WO2009101518A2

WO2009101518A2 - Gellan gum based hydrogels for regenerative medicine and tissue engineering applications, its system, and processing devices

Info

Publication number: WO2009101518A2
Application number: PCT/IB2009/000258
Authority: WO
Inventors: João Teixeira de OLIVEIRA; Rui Amandi De Sousa; Rui Luís REIS
Original assignee: Association For The Advancement Of Tissue Engineering And Cell Based Technologies & Therapies (A4Tec)
Priority date: 2008-02-15
Filing date: 2009-02-12
Publication date: 2009-08-20
Also published as: PT103970A; WO2009101518A3; EP2244753A2

Abstract

This invention refers to the processing and application of gellan gum for regenerative medicine and tissue engineering approaches, focusing processes for packaging, handling, processing of different structures, controlled anionic crosslinking of gellan gum, as well as for its combination with biomolecules and/or live cells live in order to reduce the variability in its chemical and physical properties and in the biological results produced, increasing their effectiveness in the regeneration of living tissues in cellular tests before and/ or after implantation in animals and/or humans, which will allow a more effective transfer of the potential application of gellan gum in a clinical context.

Description

DESCRIPTION

GELLAN GUM BASED HYDROGELS FOR REGENERATIVE MEDICINE AND TISSUE ENGINEERING APPLICATIONS, ITS SYSTEM, AND

PROCESSING DEVICES

OBJECT OF INVENTION

This invention refers to the processing and application of gellan gum in approaches of regenerative medicine and tissue engineering, focusing processes for packaging, handling, processing for different structures, controlled ionic crosslinking of gellan gum, as well as for its combination with bio molecules and/or live cells in order to reduce variability in physical and chemical properties and consequently in the biological results produced by gellan gum, increasing its effectiveness in the regeneration of living tissues in cell tests prior and/or after implantation in animals and/or humans . This will allow a more effective transfer of the potential medical application of gellan gum to the clinical context .

STATE OF THE ART

Hydrogels are typically defined as networks of polymer chains with great water absorbance capacity, meaning that when placed in an aqueous environment can absorb water and increase in volume through the absorption and retention of water in the polymeric mesh. Currently, hydrogels have multiple applications in the food industry, such as thickeners and food additives, as well as in the medical area, where they are used in the manufacture of contact lenses and pill coatings, just to name a few examples .

In the technical field of this invention, hydrogels have been used in the controlled release of drugs and regeneration of various types of tissue. This last field of knowledge is commonly known as tissue engineering or regenerative medicine and its most frequent approach consists in rebuilding a particular type of tissue using a processed biocompatible material that will support the growth and development of relevant cells in conditions appropriate to their cultivation.

This approach arises as a response to the inefficiency of conventional methods in the regeneration and restoration of damaged tissue functions. Conventional methods include the use of donor tissues, which are not available in most situations, and may often trigger immune rejection and transmit diseases to the host.

The use of the patient's own tissues (autologous tissues) is an option, which can eliminate some of these situations. However, some of the methods applied often give rise to morbidity at the collection site and require extended time for surgery, which is not advisable.

Another currently used approach relies on the use of prosthesis based on different types of materials such as polyethylene, silicone, or titanium that physically replace the lost tissue. This is not also the ideal solution since prosthesis present a defined lifetime which carries the need for a new surgery that will remove the old prosthesis and implant a new one, also sometimes a mismatch between the implant and the host tissue given that the prosthesis replaces but does not regenerate the lost tissue nor its included by the surrounding tissues, and finally, it can generate higher infection rates due to the surgical process .

Tissue Engineering appears as a new way to address these situations and involves the regeneration of tissues such as bone, cartilage and skin. Normally used for the cultivation of cells in polymeric supports that should create the necessary conditions for the creation of an extracellular matrix similar to the native one, making the tissue to regain its functionality and allowing its reintegration in the remaining tissue structure. Moreover, the biocompatible material used shall be biodegradable and produce non-toxic degradation products, and ideally be removed from the organism at the same rate of formation of the new tissue.

