EP4178633A1

EP4178633A1 - Tridimensional bioactive porous body for bone tissue regeneration and process for its preparation

Info

Publication number: EP4178633A1
Application number: EP21752159.0A
Authority: EP
Inventors: Luciana Sartore; Domenico Russo; Pierangelo GUIZZI; Kamol DEY; Manuel SALMERON-SANCHEZ; Elisa BORSANI
Original assignee: Fondazione Della Comunita' Bresciana Onlus; Le Rondini Citta' Di Lumezzane Onlus; Universita degli Studi di Brescia
Current assignee: Fondazione Della Comunita' Bresciana Onlus; Le Rondini Citta' Di Lumezzane Onlus; Universita degli Studi di Brescia
Priority date: 2020-07-08
Filing date: 2021-07-08
Publication date: 2023-05-17
Also published as: IT202000016576A1; WO2022009125A1

Abstract

A tridimensional bioactive porous body for the regeneration of bone tissue and processes for the production thereof are described.

Description

TRIDIMENSIONAL BIOACTIVE POROUS BODY FOR BONE TISSUE REGENERATION

AND PROCESS FOR ITS PREPARATION

FIELD OF THE INVENTION The present invention refers to a bioactive body endowed with a self-supporting tridimensional porous structure which is used in bone tissue regeneration; the invention also relates to the method for the production of the porous body.

STATE OF THE ART

In many situations it is necessary to replace or integrate portions of bone tissue of the human or animal body or facilitate the regeneration thereof.

Bone loss can result from multiple causes including trauma, blast injuries, diseases such as osteomyelitis, osteonecrosis or osteosarcoma, or surgical excisions. Such conditions often result in cavitation or complete loss of bone tissue, and bone repair or regeneration in these cases become difficult, very long-lasting, or sometimes impossible. The treatment of bone defects is still an unsolved problem in medical science since bones show limited regeneration and repair properties due to the extracellular matrix characteristics and the lack of blood and lymphatic flow essential for tissue regeneration processes.

Many techniques have been used for the purpose of attempting to repair bone defects. The most common ones contemplate bone replacement with autologous vascularized bone grafts (autotransplantation), massive allograft (generally from a cadaver), or use of resorbable or non -resorbable “artificial bone”.

Another method to promote bone regeneration is through the introduction of osteoinductive bioactive factors (bone morphogenetic proteins, plasma rich in platelets, synthetic peptides, etc.), that can be introduced into the area of bone loss through various techniques. Mechanical methods are also used to promote bone regeneration, such as distraction osteogenesis and guided or protected bone regeneration.

Despite these techniques available to physicians and surgeons, the treatment of injuries with severe bone loss, and in general the treatment of fractures that do not heal spontaneously, remain a clinical problem difficult to solve and the need remains for effective methods to promote bone tissue growth, that are as least invasive as possible.

Tissue engineering represents a possible solution for the repair and regeneration of these tissues. This branch of biomedical engineering deals with identifying systems that can be produced in laboratory and that are able, once inserted into the site of a lesion, to stimulate the regrowth of the damaged tissue. Parts that are implanted in the human or animal body in order to at least temporarily compensate for a compromised function and stimulate tissue regeneration are called in the medical field with the English term “scaffold”, which will also be used in the present description.

Various systems based on tissue engineering have been described in the medical and patent literature.

Patent application US 2005/0118230 A1 describes a fluid hydrogel to be injected into the site of a lesion obtained by reaction between a polypeptide, for example a collagen gelatin, and a long chain carbohydrate, for example dextran, hyaluronic acid, glycogen, chitosan, starch, etc amino acids and/or chelating agents of divalent ions (e.g, EDTA) can be added to the hydrogel in order to increase its mechanical consistency. This hydrogel is an injectable fluid and does not compensate for the bone structural function, which is only recovered after the bone tissue regrowth.

Patent application EP 2386321 A2 describes a “plug” consisting of two parts, a more rigid one that mimics the bone tissue and one in the form of hydrogel, adhering to the first one, with a consistency similar to chondral tissue. The stiffer part is made with polymers, such as polylactides, polyglycolides, polycarbonates, etc., optionally mixed together, and optionally added with polymethylmethacrylate having a stiffening function.

An improved system for the production of bone tissue scaffolds is described in the articles “Rational design and development of anisotropic and mechanically strong gelatin- based stress relaxing hydrogels for osteogenic/chondrogenic differentiation”, K. Dey et al., Macromol. Biosci. 2019, 1900099 and “3D gelatin-chitosan hybrid hydrogels combined with human platelet lysate highly support human mesenchymal stem cell proliferation and osteogenic differentiation”, F. Re etal. , Journal of Tissue Engineering, 2019, vol. 10, pages 1-16. The article “Effects of gamma sterilization on the physicomechanical and thermal properties of gelatin-based novel hydrogels”, K. Dey et al ., Polymer Engineering and Science, 2019, vol. 59(12), pages 2533-2540, reports the effects of gamma-ray sterilization of the material described in the two previous articles, confirming that it essentially maintains its properties unchanged following treatment.

The hydrogels described in these articles have proved to be very efficient for bone tissue regrowth; however, there is still a need in the field to have materials with modulable chemical -physical and mechanical characteristics, to allow the production of scaffolds with properties suitable for the repair needs of any specific bone defects.

The object of the present invention is to provide a tridimensional bioactive porous body for bone tissue regeneration, as well as to provide a process for the production of this porous body.

