer can be transferred to a last bath with purified water at ambient temperature for approximately 15 minutes and up to approximately 1 hour.17

[000116] In this step of the method, one can consider that the membrane layer obtained according to this chemical treatment can be defined as a membrane that is in large part disinfected, in particular virally inactivated, and decellularized.

[000117] After this part involving chemical treatments, the membrane layer is subjected to a lyophilization treatment.

[000118] On a stainless steel plate, the membrane layer is advantageously placed between two layers of support filters preferably made of cellulose or a cellulose derivative such as, for example, methylcellulose mesh in order to facilitate the steam exchanges.

[000119] If the membrane layer is defined as being the amnios, which is a particularly fine tissue, then, in order to avoid any risk of folding of the membrane layer during the lyophilization step, a screen is arranged above said membrane layer which is sandwiched between the two support filters so that approximately 1 mm separates the screen from the upper support filter.

[000120] The above-described assembly is transferred to a lyophilizer, in which a freezing step is carried out, followed by a lyophilization step, according to the following modalities: • Freezing: - The first freezing step is carried out at an acclimation temperature selected so as not to damage the structural, functional and biological integrity of the membrane layer; - the second freezing step is carried out at the final freezing temperature which is less than the acclimation temperature; • Lyophilization: - A primary lyophilization step is carried out by application of a vacuum of approximately 200 microbar and by application of a rising temperature profile; - a second lyophilization step is carried out by application of a vacuum at approximately 50 microbar and by application of a falling temperature profile.

[000121] After this lyophilization step, the membrane layer according to this treatment can be defined as a disinfected membrane, in particular a virally inactivated membrane, which is decellularized and lyophilized. The membrane layer does not have overturned edges or a gondola-shaped or embossed appearance, and its presentation is perfectly flat. A photograph by optical microscopy of a chorioamniotic membrane section treated according to the method is presented in Figure 2b; the different layers comprising it are indicated, namely: (1) denotes the amniotic membrane. (2) denotes the spongy tissue membrane. (3) denotes the chorion.18

[000122] The membrane layer obtained can advantageously be cut in this step of the method. The health professionals, for whom the final product is intended, can provide precise cutting parameters depending on their practical needs.

[000123] The virally inactivated and lyophilized membrane obtained is packaged flat under sterile conditions.

[000124] The extraction of the membranes, namely the spongy tissue membrane, can be packaged in a bottle under a vacuum.

[000125] Outside of the sterile room, a last sterilization step of the virally inactivated and lyophilized membrane layer is carried out by exposure of said membrane layer to gamma radiation at 25-32 kGray.

[000126] The product thus obtained can be distributed in order to be used as an allograft material, for example, ligament graft, fascia lata graft, tendon graft, dura mater graft, submucosal membrane graft, nerve sheath graft, cartilage graft, rotator cuff graft, conjunctival tissue graft, wound and/or ulcer treatment agents, and cicatrization and suture improvement agents.

[000127] Thus, the uses that can be considered for the product obtained according to the method comprise in particular, as indicated above, corneal ulcer repair, tendon and/or ligament repair, ligament replacement, nerve repair by sheathing, dura mater replacement, submucosal membrane replacement, rotator cuff reinforcement, conjunctival and/or corneal ulcer treatment, cutaneous ulcer and/or wound treatment, and suture improvement.

Example 1 bis: Example of an embodiment of the invention in which a membrane layer is treated according to the method so as to become an allograft material.

[000128] Reference is made advantageously to Figure 1 to visualize the important steps of the method.

[000129] A properly informed donor who consents in accordance with the requirements of the Helsinki Declaration offers as donation the placental tissue obtained during a vaginal delivery. The screening test of the donor for HIV virus, hepatitis B, C, HTLV and the treponema pallidum bacterium responsible for syphilis is negative.

[000130] The placental tissue is recovered as early as possible in the delivery room after the delivery. It is placed in a sterile box, then frozen at a temperature of -20 °C and transported to the laboratory.

