DESCRIPTION

The present patent application for industrial invention relates to a process for producing a pulverized scaffold. The field of the invention is the one of regenerative and reconstructive medicine, and particularly the field of dermatology and general plastic and/or cosmetic surgery.

Scaffolds of synthetic origin are known. However, such synthetic scaffolds have compatibility problems with the human tissue.

Scaffolds of biological origin obtained from amniotic membrane are also known. As it is well known, an amniotic membrane is a tissue of placental origin, and the cells of the epithelial layer of the membrane express the markers of the stem cells. In this regard, the amniotic membrane is used in dermatology and in plastic and cosmetic reconstructive surgery as a biomaterial used to obtain a scaffold.

When used as a scaffold, the amniotic membrane is employed in the treatment of acute and chronic wounds, as well as in the treatment of eye injuries. The amniotic membrane consists of a layer that is manipulated and sutured at the wound, inducing a proliferation of new cells.

In order to minimize rejection problems, an amniotic membrane of biological origin derived from human tissue is preferably used. However, the use of the amniotic membrane of biological origin derived from human tissue is impaired by some drawbacks and limitations.

Such limitations or drawbacks consist in the fact that the amniotic membrane of human origin cannot be sold and marketed freely and must be deposited in a Tissue Bank. Thus, a specific application must be filed to the Tissue Bank in order to use an amniotic membrane of human origin.

A further drawback lies in the fact that the human amniotic membrane cannot be used for the production of medical devices, as its commoditization is against ethical principles. Conversely, the use of an amniotic membrane of animal origin as a scaffold entails rejection issues of the human tissue, given that an amniotic membrane of animal origin has a high antigenic load.

CN107583109A discloses a method for preparing an amniotic membrane according to the preamble of claim 1 , wherein the amniotic membrane is decellularized by means of digestion with Trypsin combined with Triton X-100. The decellularized membrane is exposed in liquid nitrogen and is ground with a high-speed grinder to obtain particle fragments. CN107583109A does not disclose the defrosting of the membrane before grinding.

W02017/059089A1 discloses a method for producing particles of decellularized amniotic membrane that provides for a cryogrinding of equine amniotic membrane. W02017/059089A1 does not disclose an enzymatic digestion of the amniotic membrane or a defrosting step prior to cryogrinding.

The purpose of the present invention is to overcome the drawbacks of the prior art by devising a production process of a pulverized scaffold obtained by using an amniotic membrane that is not derived from human tissue.

A further purpose of the present invention is to eliminate the antigenic load of the amniotic membrane not derived from human tissue.

Another purpose of the invention is to provide a process for preparing a pulverized scaffold that can be used conveniently and rapidly in an operating room or in a clinic.

These purposes are achieved in accordance with the invention with the features listed in the attached independent claim 1 .

Advantageous achievements appear from the dependent claims.

The process of producing a pulverized scaffold according to the invention is defined by claim 1 .

For the sake of explanatory clarity, the description of the production process of a pulverized scaffold according to the invention continues with reference to the attached drawings, which only have an illustrative and nonlimiting value, wherein:

Fig.1 is a block diagram illustrating five steps of a production process of a pulverized scaffold according to the invention; Fig. 2 is a block diagram illustrating the machines used for the process of Fig. 1 ;

Fig. 3 illustrates two optical microscope images of a fresh amniotic membrane tissue before a decellularization step;

Fig. 4 illustrates two optical microscope images of a decellularized tissue of amniotic membrane after the decellularization step;

Fig. 5 is a histogram illustrating the viability value over time in three samples: a pulverized amniotic membrane according to the invention, a fibroblast culture without serum and a fibroblast culture with serum;

Fig. 6 are three optical microscope images illustrating the cell growth in the three samples of Fig. 5;

Fig. 7 is an optical microscope image (OM 40x) of the fresh amniotic membrane before the decellularization process;

Fig. 8A is an optical microscope image (OM 20x) of the amniotic membrane before decellularization (i.e. a fresh amniotic membrane);

Fig. 8B is an optical microscope image (OM 20x) of the amniotic membrane after the freezing-defrosting steps; and

Fig. 8C is an optical microscope image (OM 20x) of the amniotic membrane after the final pulverization step.

