TITLE

METHOD FOR DECELLULARIZING TISSUES AND ORGANS Technical field

The present invention relates to a method for decellularizing tissues, in particular dermis and skin, and organs intended for clinical use.

Prior art

The transplantation of connective tissues is an operation that is often able to save an individual’s life.

A typical example is given by skin grafting, which is used for widespread third- and second-degree burns on the body. Individuals who suffer burns over most of their body risk their lives, since the lack of the skin covering results in a loss of liquids and proteins, drastically lowers body temperature and directly exposes the body to physical and microbiological agents.

When the burns are not very widespread, it is possible to perform skin autografts by removing skin from unburnt parts of the same individual and implanting it onto the burns.

In grafts of an autologous type, the tissue necessary for grafting is taken directly from the individual who will receive the transplanted tissue.

In these types of transplantation, there are no complications due to immune and rejection reactions, since the transplanted tissue is recognized by the body and is therefore not attacked by the immune defences.

However, in some cases, autologous grafts are not practicable. In fact, for example, in children and in very widespread burns there is not enough intact skin to perform an autograft.

In these cases recourse is had to homologous or heterologous grafts, i.e. transplants in which the donor is of the same species as the recipient, but is not the recipient, or in which the donor is of a different species from that of the recipient. However, these transplants are subject to serious postoperative complications due to immunological and rejection reactions to the transplanted tissue. Rejection is a set of biological reactions whereby the body tends to refuse to accept transplanted tissue, which it recognizes as foreign.

In order to overcome this serious drawback, methods have been developed with the aim of achieving immune tolerance, that is, the biological acceptance by the recipient body of the foreign tissue grafted onto it.

A first type of method provides for the administration of large doses of immunosuppressant drugs, for example “cyclosporin”, to the individual that has undergone the transplant.

This method, though on the one hand it decreases the likelihood of rejection, on the other hand it exposes the individual receiving the transplant, who is already weakened by the operation they have undergone, to the likely contraction of infections which cannot be autonomously combated by the recipient’s body, as their entire immune system has been suppressed by the administered drugs. Another type of method developed in an attempt to prevent the rejection of transplanted tissue envisages treating the tissue taken from the donor before grafting it onto the receiving individual.

This method aims on the one hand to stabilize the protein and collagen structures of the extracellular structure of the removed tissue and on the other hand to mask or eliminate the antigen agents present in the removed tissue.

Numerous scientific studies have demonstrated the possibility of treating tissue in the above-described manner in order to render it “acellular” and thereby avoid immune or rejection reactions.

A known example of such work is given by a method for obtaining an acellular dermal implant from the skin of a human cadaver.

This method envisages eliminating the cells responsible for immune reactions from the tissue by using detergent substances such as polyoxyethylene, sodium deoxycholate and the like.

Alternatively, the use of enzymes or particular salts in place of detergent substances has been described.

The method subsequently provides for the treated tissue to be placed in a cryoprotective liquid and dehydrated. Cryofreezing allows the extracellular matrix of the tissue to be preserved intact.

The tissue thus obtained is thawed and rehydrated prior to transplantation and, once transplanted, is rapidly revascularized by the recipient’s blood. The Applicant of the present patent application has already developed a method for decellularizing tissues and/or organs, which is the subject matter of patent application W02009050571 . This method is particularly useful for treating the dermis in the case of homologous transplants and is based on a non-invasive technology that is not toxic for tissue, and which uses a trypsin-based enzyme solution capable of phagocytizing, at least partly, fibroblasts, macrophages, mast cells, and other cells responsible for immune and rejection reactions against transplants.

The decellularized dermis obtained with this method has been successfully used in diverse clinical-transplantation settings since 2009 without ever having led to adverse reactions of any type in the receiving patients, either immediately or at the various follow ups over time.

The dermis treated with this method is removed from the anatomic area of the trunk of multiorgan and/or multi-tissue donors, an area that is physiologically less rich in skin adnexa, whereas the known method is not used to treat dermis taken from other anatomical regions such as, for example, the donor’s upper or lower limbs. The known method presents limits when it comes to treating the dermis originating from areas that are physiologically rich in skin adnexa, such as, for example, the upper or lower limbs, as it does not allow for reaching a decellularization sufficient to prevent or significantly decrease rejection reactions.