SUMMARY OF THE INVENTION

These objects are achieved with the present invention, which in its first aspect relates to a tridimensional bioactive porous body obtained by reaction between:

- an aminated long chain polysaccharide having a weight average molecular weight of at least 3 kDa, selected from aminated chitosan, aminated dextran and a mixture thereof, in an amount between 1.0% and 83.0% by weight;

- a water-soluble polymer functionalized with groups capable of reacting with reactive sites of the aminated polysaccharide and/or of a polypeptide, in an amount between 11.4% and 35.0% by weight;

- a polypeptide selected from polypeptides derived from natural tissues, synthetic polypeptides and mixtures thereof, in an amount between 0% and 83.0% by weight; wherein said percentage amounts by weight refer to the sum of the weights of said aminated long chain polysaccharide, water-soluble polymer, and polypeptide.

The tridimensional bioactive porous body of the first aspect of the invention may further contain one or more additional components selected from:

- mesenchymal stromal cells deriving from bone marrow, adipose tissue or umbilical cord;

- growth factors;

- antibiotics, drugs or medicines useful in the treatment of bone lesions and/or in the regeneration of tissues;

- additives for modifying the stiffness or degradability of the porous body, such as complexes of calcium, calcium phosphate, calcium carbonate, decellularized and pulverized bone material, or hydroxyapatites.

In its second aspect, the invention relates to the processes for the production of the porous body described above.

A first possible process of the invention, carried out when the tridimensional bioactive porous body contains a polypeptide, comprises the following steps: a) dissolving the polypeptide in distilled water, at a temperature between 20 and 70

°C; b) adding the functionalized water-soluble polymer to the solution obtained in step a), and allowing the system to react at a temperature between 20 and 70 °C for a time between 5 and 30 minutes; c) adding the aminated long chain polysaccharide, selected from aminated chitosan, aminated dextran and mixtures thereof, to the solution obtained in step b), and allowing the system to react at a temperature between 20 and 70 °C for a time between 30 minutes and 2 hours, obtaining a sol; d) allowing the sol obtained in step c) to rest for a time between 30 minutes and 6 hours, obtaining a wet gel; e) freeze-drying the wet gel, through a first freezing phase at a temperature below -4 °C, followed by a sublimation phase of the water present in the system at a pressure lower than 0.1 mbar, obtaining a dry foam; f) treating the dry foam obtained in step e) at a temperature between 30 and 80 °C and a pressure lower than 0.1 mbar for a time between 1 and 10 hours, obtaining a tridimensional bioactive porous body of the invention.

A second possible process of the invention, carried out when the tridimensional bioactive porous body does not contain a polypeptide, includes the following steps: g) dissolving the aminated polysaccharide in distilled water, at a temperature between 20 and 70 °C; h) adding a functionalized water-soluble polymer to the solution obtained in step g), and allowing the system to react at a temperature between 20 and 70 °C for a time between 15 minutes and 2 hours, obtaining a sol; i) allowing the sol obtained in step h) to rest for a time between 15 minutes and 6 hours, obtaining a wet gel; j) freeze-drying the wet gel, through a first freezing phase at a temperature below -4 °C, followed by a sublimation phase of the water present in the system at a pressure lower than 0.1 mbar, obtaining a dry foam; k) treating the dry foam obtained in step j) at a temperature between 30 and 80 °C and a pressure lower than 0.1 mbar for a time between 1 and 10 hours, obtaining a tridimensional bioactive porous body of the invention.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be described with reference to the Figures, in which:

- Fig. 1 reproduces photographs, at different magnifications, of two porous bodies of the invention having different chemical compositions;

- Fig. 2 shows two graphs, with different time scales, reporting the trend over time of water absorption of two different porous bodies of the invention;

- Fig. 3 shows the trend over time of weight loss due to hydrolysis of two porous bodies of the invention;

- Fig. 4 reports the results of cell proliferation tests on two different porous bodies of the invention in different growth media;

- Fig. 5 reports the results of osteogenic differentiation and mineralization tests on two different porous bodies of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In the description and claims, unless otherwise noted, component amounts, solution concentrations and percentages are by weight.

The expression “long chain”, referred to the aminated polysaccharide used as a component of the porous bodies of the invention, means a polymeric carbohydrate having a weight average molecular weight of at least 3 kDa.

In its first aspect, the invention relates to a tridimensional bioactive porous body, endowed with sufficient mechanical consistency to be able to compensate for the function of the bone tissue until complete regrowth of the same.

The inventors observed that the use of aminated polysaccharides (aminated chitosan, aminated dextran or mixtures thereof) allows to obtain tridimensional bioactive porous bodies with better characteristics, with particular regard to the aptitude of the cells to mineralization of the same. Furthermore, it was also observed that the use of aminated polysaccharides allow to obtain tridimensional bioactive porous bodies with an increased mechanical consistency compared to those obtained with similar non-aminated polysaccharides, making it possible to obtain porous bodies useful for the purposes of the invention starting from aminated polysaccharides and water-soluble polymer only, i.e. without using the polypeptide, which is therefore only an optional component in the present invention.

The porous body is formed by reaction (cross-linking) of two or three polymeric components.

