WO2024057194A1

WO2024057194A1 - Compound for making three-dimensional structures to recreate the nervous tissue of the brain of human beings

Info

Publication number: WO2024057194A1
Application number: PCT/IB2023/059016
Authority: WO
Inventors: Donatella DI LISA; Laura PASTORINO; Elena DELLACASA
Original assignee: Bio3Dmatrix Srl
Priority date: 2022-09-12
Filing date: 2023-09-12
Publication date: 2024-03-21

Abstract

Compound for making three-dimensional structures for recreating the nervous tissue of the brain of human beings, consisting of the aggregation of a first fraction with a second fraction. The first fraction is the main part based on chitosan and comprises an acid component, while the second fraction comprises at least one or more proteins, specific growth factors of the extracellular matrix (ECM) of the human and animal brain, and a saline component.

Description

Compound for making three-dimensional structures to recreate the nervous tissue of the brain of human beings

The present invention relates to a compound for making three-dimensional structures for recreating the nervous tissue of the brain of human beings , consisting of the aggregation of a first fraction with a second fraction .

In particular , the present invention is based on the development of a compound to be used with or without cells , in making three-dimensional structures (scaffolds) so as to recreate human tissues , in particular the nervous tissue of the brain .

Preferably, the present invention sets the object of making a bio-ink for use in bioprinting for reconstructing nervous tissue of the brain , especially in biomedical applications .

Bioprinting is a process based on the additive production of three-dimensional structures , which uses biomaterials as a micro-environment for living cells , human , animal and plant cells .

3D bioprinting is a multidisciplinary technology which aims to combine engineering principles , related for example to 3D printing technology, and biology .

3D cellular bioprinting has enormous potential as regards creating native tissue micro-environments in which to culture cells , allowing the precise deposition of multiple cells in a predefined position . It can be considered the technology of the near future in the clinical field, with which it will be possible to make constructs which have predefined and customizable geometries and carry out specific functions so as to offer models capable of reproducing a certain pathological condition , so as to be able to test new drugs and develop new therapies , but also for the generation of parts of tissues or organs to be implanted directly in patients .

As known in prior art systems , 3D bioprinting involves the deposition , layer by layer , of a bio-ink so as to create 3D structures , such as tissues and organs .

Bio-ink is a bioprintable material used in 3D bioprinting processes .

The ultimate aim is to make a bio-ink which allows the printed products to have adequate mechanical properties , high cell compatibility and high model fidelity .

An ideal bio-ink should possess the desired physico-chemical properties , such as the correct mechanical , rheological , chemical and biological characteristics . Since determining the optimal formulation of a bio-ink loaded with cells is the fundamental step towards bioprinting success , to date , various natural and synthetic biomaterials with specific characteristics have been tested .

Bio-inks can be classified into two main types : scaffold-less bio-inks and scaffold-based bio-inks . In scaffold-less bio-inks , embryonic development mimics neo-tissue formation . Tissue spheroids , cellular pellets and tissue filaments are used in this approach for the fabrication of functional tissue on a large scale . Scaffold-based bio-inks consist of hydrogels or decellularized matrix-based compounds containing cells .

Scaffold-based bio-inks consist of materials which mimic the environment of the extracellular matrix so as to support the adhesion , proliferation and differentiation of living cells .

Such bio-inks have properties such that they can be deposited as filaments during an additive manufacturing process and can be both natural and synthetic .

Natural materials such as collagen , fibrin , sodium alginate and gelatin often have neutral or positive effects on cells . However , their mechanical properties are generally poor .

In contrast , synthetic materials such as polyethylene glycol (peg) , polycaprolactone (PCL) , polylactic acid (PLA) , polyurethane (PU) , polyethylene glycol dimethacrylate (PEGDMA) exhibit improved mechanical properties and controllable degradation rates . However , to be adapted to the printing process , these materials must be melted at high temperatures or dissolved in organic solvents so as to achieve a good degree of fluidity and printability, making these polymeric materials unsuitable for direct printing with living cells .