On the other hand, using dermis originating from the donor’s trunk poses some limits:

• both in terms of the overall amount (in cm ² ) of decellularized dermis produced and thus potentially distributable to the various requesting centres for clinical use (400-500 cm ² of decellularized dermis at most can be produced from the trunk area of the donor);

• and in terms of the size of the batches (in cm ² ) that it is possible to derive from the trunk area (it is possible to produce individual batches of a maximum size of 17x8 cm ² ).

There have been numerous, growing clinical demands for homologous decellularized dermis, especially in considerable sizes (also greater than the maximum dimensions of 17x8 cm ² obtainable from the trunk), for various uses in plastic surgery for the treatment of incisional hernias and vast losses of substance from the abdominal wall and in the realm of breast surgery for “one-step” breast reconstruction in patients undergoing mastectomy.

The Applicant has thus developed a new decellularization method which makes it possible to maintain a high standard of quality of the final product, but at the same time allows a satisfactory decellularization of tissues, such as the dermis of the upper and lower limbs, which are particularly rich in skin adnexa.

Summary of the invention

In a first aspect, the present invention relates to a method for decellularizing an isolated tissue and/or organ intended for clinical use, that is, for transplantation in a patient who needs it, wherein the method comprises a step of immersing a tissue and/or organ in an enzyme solution and a step of irradiating the tissue with ionizing electromagnetic radiation. The tissue and/or organ thus treated is immersed in an aqueous solution comprising: at least one antiseptic agent, at least one polymer containing at least one positively charged quaternary ammonium and at least one surfactant.

Preferably, the solution also comprises at least one stabilizing and emulsifying agent and/or at least one chelating agent.

The tissue and/or organ are maintained in the aqueous solution for at least 1 hour. In a second aspect the invention relates to the aqueous solution comprising the ingredients listed above and the use thereof to decellularize tissues and/or organs.

Brief description of the drawings

Fig. 1 shows the results of the histological analysis, by H&E staining, of the de- epidermized dermis (Ded) as such (A), Ded subjected to a known decellularization method and to the method of the invention (B), the decellularized dermis after 1 month (C), after 1 year (D), after 2 years (E) and after 3 years (F) in the aqueous solution according to the present invention;

Fig. 2 shows the results of the histological analysis of the de-epidermized dermis (Ded) decellularized with the method of the invention, by Masson’s trichrome staining (A), after 1 year (B), after 2 years (C) and after 3 years (D) in the aqueous solution according to the present invention; Fig. 3 shows the results of the histological analysis of the de-epidermized dermis (Ded) decellularized with the method of the invention, by Weigert staining (A), after 1 year (B), after 2 years (C) and after 3 years (D) in the aqueous solution according to the present invention; Fig. 4 shows the results of the analysis of cell viability of the Ded partially decellularized with the known methods and of the Ded decellularized with the method of the invention;

Fig. 5 shows the results of the cytotoxicity analysis of the Ded decellularized with the method of the invention; Fig. 6 shows the repopulating capacity of the Ded decellularized with the method of the invention;

Fig. 7 shows the peak load capacity of the Ded DEC maintained for 2 years in the solution of the invention versus the cryopreserved Derma DEC compared to the dermis decellularized with the known methods. Fig. 8 shows the tensile strength (peak stress) of the Ded DEC maintained for 2 years in the solution of the invention versus the cryopreserved Derma DEC compared to the dermis decellularized with the known methods;

Fig. 9 shows Young’s elastic modulus of the Ded DEC maintained for 2 years in the solution of the invention versus the cryopreserved Derma DEC compared to the dermis decellularized with the known methods;

Fig. 10 shows the rigidity (slope) of the Ded DEC maintained for 2 years in the solution of the invention versus the cryopreserved Derma DEC compared to the dermis decellularized with the known methods”. Detailed description of preferred embodiments of the invention

The method for decellularizing a tissue and/or organ according to the invention comprises: a) immersing an isolated tissue and/or organ in an enzyme solution capable of phagocytizing, at least partly, fibroblasts, macrophages, mast cells and other cells responsible for immune and rejection reactions in homologous and heterologous transplantations; b) irradiating the tissue and/or organ thus treated with ionizing electromagnetic radiation, c) immersing the tissue and/or organ obtained in step b) in an aqueous solution comprising: at least one polymer containing at least one positively charged quaternary ammonium, at least one antiseptic agent and at least one surfactant; d) leaving the tissue and/or organ immersed in the aqueous solution for at least 1 hour.