Hydrogels possess several features that justify their potential application in regenerative medicine and tissue engineering approaches, such as the common biocompatibility presented, resulting from their high water content, and the structural similarity with various types of soft tissue. Several hydrogels that based on their origin can be divided into synthetic and natural, have been used in this type of applications .

Although the field of regenerative medicine and tissue engineering is relatively recent (the most relevant scientific article in this area dates from 1993) , several important discoveries have been made since then and the knowledge grows at an impressive pace. Still, research is ongoing indicating that the optimal solution has not been found yet.

Tissue engineering products combine in most cases materials, biomolecules and cells, and their culturing conditions may be of several types in order to improve the effectiveness of regenerating a functional tissue.

The insufficiency at the level of results possibly relates to the fact that there are still no systems able to fulfill all the requirements associated with a product for tissue engineering.

To some extent, the effectiveness in the regeneration of tissues is jeopardized by the complex methodological and technological approaches proposed or currently under development. In these cases, the complex approaches developed or proposed as future products, in many cases, condition their effectiveness in the human or veterinary clinical context since variations in the handling of the product by the surgeon or clinician may strongly alter the therapeutic performance of the product . The correct implementation of new products for tissue engineering demands not only that the developed materials comply with the necessary requirements for this type of application, but also that the processes developed maximize the functional reproducibility and therapeutic efficacy of product.

Several patents have been written based on the use of polysaccharides able to form hydrogels that can be used in different applications. Among these, a group of the most relevant was chosen under this invention and are the following:

US Patent 6129761 of 10^th October 2000 refers to compositions of injectable hydrogels that deal with polymeric hydrogels and cells used in the medical area, commonly using alginate-based hydrogels in the treatment of craniofacial defects, reflux, and incontinentia.

US Patent 6136334 of 24^th October 2000 describes controlled systems of drug release for applications in the ophthalmologic area using injectable hydrogels that prevent the post-surgical adhesion and confer ophthalmologic protection to the cornea.

US Patent 6719797 of 13^th April 2004 describes systems for the replacement of intervertebral discs using polymers that when inserted in the disk, crosslink and form hydrogels in vivo. US Patent 6773713 of 10^th August 2004 describes approaches of injection moulding for the production of pre-shaped living tissues, which involves the suspension of cells isolated in a solution, which is then injected in a mould and induced to form a hydrogel that can be implanted later.

US Patent 6790840 of 14^th September 2004 describes methods of preparation and potential uses of hydrogels that can be reversibly crosslinked through specific agents controlling in this way important characteristics of the hydrogels such as mechanical properties and degradation rates .

US Patent 7078032 of 18^th July 2006 describes transgenic cells that overexpress biological agents of interest that are typically produced in small quantities in the cells. Inventors describe their use with different polymeric supports for the treatment of various types of pathologies, mainly focused in the area of cancer.

BRIEF DESCRIPTION OF THE FIGURES

For a better comprehension of the invention, figures are presented in annex, but not limited to, in which:

Figure 1 represents discs produced from gellan gum.

Figure 2 represents membranes produced from gellan gum. Figure 3 represents fibres produced from gellan gum.

Figure 4 represents particles produced from gellan gum.

Figure 5 represents 3D supports with non-oriented structure produced from gellan gum.

Figure 6 represents 3D supports with oriented structure produced from gellan gum.

Figure 7 graphically represents the cytotoxic evaluation of the leachables released from the discs produced from gellan gum.

Figure 8 represents staining profiles for components of the extracellular matrix of cartilage in histological sections of gellan gum systems with human articular chondrocytes encapsulated and cultivated for 8 weeks.

Figure 9 graphically i-epresents quantitative data of the production of components of the extracellular matrix of cartilage by the gellan gum systems with human articular chondrocytes encapsulated.