The production process of the pulverized scaffold according to the invention provides for a series of steps that are to be performed in the order and time described.

Referring to Fig. 1 , the production process of the pulverized scaffold according to the invention comprises the following operational steps: a. treatment of an amniotic membrane in an incubator with a decellularization enzyme; b. washing of the amniotic membrane treated in step a; c. decellularization by immersing the amniotic membrane in nitrogen vapor in order to obtain a frozen decellularized amniotic membrane; d. defrosting of the decellularized amniotic membrane; and e. pulverization of the decellularized defrosted amniotic membrane. The amniotic membrane used in the production process of the pulverized scaffold consists of an amniotic membrane of biological origin derived from animal tissue. In particular, equine amniotic membrane is used because of the following advantages:

- large size from which to obtain a good amount of product;

- easy acquisition of such a membrane;

- possibility of subjecting such a membrane to freeze-drying processes.

Fig. 3 illustrates two magnified optical microscope images of two sections of an amniotic membrane of equine origin. As it can be seen in Fig. 3, the amniotic membrane is cellularized, as indicated by the color of the cellular nuclei. In fact, the amniotic membrane has been treated with a hematoxylin- eosin dye that shows a staining of the cellular nuclei preserved in the membrane.

The cellularization of the amniotic membrane entails rejection problems.

Also with reference to Fig. 2, step a) provides for treating the amniotic membrane in an incubator (1 ) with a decellularization enzyme. Conditions of 5% carbon dioxide (CO2) and a temperature of 37°C are created in the incubator.

The amniotic membrane inside the incubator (1 ) is treated with an enzymatic solution including a sterile 2.5% trypsin 10x solution, which is appropriately diluted with a 0.9% sodium chloride (NaCI) 2x physiological solution.

The NaCI physiological solution is previously added with a mixture of antibiotics/antimycotics. Specifically, said antibiotic/antimycotic mixture is of pen-strep-Ampho.B type.

The membrane is left in the incubator for a time longer than 20 hours, preferably 24 hours. In fact, a time of at least 20 hours is necessary to completely remove the cellular nuclei responsible for immunogenicity; a shorter time would not guarantee a good removal of the cellular nuclei responsible for immunogenicity. After the completion of step a), the production process of the pulverized scaffold provides for performing step b), which consists in washing the amniotic membrane.

Said step b) is performed by washing the amniotic membrane twice in a tank (2) with a 0.9% NaCI physiological solution. The time of each washing is comprised between 10 minutes and 20 minutes.

The decellularization technique used in steps a) and b) is an enzymatic and mechanical technique, respectively. In step a), the cellular nuclei are detached with the sterile trypsin solution. In step b), the cellular nuclei that have been damaged by the incubation in the sterile trypsin solution are removed with the NaCI washings.

The incubation and washing of the amniotic membrane produce a partial decellularization. At the end of step b) a partially decellularized amniotic membrane is obtained, which is immersed in nitrogen vapor for a time comprised between 45 minutes and 2 hours in order to achieve a total decellularization.

Step c), wherein the amniotic membrane is immersed in nitrogen vapor, is performed by using a nitrogen container (3) that is commonly known as dryshipper, at a cryogenic temperature that is comprised between -190°C and -145°C, so as to freeze the amniotic membrane.

The decellularized and frozen amniotic membrane is obtained at the end of said step c).

With reference to Fig. 4, at the end of step c, the decellularized amniotic membrane is devoid of cells, unlike the amniotic membrane shown in Fig. 3. Such a finding is shown by the laboratory analysis performed. In particular, Fig. 4 shows that the decellularization of the membrane was complete. In fact, the membrane has been cleared of cells (no preserved nuclei are present according to the histological analysis), whereas the membrane matrix has remained intact.