In one embodiment, in step b) the ionizing electromagnetic radiation has a frequency between 10 ¹⁹ and 10 ²² Hz.

The enzyme solution is preferably a trypsin solution, preferably pig trypsin.

The immersion of the tissue and/or organ in the enzyme solution occurs for at least 12 hours, preferably 12 to 24 hours.

Steps a) and b) preferably take place as described in W02009050571 .

The decellularization method is a decellularization method at room temperature. In the context of the present invention, room temperature means a temperature exceeding the freezing point of the aqueous solution, preferably between 5 ^e C and 35 ^e C, more preferably between 10 ^e C and 30 ^e C, even more preferably between 15 ^e C and 25 ^e C or between 18 ^e C and 25 ^e C. The ideal temperature for maintaining the container comprising the aqueous solution and the tissue and/or organ is between 18 ^e C and 23 ^e C, preferably between 20 ^e C and 23 ^e C.

The tissue that can be decellularized with the method of the invention is chosen from:

• Skin: consisting of the epidermal layer containing all of the cellular components making it up and a thin layer of the underlying dermis (dermo- epidermal tissue);

• De-epidermized Dermis (Ded); consisting of dermis without the overlying epidermal layer.

The tissue is preferably homologous or heterologous de-epidermized dermis (Ded), i.e. originating from an individual other than the transplant recipient, of the same species (homologous) or a different species (heterologous). Preferably, the Ded is taken from the trunk or lower or upper limbs of the human or animal donor. More preferably, the Ded is removed from the lower or upper limbs of a human or animal donor.

In the case of Ded taken from the trunk or lower or upper limbs of the donor, the known techniques, in some cases, do not enable a satisfactory level of decellularization to be reached, i.e. a level of removal of the cellular appendages such as to preclude the occurrence of rejection reactions in the recipient. In this case, especially as regards the Ded taken from the upper or lower limbs of a donor, the method of the invention enables a substantial cell removal to be achieved, sufficient not to create rejection reactions in the recipient.

The organ decellularized with the method of the invention is any organ removed from a human or animal body.

In the aqueous solution, the polymer containing at least one positively charged quaternary ammonium is a polyquaternium. Polyquaternium is the INCI (International Nomenclature for Cosmetic Ingredients) name used to describe numerous polycationic polymers belonging to different chemical classes and having in common the presence of at least one quaternary ammonium, preferably several quaternary ammonium groups. Within the definition of “polyquaternium” the INCI includes at least 40 different polymers, which are indicated with a sequential numbering assigned based on the order of registration and not based on their chemical nature.

For example, polyquaternium-2 is poly[bis(2-chloroethyl) ether-alt-1 ,3-bis[3- (dimethylamino)propyl]urea]; polyquaternium-6 is poly(diallyldimethylammonium chloride); polyquaternium-48 is a polymeric quaternary ammonium salt formed from methacryloyl ethyl betaine, 2-hydroxyethyl methacrylate and methacryloyl ethyl trimethyl ammonium chloride.

Therefore, polyquaterniums are polymers of a varying chemical nature containing at least one quaternary ammonium, which can be salified, for example with a chloride, a sulfide, etc.

Preferably, the polyquaternium used in the present aqueous solution is chosen from any one of polyquaterniums from polyquaternium-1 to polyquaternium-48, i.e. from any one of polyquaterniums from 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 48 and combinations thereof. In one embodiment, the polyquaternium used in the present solution is a mixture of two or more polyquaterniums chosen from polyquaterniums 1 to 48.

The correspondence between the numbering of the polyquaterniums and the respective chemical names is known and is thus not entirely described herein.

The polymer containing at least one positively charged quaternary ammonium is present in the solution in an amount of 0.001% to 0.010% by weight, preferably 0.003% to 0.006% by weight.

The at least one antiseptic agent is chosen from: polyhexaalkylene biguanidine, preferably polyhexamethylene biguanidine or polyhexayethylene biguanidine, glycerol and a combination thereof.

The at least one antiseptic agent is present in the solution in an amount of 0.0001% to 1%, preferably 0.0001% to 0.8% by weight.

If the antiseptic agent is glycerol, it is preferably included in the solution in an amount of 0.1% to 1% by weight, preferably 0.4% to 0.8% by weight.