[000131] In the laboratory, in a sterile room, the following procedure is applied:19

[000132] The membrane layer of interest, namely the chorioamniotic membrane, is isolated from the placenta and cleaned.

[000133] The membrane layer is subjected to a succession of baths ensuring its chemical treatment. Gentle stirring at 30 rpm of the liquid medium is applied during each bath so as to ensure a homogeneous penetration of the solvents into the tissues, while avoiding breaking of the protein bridges between the chorion and the amnios of the chorioamniotic membrane.

[000134] In a first step, the membrane layer is deposited in a bath of purified water at ambient temperature for 3 hours. This step ensures the thawing of the physiological tissue as well as a first cell lysis step by osmotic pressure.

[000135] Then, the membrane layer is transferred to a decontaminating bath consisting of 70% v/v ethanol at ambient temperature for 1 hour.

[000136] A washing is carried out in purified water for 15 minutes at ambient temperature to remove the ethanol.

[000137] To ensure the second step of decontamination treatment, the membrane layer is transferred to a bath consisting of 30% w/v hydrogen peroxide at ambient temperature for 15 minutes.

[000138] Then, the membrane layer is transferred to a decontaminating bath consisting of 3% w/v hydrogen peroxide at ambient temperature for 1 hour.

[000139] The chemical action applied to the membrane layer is neutralized in two successive baths of diluted sodium hydroxide at a pH of 8.5. Each of the neutralization baths takes place at ambient temperature for 15 minutes.

[000140] In order to ensure a reequilibration of the pH and to eliminate as well as possible the organic residues detaching from the tissue of interest, the membrane layer is transferred to two successive baths in PBS solution in order to ensure its physiological reequilibration. Each of the baths takes place at ambient temperature for minutes.

[000141] Finally, the membrane layer is transferred to a last bath with purified water at ambient temperature for 15 minutes.

[000142] After this part involving the chemical treatments, the membrane layer is subjected to a lyophilization treatment.

[000143] On a stainless steel plate, the membrane layer is placed between two layers of support filters made of methylcellulose mesh in order to facilitate the steam exchanges.

[000144] The above-described assembly is transferred to a lyophilizer, in which a freezing step is carried out, followed by a lyophilization step, according to the following modalities:

[000145] Freezing:20

[000146] The first freezing step is carried out at a temperature of -10 °C for 5 minutes, then at -15 °C for 90 minutes;

[000147] the second freezing step is carried out at a temperature of -50 °C for 125 minutes;

[000148] Lyophilization:

[000149] A primary lyophilization step is carried out by application of a vacuum at 200 microbar and by application of a temperature of +10 °C for 8 hours, followed by a temperature of +25 °C for 150 minutes;

[000150] A secondary lyophilization step is carried out by application of a vacuum at 50 microbar and by application of a temperature of +35 °C for 5 hours, followed by a temperature of +25 °C for 1 hour.

[000151] After this lyophilization step, the membrane layer has no overturned edges or a gondola-shaped or embossed appearance, and its presentation is perfectly flat.

[000152] The membrane layer obtained is cut in this step of the method to 20 by cm (or 20 by 20 cm) rectangles making it suitable for use as an allograft material in ligament repair.

[000153] The virally inactivated and lyophilized membrane obtained is packaged flat under sterile conditions.

[000154] Outside of the sterile room, a last step of sterilization of the virally inactivated and lyophilized membrane layer is carried out by exposing said membrane layer to gamma radiation at 25-32 kGray.

[000155] The product thus obtained is distributed in order to be used as an allograft material for use in ligament repair.

Example 2: Demonstration of the efficacy of the viral inactivation of the membrane layer according to the method.

[000156] Measurements of the viral load in chorioamniotic membranes were carried out by injection of virus between the amnios and the chorion in order to verify the viral inactivation of the tissues according to the method.

