Hydrogel-like biomimetic material comprising regionally distributed microenvironments, method of manufacture, and uses thereof in research, industrial, and therapeutic applications

Abstract

Hydrogel-like biomimetic material comprising regionally distributed microenvironments, method of manufacture, and uses thereof in research, industrial, and therapeutic applications The hydrogel-like biomimetic material (100) has a three-dimensional distribution of biomimetic microenvironments (110), wherein each biomimetic microenvironment (110) comprises (a) a polymeric network (A) that simultaneously provides structural support and functional activity; and (b) a water-based suspension (B), wherein the water-based suspension (B) is sustained by the polymeric network (A).

Description

[0001] Hydrogel-like biomimetic material comprising regionally distributed microenvironments, method of manufacture, and uses thereof in research, industrial, and therapeutic applications

[0002] CROSS REFERECE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority from U.S. provisional patent application no. 63 / 705,782 filed October 10, 2024 and from Portuguese patent application no. 20242006561358 filed November 10, 2024, the contents of each of which are incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] The present invention lies in the field of biomaterials, bioengineering, and microbiological technologies, and more particularly in the development of programmable hydrogel-like materials that are capable of reproducing, supporting, and modulating biological microenvironments in a controlled and quantifiable manner.

[0006] The invention specifically relates to a hydrogel-like biomimetic material (100) comprising a three-dimensional distribution of biomimetic microenvironments (110). Each microenvironment comprises a polymeric network (A), a water-based suspension (B), and, in certain embodiments, viable microorganisms (C). The structural and compositional organization of these microenvironments defines regionally distributed microenvironments (RDMEs), which allow the simultaneous control of hydration, oxygen concentration, ionic strength, nutrient availability, and crosslinking density across the material.

[0007] The invention further relates to methods for manufacturing said biomimetic material (100), including directed crosslinking techniques (ionic, photochemical, enzymatic, redox, biologically induced, or field-based), and to the resulting macrostructured constructs (300) and composite systems (400) derived therefrom. The manufacturing methods disclosed herein enable the spatial and hierarchical arrangement of distinct microenvironments within a single continuous matrix, leading to reproducible and scalable materials that mimic native biological conditions.

[0008] The biomimetic materials of the invention are applicable in multiple technological and scientific contexts, including but not limited to: Research and analytical applications, such as in-vitro modeling of host-microbe interactions, microbiota cultivation, antibiotic or pro-, prebiotic testing, and high- throughput screening of microbial behavior under physiologically relevant conditions;

[0009] Industrial and bioprocessing applications, including microbial cultivation and mining, fermentation optimization, and metabolite production using bioreactors containing the macrostructured constructs of the invention;

[0010] • Therapeutic and biomedical applications, such as mucosal regeneration and or recovery, pro-, pre- and postbiotic delivery, microbiota restoration, or the development of in-situ forming and rehydratable gels for mucosal tissue repair; and

[0011] • Personal-care and diagnostic applications, including biocompatible lubricants for medical procedures, diagnostic sampling, and sexual wellness products designed to maintain mucosal hydration and physiological rheology.

[0012] In a broader context, the invention establishes a unified biomimetic hydrogel platform capable of bridging basic research, industrial production, and clinical translation, offering a reproducible, modular, and tunable system that overcomes the limitations of conventional homogeneous hydrogels.

[0013] BACKGROUND OF THE INVENTION

[0014] Hydrogels are among the most widely used biomaterials for biological and biomedical applications due to their high water content, biocompatibility, and tunable viscoelasticity. They have been explored as matrices for tissue regeneration, scaffolds for microbial or cellular culture, and mimics of complex biological fluids such as mucus. Despite these advances, conventional hydrogels remain compositionally uniform and structurally homogeneous, unable to reproduce the spatial heterogeneity, oxygen gradients, nutrient diffusion, or dynamic microbial behavior characteristic of living systems. Consequently, they offer limited physiological relevance and narrow functional scope.

[0015] Recent efforts have attempted to improve hydrogel fidelity to biological environments. For instance, mucin-based hydrogels have been proposed to reproduce mucus rheology; multilayer or gradient hydrogels have been fabricated for tissue engineering; and gel-in-gel or 3D-printed systems have been designed for probiotic delivery. Representative disclosures include US 2021 / 0032411 A1 (synthetic mucus-like gels), US 2018 / 0049977 A1 (microparticles with degradability gradients), US 2025 / 0082827 A1 (trilayer scaffolds), and EP 3209718 B1 (tissue-mimicking hydrogels for biofabrication).

[0016] The limitations of existing systems are fourfold. First, their lack of regional differentiation prevents the formation of discrete zones of hydration, oxygen, and nutrients as seen in native tissues or microbial ecosystems. Second, most rely on a single-purpose polymer network, serving only structural or carrier roles without intrinsic biofunctionality or controlled degradation. Third, crosslinking is generally homogeneous, leaving no possibility for spatial patterning or gradient programming. Fourth, available hydrogels are not designed to modulate microbial dynamics — none can deliberately maintain viability during storage and later trigger activation or growth in defined regions.

[0017] Accordingly, there remains a clear need for a single programmable material that can (i) reproduce multiple biological microenvironments within one continuous matrix, (ii) establish quantifiable gradients of key physicochemical parameters, (iii) protect and control microorganisms or bioactive entities through lag / log transitions, and (iv) be produced by simple, water-based, and scalable methods suitable for both research and therapeutic products.

[0018] The present invention fulfills these requirements by introducing a hydrogel-like biomimetic material (100) composed of a three-dimensional distribution of biomimetic microenvironments (110). Each microenvironment includes a polymeric network (A) that is simultaneously structural and functional, a water-based suspension (B) sustaining hydration and ionic balance, and, in certain embodiments, viable microorganisms (C). Through directed crosslinking — for example by controlled ionic diffusion, photochemical patterning, or sequential gelation — the material develops regionally distributed microenvironments (RDMEs) with measurable gradients of crosslinking, hydration, oxygen, and nutrients.

[0019] This architecture yields a dual-function, self-active polymeric matrix that both carries and protects biological or chemical entities while releasing functional fragments upon degradation. It enables precise control of microbial metabolic states, supports reproducible physicochemical gradients, and allows the fabrication of macro- to mesoscale constructs by scalable, eco-friendly processes. The result is a unified biomimetic hydrogel platform that bridges research, industrial bioprocessing, and therapeutic domains — overcoming the homogeneity, limited functionality, and lack of control inherent in prior hydrogel technologies. SUMMARY OF THE INVENTION

[0020] The present invention provides a hydrogel-like biomimetic material (100) designed as a programmable and multifunctional platform for reproducing, supporting, and modulating biological and synthetic microenvironments. The material integrates compositional, structural, and functional elements into a three-dimensional configuration that enables quantitative control over physicochemical parameters, microbial activity, and degradation kinetics.

[0021] The biomimetic material (100) is defined by the presence of a three-dimensional distribution of biomimetic microenvironments (110), each microenvironment comprising:

[0022] • a polymeric network (A), which provides both structural integrity and biofunctional activity;

[0023] • a water-based suspension (B), which maintains hydration, ionic balance, and serves as a carrier for bioactive molecules, nutrients, and other solutes; and

[0024] • in certain embodiments, viable microorganisms (C) suspended within phase (B) and / or adhered to the polymeric network (A).

[0025] The microenvironments (110) can be homogeneously distributed throughout the matrix or regionally distributed to form regionally distributed microenvironments (RDMEs). Each RDME corresponds to a discrete or continuous region within the material that exhibits characteristic values of hydration, crosslinking density, pH, oxygen concentration, or nutrient content. This architecture permits the creation of distinct local microconditions that interact dynamically, collectively defining the overall behavior of the material.

[0026] Directed Crosslinking

[0027] A key inventive feature of the material is the use of directed crosslinking — a fabrication strategy in which crosslinking reactions are initiated or propagated in a spatially controlled manner. Directed crosslinking can be achieved through ionic diffusion, photochemical activation, enzymatic reactions, biologically induced redox mechanisms, or electromagnetic or electrochemical fields.

[0028] Through directed crosslinking, the polymeric network (A) is formed with spatially variable crosslinking density, enabling the creation of defined regions with distinct mechanical and diffusional properties. This process produces internal gradients of oxygen, pH, or solute concentration, and establishes the physical and biochemical boundaries between microenvironments. The technique also supports the sequential or simultaneous superposition of different polymeric sub-networks, generating interpenetrating polymeric networks (IPNs) or hybrid meshes with programmable properties.

[0029] Dual-Function Polymeric Network

[0030] The polymeric network (A) is composed of one or more polymers selected from natural, semisynthetic, or synthetic materials that possess both structural and functional roles. Structurally, the network forms the backbone of the material, retaining the aqueous phase and any suspended microorganisms or molecules. Functionally, it provides functional / reactive groups (e.g., carboxylate, amine, hydroxyl, sulfate) capable of mediating biological adhesion, ionic exchange, or biochemical response.

[0031] The polymeric network also acts as a nutrient source and a chemically active component for the biological activities of the integrated microorganisms or those to which it enters in contact in any desired application.

[0032] Upon degradation, the polymeric network (A) releases not only its cargo but also its own polymeric fragments, which can act as active ingredients, prebiotics, adhesive agents, or modulators of the local environment. Thus, the polymer simultaneously behaves as a carrier, a protective matrix, and a functional component.

[0033] Preferred polymers include alginate, pectin, gellan gum, agarose, xanthan gum, carrageenan, cellulose derivatives (CMC, HPMC), and chitosan. Animal- and microbial-derived polymers such as mucin, collagen, gelatin, hyaluronic acid, heparin, and dextran may be included to impart biofunctionality. Synthetic or biodegradable polymers such as PEG, PLA, PLGA, PCL, PVA, PMMA, and polyurethanes can be incorporated to tune degradation and mechanical properties. The combination of these polymers produces a customizable mesh structure characterized by a mesh size of 18-110 nm, crosslinking density of 20-100 %, hydration level of 5-95 %, and viscoelastic moduli G' = 1.5-3000 Pa, G" = 0.3-500 Pa.

[0034] Regionally Distributed Microenvironments (RDMEs)

[0035] Within the biomimetic material (100), the microenvironments (110) are arranged spatially to form regions with specific functional logic. These may include:

[0036] • Shell regions (111) that serve as protective or diffusion-limiting layers;

[0037] • Diffuser regions (112) that regulate molecular and oxygen exchange; and • Subtypes of diffuser regions with tailored biological purposes: Bio-holder (112-H), Bioneutral (112-N), and Bio-Booster (112-B)

[0038] The spatial arrangement of these regions can be symmetrical (radial, concentric), asymmetrical (hemispherical, layered), clustered, atomized, or a combination thereof. The design can be customized according to the intended functions, such as maintaining microbial viability during storage, releasing chemically active molecules in a targeted region of the gastrointestinal tract, or mimicking mucosal gradient conditions for research models.