Alternatively, the at least one antiseptic agent is present in the solution in an amount of 0.0001% to 0.005% by weight, preferably 0.00015% to 0.003% by weight.

The at least one surfactant is preferably a non-ionic, hydrophilic surfactant. The surfactant is preferably a poloxamer. Poloxamers are a class of non-ionic polymeric surfactants with a triblock structure comprising a central hydrophobic chain of polyoxypropylene or propylene glycol bound at the sides to two hydrophilic chains of polyoxyethylene or polyethylene glycol (PEG). An example of a poloxamer that can be used in the solution of the invention is poloxamer 407, which has a PEG chain with a length of about 100 repeat units and a propylene glycol block with a length of about 56 repeat units.

The at least one surfactant is present in the aqueous solution in an amount of 0.05% to 0.5% by weight, preferably 0.1% to 0.3% by weight.

Preferably, the aqueous solution further comprises at least one ingredient chosen from: a stabilizing and emulsifying agent and/or a chelating agent. The chelating agent is preferably chosen from: EDTA and/or salts thereof, preferably EDTA sodium salt, and polyamine acetylacetonate.

The amount of chelating agent present in the solution is 0.005% to 0.5% by weight, preferably 0.01% to 0.1% by weight.

The at least one stabilizing and emulsifying agent, if present, is chosen from hydroxyalkyl cellulose, preferably hydroxymethyl cellulose.

The at least one stabilizing and emulsifying agent is present in the solution in the amount necessary (q.s., quantum sufficit) to obtain an adequate emulsion of the ingredients.

In one embodiment, the aqueous solution comprises: polyhexaalkylene biguanidine, preferably polyhexamethylene biguanidine, or glycerol, at least one polyquaternium from 1 to 48 or a mixture of a polyquaternium and a poloxamer or a mixture of poloxamers.

In one embodiment, the aqueous solution comprises: polyhexaalkylene biguanidine, preferably polyhexamethylene biguanidine in an amount of 0.0001% to 0.005% by weight, preferably 0.00015% to 0.003% by weight, at least one polyquaternium from 1 to 48 or a mixture of polyquaterniums in an amount of 0.001% to 0.010% by weight, preferably 0.003% to 0.006% by weight and at least one surfactant, preferably a poloxamer or a mixture of poloxamers, in an amount of 0.05% to 0.5% by weight, preferably 0.1% to 0.3% by weight.

In one embodiment, the aqueous solution comprises glycerol in an amount of 0.1% to 1% by weight, preferably 0.4% to 0.8% by weight, at least one polyquaternium from 1 to 48 or a mixture of polyquaterniums in an amount of 0.001% to 0.010% by weight, preferably 0.003% to 0.006% by weight, and at least one surfactant, preferably a poloxamer or a mixture of poloxamers, in an amount of 0.05% to 0.5% by weight, preferably 0.1% to 0.3% by weight.

In one embodiment, the aqueous solution comprises: polyhexaalkylene biguanidine, preferably polyhexamethylene biguanidine, or glycerol, at least one polyquaternium from 1 to 48 or a mixture of polyquaterniums, at least one surfactant, preferably a poloxamer or a mixture of poloxamers, hydroxyalkyl cellulose, preferably hydroxymethyl cellulose, and/or EDTA. In a preferred embodiment, the aqueous preservation solution comprises:

• polyhexaalkylene biguanidine, preferably, polyhexamethylene biguanidine, in an amount of 0.0001% to 0.005% by weight, preferably 0.00015% to 0.003% by weight, and/or glycerol in an amount of 0.1% to 1% by weight, preferably 0.4% to 0.8% by weight;

• at least one polyquaternium from 1 to 48 or a mixture of polyquaterniums, i.e. any one of polyquaterniums from 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 48 and combinations thereof, in an amount of 0.001% to 0.010% by weight, preferably 0.003% to 0.006% by weight;

• a non-ionic, hydrophilic surfactant, preferably a poloxamer or a mixture of poloxamers, in an amount of 0.05% to 0.5% by weight, preferably 0.1% to 0.3% by weight;

• preferably, hydroxyalkyl cellulose, more preferably hydroxymethyl cellulose, in a sufficient amount (q.s.) to obtain an adequate emulsion of the ingredients; and/or

• preferably, EDTA and/or a salt thereof, more preferably EDTA sodium salt, in an amount of 0.005% to 0.5% by weight, preferably 0.01% to 0.1% by weight.