[000157] Aliquots of membranes thus treated are collected in order to be put in solution and centrifuged. The soluble fraction is used to inoculate cells in a cell culture medium. Each virus type studied is inoculated in the appropriate cell type to enable the expression of its virulence.

[000158] The viruses that were measured are the porcine parvovirus (hereafter referred to as PPV), the pseudorabies virus (hereafter referred to as PRV) involved in21 Aujeszky's disease, the human immunodeficiency virus of type 1 (hereafter referred to as HIV-1), the hepatitis A virus (hereafter referred to as HAV).

[000159] The viral load measurements were carried out by detection of the viral production in infected cells and more particularly by observation of a specific cytopathic effect. The viruses injected in the membrane layers were subjected either to the step of chemical treatment with 70% v/v ethanol according to the method or to the step of chemical treatment with 30% w/v hydrogen peroxide, then 3% w/v hydrogen peroxide according to the method.

[000160] Each measurement of the viral load was carried out two times for each test; the measurements are recorded in the following table using the indications Run A and Run B.

[000161] The results in the following table represent the decrease of the measured viral load expressed in log. This decrease is measured by comparison of the viral load in the membrane layer before and then after the treatment indicated.

Results Virus H2O2 Treatment Ethanol Treatment Run A = 4.53 Run A = 1.43 PPV Run B = 3.94 Run B = 2.03 Run A > 2.89 Run A > 5.18 PRV Run B > 3.13 Run B > 4.83 Run A > 3.66 Run A = 1.37 HAV Run B > 4.02 Run B = 1.37 Run A > 4.52 HIV-1 Not applicable Run B > 4.50

[000162] The efficacy of the chemical treatments according to the method applied to the membrane layers and intended for the viral inactivation can be observed.

[000163] The chemical treatment with hydrogen peroxide is effective against PPV, PRV and HAV. Concerning HIV-1, the conditions of the performance of the measurements were not able to yield interpretable results.22

[000164] The chemical treatment with ethanol is effective against PRV and HIV-1 and to a lesser extent against the naked (non-enveloped) viruses such as PPV and HAV

[000165] As demonstrated, only the combination of the two chemical treatments makes it possible to ensure a viral inactivation of the membrane layers both for the enveloped viruses and for the naked viruses. This viral inactivation is defined by an at least 4 log decrease in the viral population.

Example 2 bis: Demonstration of the efficacy of the viral inactivation of the membrane layer according to the method.

[000166] Additional measurements of the viral load were carried out in chorioamniotic membranes by injection of virus between the amnios and the chorion in order to verify the viral inactivation of the tissues according to the method.

[000167] Aliquots of the membranes thus treated are collected in order to be put in solution and centrifuged. The soluble fraction is used to inoculate cells in a cell culture medium. Each type of virus studied is inoculated in the appropriate cell type in order to enable the expression of its virulence.

[000168] The viral load measurements were carried out by detection of the viral production in infected cells and more particularly by observation of a specific cytopathic effect. The viruses that were measured are the pseudorabies virus (hereafter referred to as PRV) involved in Aujeszky's disease, and the hepatitis A virus (hereafter referred to as HAV).

[000169] The viruses injected in the membrane layers were subjected to the step of chemical treatment with 30% w/v hydrogen peroxide, then 3% w/v hydrogen peroxide according to the method.

[000170] The results in the following table represent the measured decrease in the viral load expressed in log. This decrease is measured by comparison of the viral load on the membrane layer before and then after the treatment indicated. 3023 Results Virus H2O2 Treatment Run A > 4.47 PRV Run B > 4.47 Run A > 4.47 HAV Run B > 4.53

[000171] The efficacy of the chemical treatments according to the method applied to the membrane layers and intended for the viral inactivation can be observed.

[000172] The chemical treatment with hydrogen peroxide is effective against PRV and HAV.

[000173] As demonstrated, the treatment with hydrogen peroxide according to the method makes it possible to ensure a viral inactivation of the membrane layers against the PRV virus and the HAV virus. This viral inactivation is defined by an at least log 4 decrease in the viral population.