The first component of the porous body of the invention is an aminated long-chain polysaccharide selected from aminated chitosan, aminated dextran and mixtures thereof. Dextran is a branched polymer produced by fermenting glucose with lactobacilli or other fermenting bacteria. Chitosan is a linear polysaccharide obtained by deacetylation of chitin (generally extracted from crustacean exoskeleton) in a basic aqueous solution. Both dextran and chitosan are commonly used in the industry, for example in the cosmetic, pharmaceutical or food additive sectors, and are commercially available. For the purposes of the invention, dextran and chitosan are used in a form functionalized with amino groups. The preparation of polysaccharides in the aminated form occurs by activation of the polymers with 4- nitrophenylchloroformate and subsequent reaction with ethylenediamine. This component is present in the porous body in an amount between 1.0% and 83.0% by weight, and preferably between 10% and 82%, calculated on the sum of the polypeptide, water-soluble polymer and aminated polysaccharide components. If a mixture of aminated chitosan and dextran is used, the two components can be present in the mixture in any weight ratio.

The second component of the porous body of the invention is a water-soluble polymer such as polyethylene glycol (PEG), polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA); the water-soluble polymer is functionalized with groups capable of reacting with reactive sites of the aminated polysaccharide and/or the polypeptide. The preferred functionalized water-soluble polymer for the purposes of the invention is polyethylene glycol diglycidyl ether, compound of the formula: wherein n is an integer lower than 30, and preferably between 5 and 9. This component is present in the porous body in an amount between 11.4% and 35% by weight, and preferably between 14% and 30%, calculated on the sum of the aminated polysaccharide, water-soluble polymer polypeptide and optional polypeptide components.

The third, optional, component of the porous body of the invention is a polypeptide. The polypeptide can be derived from natural tissues or be synthetic; it is also possible to use a mixture of natural and synthetic polypeptides. Natural polypeptides can be of animal or plant origin. The polypeptide is preferably a gelatin produced by hydrolysis of collagen extracted from skin, bones and connective tissues of animal waste. This natural component is widely available commercially. The polypeptide can be present in the porous body in an amount between 0% and 83.0% by weight; if present, the amount thereof is preferably between 60% and 70%, calculated on the sum of the aminated polysaccharide, water-soluble polymer polypeptide and optional polypeptide components.

A preferred composition of porous body of the invention comprises 66% of polypeptide, 16% of functionalized water-soluble polymer and 18% of aminated polysaccharide, by weight.

A second preferred composition of the invention, in the case where the porous body does not include a polypeptide, comprises 19% of water-soluble polymer and 81% of aminated long-chain polysaccharide, by weight.

As discussed below with reference to the process of the invention, these components cross-link each other forming the skeleton of the tridimensional bioactive porous body.

The porous body of the invention has a number of optimal characteristics for a bone tissue regrowth scaffold:

- pore sizes in the range between a few tens of pm and about 500 pm. In various studies it was determined that these sizes, in particular between about 100 and 500 pm, are optimal for bone regrowth because they represent an ideal compromise between the need to have a size sufficient for vascularization of the regrowing tissue but not such as to make it difficult colonization of the central portion of the pores. The pore sizes can be modulated by controlling the hydrogel concentration at the time of freeze-drying and, at the molecular level, the molecular weights and the degree of functionalization of the precursor polymers. Lower molecular weights and/or a greater number of functional groups (obtained for example by reducing the PEG molecular weight, or by introducing additional functional groups in the aminated polysaccharides, or by increasing the relative amount of PEG) lead to an increase in the degree of chemical cross-linking with a consequent increase in solidity and mechanical resistance of the material;

- a porosity structure such that the pores are homogeneously distributed throughout the material, in communication and well interconnected with one another. This structure is highlighted in the photographs at different magnifications, obtained with an optical or electronic microscope, and reproduced in Fig. 1; in the figure, the three photographs in the upper row refer to a sample of the known art, obtained with non-aminated dextran, while the three photographs in the lower row refer to a sample obtained with aminated dextran: it is noted that the sample obtained from aminated dextran has a smaller mean pore size;

- as it will be discussed in greater detail in the Examples section, the dry porous bodies of the invention very quickly absorb water or physiological solution, increasing their weight up to about 630% after 20 minutes of immersion; this means that they can quickly and reversibly acquire a degree of hydration comparable to that of natural tissues and, once implanted as a scaffold, they practically immediately begin the integration process in the site of the bone defect. Furthermore, in a period of time between about 40 and 80 days, depending on the composition thereof, a scaffold of the invention begins to hydrolytically degrade, a phenomenon that can be accelerated in vivo by macrophages and proteolytic enzymes, thus leaving space that can be re-occupied by growing bone tissue;

- a porous body of the invention has excellent mechanical characteristics: it is elastic, being able to withstand numerous compression cycles, with strain up to 50% of its initial size, and re-expand to its initial size when the compressive load is released. The values of elastic modulus and maximum stress, between 0.1 and 0.6 MPa the first and between 0.01 and 0.1 MPa the second, are suitable to compensate for the compromised mechanical function during the period of bone tissue regrowth;

- finally, following dedicated tests, the scaffolds of the invention have shown excellent characteristics in relation to cell adhesion to the pore walls, stimulation of cellular colonization of the pores themselves and properties as inducers of osteogenesis.

In the pores of the tridimensional bioactive porous body of the invention, additional useful components can be introduced to promote bone tissue regrowth or to modulate the rigidity or biodegradability of the porous body itself. A first possible additive are mesenchymal stromal cells deriving from bone marrow, adipose tissue, or umbilical cord, which have the function of favouring and accelerating the osteo-inductive capacity of the scaffold. These cells can be added in amounts between 1000 and 10000 cells/mm³ of the porous body.