In step d) of the decellularization method, the tissue and/or organ is left immersed in the aqueous solution preferably for at least 1 hour, preferably for at least 2 hours, more preferably for at least 3 hours, more preferably for at least 10 days. Advantageously, the tissue and/or organ is left in the solution for a period of between 1 hour and 30 days, preferably between 2 hours and 20 days, preferably between 10 and 20 days or between 10 and 25 days.

In one embodiment, in step d) of the decellularization method, the tissue and/or the organ is left immersed in the aqueous solution preferably for at least 15 days, more preferably for at least 20 days, even more preferably for at least 30 days. The Applicant has verified, through the experiments included in the present patent application, that the Ded, preferably taken from the upper or lower limbs of a donor, and thus richer in skin adnexa, already after a few days shows an effective removal of the residual epidermal layer, and that after 20 days the fibroblasts present in the underlying dermal matrix have been sufficiently removed.

The purpose of a decellularization treatment of a tissue and/or organ is to obtain a tissue and/or organ from which fibroblasts, macrophages, mast cells, and other cells responsible for immune and rejection reactions in transplantations have been at least partly removed.

The method of the invention has shown to be very efficient in decellularizing a tissue that is very rich in skin adnexa, such as Ded from the upper or lower limbs, and has thus demonstrated to be better than the known method described in W02009050571 for this type of tissues.

The tissue cell viability measured after immersion in the aqueous solution for at least 1 hour is between 5% and 20%, preferably between 8% and 15%.

It should be noted that in order to be able to define a tissue as cellularly viable, the viability value must be greater than 50%.

At the same time, it has been demonstrated that the aqueous solution ensures a perfect biological preservation of the tissue extracellular matrix, that is to say, the extracellular matrix remains intact in terms of its protein structure, collagen matrix and in every respect except for the cells responsible for rejection reactions.

The integrity of the extracellular matrix is both biological (in the sense mentioned above) and mechanical, in the sense that the mechanical properties of the tissue, such as, for example, elasticity, tensile strength and the like, remain practically unchanged compared to a tissue that has not undergone any treatment.

It was also observed experimentally that the tissue treated with the aqueous solution assures a perfect growth of human fibroblasts (hFs), thereby enabling excellent cell regrowth (with cells belonging to the recipient) once the transplantation has taken place.

The tissue thus treated is also an excellent support for stem cells.

With particular reference to the Ded, the method of the invention has numerous advantages.

In particular, the possibility of treating a larger overall amount of Ded and obtaining an effective decellularization, as the starting tissue can also be taken from donors’ upper and/or lower limbs and effectively decellularized despite being very rich in skin adnexa. In particular, with the method of the invention it is possible to treat amounts of 1000-3000 cm ² of skin taken from the donor’s upper or lower limbs, whereas with the method of W02009050571 it was possible to decellularize only 400-500 cm ² of skin obtainable from the donor’s back, an area that is less rich in cellular appendages but at the same time has a smaller availability of tissue that can be taken.

Another advantage is the possibility of producing individual decellularized patches of a larger size (minimum 10x5 maximum 100x8 cm ² vs minimum 3x2 maximum 17x8 cm ² obtainable with the method known from W02009050571), thus overcoming the problem of the anatomical limit of the area from which to draw tissue (upper/lower limbs vs trunk).

Some major clinical implications arise from these two advantages, above all in the two main areas of application of homologous decellularized Ded, and in particular:

• in the context of emergency surgery, where it becomes possible to use a single patch of decellularized Ded of adequate size without having to suture several patches together. Suturing is presently used, given the absence of an adequately sized patch, in order to obtain a network of patches to be used in the reconstruction of an entire abdominal wall affected by an incisional hernia. The network obtained by joining several patches has less mechanical tensile strength and more scarring issues;

• in the context of plastic and breast reconstruction surgery, in particular for the application of the “one-step” technique, which provides for a single breast reconstruction operation in patients undergoing unilateral or bilateral mastectomy as a result of cancer. This surgical technique normally requires several patches of tissue for a single patient, with a size of at least of 13x7 cm ² . Thanks to the possibility of preparing much larger decellularized Ded patches, this surgical procedure can be applied more easily with the method of the present invention, with important clinical implications for the treated patients in terms of less postoperative pain, shorter hospital stays and the possibility of avoiding later operations to complete the reconstruction. Finally, the invention also relates to an aqueous solution as described above and the use thereof to decellularize a tissue and/or organ as described above, which may have already undergone a decellularization method. In the latter case, the method of the invention also comprises a pre-treatment of the tissue and/or organ that leads to a partial decellularization thereof.