In the last decade , biopolymeric hydrogels have attracted great attention in the field of tissue engineering and regenerative medicine . Biopolymeric hydrogels are characterized by biocompatibility, biodegradability and high similarity with the architecture of the extracellular matrix, thus becoming candidates for optimal biomaterials for the fabrication of 3D scaffolds for soft tissues . Hydrogels are a group of three-dimensional polymeric networks which can contain a large amount of water . Consequently, they are known as biocompatible materials and provide a cell-friendly environment thanks to their high water content and low polymer content .

In tissue engineering applications , the fabrication of 2D and 3D hydrogel -based scaffolds constitutes the critical component in many applications , since the hydrogels are permeable to nutrients , oxygen and other water-soluble compounds .

A particular class of hydrogels is that capable of carrying out the sol-gel transition under specific external stimuli and in the presence of cells : for example , by irradiation of light or following variations in temperature , ions or pH .

For example , photo-cross-linkable inks typically include an initiator which generates free radicals upon ultraviolet irradiation to initiate a cross-linking reaction , thus forming a printed product with high model fidelity and good mechanical properties .

However , such an initiator is cytotoxic and the ultraviolet irradiation step damages the cells . Ionsensitive inks such as those containing sodium alginate are generally stabilized with an ionic solution . The resulting gels have a high water content and are suitable for cells , but have poor mechanical properties . pH-sensitive inks are mostly made of collagen , which is easy to print , but the printed products often lack mechanical stability .

Another class of hydrogels sensitive to external stimuli are thermo -reactive hydrogels , also known as thermogels , which undergo a physical sol-gel transition as the temperature varies , a reversible reaction after cooling .

This type of hydrogels has advantages in that they have characteristics such as to be considered inj ectable hydrogels , and are widely used for in vivo applications (minimally invasive interventions) or as bio-inks .

In thermo-sensitive hydrogels , peptides are encapsulated at low temperature , which prevents denaturation due to the interaction of organic solvents or high temperature dissolution .

Furthermore , the biodegradable thermogel can be expelled from the body after achieving the intended purpose and the release rate of specific molecules (growth factors , drugs) can be readily adapted by modifying the starting formulation .

Extracellular matrix-based decellularized bioinks can be derived from almost all mammalian tissues . However , most organs such as heart, muscle , cartilage , bone and fat are decellularized, lyophilized and pulverized , to create a soluble matrix which can then be formed into a gel . These bio-inks have advantages with respect to other materials due to their derivation from mature tissue ; they consist of a complex mixture of decellularized extracellular matrix and proteins specific for their tissue origin .

In bioprinting, biocompatible cells and materials are used as biological ink , which can be organized in 3D space , with the aim of generating complex constructs which mimic organs and tissues .

Recent advances in biofabrication techniques have opened up the possibility of applying the 3D bioprinting methodology to human stem cells , including embryonic stem cells (h-ESCs) and induced pluripotent stem cells (h-iPSCs) .

In particular , the interest in 3D bioprinting h- iPSCs derives from their ability to ideally generate any type of cell of interest . Once incorporated into a 3D construct , h-iPSCs could maintain their plurilinear potential , or the bio-ink could deliver specific signals , inducing direct differentiation in neural , cartilage or cardiac cells .

On the market it is possible to find several bioinks representative of only some biological tissues , but as far as the nervous system is concerned, to date there is nothing specific , namely stimulus-sensitive bio-inks specific for the encapsulation of nerve cells and the development of functional 3D neuronal networks .

The need to develop a bio-ink for modelling the nervous tissue of the brain stems from the fact that pharmaceutical advances for CNS (Central Nervous System) diseases are highly hampered by the lack of adequate disease models . In fact, animal models do not faithfully represent human neuro-degenerative processes , and 2D models , although made with human cells , cannot reproduce the complexity of the nervous system in vivo .