Example 2 ter: Demonstration of the efficacy of the viral inactivation of the membrane layer according to the method.

[000174] Measurements of the viral load were carried out in amniotic membranes whose connection to their spongy tissue membranes was preserved, by injection of virus between the amniotic membrane and the spongy tissue membrane in order to verify the viral inactivation of the tissues according to the method.

[000175] Aliquots of membranes thus treated are collected in order to be put in solution and centrifuged. The soluble fraction is used to inoculate cells in a cell culture medium. Each type of virus studied is inoculated in the appropriate cell type in order to enable the expression of its virulence.

[000176] The viruses that were measured are the porcine parvovirus virus (hereafter referred to as PPV) and the human immunodeficiency virus of type 1 (hereafter referred to as HIV-1).

[000177] The viral load measurements were carried out by detection of the viral production in infected cells and more particularly by observation of a specific cytopathic effect. The viruses injected in the membrane layers were subjected to the step of chemical treatment with 30% w/v hydrogen peroxide, then 3% w/v hydrogen peroxide according to the method.24

[000178] Each measurement of the viral load was carried out two times for each test; the measurements are recorded in the following table using the indications Run A and Run B.

[000179] The results in the following table represent the decrease in the measured vial load expressed in log. This decrease is measured by comparison of the viral load on the membrane layer before and then after the treatment indicated.

Results Virus H2O2 Treatment Run A > 6.02 HIV-1 Run B > 5.90 Run A = 6.47 PPV Run B = 5.66

[000180] The efficacy of the step of chemical treatment according to the method with 30% w/v, then 3% w/v hydrogen peroxide applied to the membrane layers and intended for the viral inactivation can be observed.

[000181] The chemical treatment with hydrogen peroxide applied to amniotic membranes whose connection to their spongy tissue membranes has been preserved is effective against PPV and HIV-1.

[000182] As demonstrated, the treatment with hydrogen peroxide according to the method makes it possible to ensure a viral inactivation of the membrane layers against HIV-1 virus and PPV virus. This viral inactivation being defined by an at least 4 log decrease in the viral population.

Example 3: Demonstration of the efficacy of the preservation of the growth factors of the membrane layers produced according to the method.

[000183] Growth factor measurements by ELISA immunoassay were carried out on membrane layers before the application of the method and after the application of the method.

[000184] On membrane layers, namely on amniotic membranes, the quantity of growth factor, namely the TGF (Transforming Growth Factor) of type beta 1, is evaluated.25

[000185] Before the application of the method, a quantity of TGF beta 1 of 10 to 20 ng/g of product tested is measured.

[000186] After application of the method, this measured quantity is between 1.7 and 13.6 ng/g.

[000187] On membrane layers, namely on amniotic membranes and on chorionic membranes, the quantity of growth factor, namely EGF (Epidermal Growth Factor), is evaluated.

[000188] Before the application of the method, a quantity of EGF of approximately 0.4 to 1.6 ng/g of product tested is measured.

[000189] After application of the method, this measured quantity is between 0.01 and 0.2 ng/g.

[000190] The rates of preservation of the growth factors measured are highly variable, but they always demonstrate their presence. Indeed, numerous criteria explaining these fluctuations come into play, for example: depending on whether the amniotic membrane is located at the site of the placenta or farther away from the placenta, depending on the individual variations, or depending on the location of the extraction, since these factors are concentrated primarily in the spongy layer.

Example 3 bis: Demonstration of the efficacy of the preservation of the growth factors of the membrane layers produced according to the method.

[000191] Additional measurements of growth factors by ELISA immunoassay were carried out on membrane layers before the application of the method and after the application of the method.

[000192] The membrane layers that were subjected to additional measurements are amniotic membranes and chorioamniotic membranes.

[000193] The growth factors measured are EGF (Epidermal Growth Factor), TGF (Transforming Growth Factor) of type beta 1, and FGF (Fibroblast Growth Factor).