[0039] Macrostructured Constructs and Composites

[0040] The biomimetic material (100) can be produced in various formats, including:

[0041] • Amorphous gels (200);

[0042] • Macrostructured constructs (300) having defined geometry (e.g., beads (300-B), disks (300-D), cylinders (300-C), films (300-F), or tubes (300-T)); and

[0043] • Composite structures (400) in which one or more macrostructured constructs (300) are embedded within an amorphous gel (200).

[0044] Constructs (300) have characteristic magnitudes in the millimeter-to-centimeter range in at least one of its dimensions. They may incorporate multiple regions with different crosslinking densities, pH, or oxygen levels, yielding a multi-zonal structure analogous to natural tissues or ecological niches. Hierarchical embedding allows constructs-within-constructs, enabling controlled release or activation cascades.

[0045] Manufacturing Method

[0046] The invention also provides a method for manufacturing the hydrogel-like biomimetic material (100) and the formats derived therefrom, including amorphous gels (200), macrostructured constructs (300) and composite structures (400). The method comprises at least two sequential phases:

[0047] Phase 1 - Preparation of the biomimetic material (100):

[0048] Mixing polymeric precursors with water-based suspensions containing salts, dopants, and optional viable microorganisms. Directed crosslinking is initiated under controlled environmental conditions (e.g., temperature 4-60 °C), leading to gelation and formation of discrete microenvironments.

[0049] Phase 2 - Shaping and structuring:

[0050] The preformed biomimetic material is cast, molded, extruded, or printed into desired shapes, establishing a desired distribution of microenvironments through the biomaterial (100), and the macrostructured constructs (300). Parallel or sequential processing steps may be combined to embed constructs of one type into another, creating hierarchical embedding and multi-region composites (400).

[0051] Optional Phase 3 - Creation of a composite material (400):

[0052] Mixing, integrating, or making a controlled deposition of previously fabricated amorphous gel or macrostructured construct (300) within,

[0053] • A previously formed amorphous gel (200)

[0054] • The inputs for Phase I

[0055] And then optionally putting this newly developed composite material through a shaping Phase II.

[0056] Optional Phase 4 - Post-processing includes drying, air-drying, solvent casting, or lyophilization (water removal > 90 %), producing rehydratable or partially dehydrated products.

[0057] Functional Capabilities and Technical Effects

[0058] The biomimetic material (100) exhibits several key functional properties and technical effects:

[0059] 1. Dynamic control of microbial behavior:

[0060] The configuration of RDMEs allows selective control of microbial lag and log phases, maintaining viability under stress or triggering activation upon exposure to favorable conditions.

[0061] 2. Quantifiable internal gradients:

[0062] Gradients of oxygen (0.1-50 % available environmental oxygen), pH (4-9), hydration (5-95 %), and nutrient concentration (0.2x-2x of culture media mixtures) can be programmed, measured, and reproducible across batches. 3. Programed functional degradation:

[0063] The biomimetic material undergoes programmed self-functional degradation, wherein the material is not degraded through enzymatic activity or pH-mediated hydrolysis, but rather by a biologically programmed degradation mechanism driven by microbial metabolization.

[0064] The degradation rate is correlated with the metabolic activity of the microorganisms associated with or encapsulated within the biomimetic material.

[0065] In particular embodiments, the degradation kinetics are predetermined or tunable such that the resulting degradation products act as prebiotic agents, thereby promoting or enhancing the metabolic activity of the microorganisms, which in turn may accelerate or modulate further degradation of the biomimetic material.

[0066] In certain embodiments, the programmed self-functional degradation process additionally facilitates the controlled release of the biomimetic material’s intrinsic functional polymeric components, together with the encapsulated microorganisms and / or active agents, in a temporally and spatially regulated manner.

[0067] 4. Scalable macrostructuring:

[0068] The method supports the reproducible fabrication of constructs ranging from 0.5 mm to several centimeters, enabling integration from laboratory to industrial scale.

[0069] 5. Cross-domain adaptability:

[0070] The same material can function as a laboratory research tool, a bioprocess enhancer, a therapeutic implant, or a personal-care product, depending on composition and design.

[0071] Applications

[0072] The invention provides a single technological framework applicable to:

[0073] • Microbiome research — for cultivating complex microbial communities, mining and studying host-microbe interactions;

[0074] • Industrial bioprocesses — as carriers or enhancers for fermentation, metabolite production, production or microorganisms including but not limited to probiotics, and pre and post biotic production. • Therapeutic uses — including mucosal hydration, mucosal layer recovery, prebiotic, probiotic or postbiotic delivery, microbiota restoration, and tissue regeneration; and

[0075] • Personal-care and medical lubrication — as biocompatible, functional gels for diagnostic or sexual-wellness applications.

[0076] Technical Distinction

[0077] In contrast to prior hydrogels that are compositionally homogeneous and single-purpose, the present invention introduces a regionally differentiated, dynamically functional hydrogel-like material capable of integrating multiple bioactive and structural roles within a single, continuous construct.

[0078] By combining directed crosslinking, dual-function polymeric networks, and regionally distributed microenvironments, the invention achieves programmable control of structure, function, and biological response at both micro- and macro-scales.

[0079] BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Fig 1 : A section of material (100) composed by different regions (R) made of different types of microenvironments (110) selected from (112), (112-B), (112-N) and (112- H) and its changes over time.

[0081] Fig 2 : Conformation of material (100) in its different presentations.

[0082] Fig 3 : A presentation of the material (100) shaped as a bead (300-B) with two types of microenvironments (110).

[0083] Fig 4: Different shapes of beads (300-B).

[0084] Fig 5 : Illustration of different dispositions of regions of regionally distributed microenvironments (110).

[0085] Fig 6 : Different types of polymeric meshes creating different microenvironments (110).

[0086] Fig 7: Superposition of different polymeric meshes creating regionally distributed microenvironments (110).

[0087] Fig 8 : Interaction of bioactivity of macroconstructs (300) in the presence of microenvironments (110). Fig 9: Methodology for the production of biomimetic material (100).

[0088] Fig 10: Variations to the methodology of production of biomimetic material (100).

[0089] Fig 11: development of material for mimicking homogeneous colon mucus, gradient colon mucus, flow colon mucus, small intestine mucus.

[0090] Fig. 12: viscoelastic properties of homogeneous colon mucus.

[0091] Fig. 13: viscoelastic properties of gradient colon mucus.

[0092] Fig. 14: viscoelastic properties of flow mucus.

[0093] Fig. 15: viscoelastic properties of small intestine.

[0094] Fig. 16: viscoelastic properties of homogeneous physiologic.

[0095] Fig. 17a: viscoelastic properties of gradient cystic fibrosis.

[0096] Fig. 17b: viscoelastic properties of homogenous physiologic, homogeneous cystic fibrosis, gradient cystic fibrosis.

[0097] Fig. 18: development of material for mimicking homogeneous cervicovaginal mucus in nonovulatory conditions, gradient cervicovaginal mucus in non-ovulatory conditions, homogeneous cervicovaginal mucus in non-ovulatory conditions in MRS, gradient cervicovaginal mucus in non-ovulatory conditions in MRS, homogeneous cervicovaginal mucus in ovulatory conditions, gradient cervicovaginal mucus in ovulatory conditions, cervicovaginal mucus during pregnancy.

[0098] Fig. 19: viscoelastic properties of homogeneous cervicovaginal mucus in non-ovulatory conditions.

[0099] Fig. 20: viscoelastic properties of gradient cervicovaginal mucus in non-ovulatory conditions.

[0100] Fig. 21 : viscoelastic properties of homogeneous cervicovaginal mucus in non-ovulatory conditions in MRS.

[0101] Fig. 22: viscoelastic properties of gradient cervicovaginal mucus in non-ovulatory conditions in MRS.

[0102] Fig. 23: viscoelastic properties of homogeneous cervicovaginal mucus in ovulatory conditions. Fig. 24: viscoelastic properties of gradient cervicovaginal mucus in ovulatory conditions.

[0103] Fig. 25: viscoelastic properties of cervicovaginal mucus during pregnancy.

[0104] Fig. 26: mesh size changes through microbiota interactions.

[0105] Fig. 27: mesh size changes in Lactobacillus and Bifidobacterium.

[0106] Fig. 28: activity of microorganisms by quantification of butyrate over time.

[0107] Fig. 29: changes in microbial relative abundance or butyrate concentration vs. mesh size changes

[0108] Fig. 30: tailored microenvironmental gradients created in the biomimetic colon-like hydrogel.

[0109] Fig. 31 : tailored microenvironmental gradients created in the biomimetic colon-like hydrogel.

[0110] Fig. 32: alpha diversity through subculturing time.

[0111] Fig. 33: relative abundance genus level in beads.

[0112] Fig. 34: double subsequent crosslinking.

[0113] Fig. 35: regionally distributed Gut3Beads™ characterization.

[0114] Fig. 36: growth boosting of Akkermansia muciniphila.

[0115] Fig. 37: cervicovaginal mimicking beads for selective mining from cervicovaginal isolate.

[0116] Fig. 38: DNA quantification of V. parvula incubated with and without Beads. Statistics: B.r. 3; T.r. 3; Data expressed as mean ± SEM; 2way-Anova with Sidak's multiple comparisons test.

[0117] Fig. 39: MTT assay data of cell viability in the Beads group. Viability of V. parvula incubated with and without Beads. Timepoint 24h was set to 100%. Statistics: B.r. 3; T.r. 3; Data expressed as mean ± SEM; 2way-Anova with Sidak's multiple comparisons test. P values are expressed as **** with p < 0.05*, p < 0.01 **, p < 0.001 ***, p < 0.0001 ****

[0118] Fig. 40: macroscopic inspection showing only slight surface degradation of the beads after prolonged incubation.

[0119] Fig. 41 : biomimetic beads remained structurally stable throughout the simulated digestion sequence, with no significant mass loss.

[0120] Fig. 42: pH diffusion gradients of Gut3Beads™. Fig. 43: oxygen diffusion gradients of Gut3Beads™.

[0121] Fig. 44: percentage of attached Gut3Beads™.

[0122] Fig. 45: Principal Coordinates Analysis (PCoA) based on weighted UniFrac distances showing separation of microbial communities by donor.

[0123] Fig. 46:characterization of the changes in the community of bacteria in-vitro upon treatment with beads.

[0124] Fig. 47: Gut3Beads™ with encapsulated spermidine in standard (non-supplemented) and spermidine-loaded.

[0125] Fig. 48: encapsulated Lactobacillus plantarum and stability in pectin-based biomimetic material.

[0126] Fig. 49: vitality verification after storage and incubation (after storage at 20°C and later incubation).

[0127] Fig. 50: evaluation of the protection capacity of the microorganisms against the gastrointestinal tract.

[0128] Fig. 51: encapsulation of active therapeutic molecules within three-dimensional biomimetic material.

[0129] Fig. 52: glycerol-containing biomimetic beads.

[0130] Fig. 53: biomimetic beads storage at 4°C.

[0131] Fig. 54: biomimetic beads storage at -20°C.

[0132] Fig. 55: biomimetic beads storage at -80°C.

[0133] Fig. 56: three-dimensional gastrointestinal-like biomimetic material in bead shape.

[0134] Fig. 57: USB microscope picture taken every hour to evaluate the diffusion of the red coloured molecule contained in Biomimetic material A towards the external environment (Biomimetic material B).