The main hydrogels used for this specific application are Ma trigel and GelMa . However , Ma trigel has several limitations , mainly related to high batch- to-batch variability and low stability over time , while the photo-cross-linking process of GelMA raises concerns related to the possible impact on cells .

There are some solutions known in the prior art which , however , do not resolve the problems discussed above . For example, the document Amr Sherif M. ET AL: "Bridging defects in chronic spinal cord injury using peripheral nerve grafts combined with a chitosanlaminin scaffold and enhancing regeneration through them by co-transplantation with bone- marrow-derived mesenchymal stem cells: Case series of 14 patients", JOURNAL OF SPINAL CORD MEDICINE, vol. 37, no. 1, 23 January 2014 (2014-01-23) , pages 54-71, XP093029896, ISSN: 1079-0268, DOI: 10.1179/2045772312Y .0000000069 envisages making a compound based on a chitosan and laminin paste. The chitosan is dissolved in acetic acid and mixed with laminin. The solution described by the document does not envisage the use of a second crosslinking fraction, i.e. , it does not allow to make an injectable gel -based compound. Furthermore, such a document describes a compound for spinal cord regeneration, tissue which has particularly different mechanical characteristics with respect to the mechanical characteristics of the human brain.

In fact, the human brain has a Young’s modulus comprised between 0.1 and 1 KPa, while the spinal cord has Young’s modulus values of about 40 KPa.

Similarly, CN 110624133 refers to a "nerve matrix catheter" for peripheral nerve regeneration.

Such a document does not refer to the creation of the nervous tissues of the brain.

In particular, the sciatic nerve of the rat is used as a nerve matrix and pepsin is added. Pepsin is a proteolytic enzyme which causes the degradation of brain growth factors.

US8680182 also relates to the regeneration of spinal cord injuries and is not related to modelling an extracellular matrix of the brain. There is thus an unmet need of the systems and compounds known in the prior art , to make a compound with mechanical properties similar to the brain and, at the same time , stable and bioactive capable of supporting neuronal adhesion and growth in long-term cultures .

Furthermore , there is an unmet need of the systems and compounds known in the prior art to develop new bio-inks and new methods for 3D bioprinting so as to produce artificial constructs with adequate printability properties , high mechanical integrity, stability, insolubility in the culture medium, similar biodegradability rate with tissue regeneration , nontoxicity and non -immunogenicity and also cell adhesion promoting properties .

The present invention achieves the above obj ects by making a compound as described above , in which the first fraction constitutes the main part of the compound and is based on chitosan with the addition of an acid component , while the second fraction comprises at least one or more proteins , specific growth factors of the extracellular matrix (ECM) of the human and animal brain and a saline component .

Chitosan is a natural polymer obtained by the partial deacetylation of chitin , which is the major component of exoskeletons of crustaceans , insects and fungi . Chitosan may behave as a polycation under acidic conditions (pH < 6) , due to protonation of free amino groups , and may be physically or chemically crosslinked to produce matrices for drug delivery and cell deposition . This polysaccharide is well known for its biocompatibility, biodegradability, muco-adhesiveness , and antibacterial and antifungal activity . Interestingly, as previously demonstrated, chitosan enhances the attachment of neurons , the proliferation and extension of neurites , and exerts a powerful neuro- protective action .

The compound object of the present patent application envisages the use of chitosan as the main component .

In the field of neurobiology, chitosan is capable of sustaining and increasing neuronal adhesion , proliferation and extension of neurites , and carrying out a powerful neuro-protective action . In fact , chitosan has a structure very similar to some of the main components of the extracellular matrix of the nervous system, thus mimicking the micro- environment in which nerve cells grow naturally very well .

Chitosan hydrogels used as 3D scaffolds allow the development of active neural networks of both animal and human origin .

Therefore , a formulation based on a multicomponent and temperature-responsive biopolymeric hydrogel is made , so as to also be used as a bio-ink for the 3D bio-printing of nerve cells for the human brain .