[000194] The results are presented in the following table in the form of averages of results obtained.

[000195] The rate of preservation of the growth factors after the application of the method is also indicated in percentages.26 Amnios Amnios Rate Chorio- Chorio- Rate before after of amniotic amniotic of treatment treatment preser­ membrane membrane preser­ in ng/g in ng/g vation before after vation treatment treatment in in ng/g ng/g EGF 1.11 0.06 6 % 0.6 0.007 1% TGF 30.92 3.41 11 % 8.64 13.39 65% -31 FGF 3.21 0.02 1 % 0.71 0.15 23%

[000196] The rates of preservation of the growth factors measured are highly variable, but they always demonstrate their presence in the treated membrane.

Example 4: Demonstration of the preservation of the structural proteins of the membrane layers obtained according to the method.

[000197] The tensile strength tests on the amniotic and chorioamniotic membranes were carried out according to the method described by Tanaka in the reference "Tensile properties of amniotic membrane" (Tanaka et al,, 2010) with a calculation performed using the mathematical formula: "R=FmaxLsamplexlsample" where, R is the breaking force of the sample; Fmax is the maximum force exerted by the measurement apparatus; Lsample is the surface area of the sample; lsample is the width of the sample

[000198] The aim of these tests is to compare the tensile strengths of the untreated membranes at the time of collection and of the membranes chemically treated and lyophilized according to the method; the values obtained are collected in the following table.27 Standard Treatment Sample Breaking force (kPa) Mean deviation 9.5 Amniotic membrane 11.4 8.2 4.0 1 3.8 9.9 Chorioamniotic 9.1 5.1 13.9 Collection membrane 1 3.8 (untreated) Amniotic membrane 9.4 7.1 2 3.2 4.9 4.8 Chorioamniotic .8 1.5 membrane 2 6.9 Amniotic .1 membrane 11.4 .3 4.7 3 After 14.4 chemical Chorioamniotic treatment membrane 4 17.9 17.9 / and lyophilization Chorioamniotic membrane 5 5.9 5.9 /

[000199] These tensile strength tests demonstrate that the protein fibers of interest consisting of collagen I, laminin V and elastin are preserved after the chemical treatment, the lyophilization and the sterilization. Indeed, the differences in strength between the untreated membranes at collection time and the membranes chemically treated and lyophilized according to the method are not significant.

[000200] These observations were confirmed by immunohistochemical tests carried out on amniotic membranes and chorioamniotic membranes, in order to visualize and localize the proteins of interest, namely collagen I, laminin V and elastin. These tests were carried out on un-treated membranes at collection time and on membranes after they were subjected to all the steps of the method.

[000201] The results have demonstrated that the proteins of interest were preserved after the treatment.

Example 4 bis: Demonstration of the preservation of the structural proteins of the membrane layers obtained according to the method.

[000202] Additional tensile strength tests on amniotic and chorioamniotic membranes were carried out according to the method described by Tanaka. For this test, the calculations were performed using the formula: "R= FmaxEsamplexisample (Pa)" where,28 R is the breaking force of the sample; Fmax is the maximum force exerted by the measurement apparatus; Esample is the thickness of the sample; !sample is the width of the sample.

Breaking Standard Mean deviation Treatment Sample force (kPa) Amniotic Sample No. 1 905 membrane 631 274 before treatment 357 Sample No. 2 893 679 303 464 Chorioamniotic Sample No. 1 217 membrane 305 261 44 before treatment Sample No. 2 104 128 33 151 Brea king Standard Treatment Sample force (kPa) Mean deviation Amniotic 390 Sample No. 3 351 55 membrane 312 treated according 520 520 0 to the method Sample No. 4 381 Sample No. 5 261 299 71 Chorioamniotic 254 membrane treated according 247 to the method Sample No. 1 269 22 291