Figure 10 represents the incision sites for subcutaneous implantation of gellan gum discs in Balb/c mice. Figure 11 represents the staining of histological sections of gellan gum discs implanted in Balb/c mice in different periods.

Figure 12 represents a system for the mixing and application of gellan gum.

Figure 13 represents an alternative system for the mixing and application of gellan gum.

Figure 14 represents the alternative system of Figure 13 after the application of a dosage of gellan gum.

DETAILED DESCRIPTION OP THE INVENTION

The purpose of this invention is the processing and application of gellan gum in regenerative medicine and tissue engineering approaches. This biomaterial meets many of the prerequisites required for its potential clinical application as a product for tissue engineering and regenerative medicine, having been the study of this material scarce so far.

In this context, this invention refers to the processing and application of gellan gum in regenerative medicine and tissue engineering approaches, describing for this reason, processes for packaging, handling, processing of different structures, controlled ionic crosslinking of gellan gum, as well as combinations of this material with biomolecules and/or live cells in order to reduce variability of the biological results produced and increase the effectiveness of gellan gum in the regeneration of living tissues after implantation in animals and/or humans .

Gellan gum is a polysaccharide secreted by Sphingomonas paucimobilis that was initially described by Moorhouse et al . It is an anionic polysaccharide composed of repetitive units of glucose, rhamnose, and glucuronic acid. It exists commonly in two formulations: one with a high acyl content which is the raw product secreted by the bacteria, and another with low acyl content due to processing, which is the wider known.

Both form thermoreversible gels with different mechanical behaviors . Gellan gum has an ionotropic gelation mechanism, similar to other polysaccharides such as alginate or carrageenan, and the presence of ions is necessary for the formation of a stable hydrogel.

Gellan gum can be homogeneously dispersed in an aqueous solution at a temperature that promotes the linearization of its chains and may, by decreasing the temperature and in the presence of cations, form a hydrogel .

In addition to the gelation catalyzed by the temperature, it is possible to form gellan gum hydrogels by varying the pH of the aqueous solution. Gellan gum can be solubilised in alkaline pH and precipitate in acidic pH, with the process occurring at room temperature. Several gellan gum structures can be created using simple methods processing methods that involve control of temperature and/or Ph.

The preparation of the hydrogels can be made using:

• Gellan gum dispersions in ultrapure and distilled water with controlled mineral constitution in order to allow its packaging without the occurrence of premature gelation events;

• Crosslinking solutions that consist of a phosphate buffer with controlled constitution;

• Gellan gum dispersions in an alkaline solution, being this for example sodium hydroxide;

• Crosslinking solutions that consist in an acidic solution being this for example hydrochloric acid.

Along with the characteristics already mentioned, there are others that justify the appropriateness of gellan gum for applications in the context of regenerative medicine and tissue engineering. Besides being easily processed using simple and non harsh methods, gellan gum frequently displays a high biocompatibility degree, consists of monosaccharides that can serve as a source of carbohydrates, presents structural similarities with some tissues such as cartilage with which it shares the glucuronic acid molecule present in the extracellular matrix, and is an abundant material and economically inexpensive. In addition, gellan gum has been used for the controlled release of drugs in the ophthalmologic area, as in the case of in vivo tests with human patients.

The studies related with the use of this biomaterial in regenerative medicine and tissue engineering are scarce to date, being however under consistent development by the authors of this invention.

This invention covers the processing and application of gellan gum for applications in regenerative medicine and tissue engineering in a way not described previously.

The authors describe herein new approaches to packaging, handling, processing for different structures, controlled anionic crosslinking, and combination of gellan gum with biomolecules and/or live cells. These procedures allow reducing the variability of the biological results and increasing the effectiveness of gellan gum in the regeneration of living tissues after implantation in animals and/or humans.