In the compound object of the present invention , the first fraction based on chitosan is mixed with a second fraction containing one or more proteins , specific growth factors of the extracellular matrix (ECM) of the human and animal brain and a saline component .

Thanks to such mixing , the compound object of the present invention allows to model nerve tissues in vitro, offering a micro -environment with characteristics such as to best mimic the tissue in vivo .

The formulation of the compound object of the present invention has excellent mechanical , morphological and bioactivity properties .

The second fraction thus has a cross-linking function within the compound object of the present invention .

For this reason , as will be described below , the first and the second fraction are made separately, as two distinct fractions , and subsequently mixed , so as to ensure the aforesaid mechanical , chemical and physical properties of the compound object of the present invention .

As mentioned above , one of the main objects of the compound object of the present invention is to make a bio-ink .

Therefore , starting from the generic principle expressed by the formulation described above , a preferred embodiment envisages the addition of glycerol to give the compound object of the present invention printability and injectability characteristics .

A compound is therefore made , in particular a bioink , which promotes cellular functions such as adhesion , proliferation and maturation , having characteristics and properties such as to be extruded through a bio-printer .

The use of glycerol is fundamental for making a compound which can be used in additive manufacturing procedures .

The compound object of the present invention is liquid at a temperature between 4 ° C and 20 ° C , gels at temperatures between 20 ° C and 40 ° C and has a physiological pH .

According to an embodiment , the compound obj ect of the present invention comprises chitosan , Sodium |3- glycerophosphate pentahydrate (BGP) , ethanol , sodium hydroxide and acetic acid .

The chitosan used preferably has a molecular weight of 50-300 kDa , 70-98% degree of deacetylation , of animal or vegetable origin .

Starting from the generic formulation expressed by the independent claim attached to the present patent application , it is possible to envisage specific components within the second fraction to improve the mechanical characteristics and printability and bioactivity of the compound object of the present invention , to optimally simulate the nervous tissue of the human and animal brain .

According to a first embodiment , the second fraction comprises the proteins laminin and fibronectin .

According to an improvement , the second fraction comprises the following growth factors :

- BDNF (brain-derived neurotrophic factor) aimed at stimulating the survival and differentiation of certain neurons and synapses belonging to the central (CNS) and peripheral (PNS) nervous systems ;

- BGF (nerve growth factor) protein belonging to the neuro trophin family, aimed at stimulating nerve growth ;

- FGF polypeptides , (e . g . , aFGF , bFGF, KGF) aimed at controlling cell growth and differentiation and playing a key role in oncogenesis , neuronal development processes ; - PDGF (platelet-derived growth factor) , which acts as a regulator of growth and cell division ,

- VEGF (vascular endothelial growth factor) , an important signaling molecule involved in angiogenesis .

According to a preferred embodiment , the saline component comprises Sodium -glycerophosphate pentahydrate .

The acid component instead comprises acetic acid .

Lastly, according to a further embodiment , the first fraction is present in a percentage comprised between 75% and 85% of the total weight , while the second fraction is present in a percentage comprised between 15% and 25% .

The final chitosan percentage is equal to a value comprised between 1 .5% and 2 .5% .

Based on the characteristics described above , it is specified that the compound object of the present invention has the mechanical and rheological characteristics such as to confer sufficient stiffness to obtain self-supporting 3D structures , without the need for further components aimed at creating support structures , as for example described in the document Ku Jongbeom ET AL : "Cell-Laden Thermosensitive Chitosan Hydrogel Bioinks tor 3D Bioprinting Applications" , Applied Sciences , vol . 10 , no . 7 , 3 April 2020 (2020- 04 -03) , page 2455 , XP093032458 , DOI : 10 . 3390/appl0072455 .