[0135] Fig. 58: analysis of biomimetic material to quantify the amount of red dye released from Biomimetic material A to the Biomimetic material B. Fig. 59: evolution of the curve with steeper peak in the pure red (255 from RGD colour) demonstrated the change in the contained molecules within the polymeric network of material A.

[0136] Fig. 60: microbiota culture and community evolution.

[0137] Fig. 61: relationship between the growth of Lactobacillus and Bifidobacterium species with the changes in the polymeric network of the biomimetic material.

[0138] Fig. 62: metabolite analysis.

[0139] Fig. 63a: faecal microbiota isolates from donors.

[0140] Fig. 63b: faecal microbiota isolates from donors.

[0141] Fig. 64: tube-like biomimetic material is configured for delivery through a catheter equipped with an inflatable balloon

[0142] Fig. 65: lyophilized biomimetic material and rehydrated biomimetic material.

[0143] Fig. 66: Spherical and ellipsoidal biomimetic beads with adaptable diameter.

[0144] Fig. 67: disk-shaped biomimetic materials.

[0145] Fig. 68: tubular biomimetic materials.

[0146] Fig. 69a: films without a reservoir.

[0147] Fig. 69b: films with a central reservoir.

[0148] Fig. 70: at each timepoint, viable cells quantified by counting colony-forming units (CFU).

[0149] DETAILED DESCRIPTION OF THE INVENTION

[0150] A. COMPOSITION AND COMPONENTS

[0151] The hydrogel-like biomimetic material (100) of the present invention is formed by the three- dimensional arrangement of a plurality of biomimetic microenvironments (110), each of which comprises:

[0152] 1. a polymeric network (A) that defines the local structural and physicochemical framework; 2. a water-based suspension (B) that fills and sustains the network, maintaining hydration and ionic balance; and

[0153] 3. in certain embodiments, viable microorganisms (C) distributed within the suspension or adhered to the network.

[0154] The microenvironments (110) are discrete or continuous domains that differ in composition or degree of crosslinking, and together define the regionally distributed microenvironments (RDMEs) characteristic of the invention.

[0155] A.1 Polymeric Network (A)

[0156] A.1.1 General Structure

[0157] The polymeric network (A) is a hydrophilic, three-dimensional matrix formed by one or more polymers that are crosslinked either physically or chemically.

[0158] The resulting network retains the aqueous suspension (B) and provides a defined mesh size typically between 18 nm and 110 nm, a crosslinking density between 20 % and 100 %, preferably 34 to 100% and viscoelastic properties characterized by a storage modulus (G') of 1 .5 - 3000 Pa and a loss modulus (G") of 0.3 - 500 Pa at frequencies between 0.01 and 100 Hz. These parameters may be varied spatially by directed crosslinking to generate regions with distinct mechanical and diffusional behavior.

[0159] A.1.2 Polymer Selection

[0160] The polymeric network (A) comprises one or more polymers selected from natural, semisynthetic, or synthetic categories, provided that at least one polymer displays both structural and biofunctional activity.

[0161] (a) Saccharide-based polymers: Alginate, pectin, cellulose and its derivatives (carboxymethyl cellulose, hydroxypropyl methylcellulose, methylcellulose), chitosan, starch, xanthan gum, pullulan, agarose, guar gum, carrageenan, gellan gum, dextran.

[0162] (b) Glycosaminoglycans (GAGs): Hyaluronic acid, chondroitin sulfate, heparin.

[0163] (c) Proteins and glycoproteins: Mucin, mucin-like glycoproteins, albumin, collagen, gelatin, elastin, fibrin, silk fibroin, keratin, soy protein, whey protein, casein.

[0164] (d) Synthetic or biodegradable polymers: Polyethylene glycol (PEG), PEG-derivatives, poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinyl alcohol) (PVA), polyurethanes (PU), polyureas (PUra), poly(acrylic acid) (PAA), poly(methacrylic acid) (PMAA), ethylene-vinyl acetate (EVA), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS).

[0165] Mixtures or superpositions of two or more polymeric networks may be formed sequentially or simultaneously, yielding interpenetrating polymeric networks (IPNs) with composite behavior. In all cases, at least one polymer confers dual functionality: it provides the mechanical backbone while contributing biologically or chemically active moieties that modulate the environment.

[0166] A.1.3 Crosslinking Mechanisms

[0167] Crosslinking of the polymeric network (A) may occur through one or more of the following mechanisms:

[0168] • Ionic crosslinking between ionizable functional groups of polysaccharides and multivalent counter-ions capable of coordinating with multiple polymer chains simultaneously.

[0169] In alternative embodiments, bivalent ortrivalent cations such as Ca2+, Ba2+, Mg2+, Zn2+, Al3+, or Fe3+are employed to crosslink polysaccharides containing negatively charged groups, such as carboxylate or sulfate moieties. In a most preferred embodiment, using Ca2+ions supplied from calcium chloride, calcium gluconate, calcium carbonate + gluconolactone (GDL), calcium lactate, calcium sulfate, calcium oxide or similar sources;

[0170] • Photochemical crosslinking by exposure to ultraviolet or visible light in the presence of photoinitiators;

[0171] • Enzymatic crosslinking using oxidases, transglutaminases, or peroxidases;

[0172] • Biologically induced crosslinking: by allowing viable microorganisms to modify or generate polymeric networks through their metabolic activity, including local pH or redox changes, ion release, or secretion of polymeric or crosslinking biomolecules.

[0173] • Redox-initiated crosslinking or field-mediated polymerization; and

[0174] • Physical crosslinking through temperature or pH change, or hydrogen-bond or ionicinteraction formation. The process may be directed spatially by controlling the diffusion of crosslinking ions, the exposure to light or fields, or sequential deposition, to produce gradients or discrete zones of crosslinking density across the material.

[0175] A.1.4 Functional and Degradation Behavior

[0176] The polymeric network (A) provides both structural stability and biological functionality. Its degradability is controlled by:

[0177] • the presence or absence of microorganisms (C) within the polymeric mesh, which may secrete enzymes or metabolites that locally accelerate degradation;

[0178] • the molecular weight and chemical structure of the chosen polymers; and

[0179] • the local crosslinking density and hydration level.

[0180] The degradation profile can vary spatially within the construct — slower in shell regions (111) and faster in internal diffuser regions (112) — allowing controlled release or activation. As it degrades, the network releases both encapsulated agents and its own polymeric fragments, which may exhibit prebiotic, mucoadhesive, or anti-inflammatory activity.

[0181] A.2 Water-Based Suspension (B)

[0182] The water-based suspension (B) is retained by the polymeric network (A) and provides the fluid medium for biological and chemical activity. It is an aqueous solution or dispersion containing electrolytes, nutrients, and optional bioactive compounds.

[0183] Throughout the document this aqueous solution or dispersion will be called Water-Based Suspension.

[0184] A.2.1 Composition

[0185] The aqueous phase may comprise:

[0186] • Water, preferably deionized or sterile;

[0187] • Ionic-balancing molecules such as NaCI, KCI, CaCI2, MgCI2, phosphate, citrate, acetate, carbonate, or other physiological salts;

[0188] • Nutrient sources including sugars, amino acids, peptides, vitamins, and minerals; and

[0189] • Optional additives such as prebiotics, postbiotics, antioxidants, or short-chain fatty acids (butyrate, acetate, propionate). In specific embodiments, the water-based suspension (B) comprises or derives from known microbial culture media, including Luria Bertani (LB), De Man-Rogosa-Sharpe (MRS), Brain Heart Infusion (BHI), or Mueller Hinton (MH) formulations.

[0190] Concentrations of additives or dopants generally range from 0.01 wt % to 10 wt %, adjusted to maintain osmotic balance and diffusion compatibility with the network (A).

[0191] A.2.2 Functional Roles

[0192] The Water-Based Suspension (B) serves as:

[0193] • the hydrating element that maintains the viscoelastic nature of the gel;

[0194] • the diffusion medium for gases and solutes;

[0195] • a nutrient reservoir for microorganisms (C); and

[0196] • a carrier for optional therapeutic, probiotic, or diagnostic agents.

[0197] Its ionic composition and buffering capacity can be tailored to mimic specific environments such as gastrointestinal, cervico-vaginal, respiratory, dermal, or soil microhabitats.

[0198] A.3 Viable Microorganisms (C)

[0199] A.3.1 Nature and Classification

[0200] Microorganisms (C) may be single species, defined consortia, or natural communities. They can belong to the domains Bacteria, Archaea, and Eukaryota (Fungi, Plantae, Chromista, Protista), or Viruses (including bacteriophages), and combinations of those, provided that they are compatible with the matrix environment.

[0201] In certain embodiments, the viable microorganisms (C) comprise microbial consortia that are laboratory-assembled, isolated from environmental or industrial sources, or obtained from donor-derived samples. In preferred embodiments, the donors are human, and the microbial material is derived from mucus, fecal, or cervico-vaginal samples representative of native microbiota.

[0202] In certain embodiments, the viable microorganisms (C) are selected, in single strain or consortia, from the following categories:

[0203] According to native environments for the microorganisms, representative categories include: • Human associated microbiota species: Lactobacillus, Bifidobacterium, Bacteroides, Streptococcus, Akkermansia, Faecalibacteriunr,

[0204] • Industrial and environmental species: Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus niger, Cupriavidus necator,

[0205] • Agricultural or bioremediation species: Pseudomonas fluorescens, Azospirillum brasilense, Rhizobium leguminosarunr, and

[0206] • Viral and phage elements: bacteriophages targeting the above bacterial hosts.

[0207] According to activity, representative categories include:

[0208] • Remodeling and maintaining the structural characteristics of the microenvironments (110) for extended periods of time. These microbial active agents may be defined as: i. Biopolymer producing microorganisms, which are species that contribute to the remodeling of the microenvironment structure by the synthesis and secretion of naturally occurring polymers: exopolysaccharides (EPS), biofilms, or specific structural components like capsules. Microorganisms can be selected from:

[0209] • Bacteria: Pseudomonas aeruginosa, Streptococcus mutans, Bacillus subtilis, Escherichia coli (EPS producing strains), Acetobacter xylinum (cellulose producer), Lactobacillus rhamnosus (exopolysaccharide producer), Klebsiella pneumoniae (capsule-forming), Leuconostoc mesenteroides (dextran-producing), Xanthomonas campestris (xanthan gum), Akkermansia Muciniphila.

[0210] • Fungi: Aspergillus niger, Penicillium chrysogenum, Rhizopus oryzae, Trichoderma reesei, Saccharomyces cerevisiae (yeast producing EPS), Cryptococcus neoformans (capsule-forming).

[0211] • Algae and Cyanobacteria: Chlorella vulgaris (polysaccharide producer), Anabaena variabilis, Nostoc punctiforme, Craspedostauros australis Cox. ii. Microorganisms that are used to degrade the structural components of the microenvironments (110). These microorganisms can be included in the microenvironments (110) with the purpose of degrading the structure and progressively releasing the embedded nutrients forthem to be available to the species of interest. Microorganisms can be selected from:

[0212] • Bacteria: Pseudomonas putida, Sphingomonas paucimobilis, Rhodococcus erythropolis (degrades synthetic polymers), Bacillus licheniformis (protease producer), Clostridium thermocellum (cellulase producer), Bacillus cereus (lipase producer), Acinetobacter baylyi (polymer-degrading).