[000203] These tensile strength tests demonstrate that the protein fibers of interest, namely collagen I, laminin V and elastin, are preserved after the chemical treatment, the lyophilization and the sterilization. Indeed, the differences in the strength measurements between the un-treated membranes at collection time and the membranes chemically treated and lyophilized according to the method are not significant. The mechanical strength of the membranes is preserved. The great variability of the results obtained is due to the physiological nature of the samples which makes the reproducibility of the tests very difficult. 1529 261174/2

Claims (31)

1. A method for preparing an allograft material forming a sterile, lyophilized and virally inactivated membrane obtained from mammalian placenta and consisting of at least one membrane layer, said method including at least two viral inactivation steps and at least one lyophilization step, and wherein: • the first viral inactivation step is carried out by treating said at least one membrane layer with a first viral inactivation agent selected from the family of the alcohols; • a second step of viral inactivation is carried out after said first step of viral inactivation, by treating said at least one membrane layer with a second viral inactivation agent selected from the family of the peroxides, wherein the second step of viral inactivation of said at least one membrane layer comprises two substeps of treatment with the second viral inactivation agent selected from the family of the peroxides: • a first substep of treatment being carried out with a peroxide solution at a concentration of more than 10% w/v for less than 20 minutes; and • a second treatment substep being carried out with a peroxide solution at a concentration of less than 5% w/v for a duration of more than 50 minutes

2. The method according to claim 1, wherein the membrane layer is selected from the amnios, the chorion, and the chorioamniotic membrane in which the amnios and the chorion are connected to one another by protein bridges.

3. The method according to claim 1, wherein the membrane layer is the spongy tissue membrane.

4. The method according to any one of the preceding claims, wherein the first viral inactivation agent is ethanol.

5. The method according to any one of the preceding claims, wherein the first viral inactivation agent is 70% v/v ethanol.

6. The method according to any one of the preceding claims, wherein the first viral inactivation step is carried out by treating said at least one membrane layer with 70% v/v ethanol for approximately 60 minutes.

7. The method according to any one of the preceding claims, wherein the second viral inactivation agent is hydrogen peroxide. 02581797\69-0130 261174/2

8. The method according to any one of the preceding claims, wherein the second viral inactivation agent is hydrogen peroxide in a form selected from an aqueous solution and a gas.

9. The method according to any one of the preceding claims, wherein the second viral inactivation agent is hydrogen peroxide in the form of an aqueous solution at a concentration between 3% and 30% w/v.

10. The method according to any one of the preceding claims, wherein the second step of viral inactivation of said at least one membrane layer comprises two substeps of treatment with hydrogen peroxide: • a first substep of treatment being carried out with a 30% w/v hydrogen peroxide solution for approximately 15 minutes; and • a second substep of treatment being carried out with a 3% w/v hydrogen peroxide solution for approximately 60 minutes.

11. The method according to any one of the preceding claims, which further comprises at least one step wherein the at least one membrane layer is thawed before the at least two steps of viral inactivation.

12. The method according to any one of the preceding claims, which further comprises at least one step wherein the at least one membrane layer is thawed under conditions causing cell lysis.

13. The method according to any one of the preceding claims, which further comprises at least one step wherein the at least one membrane layer is washed with purified water between the steps of viral inactivation.

14. The method according to any one of the preceding claims, which further comprises at least one step of adjustment of the pH of the solution containing the at least one membrane layer after the steps of viral inactivation.

15. The method according to any one of the preceding claims, which further comprises at least one step of adjustment to 8.5 of the pH of the solution containing the at least one membrane layer.

16. The method according to any one of the preceding claims, which further comprises at least one step of placing the at least one membrane layer in a buffer after a step of adjustment of the pH of the solution containing the at least one membrane layer. 02581797\69-0131 261174/2

17. The method according to any one of the preceding claims, wherein the steps of thawing, viral inactivation, washing, pH adjustment and placement in a buffer are carried out at ambient temperature.

18. The method according to any one of the preceding claims, which further comprises a step of lyophilization of said at least one virally inactivated membrane layer.