In view of the advantageous characteristics related to the compound described above , the present invention further relates to a method for producing a compound for making three-dimensional structures for recreating the nervous tissue of the brain of human beings , which method envisages preparing a first fraction and a second fraction , which two fractions are mixed to make the compound .

In particular , the first fraction is obtained by dissolving chitosan powder in an acid-based solution , while the second fraction is obtained by mixing one or more proteins , specific growth factors of the extracellular matrix (ECM) of the human and animal brain and a saline component .

Preferably, the acid-based solution comprises acetic acid .

In particular , the method object of the present invention allows to prepare a bio-ink based on the compound object of the present invention .

As with the compound, the bio-ink consists of two fractions , a first fraction and a second fraction , made separately and then mixed together .

The first fraction is the main chitosan-based polymeric component and is obtained by dissolving the chitosan powder (2-5% , w/v) in an acetic acid solution (0 . 1-1M molarity) .

The solution is kept stirring at room temperature over a period of 12 hours .

The first fraction is then sterilized in an autoclave at 120 ° C for 20 minutes , and then stored at 4 ° C .

The second fraction is the gelling solution with a base obtained by mixing BGP (5-30%) , glycerol , laminin , fibronectin , BDNF, NGF , NT-3 , PDGF in culture medium, for nerve cells .

Neurobasal , Brainphys , Essential 8 Flex medium and Dulbecco ’ s Modified Eagle Medium can be used as the culture medium. The second fraction is sterilized by means filtration through 0 .2 pm syringe filters .

The second fraction must be stored at 4 ° C .

The procedure just described is related to a sterilization methodology which allows to obtain the rheological properties which allow to identify the optimal concentrations of the various components so as not to have a negative impact on bi ©compatibility and bioactivity with respect to the neuronal cells of the brain .

The thermo-sensitive bio-ink is obtained by mixing the first fraction with the second fraction . In detail , the second fraction is added drop-wise to the first fraction , while this is kept under stirring . The production process of the thermo-sensitive bio-ink must be carried out at a temperature of 4 ° .

The gelling mechanism is temperature-dependent and involves the heat-induced transfer of protons from chitosan to glycerol phosphate , which reduces the repulsive forces between the positively charged ammonium groups and allows the interaction of the chitosan chains .

The thermo-sensitive bio-ink has a final concentration 1- 2% (w/v) of chitosan .

In the thermo-sensitive bio-ink , nerve cells are added in such a concentration as to obtain cell densities ranging from 6 , 500 cel Is /microlitre to 100 , 000 cel Is /microlitre depending on the different applications .

The suspension of cells in the thermo-sensitive bio-ink is placed at 37 ° in an incubator for 30-40 min so as to obtain the complete gelling of the bio-ink and the formation of a three-dimensional scaffold capable of supporting the growth of a three-dimensional neuronal network .

The thermo-sensitive compound can be used by different technologies : 3D bioprinting or manual deposition .

The first technology (3D bioprinting) envisages the use of syringes filled with bio-ink and installed in the bio-printer . For this purpose , the suspension of cells in the bio-ink , object of the following invention , is loaded into the syringes . The syringes are placed at 37 ° to trigger the gelling process . Once gelled, the syringes are ready to be installed and used with bioprinters .

The second technology (manual deposition) envisages the use of polydimethylsiloxane rings with a physical confinement function within which the cell suspension is deposited in the thermo-sensitive compound, object of the following invention . The aforesaid are placed at 37 ° to trigger the gelling process .

These and further objects of the present invention are achieved by a bottling device according to the appended independent claim and the sub-claims .

Optional features of the compound of the invention are contained in the appended dependent claims , which form an integral part of the present disclosure .