These processes can be reproduced using two supplementary systems, and both can be incorporated in the same device :

• Processing system for mixture and dosage of gellan gum (extruders/injection) that monitors parameters such as temperature and pH and enables their adjustment in real time ;

• Adjustment system of the gellan gum composition that defines the relationships between the volume/concentration of the gellan gum solution and the volume/concentration of the crosslinking solution.

With these two systems different structures can be manufactured such as membranes, particles, fibres, or scaffolds. These hydrogels may also be directly injected to fill in cavities or defects with different geometries in animals and/or humans .

The hydrogels produced may be constituted only by the material, the material combined with different types of biomolecules, the material combined with different types of cells, or the material combined with different types of biomolecules and different types of cells.

These hydrogels can be combined with any type of biomolecule used in the regeneration of musculoskeletal tissues and these biomolecules include angiogenic factors, antibiotics, anti-inflammatory drugs, growth factors, and agents that promote cell differentiation, among others .

The hydrogel can be combined with cells used in the regeneration of musculoskeletal tissues, being these autologous, originated from a donor, or from a pre- established cell line, being examples of these: chondrocytes, or cells that can originate chondrocytes, bone cells or cells that can originate bone cells, muscle cells or cells that can originate muscle cells.

The novelty of this invention in view of the current state-of-the-art lies in the following points:

• Use of a specific material, gellan gum, for regenerative medicine and tissue engineering approaches;

• Strict control of the gelation parameters and consequently of the properties of gellan gum based on the use of the processing system for mixture and dosage

(extruders/injection), which monitors parameters such as temperature and pH and enables their adjustment in realtime, and on the adjustment system for the composition of gellan gum that defines the relationships between the volume/concentration of gellan gum solution and the volume/concentration of crosslinking solution, solution reticulation, and on the incorporation of both in the same device :

• Development of systems that reduce the direct handling of the gellan gum hydrogels by the surgeon or clinician in a way to reduce the variability in physical and chemical properties and also on the biological results produced, which will allow a more effective transfer of gellan gum applications to the clinical scenario.

The system for mixing and applying gellan gum application will be described in detail further on with the assistance of figures 12 and 13 in order to provide a better understanding of invention.

The system for mixing and applying gellan gum represented in Figure 12 consists of a cylinder (1) in which the gellan gum is mixed with the use of a mixing shaft (2) that is semi- flexible and concentrically aligned with the longitudinal axis of the cylinder (1) that has several collects one or more blades for agitation (3) . The blades for agitation (3) shall ensure the mixture of gellan gum with biomolecules and/or cells by rotation of the mixture shaft (2) . Parameters such as temperature and pH of hydrogel can be measured through monitoring sensors (4) placed on the cylinder (1) in direct contact with the hydrogel linked to a driver device (10) that also contains an engine at the top responsible for the rotation of the semi- flexible shaft (2) . The temperature of the cylinder can be adjusted in real-time through a heating coat (5) placed on the cylinder (1) . The dosage/injection of gellan gum or its mixture with biomolecules and/or cells is made through the movement of the piston (6) over the longitudinal axis of the cylinder (1) . The adjustment of the composition of the gellan gum, which defines the relationships between the volume/concentration of gellan gum solution and the volume/concentration of the crosslinking solution, can be accomplished by feeding orifices (7) annexed to the cylinder (1) for addition of gellan gum solution and/or crosslinking solution before or during the mixture of gellan gum with biomolecules and/or cells . The system for the mixture and application of gellan gum represented by Figure 13 consists of a cylinder (1) where the gellan gum mixture is performed by using a semi- flexible shaft (2) concentrically aligned with the longitudinal axis of the cylinder (1) that collects an extensible and asymmetric bellow (8) . The extensible and asymmetric bellow (8) assures the mixture of gellan gum with biomolecules and/or cells through the rotation of the mixture shaft (2) . The extensible and asymmetric bellow (8) has several sprocket configurations (9) that make deformation by extension or compression of the bellow (8) . Parameters such as temperature and pH can be measured through monitoring sensors (4) placed on the cylinder (1) in direct contact with the hydrogel linked to a driver device (10) that also contains an engine at the top responsible for the rotation of the semi-flexible shaft (2) . The temperature of the cylinder can be adjusted in real time through a heating coat (5) placed on the cylinder (1) . The dosage/injection of gellan gum or its mixture with biomolecules and/or cells is made through the movement of the piston (6) over the longitudinal axis of the cylinder (1) . Adjustment of the composition of gellan gum can be accomplished by feeding orifices (7) annexed to the cylinder (1) for addition of gellan gum solution and/or crosslinking solution before or during the mixture of gellan gum with biomolecules and/or cells .