Examples

All of the experiments described herein were carried out with the following preservation solution:

• Glycerol 0.25%

• Polyquaternium 0.004%

• EDTA O.01%

• Poloxamer 0.18%

• Hydroxyethyl cellulose (q.s.)

Amount: 10-30 ml solution per tissue sample.

Glycerol: 25 mI in 10 ml and 75 mI in 30 ml.

Preservation at room temperature of De-epidermized Dermis (Ded), previously subjected to a decellularization method to induce both the full removal of the cellular component and its preservation at room temperature.

The de-epidermized dermis (Ded) obtained from the upper or lower limbs of a donor has a larger cellular component than the Ded obtained from the donor’s trunk. For this reason, the decellularization method presently used for the Ded obtained from upper or lower limbs, as described in W02009050571 , is not capable of completely removing the cellular component. For this reason, the Ded obtained from upper or lower limbs cannot presently be taken into consideration as the starting tissue to be decellularized and used as a scaffold in a clinical setting. The Ded obtained from the upper or lower limbs of a donor was subjected to the method described in W02009050571 , with the addition of a further step of maintaining it in the solution for 1 month in order to induce the removal of the residual cellular component and thereby obtain fully decellularized Ded. For the purpose of evaluating whether the decellularized Ded is capable of maintaining its characteristics over time, analyses were also performed after 1-2 years of preservation in the solution.

The Ded was thus maintained in the solution for:

• 1 month, in order to assess cell removal and, consequently, the production of fully decellularized Ded (Ded DEC 1 month in SS)

• 1-3 years, in order to assess whether the characteristics of the fully decellularized Ded are maintained over time.

Once the experimental period was over, the tissue was subjected to the following analyses:

• Histological analysis and H&E staining

• Analysis of cell viability

• Microbiological analysis: plate culture and LAL test

Results of the experimentation Histological analysis and H&E staining

As expected, the Ded has numerous cells, mainly present in the skin adnexa (Fig. 1 A). The application of the decellularization method - the subject matter of patent application W02009050571 - on the Ded enables a good removal of the cellular component, but not a total removal (Fig. 1 B). The partially decellularized Ded (Ded DEC) maintained in the solution of the invention for 1 month wholly loses its residual cellular component, while at the same time maintaining its structural characteristics intact (Fig. 1 C). The partially decellularized Ded (Ded DEC) maintained in the solution for 1 year (Fig. 1 D) and 2 years (Fig. 1 E), as expected, shows no residual cellular component and maintains structural characteristics compatible with clinical use. The Ded DEC maintained in the solution for 3 years shows to be partially deteriorated, but the structural characteristics maintained are nonetheless potentially compatible with clinical use (Fig. 1 F).

Histological analysis and Masson’s trichrome staining

The specific Masson’s trichrome staining of the samples identifies a maintenance of the collagen fibres of the Ded DEC compatible with clinical use for up to 1 -2 years of preservation in the solution (Fig. 2B-C). The specific Masson’s trichrome staining of the samples identifies collagen fibres of the Ded DEC - preserved in the solution for 3 years - which have partially deteriorated. The structural characteristics maintained are nonetheless potentially compatible with clinical use (Fig. 2D).

Histological analysis and Weigert staining

The specific Weigert staining of the samples identifies a maintenance of the elastic fibres of the Ded DEC up to 1-2 years of preservation in the solution (Fig. 3B-C). The specific Weigert staining of the samples identifies a loss of the elastic fibres of the Ded DEC preserved in the solution for 3 years (Fig.3D).

Analysis of cell viability

The analysis of cell viability, performed by means of an MTT assay, shows that the cell viability of the Ded is reduced in the partially decellularized Ded (Ded DEC) following the application of the decellularization method of W02009050571 (Fig. 4). The cell viability of the Ded DEC is completely removed after the tissue has been maintained in the solution for 1 month (sample: Ded DEC 1 month in SS, solution) and, as expected, this is maintained after 1-2 years of the tissue in the solution (samples: Ded DEC 1 yr in SS and Ded DEC 2 yr in SS) (Fig. 4).