On the basis of what was disclosed previously, starting from the composition disclosed, two formulations were validated, respectively called Fl and F2 , with two different initial contractions of chitosan but with a final concentration in the range between 1- 2% . The figures attached to the present patent application are thus aimed at illustrating the different mechanical , chemi cal -physical properties and biocompatibility of the two formulations , in particular : figures la to lc illustrate some diagrams aimed at describing the rheological properties of the formulations Fl and F2 ;

Figures 2a and 2b illustrate the evaluation of the injectability of formulations Fl and F2 ; figures 3a and 3b illustrate the morphology of formulations Fl and F2 by scanning electron microscopy; figures 4a and 4b show two graphs aimed at illustrating the mechanical characterization of the compound object of the present invention ; figures 5a and 5b illustrate two images acquired by means of confocal microscopy, in which the ability of both formulations (Fl and F2 ) to support the formation of the neural and glial network is demonstrated .

In particular , figures la to lc illustrate the rheological properties : figure la shows the temperature dependence of the elastic and viscous moduli (G ’ , G ’ ’ ) of formulations Fl and F2 as the temperature varies from 4 to 40 ° C at a rate of l ° C/min , while figures lb and lc show the variation of the elastic modulus (G ’ ) and the viscous modulus (G ’ ’ ) as a function of time at 37 ° , respectively, of Fl and F2 .

As illustrated in figures la to lc , both chitosan- based solutions Fl and F2 have a G" (viscous modulus) lower than G ’ (elastic modulus) at low temperature (Fig . 1A) and this fact is conventionally considered as a typical condition of a liquid phase sample . With the increase in temperature, the graph of G' , which initially shows a slight decrease up to 20-25 °C, beginning an exponential increase with the intersection between G’ and G" which can be attributed to the formation of a dispersed internal network and thus to a gelling process. This phenomenon is most evident in formulation Fl, which with a G’ value of 0.005 kPa at 4 °C and a minimum of 0.002 kPa (22 °C) reaches a value of 0.4 kPa at 40°C, with an increase of about 200 times, while formulation F2 with 0.01 kPa at 4 °C and a minimum of 5 Pa reaches 0.2 kPa at 40°C. The slower gelling kinetics for F2 is also evidenced by the trend of the storage modulus over time obtained with an isothermal test at 37 °C on samples prepared and stored at 4 °C for different times up to 30 days (Fig. IB- C) . While the initial slope of the curves for non-aged solutions of F2 is very high and, after 5 minutes, no differences in the values of the storage modulus can be appreciated between the samples stored at 4 °C below 24 h (FIG. 10) , Fl shows a slower evolution of the elastic modulus and thus of the network .

This fact can be observed not only as a result of the rise in the modulus of G' already in the early stages during the isothermal test at 37 °C, but also in the trend of the G’ curves, which gradually increase as the samples are stored at 4 °C (FIG. IB) . Furthermore, it can be seen that the final gel stiffness measured after 30 days is slightly higher for Fl (8 kPa) with respect to F2 (5 kPa) .

Figures 2a and 2b illustrate the evaluation of the injectability of formulations Fl and F2 , showing a graph of the force as the printing speed varies, figure 2a, and of the force as the needle dimensions vary, figure 2b.

To evaluate injectability, the maximum force required to extrude each formulation was measured by loading the syringe with the formulation and immersing the syringe needle in a saline solution at 37 °C, varying the injection rate (fig. 2a) and varying the needle size (fig. 2b) . Increasing the injection rate, the maximum force required increased in a range of 1.8- 3 N, (fig. 2a) ; furthermore, decreasing the needle size, the maximum force required increased in a range of 2.5-6 N, (fig. 2b) , for both formulations. In particular, formulation F2 is extruded, creating a linear and continuous filament.

Figures 3a and 3b illustrate the morphology of the compound object of the present invention by means of scanning electron microscopy, respectively of formulation Fl and formulation F2 and it is possible to note how the microstructures have a lower porosity comprised between 2 and 100pm.

Figures 4a and 4b illustrate the mechanical characteristics of formulations Fl and F2 , with and without encapsulated cells, with particular reference to the elastic modulus at DIV 1, figure 4a, and at DIV 20, figure 4b.