[0213] • Fungi: Aspergillus fumigatus, Trametes versicolor (lignin and complex polymer degrader), Phanerochaete chrysosporium (ligninase producer), Pleurotus ostreatus (white rot fungus), Candida antarctica (lipase producer), Fusarium oxysporum (cellulase and pectinase producer).

[0214] • Actinobacteria: Streptomyces coelicolor, Streptomyces griseus (chitinase and protease producer), Nocardia asteroides (hydrocarbon degrader).

[0215] • Yeasts: Yarrowia lipolytica (lipid-degrading), Candida tropicalis (hydrocarbon degrader).

[0216] • Providing bioactive cues for the microorganisms that reside in the microenvironment (110). These cues can come from: i. Molecules resulting from the microbial metabolic activity with prebiotic potential. These metabolic products can serve as a nutrient source for another microorganism in a symbiotic interaction. These producer microorganisms can be included in the structure with the purpose of metabolizing the available nutrients within the microenvironments (110) and secrete specific components that allow the growth of different species of microbes. ii. Presence of prebiotics or structural proteins that are available for the nutrition of desired targeted microorganisms to thrive within the microenvironment.

[0217] • The delivery of microorganisms and molecules of biologic interest. i. Different regionally distributed microenvironments (110) of the biomimetic material (100) may encapsulate microorganisms and / or biologically active molecules, each microenvironment being formulated to degrade at a predetermined rate, thereby enabling the programmed and sequential release of the encapsulated contents. ii. Microbiome recovery: The intrinsic components of the microenvironments (110) can be used upon degradation for feeding bacteria cultures and propelling their growth, such as dissolved peptides, sugars or proteins within the structure of the microenvironments (110). iii. Modulation of the behavior of communities of bacteria: Existing prebiotics (precursors for bacteria metabolism) can be included within the different microenvironments of the microenvironments (110) to be released upon degradation of the structure in the recipient of interest.

[0218] According to taxonomic classification, representative categories include: • Bacteria selected from: o Phylum: Actinomycetota

[0219] ■ Streptomycetaceae-. Streptomyces griseus, Streptomyces coelicolor

[0220] ■ Nocardiaceae Rhodococcus erythropolis, Nocardia asteroids

[0221] ■ Propionibacteriaceae Propionibacterium freudenreichii

[0222] ■ Bifidobacteriaceae'. Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium adolescentis, Bifidobacterium infantis, Bifidobacterium breve, Bifidobacterium bifidum o Phylum: Bacteroidota

[0223] ■ Prevotellaceae'. Segatella copri

[0224] ■ Bacteroidaceae Bacteroides acidifaciens, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides fragilis

[0225] ■ Tannerellaceae : Para bacteroides goldsteinii o Phylum: Bacillota

[0226] ■ Oscillospiraceae-. Faecalibacterium prausnitzii, Acetivibrio thermocellus, Ruminococcus

[0227] ■ Clostridiaceae-. Clostridium sporogenes, Clostridium butyricum

[0228] ■ Christensenellaceae-. Christensenella minuta

[0229] ■ Anoxybacillaceae-. Geobacillus stearothermophilus, Paenibacillus polymyxa

[0230] ■ Bacillaceae-. Bacillus megaterium, Bacillus coagulans, Bacillus subtilis, Bacillus licheniformis, Bacillus cereus

[0231] ■ Enterococcaceae’. Enterococcus faecium

[0232] • Hafniaceae Hafnia alvei

[0233] ■ Lachnospiraceae Anaerobutyricum hallii

[0234] • Streptococcaceae Lactococcus lactis, Streptococcus mutans, Streptococcus salivarius, Streptococcus thermophilus

[0235] ■ Lactobacillaceae-. Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum, Lactiplantibacillus plantarum, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus salivarius, Lacticaseibacillus paracasei, Lacticaseibacillus casei, Limosilactobacillus reuteri, Limosilactobacillus fermentum, Limosilactobacillus oris, Leuconostoc mesenteroides o Phylum: Verrucomicrobiota ■ Akkermansiaceae-. Akkermansia muciniphila o Phylum: Pseudomonadota

[0236] ■ Acetobacteraceae’. Komagataeibacter xylinus

[0237] ■ Sphingomonadaceae'. Sphingomonas paucimobilis

[0238] ■ Paracoccaceae’. Cereibacter sphaeroides

[0239] ■ Methylobacteriaceae-. Methylorubrum extorquens

[0240] ■ Moraxellaceae Acinetobacter baylyi

[0241] ■ Yersiniaceae Serratia marcescens

[0242] ■ Enterobacteriaceae'. Klebsiella pneumoniae, Escherichia coli, Escherichia coli Nissle 1917

[0243] ■ Lysobacteraceae Xanthomonas campestris

[0244] ■ Pseudomonadaceae'. Azotobacter vinelandii, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa

[0245] • Fungi selected from: o Phylum: Ascomycota

[0246] ■ Dipodascomycetes'. Yarrowia lipolytica

[0247] ■ Dothideomycetes'. Alternaria alternata

[0248] ■ Pichiomycetes'. Candida tropicalis, Komagataella pastoris, Candida albicans

[0249] ■ Sordariomycetes'. Beauveria bassiana, Trichoderma reesei, Trichoderma harzianum, Fusarium venenatum, Fusarium oxysporum, Neurospora crassa

[0250] ■ Eurotiomycetes'. Monascus purpureus, Penicillium roqueforti, Penicillium chrysogenum, Aspergillus fumigatus, Aspergillus terreus, Aspergillus oryzae, Aspergillus niger

[0251] ■ Saccharomycetes'. Saccharomyces cerevisiae, Saccharomyces cerevisiae var boulardii, Nakaseomyces glabratus, Cyberlindnera jadinii o Phylum: Basidiomycota

[0252] ■ Ustilaginomycetes-. Moesziomyces antarcticus

[0253] ■ Agaricomycetes'. Phanerodontia chrysosporium, Phanerochaete sordida, Trametes versicolor, Pleurotus ostreatus

[0254] ■ Tremellomycetes'. Cryptococcus neoformans o Phylum: Mucoromycota

[0255] ■ Mucoromycetes'. Mucor circinelloides, Rhizopus arrhizus, Rhizopus stolonifer • Virus (and bacteriophages) selected from: o Phylum: Duplodnaviria

[0256] ■ Caudoviricetes'. Escherichia phage Lambda, Salmonella phage P22, Mycobacterium phage D29, Escherichia phage T4 o Phylum: Riboviria

[0257] ■ Vidaverviricetes'. Pseudomonas phage phi6

[0258] ■ Leviviricetes'. Escherichia phage MS2 o Phylum: Monodnaviria

[0259] ■ Faserviricetes'. Escherichia phage M13 o Phylum: Varidnaviria

[0260] ■ Tectiliviricetes

[0261] ■ Enterobacteria phage PRD1 o Phylum Unclassified:

[0262] ■ Phage phiX174

[0263] In certain embodiments, the viable microorganisms are selected from fastidious (hard to culture) microorganisms such as anaerobic syntrophes. In preferred embodiments selected from the set: Campylobacter, Faecalibacterium prausnitzii, Akkermansia muciniphila, Bacteroides, and Bifidobacterium.

[0264] Microorganisms may be included as active viable entities, as spores, or as inactivated or lysed components when the functional role is limited to enzymatic or structural contribution.

[0265] A.3.2 Spatial Distribution and Function

[0266] Microorganisms may be:

[0267] • uniformly dispersed within the aqueous phase (B);

[0268] • immobilized or adhered to the polymeric network (A);

[0269] • selectively located within specific microenvironment types (e.g., 112-H, 112-N, 112- B); or

[0270] • selectively located within specific regional distributions of microenvironments (110) In preferred embodiments, their metabolically quiescent (lag phase), active (log phase), or dormant are controlled by:

[0271] • Their concentration and localization within each microenvironment (110)

[0272] • Their communication / interaction with contiguous / adjacent microenvironments (100)

[0273] • Their interaction with the application site when they reach the target upon use.

[0274] The local environment — controlled by crosslinking density, nutrient level, and oxygen availability — allows reproducible control of microbial phase transitions.

[0275] A.4 Additives, Dopants, and Functional Modifiers

[0276] The biomimetic material (100) may further contain additives or dopants that adjust mechanical, chemical, or biological performance, including:

[0277] • Mucin or mucin-like glycoproteins (0.1-10 wt %), to modulate rheology, adhesion, and degradation kinetics;

[0278] • Proteins or peptides, such as albumin, collagen fragments, lectins, integrins, cadherins, selectins, antibodies, fibronectin, glutathione, elastin, or lysozyme, to influence biocompatibility and enzymatic activity;

[0279] • Prebiotic or postbiotic compounds, including fructooligosaccharides, galactooligosaccharides, polyphenols, lactulose, lactosucrose, isomaltooligosaccharides, soybean oligosaccharides, xylo-oligosaccharides, gentio- oligosaccharides, human milk oligosaccharides, arabinogalacto-oligosaccharides, arabino-oligosaccharides, rhamnogalacturono-oligosaccharides, arabinoxylooligosaccharides, galacturono-oligosaccharides, pectin, lacto-N-neotetraose, resistant starch, gum arabic, guar gum, hemicellulose, lactose, maltodextrins, cocoa-derived flavonols, resveratrol, flavanols, procyanidins, flavanones, 3-O-methylgallate, gallocatechin gallate, epigallocatechin, theaflavins, theasinensis, thearubigins, quercetin, sinapic acid, chlorogenic acid, ferulic acid, kaempferol, coumaric acid, hydroxycinnamates acid, proanthocyanidins, tannins, catechins, cyanidin, delphinidin, hydroxybenzoic acid, hydroxycinnamic acid, stilbenes, rutin, ellagitannins, proanthocyanidins, myricetin, naringenin, hesperidin, isoflavones, genistein, daidzein, glycitein, anthocyanins, lignans, secoisolariciresinol, ammoresinol, ostruthin, red fruit extracts, pomegranate extract, green tea, black tea, oolong tea, grape pomace, ellagic acid, gallic acid, mangiferin, jabuticaba, castalagin, vescalagin, procyanidin A, saponins of ginseng, red ginseng, notoginseng, Gynostemma pentaphyllum, sulforaphanes, iberin, inulin, carrot, resistant dextrin, SCFAs, acetate, butyrate, propionate, polyamines, spermidine, putrescine, spermine, histamine, tyramine, cadaverine, bacteriocins (classes I, II, Ila, lib, lie, lid, III, IV), polypetides, bacitracin, nisin, reuterinm, poly-unsaturated fatty acids (PUFAs), arachidonic acid, docosahexaenoic acid, linoleic acids, linolenic acids, vitamin K, vitamin B, biotin, cobalamin, folate, niacin, pantothenic acid, pyridoxine, riboflavin, thiamine, nicotinic acid, panthothenic acid, eicosapentanoic acid, minerals, omega-3 PUFAs, omega-6 PUFAs, cell wall fragments, bacterial lipoteichoic acid, bacterial lysates, peptidoglycans, exopolysaccharides, cell-free supernatants, capsular polysaccharides, extracellular vesicles, polysaccharide A, bile acids, deoxycholic acid, lithocholic acid, taurine, tryptophan metabolites / indole derivatives (indol-3-aldehyde, indol-3-acetic acid, indolelactic acid, indolpropyl acid, indolacrylic acid), trimethylamine, trimethylamine N-oxide, triptamine, p-cresol, ammonia, hydrogen sulfide, serotonin, dopamine precursors, gamma-aminobutyric acid (GABA), branched-chain amino acids, serine-threonine peptides (STp), sactipeptides, adenosine triphosphate (ATP);