Advantageously, the decellularization of the equine amniotic membrane allows to eliminate its antigenic load, thus removing the cellular components that are responsible of a possible rejection of the amniotic membrane by the recipient subject.

MTT assays (measurement of cytotoxicity of drugs) were performed on the initial cellularized amniotic membrane and on the decellularized amniotic membrane. The MTT assay shows the enzymatic-metabolic activity of the cells in vitro.

The MTT assays gave the results shown in Table 1 below:

Table 1

The fresh amniotic membrane tissue has a viability value (OD) = 0.616 (i.e. a 3.425 percentage). In contrast, the decellularized tissue of the amniotic membrane has a viability value (OD) = 0.027.

The results in Table 1 confirm the decellularization of the amniotic membrane by means of the process according to the invention, with special reference to the enzyme-metabolic activity of the cells in a tissue. It must be considered that a viable tissue has a reference viability percentage greater than or equal to 50%. Conversely, in the specific case, the decellularized amniotic membrane has a viability percentage of 0.1 %, which indicates that the tissue is not viable, that is to say, the tissue is decellularized.

Step d) antecedes the pulverization of the decellularized equine amniotic membrane.

Step d) provides for defrosting the amniotic membrane by immersing the amniotic membrane in a 0.9% NaCI physiological solution contained in a container and for placing the container in an air-flow hood (4) that ensures the sterility of the entire operation.

The defrosting in air-flow hood is advantageously performed to maintain a sterility of the decellularized amniotic membrane. The air-flow hood ensures a total sterility, being classified as A according to the Good Manufacturing Practice (GMP) Standards.

No blood vessels remain in the defrosted amniotic membrane; however, impressions of the vascular channels where the vessels pass may remain histologically.

The pulverization step e) of the decellularized amniotic membrane provides for cryo-milling by means of a grinding device (5).

The grinding device (5) comprises a grinding container, a ball arranged in the grinding container, and a vibrator that puts the grinding container into vibration. Such a type of grinding device is commercially available under the name "CryoMill grinder, MM 500 nano chef."

The decellularized amniotic membrane is placed in the grinding container, wherein it is frozen by means of liquid nitrogen. The first freezing during step c), the following defrosting during step d) and the second freezing during step e) guarantee a complete decellularization of the amniotic membrane.

Specifically, the grinding container with the decellularized amniotic membrane is immersed in liquid nitrogen until the decellularized amniotic membrane is completely frozen. Also the grinding ball is immersed in liquid nitrogen for freezing and then is placed in the grinding container with the frozen amniotic membrane.

The vibrator is operated in such a way that the grinding container vibrates at a frequency of 30 oscillations per second. In such a way, the ball is moved in the grinding container, pulverizing the frozen amniotic membrane.

The cryo-milling process takes about 1 -3 minutes. The entire cryo- milling process is performed in a sterile environment.

The grinding container is brought to a sterile environment in order to take the pulverized decellularized amniotic membrane. The taking of the pulverized decellularized amniotic membrane is performed after waiting for a few minutes using a sterile pipe cleaner. Such a taking of the pulverized decellularized amniotic membrane is performed without letting the amniotic membrane powder to defrost. The taking of the amniotic membrane powder is performed in a sterile environment, in an air flow hood wherein the membrane powder is not aspirated because the laminar flow is at the top of the hood and ensures the total sterility of the process and thus of the final product.

In the specific example, a pulverized scaffold comprising the pulverized decellularized amniotic membrane in a quantity equal to 4.23g was obtained with a 3x3 cm ² patch of amniotic membrane.

The advantages of the pulverized scaffold production process according to the invention, which provides for obtaining a biocompatible and bioactive pulverized scaffold, are evident.

Advantageously, the pulverized scaffold obtained according to the present invention is ready for use in the operating room or in a clinic; in fact, it can be immersed in a 0.9% NaCI physiological solution under sterile conditions and can be employed for topical and/or infiltrative use.