A second useful additive consists in growth factors, that is proteins specialized in stimulating cell proliferation and differentiation. Particularly useful for the purposes of the invention are the bone morphogenetic protein (BMP), which stimulates the differentiation of osteoblasts, and the vascular endothelial growth factor (VEGF) which stimulates the growth of vessels and therefore promotes the vascularization of the regrowing bone tissue.

A third possible additive are drugs or medications useful in the treatment of bone lesions and/or tissue regeneration, such as antibiotics and platelet growth factors. The amount of these additives depends on both the recipient (age, body weight, etc.) and the type of medicine, and it is a parameter that can be easily determined by medical personnel.

Finally, additives capable of modifying the rigidity or biodegradability of the porous body can be added to the porous body. These additives can be complexes of calcium, calcium phosphate, calcium carbonate, decellularized and pulverized bone material, or nanometric hydroxyapatite powders.

In its second aspect, the invention relates to processes for the preparation of the tridimensional bioactive porous bodies described above.

According to a first possibility, the process of the invention is directed to the production of a tridimensional bioactive porous body containing a polypeptide; according to this first embodiment, the process of the invention includes steps a) to f).

In the first step, a), the polypeptide component is dissolved in distilled water. The amount of water is between 5 and 20 ml, preferably between 8 and 12 ml, per gram of polypeptide used; lower amounts of water lead to dissolution issues, also of the additional polymeric components added afterwards in steps b) and c), while higher amounts of water lead to wet gels with a too low fraction of solids, and subsequently too rarefied dry foams. The dissolution of the polypeptide takes place at a temperature between 20 and 70 °C, preferably between 40 and 50 °C, preferably under stirring (for example magnetic stirring).

In the second step of the process, b), the functionalized water-soluble polymer is added to the solution obtained in step a), in an amount determined by the initial amount of polypeptide and such as to obtain the desired polypeptide/water-soluble polymer ratio within the ranges indicated above. The water-soluble polymer is added to the polypeptide solution slowly, for example dropwise, keeping the system at a temperature in the same range indicated above for step a), and preferably (for process simplicity) at the same temperature as step a). Once the addition of the water-soluble polymer to the polypeptide solution is completed, the system is allowed to react at the same temperature as step a) for a time between 5 and 30 minutes.

At the end of this period, step c) is carried out, wherein the aminated long-chain polysaccharide, selected from aminated chitosan, aminated dextran or a mixture thereof, is added to the mixture obtained. The aminated polysaccharide is also added in an amount determined by the initial amount of polypeptide and such as to obtain desired values of weight ratios between polypeptide and aminated polysaccharide and between water-soluble polymer and aminated polysaccharide, within the ranges indicated above. The aminated polysaccharide may be added in the form of a powder or, preferably, in the form of an aqueous solution thereof. Among the additional and optional components of the porous body of the invention, additives to modify the stiffness or degradability thereof can be added at this stage (or at the end of the process); these components can be one or more from calcium complexes and powders of calcium phosphate, calcium carbonate, decellularized and pulverized bone material or hydroxyapatites. The other optional components, if present, are instead added at the end of the process, as described below.

The three-component system thus obtained is allowed to react at a temperature between 20 and 70 °C for a time between 30 minutes and 2 hours, preferably at a temperature between 40 and 50 °C for a time between 30 minutes and one hour, obtaining a sol.

In the subsequent step d), the sol obtained in step c) is allowed to rest at room temperature until a wet gel is obtained; gel formation generally takes a time between 30 minutes and 6 hours. For this step, the sol can be left in the original container in which the solution of step a) was prepared, or transferred into a container with desired characteristics, for example of shape (to obtain a final porous body having shape and size as similar as possible to the scaffold to be produced, and thus reduce waste) or thermal conduction (in view of the subsequent freeze-drying).

In step e) the wet gel thus obtained is then dried by freeze-drying. This technique is widely known in various sectors of the industry, for example the food industry, and does not require an extended description. In short, freeze-drying comprises a freezing phase at temperatures below -4 °C and a subsequent sublimation phase of the solidified water at a pressure lower than 0.1 mbar. For simplicity, the freezing of the hydrogel can be achieved by placing the container in contact with liquid nitrogen, for example, only through the lower part of the container (preferred mode) or through all its sides except the upper open one or by directly immersing the hydrogel of the desired form in nitrogen. The mode of cooling of the hydrogel influences the shape and direction of the pores in the final porous body: the inventors observed that if the cooling occurs simultaneously through multiple sides of the hydrogel (and therefore of the container in which it is located), the orientation of the pores in the porous body is essentially random and isotropic, while when the cooling occurs through the lower wall of the container, the pores of the final porous body will be in the form of channels orthogonal to said wall, therefore with a strong porosity anisotropy; this characteristic can be exploited to produce bodies with isotropic or anisotropic mechanical characteristics. The removal of solidified water by sublimation is achieved by placing the frozen hydrogel in a chamber that is evacuated down to a pressure lower than 0.1 mbar, continuing the treatment for a time between 30 minutes and 24 hours.

Finally, in the last step of the process, f), the dry foam extracted from the freeze dryer is placed in a pressure tight oven and treated at a temperature between 30 and 80 °C, at a pressure lower than 0.1 mbar for a time between 1 and 10 hours, for example 3 or 4 hours. In this step, the temperature and moisture removal due to the low pressure favour the last condensation reactions between reactive groups still present in the dry foam, leading to the formation of the tridimensional bioactive porous body of the invention.