The description of this invention is complemented through the following examples that are intended to provide a better understanding of the same, although these examples should not be addressed with a restrictive nature .

Example 1 - Production of discs and membranes of gellan gum using temperature-dependent processes

Gellan gum was processed using temperature dependent methods giving rise to various structures . Discs and membranes of gellan gum were produced as follows. Gellan gum was mixed with distilled water and kept under constant agitation at room temperature, resulting in a final concentration of 0.7% weight on volume (w/v) . The mixture was progressively heated to the temperature of 9O⁰C and maintained at this temperature for 20-30 minutes. After this, the complete and consistent dispersion of gellan gum was observed. Calcium chloride was then added to the mixture for a final 0.03% (w/v) concentration and the temperature was gradually adjusted to 50⁰C. The mixture was cast in a cylindrical mould and allowed to rest during 2-5 minutes at room temperature until the formation of a stable gel was observed. Gellan gum discs were produced using a cutter (diameter 6+0.01 mm x height 5.5 +0.46 mm) . (Figure 1)

Regarding the production of membranes of gellan gum the procedure was as follows . The mixture was cast into Petri dishes and allowed to rest for 2-5 minutes at room temperature until the formation of a solid gel was obtained. The dishes were then placed in an oven at 37⁰C for 90 minutes. Afterwards, the dishes were collected from the oven and the membranes detached with the assistance of a spatula. (Figure 2)

Example 2 - Production of fibres and particles of gellan gum using pH dependent processes

Gellan gum was processed using temperature- dependent methods giving rise to different structures. Fibres and particles of gellan gum were produced in the following way. Gellan gum was mixed with a sodium hydroxide 0.10 M solution and stirred at room temperature resulting in a solution with a concentration of 4% (w/v) . Gellan gum fibres were produced by extruding the mixture through a needle into a solution of L-ascorbic acid (20% v/v) with a flow rate of 0.2 mL/min, using a 21 gauge needle. The fibres were immersed in distilled water, press fitted into cylindrical moulds and kept in an oven at 37 ⁰C for 24 hours. After this period, the fibres were removed from moulds with a spatula. (Figure 3)

Concerning the particles of gellan gum, the procedure was the following. The mixture was extruded drop-by-drop through a needle into a solution of L- ascorbic acid 20% (v/v) under a continuous flow rate of 0.8 mL/min, using a 21 gauge needle. The particles were collected from the solution of immersed in distilled water. (Figure 4) Example 3 - Production of scaffolds of gellan gum using simple materials processing technologies

Gellan gum has been processed in the form of discs as described in example 1 using temperature- dependent methods, although this example can also be used for structures processed using pH dependent methods. Scaffolds of gellan gum were produced with different profiles of porosity, being ones non-oriented and others oriented at the micrometric scale.