19. The method according to any one of the preceding claims, which comprises a step of lyophilization of said at least one virally inactivated membrane layer wherein said at least one virally inactivated membrane layer is sandwiched between two substantially flat supports.

20. The method according to any one of the preceding claims, which comprises a step of lyophilization of said at least one virally inactivated membrane layer wherein said at least one virally inactivated membrane layer is sandwiched between two substantially flat supports which are permeable to gas and whose surface in contact with the membrane layer is substantially hydrophobic.

21. The method according to any one of the preceding claims, which comprises a step of lyophilization of said at least one virally inactivated membrane layer wherein said at least one virally inactivated membrane layer is sandwiched between two substantially flat supports made of cellulose, whose surface in contact with the membrane layer is substantially hydrophobic.

22. The method according to any one of the preceding claims, which comprises a step of lyophilization of said at least one virally inactivated membrane layer, step wherein said at least one virally inactivated membrane layer is subjected to a lyophilization under the following conditions: • freezing in two steps: - the first freezing step being carried out at an acclimation temperature selected so as not to damage the structural, functional and biological integrity of the membrane layer; - the second freezing step being carried out at the final freezing temperature which is below the acclimation temperature; • a lyophilization in two main steps, referred to as primary step and secondary step: - the primary lyophilization step being carried out by application of a vacuum at approximately 200 microbar and of a rising temperature profile; 02581797\69-0132 261174/2 - the secondary lyophilization step being carried out by application of a vacuum at approximately 50 microbar and of a falling temperature profile.

23. The method according to any one of the preceding claims, which further comprises a step of sterilization of the virally inactivated and lyophilized membrane layer, by exposing the latter to gamma radiation at 25-32 kGray.

24. Virally inactivated and lyophilized allograft material which can be obtained by any one of the preceding method claims 1 to 23, wherein it is obtained from placenta and consists of at least one membrane layer selected from the amnios, the chorion, and the chorioamniotic membrane in which the amnios and the chorion are connected to one another by protein bridges.

25. Virally inactivated and lyophilized allograft material according to claim 24, presented in a form which can be used as a graft selected from the group consisting of ligament graft, fascia lata graft, tendon graft, dura mater graft, submucosal membrane graft, nerve sheath graft, rotator cuff graft, conjunctival tissue graft, wound and/or ulcer treatment agents, and suture improvement agents.

26. Virally inactivated and lyophilized allograft material according to claim 24, wherein under a vacuum it has a residual water content at ambient temperature of less than 5%.

27. Virally inactivated and lyophilized allograft material according to either claim 24 or 25, or which can be obtained by the method according to any one of claims 1 to 23, for use as a graft, selected from the group consisting of corneal ulcer repair, tendon and/or ligament repair, ligament replacement, nerve repair by sheathing, dura mater replacement, submucosal membrane replacement, rotator cuff reinforcement, conjunctival and/or corneal ulcer treatment, cutaneous ulcer and/or wound treatment, and suture improvement.

28. Virally inactivated and lyophilized injectable material, which can be obtained according to any one of the preceding method claims 3 to 23, wherein it is obtained from placenta and consists of at least one membrane layer, namely the spongy tissue membrane.

29. Virally inactivated and lyophilized injectable material, which can be obtained according to claim 28, which is in a form suitable for a treatment by injection, selected from the group consisting of the treatment by intra-articular injection of the pre- arthritic or arthritic condition and ligament, meniscus, or cartilage repair. 02581797\69-0133 261174/2

30. Virally inactivated and lyophilized injectable material according to claim 28, wherein under a vacuum it has a residual water content at ambient temperature of less than 5%.

31. Virally inactivated and lyophilized injectable material according to either claim 28 or 29, or which can be obtained according to the method of any one of claims 3 to 23 and which is in a form suitable for use in a treatment by injection, selected from the group consisting of the treatment by intra-articular injection of the pre-arthritic or arthritic condition and ligament, meniscus or cartilage repair. For the Applicants, REINHOLD COHN AND PARTNERS 02581797\69-01