In a second embodiment thereof, the process of the invention is directed to the production of a tridimensional bioactive porous body in which there is no polypeptide; in this second embodiment, the process of the invention includes steps from g) to k).

In step g), the aminated polysaccharide is dissolved in distilled water at a temperature between 20 and 70 °C, preferably under stirring (for example magnetic stirring). The amount of water is between 5 and 25 ml, preferably between 10 and 20 ml, per gram of aminated polysaccharide used.

In step h), the functionalized water-soluble polymer is added to the solution thus obtained, in an amount determined by the initial amount of aminated polysaccharide and such as to obtain the desired aminated polysaccharide/water-soluble polymer ratio within the above ranges. The resulting system is allowed to react at a temperature between 20 and 70 °C for a time between 30 minutes and 2 hours, obtaining a sol.

In step i) the sol obtained in the previous step is allowed to rest for a time between 30 minutes and 6 hours, obtaining a wet gel. Also in this case, the sol can be left in the initial container of step g), or transferred into a different container, as indicated above in the case of step d).

Step j) consists in freeze-drying the wet gel, through a first freezing phase at a temperature below -4 °C followed by a sublimation phase of the water present in the system at a pressure lower than 0.1 mbar, obtaining a dry foam; this step corresponds to step e) of the first embodiment of the process, and is carried out in the same way.

Finally, in step k) the dry foam obtained in step j) is treated at a temperature between 30 and 80 °C and a pressure lower than 0.1 mbar for a time between 1 and 10 hours, obtaining a tridimensional bioactive porous body of the invention (similarly to what described above for step f)).

The porous body obtained according to any one of the two embodiments of the process described above is preferably sterilized, and can be stored in gas-tight containers or bags for periods of months or even a few years. Before use, the operator can extract the porous body from the container, possibly shape it to adapt it to the implant site, and rehydrate it with water or physiological solution before implantation in the body. In this phase, it is possible to add some additional components mentioned above (dissolved in water or physiological solution, or at a later stage), i.e. mesenchymal stromal cells from bone marrow, growth factors, drugs or medicines useful in the treatment of bone lesions and/or in tissue regeneration, while additives capable of modifying the stiffness or biodegradability of the porous body, if used, can be added in this phase or in step c), as described above.

The invention will be further illustrated by the following examples.

Methods, instruments and materials

An electromechanical dynamometer (Instron Model 3366) was used for the mechanical characterizations (compression tests).

The molecular weight of polymers was determined by gel permeation chromatography (GPC) (Erma Inc. chromatograph) and Shodex KF columns. The calibration curve was obtained with 16 narrow distribution polystyrene standards (Polymer Laboratories) with molar mass between 3.18 x 10⁶ and 162 g/mol.

An Eclipse 90i microscope (Nikon Instruments Europe BV) and a Leica CM 1860 ultraviolet (UV) cryostat were used for the biological characterizations.

The following materials were used in the tests:

- Type A Gelatin, pharmaceutical grade, 280 bloom, viscosity 4.30 mPs, Italgelatine, Cuneo, Italy;

- Polyethylene glycol diglycidyl ether, molecular weight 526 Da, Sigma-Aldrich Co, Milan, Italy;

- Dextran, Mw 70,000 Da, catalogue no. 31390 Sigma-Aldrich Co, Milan, Italy;

- 4-Nitrophenyl chloroformate Sigma-Aldrich Co, Milan, Italy;

- Ethylenediamine Sigma-Aldrich Co, Milan, Italy;

- Growth medium with 10% fetal bovine serum (GM FBS): Dulbecco’s modified Eagle’s medium (DMEM), 1-glutamine, penicillin-streptomycin, sodium pyruvate (Sigma- Aldrich Co, USA), amphotericin and non-essential amino acids (Gibco, ThermoFisher Scientific, USA), fetal bovine serum (Sigma-Aldrich Co, USA);

- Growth medium with 5% platelet lysate (GM HPL): Dulbecco’s modified Eagle’s medium (DMEM), L-glutamine, penicillin-streptomycin, sodium pyruvate (Sigma-Aldrich Co, USA), amphotericin and non-essential amino acids (Gibco, ThermoFisher Scientific, USA);

- Osteogenic medium with 10% fetal bovine serum (OM FBS): Dulbecco’s modified Eagle’s medium (DMEM), L-glutamine, penicillin-streptomycin, sodium pyruvate (Sigma- Aldrich Co, USA), amphotericin and non-essential amino acids (Gibco, ThermoFisher Scientific, USA), fetal bovine serum, dexamethasone, L-ascorbic, NaHiPCri (Sigma-Aldrich Co, USA);

- Osteogenic medium with platelet lysate (OM HPL): Dulbecco’s modified Eagle’s medium (DMEM), L-glutamine, penicillin-streptomycin, sodium pyruvate (Sigma-Aldrich Co, USA), amphotericin and non-essential amino acids (Gibco, ThermoFisher Scientific, USA), dexamethasone, L-ascorbic, NaH₂P0₄ (Sigma-Aldrich Co, USA);

- Phosphate buffered saline (PBS) (Sigma-Aldrich Co, USA);

- 4% Paraformaldehyde (PFA) (Sigma-Aldrich Co, USA);

- 4’,6-Diamidine-2-phenylindole (DAPI) (Vector Laboratories, USA);

- Mesenchymal stromal cells from bone marrow (BM-hMSCs) and from adipose tissue (AT-hMSCs) (PromoCell, Germany).