With particular reference to figures 4a and 4b, the elastic modulus E ’ , equal to about 6 kPa for a predeformation of 10%, does not show significant differences between the two concentrations when 1 day is maintained in the incubator at 37 °C, but shows a slightly lower value for the formulation F2 (9 kPa versus 12 kPa) when the pre-deformation applied is 20%, i.e. , when the internal stiffness is studied, i.e. , linked to the response of the internal layers of the network . If the elastic modulus E ’ remains approximately at the same values after 20 days for F2 , an important growth occurs in Fl up to 15 kPa and 30 kPa , respectively at 10% and 20% pre-deformation , corresponding to an increase in stiffness of about three times . The mechanical characterization of the gels after cell plating, figure 4b, shows that already after 1 day the elastic modulus is weakly reduced in formulation Fl , with respect to the sample without cells and in particular this behaviour is observed for a pre-deformation of 20% , since the presence of the cells interferes with the conformation of the hydrogel network and consequently with the mechanical properties . This behaviour can be observed even after 20 days of incubation . As regards formulation F2 , a different trend of the variation of the mechanical properties can be observed : the hydrogel network is positively influenced by the presence of neuronal cells , and shows an increase of the elastic modulus of more than three times , from 7 kPa to 23 kPa in 10% predeformation and from 12 kPa to 37 kPa in 20% predeformation .

Yield stress is considered the minimum stress required for a bio-ink to start flowing through a printing system. It is a very important parameter for the characterization of a bio-ink , as the mechanical integrity and printing fidelity of an ink can be improved if an optimal Yield stress is achieved . Yield stress increases proportionally with printability and is correlated with weak interactions between bio-ink components . The evaluation of yield stress was carried out on both formulations using a rheometer . The tests were conducted in two different conditions :

• sol-gel phase bio-ink

• gelled bio-ink

An increasing strain from 1% to 1000% was then applied and it was observed that with the increase in strain , the loss modulus (G") begins to dominate the elastic modulus (G' ) , indicating a transition from a gel-like to a fluid-like behaviour necessary for ink extrusion . The cross point between the two curves G" and G' thus represents the yield stress .

Shear-thinning is the most common behaviour which can be observed in a non-Newtonian fluid and consists of varying the viscosity of the fluid when a shear stress is applied . It is independent of time , it is also called pseudo-plasticity and the fluids which show this behaviour are characterized by an apparent viscosity, which tends to decrease with increasing shear stress .

Such behaviour is ideal for solutions to be used as bio-ink and is due to the reorganization of the polymers following the application of a shear stress ; in particular , as the shear stress increases , a greater degree of disentanglement of the polymeric chains is observed, which translates into a decrease in viscosity .

In fact , this facilitates the extrusion of highly viscous hydrogels . It has been demonstrated through rheological studies how solutions of chitosan in 0 . 1 Molar acetic acid have the characteristics of a nonNewtonian fluid, in particular it has been studied how there is a shear-thinning behaviour which leads to a decrease in the viscosity of the material with the application of an increasingly higher shear rate .

Two different shear rates can be used to simulate the extrusion force during printing : a first low value which simulates the static conditions before and after extrusion and a second higher value , close to the dynamic conditions of extrusion from a needle . Some studies conducted on alginate have shown that the application of a shear rate of 100 Hz reduces the viscosity of the material by up to 97% , with a recovery of 79% , 83% and 85% after 10 , 20 and 30 seconds , respectively .

To allow the material to recover its viscosity characteristics and to improve the printability thereof , a waiting time of about 30 seconds between the printing of two different layers is recommended . The evaluation of the shear thinning characteristics of the solutions was carried out under the same conditions already used for the yield stress tests so as to verify any differences in behaviour between the material being gelled and the fully gelled material .