Microbiological analysis

Plate culture : the microbiological analysis was performed on portions of Ded (1 cm ² ) that was partially decellularized (Ded DEC) and partially decellularized Ded preserved in the solution for 1-2 years (Ded DEC 1 year in the solution and Ded DEC 2 years in the solution), incubated at 37°C on COS and Sabouraud plates for 3 and 14 days, respectively, in order to determine whether there was any growth of bacteria and/or fungi. The microbiological analysis identified the absence of bacterial and fungal contamination in the partially decellularized Ded (Ded DEC) and in the partially decellularized Ded preserved for 1-2 years in the solution

LAL test: the LAL test was carried out to evaluate the possible presence of bacterial endotoxins, released by the tissue, in the preservation liquid. The results show that no presence of bacterial endotoxins was identified in the solution used to preserve the decellularized Ded for 1-2 years. Cytotoxicity analysis on primary cultures of human fibroblasts

The cell cytotoxicity analysis was performed by treating primary cultures of human fibroblasts (hFs) with an extract obtained from 3 days of incubation, at +4°C, of the decellularized Ded, maintained in the solution for 1 -2 years, with RPM1 1640 culture medium + 1% antibiotics, in a tissue/RPMI ratio of 25cm ² /100 ml. The cells were treated with the extract for 2, 24 and 72h and were then subjected to an MTT assay to evaluate their viability (samples: SS 1 yr, SS 2yr). The results demonstrate that the extract is not toxic for cells (Fig. 5).

Repopulation

The repopulating capacity of the Ded DEC preserved for 1-2 years in the solution was analysed by culturing primary cultures of human fibroblasts on it in vitro, after it had been previously washed and dried on gauze. The MTT assay identifies a repopulating capacity of the Ded DEC preserved for 1-2 years in the solution with viable cells (samples: Ded DEC 1 yr in the solution + hFs, Ded DEC 2yr in the solution + hFs) that is comparable to that of the control, i.e. cryofrozen Ded DEC (sample: Ded DEC CRYO + hFs) (Fig. 6). It should in fact be underscored that the greater viability identified in the Ded DEC CRYO sample is to be attributed to its partial decellularization and hence to the maintenance of viable cells in the initial sample subjected to repopulation.

Mechanical tests

The mechanical tests were performed at the “Surgical Sciences and Technologies” department of the Rizzoli Orthopaedic Institute in Bologna using instruments validated for clinical use. In particular, the mechanical tests were carried out on Ded DEC preserved for 2 years in the solution, in order to assess its biomechanical properties. Cryopreserved Derma DEC was used as a control. In particular, the thickness, width, length and area (mm ² ) of every sample were measured using a digital calliper. The tensile tests were carried out by securing the lower and upper part of the samples to the lower fixed grip and upper movable grip of the tensile testing apparatus. The peak load, tensile strength (peak stress), Young’s elastic modulus and rigidity (slope) were measured for each sample. The results demonstrate that the Ded DEC maintained for 2 years in the solution has a significantly lower (p<0.5) peak load capacity than the cryopreserved Derma DEC (Fig. 7), a significantly lower (p<0.5) tensile strength than the cryopreserved Derma DEC (Fig. 8), a Young’s elastic modulus comparable to that of the cryopreserved Derma DEC (Fig. 9) and a significantly higher (p<0.5) rigidity than the cryopreserved Derma DEC (Fig. 10).

Conclusions of the experimentation

The solution is capable of removing the residual cellular component from the Ded previously subjected to the decellularization method, while maintaining a structural integrity thereof compatible with clinical use and sterility after 1-2 years.

The Ded DEC maintained in the solution for 3 years is partially deteriorated; the structural characteristics maintained are nonetheless potentially compatible with clinical use (Fig. 1 F). The Decellularized Ded thus preserved is not cytotoxic, easily repopulates and, based on a macroscopic analysis, seems to maintain its biomechanical properties, though specific mechanical tests are necessary to support such considerations.

These results suggest the possibility of expanding the availability of decellularized dermis in cases where Derma, which nonetheless remains the preferred starting tissue, is not sufficiently available to satisfy the growing clinical demand.

The Ded DEC maintained in the solution for 2 years has some biomechanical characteristics that are inferior (peak load, tensile strength), others that are comparable (Young’s elastic modulus) and others that are superior (rigidity) to those of the cryopreserved Derma DEC.