EXAMPLE 1 (COMPARATIVE)

This example refers to the preparation of a porous body made according to the known art.

6 g of gelatin were dissolved in 60 ml of distilled water at 45 °C under magnetic stirring, followed by dropwise addition of polyethylene glycol diglycidyl ether (1.4 g). The reaction mixture was kept under magnetic stirring for 10 minutes. 25 g of a 7% by weight solution of dextran in distilled water were then added, and the system was kept under stirring for another 45 minutes at 45 °C.

The mixture was then poured into a glass crystallizer obtaining complete gelling after one hour at room temperature. The resulting wet gel was cut into rectangular bars (5 cm x 1 cm x 1 cm) and frozen by cooling (through complete immersion) the crystallizer in liquid nitrogen.

The frozen gel was then dried by sublimation of the ice present in its pores, with a treatment at 0.1 mbar for 24 hours. A material having the appearance of a dry and porous sponge was obtained. This material was finally treated with a post-polymerization process at 45 °C for 3 hours in an oven at a pressure of about 0.1 mbar to complete the cross-linking reactions.

In the following characterization tests, the material obtained is referred to as G/PEG/Dx.

EXAMPLE 2

This example refers to the preparation of a first porous body of the invention.

The procedure of Example 1 was repeated, with the only difference that aminated dextran was used instead of dextran. The aminated dextran was prepared as described below.

17 g of dextran were dissolved in 100 ml of chloroform previously dried with CaEE and the solution was cooled in an ice bath. 4 g of 4-nitrophenylchloroformate, and finally triethylamine up to pH 8 (2.8 ml), were added slowly and under stirring. The solution was brought back to room temperature and kept under stirring in the dark for a further 24 hours. The 4-nitrophenyl dextran derivative was isolated by precipitation in ether and subsequently filtered and dried.

10 g of 4-nitrophenyl dextran derivative obtained in the previous step were added, very slowly (about 2 hours) and under vigorous stirring, to 200 ml of ethylenediamine in a flask. The solution was kept under stirring for a further 24 hours at room temperature. The product was isolated by precipitation in a 4/1 ethanol/ether mixture, filtered, washed, and dried.

In the following characterization tests, the material obtained is referred to as G/PEG/DxN.

EXAMPLE 3

This example refers to the preparation of a second porous body of the invention, consisting exclusively of an aminated polysaccharide and polyethylene glycol diglycidyl (PEGDGE).

5 g of aminated dextran were dissolved in 30 ml of distilled water at 45 °C and kept under magnetic stirring for 2 hours. 1.4 g of polyethylene glycol diglycidyl ether were added dropwise to the solution, and the reaction mixture was kept under gentle stirring at 45 °C for a further 40 minutes.

The mixture was then diluted with 30 ml of distilled water, poured into a glass crystallizer, and kept for 30 minutes at room temperature, for a further 30 minutes in the refrigerator at -4 °C, and finally frozen by complete immersion of the crystallizer in liquid nitrogen.

The frozen gel was then dried by sublimation of the ice present in its pores, with a treatment at 0.1 mbar for 24 hours. A material having the appearance of a dry and porous sponge was obtained. This material was treated with a post-polymerization process at 45 °C for 3 hours in an oven at a pressure of about 0.1 mbar to complete the cross-linking reactions.

EXAMPLE 4

The morphology of the G/PEG/Dx and G/PEG/DxN samples obtained in Examples 1 and 2 was evaluated.

The two samples were examined and photographed under optical and electronic microscopes. The images obtained are reproduced in Fig. 1: the three images in the upper row refer to the G/PEG/Dx sample, the three images in the lower row to the G/PEG/DxN sample; in each row in Fig. 1, the first two images were obtained with an optical microscope, the last one with an electronic microscope.

Pores and channels homogeneously distributed throughout the material are observed; the pores are in communication and well interconnected with each other.

As it can be seen in the figures, the G/PEG/Dx sample has larger porosities; in all cases the pore maximum size is not greater than 500 pm.

The pore sizes in the two samples are shown in Table 1. Table 1

The two samples were also subjected to sterilization tests by gamma irradiation at a dose of 25 kGy (ISO 11137 method): no significant changes were measured with respect to the initial characteristics.

EXAMPLE 5

In this test, the properties of water absorption and hydrolytic degradation of the samples of the invention were evaluated.

From the samples prepared in Examples 1 and 2, two discs with a thickness of about 1 mm were obtained, having a weight of 150 mg (sample G/PEG/Dx) and 155 mg (sample

G/PEG/DxN), respectively.

The two samples were introduced into containers containing distilled water, thermostated at 37 °C, and extracted after times of 10 minutes, 1 hour, 3 hours, 5 hours, 1 day, 3 days, 7 days, 14 days, and 21 days, to measure weight gain. The data obtained were plotted in two graphs in Fig. 2, as Curve 1 for the G/PEG/Dx sample and as Curve 2 for the G/PEG/DxN sample, respectively, showing the percentage increase by weight (with respect to the initial dry weight) as a function of time; in the figure, the graph on the left shows the weight gain trend in the first 20 hours of testing, the graph on the right shows the trend in the first four weeks. As it can be seen in the figure, the G/PEG/DxN sample has a lower water absorption than the G/PEG/Dx one, due to its higher cross-linking and reduced porosity. Both samples absorb water until they reach a weight between 5.5 and 6 times the initial weight already after 10 minutes of immersion, and up to about 8 times the initial weight after 3 weeks.

