Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/52—Hydrogels or hydrocolloids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/14—Macromolecular materials
  • A61L27/20—Polysaccharides
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J19/14—Production of inert gas mixtures; Use of inert gases in general
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J3/00—Processes of treating or compounding macromolecular substances
  • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
  • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
  • C08J3/075—Macromolecular gels
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
• • D—TEXTILES; PAPER
  • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
  • D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
  • D01D5/00—Formation of filaments, threads, or the like
  • D01D5/06—Wet spinning methods
• • D—TEXTILES; PAPER
  • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
  • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
  • D01F9/00—Artificial filaments or the like of other substances; Manufacture thereof; Apparatus specially adapted for the manufacture of carbon filaments
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J3/00—Processes of treating or compounding macromolecular substances
  • C08J3/12—Powdering or granulating
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J3/00—Processes of treating or compounding macromolecular substances
  • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules

Definitions

• 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 .
• 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.
• 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 .
• hydrogels have been used in the controlled release of drugs and regeneration of various types of tissue.
• tissue engineering or regenerative medicine 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.
• 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.
• tissues such as bone, cartilage and skin.
• 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 .
• 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.
• 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.
• 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.
• 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.
• 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.
• 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 .
• Gellan gum hydrogels 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 an alkaline solution being this for example sodium hydroxide
• Crosslinking solutions that consist in an acidic solution being this for example hydrochloric acid.
• gellan gum 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.
• This invention covers the processing and application of gellan gum for applications in regenerative medicine and tissue engineering in a way not described previously.
• 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.
• 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.
• 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.
• compositions 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 :
• 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 .
• 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 9O 0 C 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 50 0 C. 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)
• 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 0 C for 24 hours. After this period, the fibres were removed from moulds with a spatula. ( Figure 3)
• 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.
• gellan gum discs previously produced and described in example 1 were placed in a -8O 0 C freezer for at least 4 hours, quickly transferred to a freeze-dryer, and lyophilized for 2 days. ( Figure 6)
• 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 6.6xlO 4 cells/well (200 ⁇ l/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 (CO 2 ) . After this period, the culture medium was replaced by the previously collected extracts (gellan gum and latex) using culture medium as the negative control.
• CO 2 carbon dioxide
• the cells were incubated with the MTS reagent (using growing medium without red phenol to avoid interference in further readings) for 3 hours at 37 0 C in a wet atmosphere with 5% (v/v) of CO 2 .
• 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) .
• Example 5 In vitro tests using primary cultures of human articular chondrocytes
• 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 ⁇ g/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) .
• FBS fetal bovine serum
• FGF-2 basic fibroblast growth factor 2
• 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 90 0 C 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 41-42 0 C 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 37 0 C in a wet atmosphere with 5% (v/v) of CO 2 using expansion medium for 7 days, and afterwards replacing FGF-2 by insulin (1 ⁇ g/ml) and L-ascorbic acid (50 ⁇ g/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) .
• HE haematoxylin-eosin
• toluidine blue toluidine blue
• alcian blue alcian blue
• safranin-0 stainings concerning the histological analyses
• Figure 8 semi-quantitative real-time PCR for Sox9, collagen type I, collagen type II, and aggrecan, using GAPDH as the housekeeping gene
• 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.

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