[0280] • Nanofillers or reinforcing agents, such as silica, fumed silica, cellulose nanofibers, metal oxide nanomaterials, carbon nanotubes, small-walled carbon nanotubes, multiwalled carbon nanotubes, carbon black, clay-based nanofillers, quantum dots, boron nitrate, zeolite, kaolinite, bentonite, nanosilica, nanosilicate, nanoclay, nanoalumina, cellulose nanocrystals, metallic nanoparticles (e.g. silver, gold, calcium, iron, cobalt, nickel, and their oxides), montmorillonite, bioceramics, graphene derivatives, functionalized graphene, graphite, silicate, coral, nacre, bisphosphonate- functionalized materials, nanoscale modified titanium, magnesium-based alloys;

[0281] • Optional therapeutic or diagnostic molecules, such as anti-inflammatory agents, antimicrobials, antimicrobial peptides, caffein, aldesulfone sodium, amikacin, aminosalicylate calcium, amoxicillin, amoxicillin-clavulanic acid, ampicillin, ampicillinsulbactam, arbekacin, astromicin, azithromycin, azlocillin, aztreonam, bacitracin, bedaquiline, bekanamycin, besifloxacin, bicozamycin, bipyridinium, brodimoprim, carbadox, carumonam, cefacetrile, cefaclor, cefadroxil, cefalexin, cefalorium, cefaloridine, cefalotin, cefamandole, cefapirin, cefatrizine, cefazedone, cefazolin, cefbuperazone, cefcapene, cefdinir, cefditoren, cefepime, cefetamet, cefixime, cefmenoxime, cefodizime, cefoperazone, cefoperazone-sulbactam, cefoselis, cefotaxime, cefotiam, cefoxitin, cefpirome, cefpiramide, cefpodoxime, cefprozil, cefquinome, cefradine, cefroxadine, ceftazidime, ceftazidime-avibactam, cefteram pivoxil, ceftibuten, ceftiofur, ceftizoxime, ceftolozane, ceftolozane-tazobactam, ceftriaxone, ceftriaxone-sulbactam, ceftaroline, ceftobiprole, cefuroxime, chloramphenicol, chlorhexidine, clindamycin, clomocycline, cloxacillin, clofazimine, colistin, demeclocycline, delafloxacin, daptomycin, dirithromycin, doxycycline, enramycin, enrofloxacin, enoxacin, ertapenem, ethambutol, ethionamide, fidaxomicin, flurithromycin, florfenicol, fleroxacin, fluconazole, flumequine, fusidic acid, gamithromycin, garenoxacin, gatifloxacin, gemifloxacin, gentamicin, isepamicin, josamycin, kanamycin, kitasamycin, laidlomycin, lasalocid, levofloxacin, levonadifloxacin, lincosamides, lomefloxacin, lymecycline, maduramicin, meropenem, midecamycin, minocycline, monensin, moenomycin, moxifloxacin, mupirocin, netilmicin, nitarsone, nitrofural, nitrofuran, nedaplatin, neomycin, nemonoxacin, ofloxacin, oleandomycin, oxolinic acid, oxytetracycline, paromomycin, pazufloxacin, pefloxacin, phenoxymethylpenicillin, pirlimycin, pivampicillin, pipemidic acid, piperacillin, piperacillin-tazobactam, pradofloxacin, prulifloxacin, procaine benzylpenicillin, quinolamine, quinazolindiamine, rifabutin, rifampin, rifapentine, rifaximin, rokitamycin, rosoxacin, roxithromycin, salicylanilide, salinomycin, semduramicin, sitafloxacin, sisomicin, spectinomycin, streptoduocin, streptomycin, sulfadiazine, sulfadimethoxine, sulfadimidine, sulfafurazole, sulfaguanidin, sulfaisodimidine, sulfalene, sulfamazone, sulfamerazine, sulfamethazine, sulfamethizole, sulfamethoxazole, sulfamethoxypyridazine, sulfametomidine, sulfametoxydiazine, sulfametrole, sulfamoxole, sulfanilamide, sulfaperin, sulfaphenazole, sulfapyridine, sulfaquinoxaline, sulfathiazole, sulfathiourea, talampicillin, tazemetostat, telithromycin, temocillin, tetracycline, tiamulin, tildipirosin, tigecycline, tilmicosin, tinidazole, tobramycin, tosufloxacin, tylosin, tylvalosin, valnemulin, vancomycin, anticancer, 2-hydroxycinnaldehyde, 2- benzoyloxycinnamaldehyde, doxorubicin, temozolomide, cyclophosphamide, methotrexate, bleomycin, mitomycin, vinblastine, vincristine, paclitaxel, daratumumab, nivolumab, osimertinib, curcumin, epigallocatechin gallate, genistein, Abemaciclib, Abiraterone, ABVD, AC, Acalabrutinib, Actinomycin D, Afatinib, Aflibercept, FOLFIRI (folinic acid, fluorouracil, irinotecan), Alectinib, Alpelisib, Fulvestrant, Anastrozole, Apalutamide, Ara-C (Cytarabine), Arsenic trioxide, Tretinoin, Asciminib, Asparaginase, Atezolizumab, Bevacizumab, Nab-paclitaxel, Avelumab, Axitinib, Azacitidine, BEACOPDac, BEACOPP, BEAM, Belzutifan, Bendamustine, Bevacizumab, Bexarotene, Bicalutamide, Bleomycin, Etoposide, Platinum, Blinatumomab, Bortezomib, Cyclophosphamide, Dexamethasone, Bosutinib, Brentuximab, Brigatinib, Buserelin, Busulfan, CAPE-OX, CAPOX, CAPTEM, CAV, CCNU, CHOP, CPX-351 , Cabazitaxel, Cabozantinib, Capecitabine, Carboplatin, Etoposide, Paclitaxel, Carfilzomib, Carmustine, Cemiplimab, Ceritinib, Cetuximab, Chlorambucil, Cisplatin, Fluorouracil, Ifosfamide, Trastuzumab, Cladribine, Glofitamab, Crizotinib, Cyclophosphamide, Thalidomide, Dexamethasone, Cyproterone acetate, Cytarabine, Dacarbazine, Dabrafenib, Trametinib, Dacomitinib, Dactinomycin, Daratumumab, Lenalidomide, Darolutamide, Dasatinib, Daunorubicin, Midostaurin, Degarelix, Denosumab, Dexamethasone, Docetaxel, Nintedanib, Cisplatin, Fluorouracil, Doxorubicin, Ifosfamide, Durvalumab, Elacestrant, Encorafenib, Binimetinib, Entrectinib, Enzalutamide, Epcoritamab, Epirubicin, Oxaliplatin, Eribulin, Erlotinib, Etoposide, Everolimus, Exemestane, FLAG, FLAG-lda, FOLFIRINOX, FOLFOX, FOLFOXIRI, Faslodex (Fulvestrant), Fludarabine, Cyclophosphamide, Rituximab, Fluorouracil, Mitomycin C, Docetaxel, Flutamide, Folinic acid, Irinotecan, Fotivda (Tivozanib), Futibatinib, Gefitinib, Gemcitabine, Capecitabine, Cisplatin, Paclitaxel, Daunorubicin, Gemtuzumab ozogamicin, Gilteritinib, Glofitamab, Goserelin, Hydroxycarbamide, Hydrocortisone, Ibandronic acid, Ibrutinib, Idarubicin, Ifosfamide, Imatinib, Imiquimod, Inotuzumab ozogamicin, Ipilimumab, Nivolumab, Irinotecan, Ivosidenib, Azacitidine, Ixazomib, Lanreotide, Larotrectinib, Lenalidomide, Lenvatinib, Letrozole, Leuprorelin, Lomustine, Loncastuximab tesirine, Lorlatinib, Lutrate (Triptorelin), Medroxyprogesterone acetate, Megestrol acetate, Melphalan, Mercaptopurine, Methotrexate, Cytarabine, Thiotepa, Rituximab, Methylprednisolone, Midostaurin, Mitomycin C, Mitotane, Mitoxantrone, Mobocertinib, Morphine, Nab-paclitaxel, Nelarabine, Neratinib, Nilotinib, Niraparib, Nivolumab, Relatlimab, Obinutuzumab, Octreotide, Olaparib, Osimertinib, Oxaliplatin, Capecitabine, Paclitaxel, Carboplatin, Palbociclib, Pamidronate disodium, Panitumumab, Panobinostat, Pazopanib, Peginterferon alfa-2a, Pegylated liposomal doxorubicin, Pembrolizumab, Pemetrexed, Carboplatin, Cisplatin, Pemigatinib, Pentostatin, Pertuzumab, Polatuzumab vedotin, Bendamustine, Rituximab, Pomalidomide, Dexamethasone, Ponatinib, Prednisolone, Procarbazine, Lomustine, Vincristine, R-CHOP, R-CVP, R-DHAP, R-ESHAP, R-GDP, R-ICE, Raloxifene, Raltitrexed, Regorafenib, Relugolix, Ribociclib, Risedronate sodium, Rituximab, Rucaparib, Ruxolitinib, Sacituzumab govitecan, Selpercatinib, Sorafenib, Sotorasib, Streptozocin, Sunitinib, Talazoparib, Talimogene laherparepvec, Tamoxifen, Tebentafusp, Teclistamab, Temozolomide, Tepotinib, Thiotepa, Tivozanib, Topotecan, Trabectedin, Trastuzumab, Pertuzumab, Trastuzumab deruxtecan, Trastuzumab emtansine, Treosulfan, Trifluridine, Tipiracil, Triptorelin, Tucatinib, Vemurafenib, Venetoclax, Vinblastine, Vincristine, Vinorelbine, Vismodegib, Vorinostat, Votrient (Pazopanib), Vyxeos (Liposomal daunorubicin, Cytarabine), Zanubrutinib, Zoledronic acid, Zydelig (Idelalisib), Zynlonta (Loncastuximab tesirine), Abecma (Idecabtagene Vicleucel), Abiraterone Acetate, Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVE, ABVE-PC, AC-T, Actemra (Tocilizumab), Adagrasib, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adstiladrin (Nadofaragene Firadenovec-vncg), Afamitresgene Autoleucel, Afinitor (Everolimus), Akeega (Niraparib Tosylate Monohydrate and Abiraterone Acetate), Akynzeo (Netupitant and Palonosetron Hydrochloride), Aldesleukin, Alemtuzumab, Alimta (Pemetrexed Disodium), Alkeran for Injection (Melphalan Hydrochloride), Alkeran Tablets (Melphalan), Aloxi (Palonosetron Hydrochloride), Alunbrig (Brigatinib), Alymsys (Bevacizumab), Ameluz (Aminolevulinic Acid Hydrochloride), Amifostine, Aminolevulinic Acid Hydrochloride, Amivantamab-vmjw, Amtagvi (Lifileucel), Anktiva (Nogapendekin Alfa Inbakicept-pmln), Aprepitant, Aranesp (Darbepoetin Alfa), Arzerra (Ofatumumab), Asparaginase Erwinia Chrysanthemi, Asparaginase Erwinia Chrysanthemi (Recombinant)-rywn, Asparlas (Calaspargase Pegol-mknl), Atezolizumab and Hyaluronidase-tqjs, Aucatzyl (Obecabtagene Autoleucel), Augtyro (Repotrectinib), Avapritinib, Azedra (lobenguane I 131), Balversa (Erdafitinib), Beleodaq (Belinostat), BEP, Besremi (Ropeginterferon Alfa-2b-njft), Binimetinib, Bizengri (Zenocutuzumab-zbco), Bleomycin Sulfate, Brexucabtagene Autoleucel, Breyanzi (Lisocabtagene Maraleucel), Brukinsa (Zanubrutinib), BuMel, Busulfex (Busulfan), Cablivi (Caplacizumab-yhdp), Cabozantinib-S-Malate, CAF, Calaspargase Pegol-mknl, Camcevi (Leuprolide Mesylate), Campath (Alemtuzumab), Camptosar (Irinotecan Hydrochloride), Capivasertib, Caplacizumab-yhdp, Capmatinib Hydrochloride, CARBOPLATIN- TAXOL, Carmustine Implant, Carvykti (Ciltacabtagene Autoleucel), CEM, Cemiplimab-rwlc, Cervarix (Recombinant HPV Bivalent Vaccine), CHLORAMBUCILPREDNISONE, Ciltacabtagene Autoleucel, Clofarabine, CMF, Cobimetinib Fumarate, Columvi (Glofitamab-gxbm), Cometriq (Cabozantinib-S-Malate), COPDAC, Copiktra (Duvelisib), COPP, COPP-ABV, Cosibelimab-ipdl, Cotellic (Cobimetinib Fumarate), Cyramza (Ramucirumab), Dacarbazine, Dacogen (Decitabine), Danyelza (Naxitamab- gqgk), Daratumumab and Hyaluronidase-fihj, Daurismo (Glasdegib Maleate), Decitabine, Decitabine and Cedazuridine, Defibrotide Sodium, Denileukin Diftitox- cxdl, Dexrazoxane Hydrochloride, Dinutuximab, Doxorubicin Hydrochloride Liposome, Duvelisib, Eflornithine Hydrochloride, Elahere (Mirvetuximab Soravtansine-gynx), Elotuzumab, Elranatamab-bcmm, Elzonris (Tagraxofusp-erzs), Emapalumab-lzsg, Enasidenib Mesylate, Enfortumab Vedotin-ejfv, Ensartinib Hydrochloride, Epcoritamab-bysp, Epoetin Alfa, Erdafitinib, Etoposide Phosphate, Etopophos (Etoposide Phosphate), Evista (Raloxifene Hydrochloride), Exemestane, Fam- Trastuzumab Deruxtecan-nxki, Fedratinib Hydrochloride, Filgrastim, Fostamatinib Disodium, Fruquintinib, Fulphila (Pegfilgrastim), Fyarro (Sirolimus Protein-Bound Particles), Gavreto (Pralsetinib), Glasdegib Maleate, Gliadel Wafer (Carmustine Implant), Glucarpidase, Granisetron, Halaven (Eribulin Mesylate), Hepzato (Melphalan Hydrochloride), Hycamtin (Topotecan Hydrochloride), Hyper-CVAD, Ibritumomab Tiuxetan, Iclusig (Ponatinib Hydrochloride), Idecabtagene Vicleucel, Idelalisib, Ifex (Ifosfamide), IL-2 (Aldesleukin), Imdelltra (Tarlatamab-dlle), Imetelstat Sodium, Imjudo (Tremelimumab-actl), Inavolisib, Inotuzumab Ozogamicin, Inqovi (Decitabine and Cedazuridine), Interferon Alfa-2b, lobenguane I 131 , Irinotecan Sucrosofate, Isatuximab-irfc, Ixabepilone, Jakafi (Ruxolitinib Phosphate), Jaypirca (Pirtobrutinib), Jelmyto (Mitomycin), Jemperli (Dostarlimab-gxly), Kimmtrak (Tebentafusp-tebn), Kisqali Femara Co-Pack (Ribociclib Succinate and Letrozole), Koselugo (Selumetinib Sulfate), Krazati (Adagrasib), Kymriah (Tisagenlecleucel), Lazertinib Mesylate Hydrate, Lifileucel, Lisocabtagene Maraleucel, Loqtorzi (Toripalimab-tpzi), Lurbinectedin, Luspatercept-aamt, Lutetium Lu 177 Vipivotide Tetraxetan, Margenza (Margetuximab-cmkb), Margetuximab-cmkb, Mechlorethamine Hydrochloride, Mesna, Methylnaltrexone Bromide, Mirvetuximab Soravtansine-gynx, Mogamulizumab-kpkc, Momelotinib Dihydrochloride Monohydrate, Mosunetuzumab- axgb, Nadofaragene Firadenovec-vncg, Naxitamab-gqgk, Necitumumab, Neratinib Maleate, Netupitant and Palonosetron Hydrochloride, Nirogacestat Hydrobromide, Nogapendekin Alfa Inbakicept-pmln, Obecabtagene Autoleucel, Ofatumumab, Olutasidenib, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ondansetron Hydrochloride, Onivyde (Irinotecan Sucrosofate), Onureg (Azacitidine), Opdualag (Nivolumab and Relatlimab-rmbw), Orserdu (Elacestrant Dihydrochloride), Osimertinib Mesylate, Paclitaxel Albumin-stabilized Nanoparticle Formulation, Pacritinib Citrate, Padcev (Enfortumab Vedotin-ejfv), Palifermin, Palonosetron Hydrochloride, Pamidronate Disodium, Pexidartinib Hydrochloride, Pirtobrutinib, Pluvicto (Lutetium Lu 177 Vipivotide Tetraxetan), Polatuzumab Vedotin-piiq, Pralatrexate, Pralsetinib, Prednisone, Propranolol Hydrochloride, Provenge (Sipuleucel-T), Plerixafor, Qinlock (Ripretinib), Quizartinib Dihydrochloride, Radium 223 Dichloride, Ramucirumab, Rasburicase, Ravulizumab-cwvz, RebLozyl (Luspatercept-aamt), Relistor (Methylnaltrexone Bromide), Repotrectinib, Retifanlimab-dlwr, Revumenib Citrate, Ripretinib, Rituximab and Hyaluronidase Human, Rolapitant Hydrochloride, Romidepsin, Romiplostim, Ropeginterferon Alfa- 2b-njft, Rubraca (Rucaparib Camsylate), Rucaparib Camsylate, Rydapt (Midostaurin), Rytelo (Imetelstat Sodium), Sacituzumab Govitecan-hziy, Sarclisa (Isatuximab-irfc), Sclerosol Intrapleural Aerosol (Talc), Selinexor, Selumetinib Sulfate, Siltuximab, Sipuleucel-T, Sirolimus Protein-Bound Particles, Sonidegib, Sunitinib Malate, Sylvant (Siltuximab), Tabrecta (Capmatinib Hydrochloride), Tafasitamab-cxix, Tagraxofusp- erzs, Talquetamab-tgvs, Talvey (Talquetamab-tgvs), Talzenna (Talazoparib Tosylate), Tarlatamab-dlle, Tasigna (Nilotinib), Tazemetostat Hydrobromide, Tazverik (Tazemetostat Hydrobromide), Tecartus (Brexucabtagene Autoleucel), Teclistamab- cqyv, Tecvayli (Teclistamab-cqyv), Temsirolimus, Tepmetko (Tepotinib Hydrochloride), Tisagenlecleucel, Tisotumab Vedotin-tftv, Tivdak (Tisotumab Vedotin- tftv), Toremifene, Toripalimab-tpzi, Tovorafenib, TPF, Trametinib Dimethyl Sulfoxide, Trastuzumab and Hyaluronidase-oysk, Tremelimumab-actl, Trifluridine and Tipiracil Hydrochloride, Triptorelin Pamoate, Truqap (Capivasertib), Turalio (Pexidartinib Hydrochloride), Uridine Triacetate, Valrubicin, Vandetanib, Vanflyta (Quizartinib Dihydrochloride), Varubi (Rolapitant Hydrochloride), Vimseltinib Dihydrate, Vinblastine Sulfate, Vincristine Sulfate, Vinorelbine Tartrate, Vorasidenib Citrate, Votrient (Pazopanib Hydrochloride), Vyloy (Zolbetuximab-clzb), Xatmep (Methotrexate Sodium), Xeloda (Capecitabine), XELIRI, XELOX, Xofigo (Radium 223 Dichloride), Xospata (Gilteritinib Fumarate), Xpovio (Selinexor), Yescarta (Axicabtagene Ciloleucel), Yonsa (Abiraterone Acetate), Zaltrap (Ziv-Aflibercept), Zanidatamab-hrii, Zejula (Niraparib Tosylate Monohydrate), Zenocutuzumab-zbco, Zepzelca (Lurbinectedin), Zevalin (Ibritumomab Tiuxetan), Ziextenzo (Pegfilgrastim), Ziihera (Zanidatamab-hrii), Zolbetuximab-clzb, Zometa (Zoledronic Acid), Zyclara (Imiquimod), Zynyz (Retifanlimab-dlwr), vitamins (retinol, thiamin, riboflavin, niacin, pantothenic acid, vitamin B6, biotin, folate, folic acid, vitamin B12, ascorbic acid, vitamin D, vitamin E, vitamin K, calcium, iodine, iron, beta-carotene, chromium, copper, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, zinc, sodium chloride, small proteins, streptokinase, DNase, tissue plasminogen activator, recombinant human insulin , erythropoetin, follicle-stimulating hormone, blood factors, interferons, interleukins, growth hormone, insulin-like growth factor, therapeutic monoclonal antibodies, pramlintide acetate, pegvisoman, mecasermin, protein C concentrate, alphal -proteinase inhibitor, filgrastim, sargramostim36, 37, oprelvekin, human chorionic gonadotropin, lutropin-alpha, interleukin-2, denileukin diftitox, interferon alfacon 1 , interferon-a2a, interferon-a2b, interferon-an3, interferon- pia, interferon-pi b, interferon-y1 b, salmon calcitonin, teriparatide, exenatide, octreotide, dibotermin-a, recombinant human bone morphogenic protein 7, histrelin acetate, palifermin, becaplermin, nesiritide, lepirudin, anakinra, enfuvirtide, [3- glucocerebrosidase, alglucosidase-a, laronidase, idursulfase, galsulfase, agalsidase- P, lactase, pancreatic enzymes, adenosine deaminase, tissue plasminogen activator, factor Vila, drotecogin-a, trypsin, botulinum toxin type A, botulinum toxin type B, collagenase, human deoxyribonuclease I, hyaluronidase, papain, L-asparaginase, rasburicase, antithrombin III, RNA and DNA, dyes, fluorescein, crystal violet, methylene blue, fuchsin, propidium iodide, malachite green, rhodamine, tetramethylrosamine, acid orange, LPS-selective fluorescent probes, ethidium bromide, dUTP-conjugated probes, DAPI (4',6-diamidino-2-phenylindole), 7-AAD (7- aminoactinomycin D), Hoechst 33258 (33342, 34580), YOYO-1 / DiYO-1 / TOTO- 1 / DiTO-1 , acridine orange, auramine-rhodamine, auramine O, calcofluor white, fluorescein isothiocyanate (FITC), Alexa fluor dyes, BODIPY dyes, cyanine dyes, SYBR green, Fura-2, styryl-based dyes, DiOC6(3), fluorescent ceramide analogs, sodium-binding benzofurzanisophthalate, potassium-binding benzofurzanisophthalate, nile red, nile blue, mCherry, or imaging markers.