A hydrolytic degradation test of the material was carried out on two other discs obtained from the samples prepared in Examples 1 and 2.

The two discs were immersed in distilled water maintained at 37 °C. After immersion times of 1, 7, 14, 21 and 28 days, the samples were extracted from the bath, dried completely and weighed, to measure the weight loss due to solubilization of the material in water. The results are reported in Fig. 3, as Curve 1 for the G/PEG/Dx sample and as Curve 2 for the G/PEG/DxN sample, respectively; also in this case, the G/PEG/DxN sample exhibits a lower (and therefore slower) degradation compared to the G/PEG/Dx sample.

EXAMPLE 6

The mechanical properties of the materials of the invention were evaluated.

The mechanical properties of the samples produced in Examples 1 and 2 were evaluated on fully rehydrated materials (to simulate the behaviour of scaffolds produced with these materials once implanted in the site of a bone lesion). The tests were performed by subjecting the samples to 10 compression cycles, with a strain of 50%, and subsequent load release with an Instron Model 336 electromechanical dynamometer (ESISTRON, Norwood, Massachusetts, USA). The results of the stress/strain tests show that, after the first training cycles, both samples show a stable mechanical behaviour characterized by almost constant values of energy dissipated at each cycle. From the same tests, the values (measured in MPa) of the elastic modulus and the stress corresponding to a strain of 50% were obtained for the two samples; these values are reported in Table 2.

Table 2

From the values shown in the table, it can be seen that the hydrogel obtained from aminated dextran has greater stiffness and greater resistance to compression (with increases of approximately 60% and 35%, respectively, in the two values).

The mechanical characteristics are kept at a high level even during the degradation of the material. EXAMPLE 7

In this test, the cell regrowth properties on scaffolds prepared with materials of the invention were evaluated.

Scaffolds obtained from samples prepared in Examples 1 and 2 were seeded with commercial bone marrow mesenchymal stromal cells (BM-hMSCs) in the culture step 3, and at a concentration of 1,000,000 cells/ml. The scaffolds were arranged in special plates and placed in an incubator at 37 °C with 5% CO2 for 28 days in four different growth media, i.e. GM FBS, GM HPL, OM FBS and OM HPL.

28 days after seeding, the scaffolds were washed with PBS, fixed with 4% PFA, embedded in paraffin and cut by microtome. The sections were placed on slides for DAPI staining with the aim of highlighting the cell nuclei, and the images were subsequently analysed by means of a fluorescence microscope for counting the nuclei; the results are reported in Fig. 4, in the form of histograms. Excellent cell proliferation and homogeneous distribution in the material are highlighted both in the presence of fetal bovine serum and platelet lysate. Visual analysis under the microscope also shows that the cells are well adherent to the scaffold material and developed.

EXAMPLE 8

The osteogenic differentiation properties of mesenchymal stromal cells from bone marrow were evaluated in this test.

Scaffolds obtained from the samples prepared in Examples 1 and 2 were seeded with BM-hMSCs in the culture step 3, at a concentration of 1,000,000 cells/ml. Each scaffold was placed in an incubator at 37 °C with 5% CO2 for 28 days in the presence of the four different growth media, GM FBS, GM HPL, OM FBS and OM HPL.

After 28 days, the scaffolds were washed with PBS, fixed with 4% PFA, embedded in paraffin and cut by microtome. The sections were treated with Von Kossa stain (silver nitrate solution) to highlight the calcium deposits, and were then analysed in order to determine the percentage of the area affected by the presence of calcium (Fig. 5). The analyses showed the presence of important and homogeneously distributed calcium and phosphorus deposits in the scaffolds. In the deposits, the presence of hydroxyapatite, one of the main components of healthy bone tissue, was recognised. Furthermore, such hydroxyapatite is much more abundant and of better quality in the scaffolds obtained from Example 2.

COMMENTS ON THE RESULTS

The results of the tests confirm that the porous bodies have an excellent set of properties in view of the application as a scaffold for bone regrowth.

Firstly, all the reagents used are biocompatible and widely used for biomedical applications, and the production of these porous bodies does not require the use of additives or catalysts, thus avoiding possible risks of non-biocompatibility or toxicity; all the reagents used in the production of these bodies are also water-soluble so that no other solvent is required.

The water absorption and degradation tests following hydrolysis indicate that the materials of the invention are highly wettable by aqueous solutions, and can therefore be easily impregnated by body fluids to initiate the cell colonization process; furthermore, these materials degrade over the course of a few months; the overall result is that scaffolds made with the porous bodies of the invention provide optimal support for cell colonization starting immediately after implantation in the body, while over a span of a few months they are reabsorbed, leaving space for new bone tissue formation.

Mechanical tests show that the material of these scaffolds does not break even after extensive and repeated strains, and it can be subjected to a large number of compression and decompression cycles, immediately recovering the shape and keeping the dissipated energy almost constant. The mechanical characteristics are kept at a high level even during the degradation of the material. Thanks to these properties, a scaffold of the invention provides a stable support to the cells for the entire time of bone regrowth.

Finally, cell regrowth and osteogenic differentiation tests confirm the properties of the scaffolds of the invention that favour the osseointegration of prosthetic implants by reducing bone healing times. Furthermore, the formation of calcium and phosphorus deposits in which the signal of hydroxyapatite, which is one of the fundamental components of bone tissue, was identified, is much more abundant and qualitatively better in scaffolds obtained from aminated polysaccharides.