Other biomaterials and/or tissues of animal, human, synthetic or mixed origin can be subsequently added to the pulverized scaffold.

The pulverized scaffold obtained according to the production process of the invention can be used in both ophthalmology and wound care.

Experimental trials conducted by the applicant reported that the use of the pulverized scaffold achieved positive effects on fibroblast growth.

In order to evaluate the effect of the pulverized decellularized tissue on the growth of human fibroblasts, the cellular viability was measured at 2, 24, and 72 hours. Fibroblasts with and without serum were cultured as controls.

The graph in Fig. 5 shows the viability values over time:

AM DEC is the decellularized pulverized tissue according to the invention,

CTR is the control consisting of fibroblasts without serum, and

CTR+ is the control consisting of fibroblasts with serum.

The graph shows that the pulverized tissue has a positive effect on the growth of fibroblasts. In fact, the growth of the AM DEC sample maintains a trend similar to the control, up to 72 hours, where the viability value exceeds that of the control. With reference to Fig. 6, by observing the cell growth in pulverized decellularized tissue (AMDEC), the control without serum (CTR) and the control with serum (CTR+) with the optical microscope, it can be seen that the morphology of the fibroblasts cultured with the pulverized decellularized tissue (AM DEC) is better than that of the control without serum (CTR) and comparable to that of the control with serum (CTR+).

The described method is effective in removing the cellular components contained in the amniotic membrane only and exclusively if the indicated steps are followed in the prescribed time sequence.

In particular, laboratory data showed that the freezing and defrosting of the tissue must occur prior to pulverization and are indeed preparatory to tissue pulverization.

The freezing-defrosting-pulverization steps must necessarily occur in this time sequence. In fact, it was shown that, during the transition from the freezing step to the defrosting step, the amniotic membrane is subjected to such a thermal shock that, at histomorphological level, the extracellular membrane is damaged and then the cellular nucleus is completely degraded. Indeed, the cellular nucleus appears necrotic and in an evident degenerative state, whereas the cytoplasmic membrane is also almost completely damaged.

The subsequent pulverization of the tissue is the successive essential step to complete the decellularization. In such a specific case, in fact, the tissue that is already damaged in the cellular component due to the thermal shock is subjected to a subsequent mechanical shock, which is capable of completely removing the necrotic nuclei already in an evident degenerative state from the previous steps.

Fig. 7 shows a macroscopic image of an amniotic membrane before the decellularization process. Such an image indicates that the initial tissue is intact, without any damage or injury, and this is very important and should be not taken for granted.

The amniotic membranes may not always be totally intact. In fact, a damaged/perforated/fragmented amniotic membrane would not be suitable for the decellularization process according to the invention because a decellularized tissue would be definitely obtained, but not preserved in terms of matrix and tissue architecture.

The decellularization method according to the invention has a twofold advantage:

- the advantage of totally eliminating the cellular component; and

- the advantage of keeping the architectural and fibrous structure of the tissue unaltered.

Such an aspect has an important consequence also from a clinical point of view, since a non-intact tissue - in such a case an amniotic membrane that is not structurally intact - will not guarantee the same clinical efficacy compared to a patient that receives an intact tissue.

Fig. 8A shows the amniotic membrane before the decellularization. Following to staining with hematoxylin-eosin, it is possible to see an abundant cellular component with preserved nuclei and a homogeneous, compact matrix.

Fig. 8B shows the amniotic membrane after undergoing freezingdefrosting. Following to staining with hematoxylin-eosin, it can be seen that the membrane is partially decellularized, as remnants of small cellular nuclei remain in an evident necrotic degenerative state.

Fig. 8C shows the amniotic membrane after undergoing the final pulverization step. Following to staining with hematoxylin-eosin, it can be seen that the amniotic membrane is completely decellularized, devoid of necrotic cells, and at the same time characterized by a compact and homogeneous matrix and architectural and fibrous structure. The structural integrity of the fragment of decellularized amniotic membrane is also evident microscopically with the hematoxylin-eosin staining.