Based on studies in the literature regarding printing hydrogels , two steps were then carried out : two minutes in which the applied strain is equal to 1% (simulates the pre- and post-press step) and 30 seconds in which the strain is equal to 250% (simulates the step during printing) . These steps were repeated alternately, 2 times each . It was thereby possible to evaluate the immediate recovery abilities of the viscosity characteristics of the material .

From the graphs shown in Figure 4a , it is evident that the newly formed solution Fl is in the liquid phase , with the modulus G" initially greater than G' ; the gelling process at 37 ° C begins after 180 seconds until it reaches a complete gelling after 25 minutes .

Figures 5a and 5b illustrate , in both formulations Fl and F2 , the neuronal cells , identified with the number 10 , and the glia cells , identified with the number 20 , which have a morphology similar to that which can be observed in the tissue in vivo . The neuronal cells have a very different spherical shape from that observed in traditional two-dimensional cultures ; the glia have a filiform shape . Moreover , it can be seen how both formulations , object of this patent , have demonstrated the ability to support the growth and formation of three-dimensional networks of nerve cells , highly connected and maintained in medium long term.

While the invention is subject to various modifications and alternative constructions , some preferred embodiments have been shown in the drawings and described in detail .

It should be understood, however , that there is no intention to limit the invention to the specific illustrated embodiment but , on the contrary, the aim is to cover all the modifications , alternative constructions and equivalents falling within the scope of the invention as defined in the claims .

The use of "for example" , "etc . " , "or" refers to non-exclusive non-limiting alternatives , unless otherwise stated .

The use of "includes" means "includes but is not limited to" , unless otherwise stated .

Claims

1 . Compound for making three-dimensional structures for recreating the nervous tissue of the brain of human beings , consisting of the aggregation of a first fraction with a second fraction , characterized in that the first fraction is the main part based on chitosan and comprises an acid component , while the second fraction comprises at least one or more proteins , specific growth factors of the extracellular matrix (ECM) of the human and animal brain , and a saline component .

2 . Compound according to claim 1 , wherein said second fraction envisages the addition of glycerol .

3 . Compound according to claim 1 or claim 2 , wherein the second fraction comprises the proteins laminin and fibronectin .

4 . Compound according to one or more of the preceding claims , wherein the second fraction comprises the growth factors BDNF (brain-derived neurotrophic factor) , BGF (nerve growth factor) , FGF polypeptides , PDGF (platelet-derived growth factor) , VEGF (vascular endothelial growth factor) .

5 . Compound according to one or more of the preceding claims , wherein said saline component comprises Sodium -glycerophosphate pentahydrate .

6 . Compound according to one or more of the preceding claims , wherein the first fraction is present in a percentage comprised between 75% and 85% of the total weight , while the second fraction is present in a percentage comprised between 15% and 25% , the final chitosan percentage being equal to a value comprised between 1 .5% and 2 .5% .

7 . Method for preparing a compound for making three-dimensional structures for recreating the nervous tissue of the brain of human beings , which method envisages preparing a first fraction and a second fraction , which two fractions are mixed to make the compound, characterized in that the first fraction is obtained by dissolving chitosan powder in an acidbased solution , while the second fraction is obtained by mixing BGP , one or more proteins , specific growth factors of the extracellular matrix (ECM) of the human and animal brain and a saline component .

8 . Method according to claim 7 , wherein the solution of chitosan and acetic acid is kept under stirring at room temperature for a period comprised between 10 and 14 hours , the first fraction being subsequently sterilized in autoclave at a temperature comprised between 100 and 140 °C for a period comprised between 15 and 25 minutes , the first fraction being subsequently stored at a temperature comprised between 2 ° C and 6° C .

9. Method according to claim 7 or claim 8 , wherein the second fraction is sterilized by filtration through syringe filters , the second fraction being subsequently stored at a temperature comprised between 2 ° C and 6°C .

10 . Method according to one or more of claims 7 to 9 , wherein the mixing between the first fraction and the second fraction occurs through the addition of the second fraction drop-wise into the first fraction , the first fraction being kept under stirring during the addition .