Claims

1. Tridimensional bioactive porous body obtained by reaction between:

2. Tridimensional bioactive porous body according to claim 1, wherein said water-soluble polymer is selected from polyethylene glycol (PEG), polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA).

3. Tridimensional bioactive porous body according to claim 1, wherein said water-soluble polymer is polyethylene glycol diglycidyl ether, compound of formula: wherein n is an integer lower than 30.

4. Tridimensional bioactive porous body according to claim 3, wherein n is between 5 and

9.

5. Tridimensional bioactive porous body according to any one of the preceding claims, wherein said polypeptide is of animal or plant origin.

6. Tridimensional bioactive porous body according to any one of the preceding claims, wherein said polypeptide is a gelatin produced through the hydrolysis of collagen extracted from skin, bones, and connective tissues of animal waste.

7. Tridimensional bioactive porous body according to any one of the preceding claims, wherein said aminated long chain polysaccharide is present in an amount between 10% and 82% with respect to the sum of the weights of said aminated long chain polysaccharide, water-soluble polymer, and polypeptide.

8. Tridimensional bioactive porous body according to any one of the preceding claims, wherein said water-soluble polymer is present in an amount between 14% and 30% with respect to the sum of the weights of said aminated long chain polysaccharide, water- soluble polymer, and polypeptide.

9. Tridimensional bioactive porous body according to any one of the preceding claims, wherein the polypeptide is present in an amount between 60% and 70% with respect to the sum of the weights of said aminated long chain polysaccharide, water-soluble polymer, and polypeptide.

10. Tridimensional bioactive porous body according to any one of the preceding claims comprising 18% of aminated long chain polysaccharide, 16% of water-soluble polymer and 66% of polypeptide, by weight with respect to the sum of the weights of said aminated long chain polysaccharide, water-soluble polymer, and polypeptide.

11. Tridimensional bioactive porous body according to any one of the preceding claims comprising 19% of water-soluble polymer and 81% of aminated long chain polysaccharide, by weight with respect to the sum of the weights of said water-soluble polymer and aminated long chain polysaccharide.

12. Tridimensional bioactive porous body according to any one of the preceding claims, further containing one or more additional components selected from:

- growth factors;

- antibiotics, drugs or medicines useful in the treatment of bone lesions and/or regeneration of tissues;

- additives for modifying the stiffness or degradability of the porous body, selected from complexes of calcium, calcium phosphate, calcium carbonate, decellularized and pulverized bone material, and hydroxyapatites.

13. Process for the production of a tridimensional bioactive porous body of any one of claims 1 to 11, comprising the following steps: a) dissolving a polypeptide in distilled water, at a temperature between 20 and 70 °C; b) adding a water-soluble polymer to the solution obtained in step a), and allowing the system to react at a temperature between 20 and 70 °C for a time between 5 and 30 minutes; c) adding an aminated long chain polysaccharide, selected from aminated chitosan, aminated dextran and mixtures thereof, to the solution obtained in step b), and allowing the system to react at a temperature between 20 and 70 °C for a time between 30 minutes and 2 hours, obtaining a sol; d) allowing the sol obtained in step c) to rest for a time between 30 minutes and 6 hours, obtaining a wet gel; e) freeze-drying the wet gel, through a first freezing phase at a temperature below -4 °C, followed by a sublimation phase of the water present in the system at a pressure lower than 0.1 mbar, obtaining a dry foam; f) treating the dry foam obtained in step e) at a temperature between 30 and 80 °C and a pressure lower than 0.1 mbar for a time between 1 and 10 hours, obtaining a tridimensional bioactive porous body of the invention.

14. Process according to claim 13, wherein in step a) it is used an amount of water between 5 and 20 ml per gram of polypeptide used.

15. Process for the production of a tridimensional bioactive porous body of any one of claims 1 to 11, comprising the following steps: g) dissolving an aminated long chain polysaccharide selected from aminated chitosan, aminated dextran and a mixture thereof in distilled water, at a temperature between 20 and 70 °C; h) adding a water-soluble polymer to the solution obtained in step g), and allowing the system to react at a temperature between 20 and 70 °C for a time between 30 minutes and 2 hours, obtaining a sol; i) allowing the sol obtained in step h) to rest for a time between 30 minutes and 6 hours, obtaining a wet gel; j) freeze-drying the wet gel, through a first freezing phase at a temperature below -4 °C, followed by a sublimation phase of the water present in the system at a pressure lower than 0.1 mbar, obtaining a dry foam; k) treating the dry foam obtained in step j) at a temperature between 30 and 80 °C and a pressure lower than 0.1 mbar for a time between 1 and 10 hours, obtaining a tridimensional bioactive porous body of the invention.

16. Process according to any one of claims 13 to 15, further comprising the steps of rehydrating the porous body with water or a physiological solution and/or adding at least one additional component selected from: mesenchymal stromal cells deriving from bone marrow, adipose tissue, or umbilical cord; growth factors; drugs or medicines useful in the treatment of bone lesions and/or regeneration of tissues; and modifiers of the stiffness or biodegradability of the porous body.

17. Process according to claim 16, wherein said growth factors are selected from bone morphogenetic protein (BMP) and vascular endothelial growth factor (VEGF); said drugs or medicines are selected from antibiotics and platelet growth factors; and said modifiers of stiffness or biodegradability of the porous body are selected from complexes of calcium, calcium phosphate and nanometric hydroxyapatite powders.