For the production of non-oriented structures, the gellan gum discs previously produced and described in example 1 were immersed in liquid nitrogen during 1-2 minutes and quickly transferred to a freeze-dryer and lyophilized for 2 days. (Figure 5)

For the production of oriented structures, gellan gum discs previously produced and described in example 1 were placed in a -8O⁰C freezer for at least 4 hours, quickly transferred to a freeze-dryer, and lyophilized for 2 days. (Figure 6)

The characterization of the microstructure was made through micro-computed tomography (μCT) . 3D virtual models (diameter 3mm, height 1mm) were created from representative regions of the gellan gum structures. Example 4 - Assessment of the cytotoxicity of gellan gum hydrogels

The evaluation of the possible cytotoxicity of the gellan gum structures was conducted using a standard test MTS (3-4, 5-dimetiltiazol-2-IL) -5(3) -carboximetoxifenil-2

(4-sulfofenil) -h-tetrazolium) in accordance with ISO/EN 10993 part 5. This test determines if the cells are metabolically active when in contact with the extracts collected from the hydrogels after immersion times in aqueous medium and is commonly used in cell viability tests. Latex was used as a positive control for cell death due to its high cytotoxicity, and culture medium was used as a negative control for cell death. The cells used in the test are derived from a rat pulmonary fibroblasts cell line (L929) , acquired from the European Bank for Cell Culture (ECACC) and have been cultivated in monolayer in Dulbecco Modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum and 1%

(v/v) mixture of antibiotic - antimycotic.

Discs of gellan gum processed as described in example 1 were incubated in culture medium for 24 hours at 37° C under constant stirring, being the same procedure conducted for the latex. Cultured L929 cells were tripsinised using a mixture of trypsin-EDTA and cultured at a density of 6.6xlO⁴ cells/well (200 μl/well) in 96- well plates. The plates were incubated for 24 hours at 37° C in a wet atmosphere with 5% (v/v) of carbon dioxide (CO₂) . After this period, the culture medium was replaced by the previously collected extracts (gellan gum and latex) using culture medium as the negative control. After 72 hours, the cells were incubated with the MTS reagent (using growing medium without red phenol to avoid interference in further readings) for 3 hours at 37⁰C in a wet atmosphere with 5% (v/v) of CO₂. The culture medium with MTS reagent was then transferred to new wells. The optical density, which is directly proportional to the cellular activity since it reflects the mitochondrial activity, was read in a microplate reader at 490 nm (Figure 7) . Statistical analyses of the obtained data were carried using a parametric test, t-test for two samples assuming unequal variances considering n = 3.

Example 5 - In vitro tests using primary cultures of human articular chondrocytes

The samples for the isolation of human chondrocytes were removed from the femoral head of femurs from adult patients (40-65 years) that have been subjected to reconstructive surgical procedures. The procedure was conducted under informed consent of the patient and in accordance with an established protocol with the Hospital S. Marcos, Braga, Portugal that was previously approved by its Ethics Committee. The human chondrocytes were isolated by enzymatic digestion and cultivated in expansion culture medium [DMEM, 10 mM HEPES buffer pH 7.4, 10000 units/ml penicillin/10000 μg/ml streptomycin,20 mM L-alanine glutamine, Ix MEM non-essential amino acids, 10% (v/v) fetal bovine serum (FBS) , and supplemented with 10 ng/ml of basic fibroblast growth factor 2 (FGF-2) . The cells were expanded until an appropriate number to be encapsulated in the gellan gum hydrogels . The procedure for the encapsulation of the human articular chondrocytes in the gels and posterior culture is the following.

Gellan gum was mixed with distilled water and kept under constant stirring at room temperature, resulting in a final concentration of 1% (w/v) . The mixture was progressively heated to the temperature of 90⁰C and maintained at this temperature for 20-30 minutes. A full and consistent dispersion of gellan gum was observed after this time. Calcium chloride was added for a final concentration of 0.03% (w/v) and the temperature gradually adjusted to 41-42°C always under constant stirring. Human chondrocytes at passage 2 (P2) were collected by tripsinisation, mixed with expansion medium and centrifuged at 20Og for 7 min. The supernatant was discarded and the cells resuspended in phosphate buffered saline (PBS) , counted using a hemocytometer and centrifuged at 20Og for 7 min. The supernatant was discarded and the pellet of chondrocytes kept at the bottom of tube. The gellan gum solution previously prepared and kept under constant stirring at a temperature of 41-42⁰C was added to the pellet of chondrocytes and this mixture resuspended to obtain a homogeneous cell distribution, with a final concentration of 8xlO^s cells/ml. Gellan gum discs with encapsulated chondrocytes were produced by transferring the mixture to sterile polystyrene moulds, and allowing the mixtui-e to rest at room temperature for 1-2 minutes to enable the gelation. Then, discs (diameter 6+0.01 mm x height 5.5 ±0.46 mm) were cut using a cutter.