[0282] A.5 Design Parameters and Physical-Chemical Ranges

[0283] Key physicochemical parameters of the biomimetic material (100) include:

[0284] These parameters can be varied spatially within a construct to reproduce natural gradients or to create specific functional zones.

[0285] Degradation Rule

[0286] In certain embodiments, the biomimetic material follows a Programmed Self-Functional Degradation Rule, wherein degradation proceeds according to a biologically programmed sequence rather than through passive enzymatic or pH-mediated hydrolysis.

[0287] The degradation process is initiated and sustained by microbial metabolization of specific components of the biomimetic material. The rate and extent of degradation are dynamically linked to the metabolic activity of the microorganisms associated with or encapsulated within the material. When in presence of a complex community of microbiota capable of metabolizing the components of the biomimetic material (e.g Akkermancia muciniphila as mucin-degrader bacteria, or Lactobacilli alginate or pectin metabolizers), the biomimetic material structural components will be fully degraded after 72h of inoculation and incubation. In sterile conditions such same formulations maintain structural integrity for up to 6 months at room temperature.

[0288] Pursuant to the Degradation Rule, the kinetics of degradation are predetermined such that the degradation products act as prebiotic agents, stimulating microbial metabolism and thereby feedback-regulating the degradation process — either accelerating or modulating it in response to biological activity.

[0289] The Degradation Rule further governs the controlled and sequential release of:

[0290] (i) the material’s own functional polymeric components, and

[0291] (ii) encapsulated microorganisms and / or active agents, in a temporally and spatially regulated manner.

[0292] In this way, the material’s degradation and functional output are self-regulated through the biological activity of the microorganisms, establishing a closed-loop degradation-activation cycle within the biomimetic system.

[0293] A.6 Interdependence of Components

[0294] The components (A), (B), and (C) act synergistically:

[0295] • The polymeric network (A) provides the framework that retains (B) and protects (C).

[0296] • The aqueous phase (B) maintains hydration and facilitates diffusion.

[0297] • The microorganisms (C) can locally alter (A) through enzymatic degradation or metabolite production, thereby self-modifying the microenvironment.

[0298] This interdependence results in a dynamic and responsive material whose structure and function co-evolve over time and can be tailored for specific research, industrial, or therapeutic outcomes. B. STRUCTURAL AND PHYSICO-CHEMICAL FEATURES

[0299] The biomimetic material (100) of the invention is distinguished from conventional hydrogels by the presence of a three-dimensional spatial organization of biomimetic microenvironments (110).

[0300] Each microenvironment represents a discrete or semi-continuous domain within the material and collectively they define the regionally distributed microenvironments (RDMEs) that impart heterogeneous yet programmable properties across the construct.

[0301] The architecture of the material is controlled through the method of creating a superposition of polymeric networks by directed crosslinking, achieving the arrangement of shell and diffuser regions, and the establishment of controlled physicochemical parameters such as hydration, crosslinking, oxygen, and nutrients, which can be presented in gradients.

[0302] B.1 Regional Distribution of Microenvironments

[0303] In a preferred embodiment, the Macrostructured Construct (300) is composed of a 3D distribution of two or more different types of biomimetic microenvironments (110).

[0304] In a presentation of the invention, the distribution of the biomimetic microenvironments (110) is homogeneous through the Macrostructured Construct (300).

[0305] In a presentation of the invention, the distribution of the biomimetic microenvironments (110) is a regional distribution through the Macrostructured Construct (300).

[0306] B.1.1 Spatial Organization

[0307] The microenvironments (110) are arranged within the bulk of the biomimetic material in discrete geometrical or geographical regions that may be configured according to functional purpose.

[0308] The regional distribution can assume any of the following patterns:

[0309] • External or internal (microenvironments positioned near the surface or within the interior core);

[0310] • Symmetric or asymmetric (homogeneous or anisotropic across axes);

[0311] • Radial or concentric, forming multilayer shells around a central nucleus;

[0312] • Clustered, forming islands of localized enrichment; • Atomized, where small microdomains are heterogeneously distributed throughout a continuous matrix;

[0313] • Hemispherical or gradient hemispheres for directional diffusion; or

[0314] • Combinations of any of the above.

[0315] Each region may be composed of a distinct subtype of microenvironment (e.g., 112-H, 112- N, 112-B), thereby creating a functional landscape inside the construct that defines its mechanical, biological, and diffusional behavior.

[0316] The dimensions of individual microenvironments are not limited to a fixed scale and may vary continuously; what defines them is the persistence of distinct physicochemical parameters rather than absolute size.

[0317] B.1.2 Functional Logic of RDMEs

[0318] Every RDME is tailored for a specific task. In different embodiments of the invention, the biomimetic microenvironments (110) are configured as a shell (111) or as a diffuser (112) type of microenvironments for diffusion and degradation control, where:

[0319] • Shell (111) regions (R) - dense or crosslink-rich zones acting as protective or diffusionlimiting barriers.

[0320] • Diffuser (112) regions (R) - porous zones enabling exchange of solutes, gases, and metabolites. Within Diffuser (112) regions (R), specialized subtypes exist for controlling the metabolic activity of selected microorganisms. These selected microorganisms can be in single strain, consortia, or from environmental or from donor samples. These selected microorganisms may be included within the material as Optionally viable microorganisms (C), or may be present in the local ecosystems where the biomimetic material (100) is deposited, or may be deposited in / on the biomimetic material for further use. The diffuser types of microenvironment (112) are:

[0321] • Bio-holder (112-H): Limits the metabolic activity of selected microorganisms.

[0322] In certain embodiments, the Bio-holder (112-H) is configured to limit the metabolic activity of selected microorganisms.

[0323] In some embodiments, the Bio-holder (112-H) maintains the microorganisms in a lag phase or quiescent state by restricting nutrient availability to an AUG 0-72 value of about 2 to 3 mg mL-1, and by maintaining a water content of less than about 15%, without supplementation with bioactive molecules.

[0324] In other embodiments, the Bio-holder (112-H) maintains the microorganisms in a logarithmic growth phase through the prior degradation of regionally distributed microenvironments, thereby consuming all available nutrients.

[0325] In preferred embodiments, when provided in a water-based suspension (B), the Bioholder (112-H) further comprises one or more environmental stabilizers selected from the group consisting of glycerol, trehalose, L-cysteine, and combinations thereof.

[0326] • Bio-neutral (112-N): Sustains microorganisms under near-physiological conditions.

[0327] In certain embodiments, the Bio-neutral (112-N) sustains microorganisms under near- physiological conditions similar to those present in their natural environment by providing nutrient availability corresponding to an AUG 0-72 value of about 15 to 20 mg mL-1, and a water content of about 15% to 30%.

[0328] • Bio-booster (112-B): Boost the metabolic activity of selected microorganisms. Promotes growth and metabolic activation through higher nutrient and oxygen availability.

[0329] In certain embodiments, the Bio-booster (112-B) enhances or boosts the metabolic activity of selected microorganisms by increasing nutrient and oxygen availability. In some embodiments, the Bio-booster (112-B) accelerates microorganisms in the lag phase by providing nutrient availability corresponding to an AUC 0-72 value of greater than about 80 mg mL-1and a water content of greater than about 50%, with supplementation of bioactive molecules.

[0330] In other embodiments, the Bio-booster (112-B) induces a transition from the logarithmic phase to a lag or stationary state by programmed degradation of regionally distributed microenvironments of a biomimetic material (110) encapsulating bioactive molecules; wherein the microenvironments (110) are formulated to degrade in relationship with microorganisms growth from 48 h to 2 weeks.

[0331] The interplay of these regions creates complex functional behavior such as sequential microbial activation or gradient-dependent diffusion of active compounds.

[0332] B.1.3 Lag / Log Phase Control and Dynamic Function

[0333] A unique technical effect of the invention is its capacity to control the growth phase of microorganisms within the same material. This is achieved by spatial distribution of microenvironments and gradient formation:

[0334] In a preferred embodiment:

[0335] • Outer shell (111) - dense, low-nutrient, low-O2zone maintaining microbes in lag phase;

[0336] • Inner diffuser (112-B) - nutrient-rich, higher-hydration zone inducing log phase;

[0337] • Intermediate diffuser (112-N) - transition zone for sustained metabolic activity.

[0338] Diffusion and degradation kinetics govern phase transitions and enable cascade activation of microbial communities, supporting complex consortia behavior. This mechanism is particularly advantageous for probiotic delivery, bio-reactivation after storage, and in-vitro microbiome modeling.

[0339] In an exemplary configuration, for external or internal arrangements — whether radial, concentric, or hemispherical gradient — the outer region may represent between 4% and 75% of the total volume, while the inner region may represent between 25% and 96%. Each region (R) is composed of a distinct type of microenvironment, allowing functional interactions between adjacent regions. In another exemplary configuration, for clustered or atomized distributions, the clusters may represent up to 85% of the total volume of the biomimetic material, while the remaining 15- 25% corresponds to the biomimetic matrix (Material B). Each region (R) is likewise constituted by a different type of microenvironment, enabling controlled diffusive and biological interactions within the composite structure.

[0340] B.2 Physicochemical Gradients

[0341] The biomimetic material (100) of this invention is characterized by the presence of discrete or continuous physicochemical gradients intentionally programmed within and between regionally distributed microenvironments (RDMEs). These gradients define local diffusion profiles and biological responses, reproducing the spatial heterogeneity and functional stratification of natural tissues and mucosal layers. The gradients may be established along one or multiple spatial axes (radial, axial, or planar) and are stabilized by the composition of the polymeric network (A), the type and concentration of crosslinking agents, and the duration or sequence of the crosslinking process.