The gellan gum hydrogels with human articular chondrocytes were cultured at 37⁰C in a wet atmosphere with 5% (v/v) of CO₂ using expansion medium for 7 days, and afterwards replacing FGF-2 by insulin (1 μg/ml) and L-ascorbic acid (50 μg/ml) during the remaining periods of culture that extended to 8 weeks .

The gellan gum hydrogels with encapsulated chondrocytes were analyzed using histological and molecular methods to evaluate cartilage-like tissue formation and the deposition of typical components from its extracellular matrix, namely using haematoxylin-eosin (HE) , toluidine blue, alcian blue, and safranin-0 stainings concerning the histological analyses (Figure 8), and semi-quantitative real-time PCR for Sox9, collagen type I, collagen type II, and aggrecan, using GAPDH as the housekeeping gene (Figure 9) .

Example 6 - Subcutaneous implantation of gellan gum discs in Balb/c mice for evaluating the inflammatory response

Gellan gum discs were prepared in a sterile environment as described in example 1, using a final concentration of 1% (w/v) . The discs were subcutaneousIy implanted in Balb/c mice (2-3 months old, approximately 20 g) for 3 weeks, having been implanted 4 discs per animal. The procedure used the following. The mice were subjected to trichotomy in the incision area and subsequently a mixture of 5:1 composed of Imalgene^® 1000 and Domitor^® in physiological serum was administered intramuscularly. After confirmation of anesthesia/analgesia, two incisions (up to a maximum of 1.5 cm) were performed in the interscapular and lumbar areas and lateral pockets for inserting the gellan gum discs were created (Figure 10) . One gellan gum disc was implanted per pocket and the incisions were posteriora sutured. The animals were transferred to heating chambers for recovery from anesthesia and afterwards returned to adequate compartments. After the periods defined, the animals were euthanized by exposition to an oversaturated carbon dioxide environment and the gellan gum hydrogels collected surgically. These were subsequently examined by histological methods, namely using HE staining, Figure 11, where the results for 7 days (left) , 14 days (centre) , and 21 days (right) are presented.

Any person with the adequate expertise in the field may be able to make changes not described in this request, in which the application of those changes in a determined structure or manufacturing process requires the claimed matter in the following claims; these structures must therefore be understood within the scope of this invention.

Claims

1. Gellan gum hydrogels characterized in that it has varied degrees of acylation and allowing the incorporation of chemical modifications in order to incorporate biomolecules and/or live cells, being these hydrogels able to be processed in different morphologies such as particles, membranes, fibres, discs, scaffolds, or directly injected to fill holes or defects of different geometries in animals and/or humans.

2. Gellan gum hydrogels according to claim 1, characterized in that its preparation comprises:

• dispersions of gellan gum in ultrapure and distilled water with controlled mineral in order to enable their packaging without the occurrence of premature gelation events,

• crosslinking solutions which consist of a salt phosphate buffer with controlled composition.

3. Gellan gum hydrogels according to claim 1, characterized in that its preparation comprises:

• dispersions of gellan gum in an alkaline solution, which may be, for example, sodium hydroxide;

• crosslinking solutions that consist of an acidic solution, which may be, for example, hydrochloric acid.

4. Procedure for processing and adjusting gellan gum hydrogels characterized in that:

• processing system for the mixture and dosage (extrusion/injection) of gellan gum that monitors parameters such as temperature, pH and allows their adjustment in real-time

• adjustment system of the composition of gellan gum composition that allows to vary the relations between the volume/concentration of gellan gum solution and the volume/concentration of crosslinking solution, by adding solutes, solvents and agents crosslinking agents.

5. Gellan gum hydrogels according to the claims 1 to 4 , characterized in that the hydrogel is combined with any type of biomolecules used in the . regeneration of musculoskeletal tissues, and these factors including angiogenic factors, antibiotics, anti-inflammatory drugs, growth factors and promoters of cell differentiation.

6. Gellan gum hydrogels according to claims 1 to 5, characterized in that the hydrogel is combined with cells used in the regeneration of musculoskeletal tissues, that may be autologous, come from a donor or a pre-established cell line, being examples of these: chondrocytes, or cells that can originate chondrocytes, bone cells or cells that can originate bone cells, muscle cells or cells that can originate muscle cells .

7. Gellan gum hydrogels characterized in that it is applied in the area of tissue engineering and regenerative medicine into products for research in the field of tissue engineering and regenerative medicine, pre-clinical tests or as products for musculoskeletal tissue engineering and regenerative medicine.

8. Method for processing and adjustment of gellan gum hydrogels, according to claim 4, characterized in that it defines the relations between the volume/concentration of the gellan gum solution and the volume/concentration of the crosslinking solution through feeding orifices (7) annexed to the cylinder (1) for addition of gellan gum solution and/or crosslinking solution before or during the mixture of gellan gum with biomolecules and/or cells .

9. Device for processing gellan gum hydrogels characterized in that it incorporates:

• a processing system for the mixture and dosage (extrusion/injection) of gellan gum that monitors parameters such as temperature, pH and allows their adjustment in real-time,

• a system of adjustment of the composition of gellan gum that defines the relationships between the volume/concentration of gellan gum solution and the volume/concentration of the crosslinking solution,

10. Device for processing gellan gum hydrogels according to claim 9, characterized in that it comprises:

• a cylinder (1) for the mixture of gellan gum through a semi-flexible mixture shaft (2) concentrically aligned with the longitudinal axis of the cylinder

(1) with one or more blades for agitation (3) ,

• a mixture shaft (2) that through its movement of rotation and consequently of the blades for agitation (3), ensures the mixture of the biomolecules with gellan gum.

• a mixture shaft (2) that through its movement of rotation and consequently of the blades for agitation (3), ensures the dispersion of cells in the gellan gum

• a controlling equipment (10) of parameters such as temperature and the pH of hydrogel measured through monitoring sensors (4) incorporated on the cylinder

(1) in contact with the hydrogel,

• a heating coat (5) located in the cylinder (1), which enables the adjustment of the temperature in real-time

• a piston (6) that allows the dosage of gellan gum or its mixture with biomolecules and/or cells through the movement along the longitudinal axis of the cylinder (1) .

11. Device for processing gellan gum hydrogels according to claim 9, characterised in that it comprises:

(1) , with a retractable semi-rigid and non symmetrical bellow (8)

• a mixture shaft (2) that through the movement of rotation and consequently the retractable semi-rigid and non symmetrical bellow (8) ensures the mixing of biomolecules with gellan gum

• a mixture shaft (2) that through the movement of rotation and consequently the retractable semi-rigid and non symmetrical bellow (8) ensures the mixing of cells with gellan gum

(1) in contact with the hydrogel

• a heating coat (5) located in the cylinder (1) , which enables the adjustment of the temperature in real-time a piston (6) that allows the dosage (injection of gellan gum or its mixture with bioraolecules and/or cells through the movement along the longitudinal axis of the cylinder (1) .

several sprocket configurations (9) in the extensible and asymmetric bellow (8), that facilitate its deformation through extension and compression of the bellow (8) .