[0342] Preferably, the gradients are generated through directed crosslinking mechanisms, including controlled ionic diffusion, localized photopolymerization, enzymatic or redox reactions, or sequential deposition of polymeric precursors differing in ionic strength, pH, polymer concentration, or crosslinker type. The combination of these factors enables the formation of defined zones with distinct hydration levels, oxygen tension, and nutrient availability. The resulting gradients are quantifiable and reproducible across batches, measured by microelectrodes, pH microsensors, or confocal imaging for industrial applications.

[0343] B.2.1 Oxygen Gradients

[0344] Oxygen gradients arise naturally through the combination of polymer crosslinking density and oxygen-consuming reactions during fabrication. Oxygen concentration may vary from hypoxic (< 1 % O2; < 12 pmol / L O2) to normoxic (5 - 12 % O2; 62 - 148 pmol / L O2) or ambient (= 21 % O2; 260 pmol / L O2) and up to hyperoxic (> 30 % O2; > 370 pmol / L O2) depending on design.

[0345] Typical measurable ranges include 290 pmol L“1(surface) to 195 pmol L“1(core) - 0.0013 mM - 0.4 mM dissolved oxygen. Local oxygen partial pressure can be tuned to maintain aerobic, microaerophilic, or anaerobic zones within the same construct, supporting mixed microbial populations. B.2.2 pH Gradients

[0346] Regional pH values are tuned between 4.0 and 9.0 by varying the ratio of polymers, acidifying agents and culture medium (e.g., pectin, glucono delta-lactone, MRS, LB, MH, PYG media) and the concentration of buffering salts within the water-based suspension (sodium hydroxide and chloridric acid) (B). This tunability allows replication of site-specific mucosal conditions — such as gastric (pH 3.5-6.0), intestinal (6.0-7.5), or cervico-vaginal (3.8-4.5) — and enables controlled protonation states that affect crosslinking, mucoadhesion, and microbial metabolism.

[0347] B.2.3 Hydration and Water-Activity Gradients

[0348] Hydration gradients (5 - 95 wt %) regulate water activity, osmotic balance, and diffusion coefficients (10“6- 10“9cm2s“1). Outer shell regions (111) typically present reduced hydration (< 35 %) to enhance structural integrity and minimize oxygen permeability, while inner diffuser zones (112-B) exhibit higher hydration (> 70 %) to sustain metabolic activity. These programmed differences enable control over microbial lag-to-log transitions and influence degradation kinetics according to the Programmed Self-Functional Degradation Rule.

[0349] In possible embodiments, in case of execution of Phase 4 - Post-processing, including drying, air-drying, solvent casting, or lyophilization, the hydration level will be near to zero (0% - 20% (w / w)).

[0350] B.2.4 Nutrient and Crosslinking Gradients

[0351] Nutrient concentration within the biomimetic material can be adjusted between 0.2* and 2* relative to standard culture media, producing zones of nutrient limitation or enrichment corresponding to Bio-holder (112-H) or Bio-booster (112-B) regions, respectively.

[0352] Crosslinking density can differ by 34 - 80 % between regions, within an absolute range of 20 - 100%. Such variation, achieved by controlled ion diffusion or sequential polymer deposition, establishes a mechanical gradient (G' « 1.5-3000 Pa) that governs both diffusional flux and degradation rate.

[0353] Collectively, these gradients of oxygen, pH, hydration, nutrient content, and crosslinking density confer the biomimetic material with quantifiable regionally distributed microenvironments. Each gradient contributes to the overall functional hierarchy — controlling microbial activity, diffusion, degradation, and mechanical behavior in a predictable and reproducible manner. These gradients are quantifiable by standard analytical methods (oxygen microelectrodes, confocal, pH profiling, rheometry) and provide reproducibility for industrial testing, production and delivery.

[0354] B.3 Physico-Chemical Characterization and Functional Integration Measured properties of the biomimetic material (100), within each individual biomimetic microenvironment (110) and / or region include:

[0355] The functional integration of these parameters, and the selection of its compositions and components, allows the biomimetic material (100) to behave as a living-like system.

[0356] Hydration affects both microbial metabolism and viscoelasticity; crosslinking density defines mechanical support; oxygen and nutrient gradients regulate bioactivity; and degradation kinetics determine release and lifetime. Together, these features confer predictable and programmable performance across research, industrial, and therapeutic contexts.

[0357] B.4 Universal / multipurpose configuration of the invention

[0358] In a preferred presentation of the invention, the Hydrogel-like Biomimetic Material (100) is conformed by a 3D distribution of biomimetic microenvironments (110) that present the following configuration:

[0359] B.5 MUCUS MIMICKING Hydrogel-like Biomimetic Material (100)

[0360] In different embodiments of the invention, the Hydrogel-like Biomimetic Material (100) is conformed by a 3D distribution of biomimetic microenvironments (110) that mimics mucus layers. More preferably, human mucus layers.

[0361] In possible embodiments, the biomimetic microenvironments (110) mimic respiratory mucus:

[0362] In possible embodiments, the biomimetic microenvironments (110) mimic gastrointestinal mucus:

[0363] In possible embodiments, the biomimetic microenvironments (110) mimic cervicovaginal mucus:

[0364] C. MACRO SHAPES

[0365] The biomimetic material (100) may be shaped or be structured as an Amorphous biomimetic material (200), Macrostructured constructs (300), or Composite materials. C.1 Amorphous biomimetic material (200)

[0366] In certain embodiments, the biomimetic material (100) is provided in the form of a self- supporting amorphous biomimetic material (200), which assumes the shape of its containing vessel or mold.

[0367] In a possible presentation, the base presentation of the amorphous biomimetic material (200) is obtained as the direct result of Phase I of the manufacturing method of the biomimetic material (100), wherein the crosslinking process is completed without subsequent shaping or molding.

[0368] In other embodiments, the amorphous biomimetic material (200) is obtained by combining the results of two or more parallel executions of Phase I, each employing distinct compositions or crosslinking parameters, such that their mixing generates a heterogeneous or composite amorphous matrix, thereby forming a superposition of polymeric networks (A) with interpenetrating or layered structural characteristics.

[0369] In further embodiments, the amorphous biomimetic material (200) is produced through sequential iteration of two or more Phase I processes, wherein the output of a previous phase is reintroduced as the input of a subsequent one, thereby forming a superposition of polymeric networks (A) with interpenetrating or layered structural characteristics.

[0370] In preferred embodiments, the amorphous biomimetic material (200) is extrudable, allowing its deposition or direct application through manual or automated devices, such as syringetype applicators or extrusion systems.

[0371] In additional embodiments, the amorphous biomimetic material (200) is adapted for additive manufacturing, being 3D printable or suitable for layer-by-layer deposition techniques that enable the fabrication of customized geometries, gradients, or structural patterns.

[0372] C.2 3D macrostructured constructs (300)

[0373] In certain embodiments, the biomimetic material (100) is shaped as a macrostructured construct (300).

[0374] In such embodiments, the macrostructured construct (300) is composed of a three- dimensional distribution of biomimetic microenvironments (110), corresponding in composition and functional logic to those of the biomimetic material (100).

[0375] The construct (300) is characterized by dimensions within the millimeter-to-centimeter scale, maintaining both mechanical stability and diffusional functionality.

[0376] In preferred embodiments, at least one of the construct’s dimensions is within a range from 0.5 mm to 200 mm, depending on its geometry and intended application.

[0377] In further preferred embodiments, the constructs (300) are obtained through Phase II of the method for fabricating the biomimetic material (100), wherein the preformed hydrogel-like biomimetic material (100) is shaped, molded, or otherwise structured into a defined macrogeometry.

[0378] In a preferred embodiment the Macrostructured Construct (300) is shaped like a bead (300-

[0379] B), selected from spherical, ellipsoidal, pear-like, bean-like, comet-like, drop-like, Prince Rupert’s drop-like, wobbly-shaped, or egg-like geometries. In a preferred embodiment the bead (300-B) has a diameter between 0.5 mm and 20 mm. Preferably, 1 mm to 10 mm, more preferably 2 mm to 7 mm, more preferably 3 mm to 6 mm.

[0380] In a preferred embodiment the Macrostructured Construct (300) is shaped like a disk (300-D). The disk (300-D) may have a base / transversal cut of any desired 2D shape, including circles, squares, rectangles, regular and irregular polygons, with or without cured edges. In a preferred embodiment having a thickness of 2 mm to 30 mm and a diameter of 5 mm to 200 mm. Preferably a thickness of 3 mm to 10 mm and a diameter of 10 mm to 150 mm. Preferably a thickness of 4 mm to 6 mm and a diameter of 30 mm to 50 mm

[0381] In a preferred embodiment the Macrostructured Construct (300) is shaped like a cylinder (300-

[0382] C). The cylinder (300-C) may have a transversal cut of any desired 2D shape, including circles, squares, rectangles, regular and irregular polygons, with or without cured edges. The cylinder (300-C) may also be disposed in a straight, angled, S-like, spiral, or coiled shape, as a whole or by segments. In a preferred embodiment, the cylinder (300-C) has a diameter of 2 mm to 30 mm and a height of 5 mm to 200 mm. Preferably, diameter of 2 mm to 4 mm and a height between 50 to 100 mm.

[0383] In a preferred embodiment the Macrostructured Construct (300) is shaped like a film (300-F). The film(300-F) may have a transversal cut of any desired 2D shape, including circles, squares, rectangles, regular and irregular polygons, with or without curved edges. In a preferred embodiment, the film has a thickness of 2 mm to 30 mm, a width of 5 mm to 200 mm, and a length of 5 mm to 200 mm. Preferably, having a thickness of 2 mm to 4mm, a width of 30 mm to 100 mm, and a length of 30 mm to 100 mm.

[0384] In a preferred embodiment, the macrostructured construct (300) is shaped like a tube (300- T). The tube (300-T) may have a hollow or semi-hollow cylindrical geometry, optionally open at one or both ends, and may include straight, curved, coiled, or branched configurations. The transversal section of the tube (300-T) may be circular, oval, polygonal, or irregular, with smooth or textured internal and external surfaces. In a preferred embodiment, the tube (SOO- T) has an outer diameter between 2 mm and 30 mm, an inner diameter between 1 mm and 25 mm, and a length between 5 mm and 200 mm. More preferably, the tube (300-T) has an outer diameter of 3 mm to 10 mm, an inner diameter of 2 mm to 8 mm, and a length of 20 mm to 100 mm.

[0385] C.3 Composite materials (400)

[0386] In certain embodiments, the invention provides composite materials (400) in which one or more macrostructured constructs (300) are dispersed, embedded, or otherwise integrated within a continuous amorphous biomimetic matrix (200).

[0387] These composite configurations enable the coexistence of distinct structural hierarchies and multiple types of biomimetic microenvironments (110) within a single unified system.

[0388] The composite materials (400) may comprise between 10 vol % and 70 vol % of macrostructured constructs (300) distributed within the amorphous phase. The constructs may be randomly dispersed or spatially arranged in planned geometries, layers, or clusters to achieve specific diffusion dynamics or functional sequences. The amorphous phase (200) functions as an interfacial and connective medium, ensuring continuity of hydration and diffusion while maintaining the individual integrity and regional distribution of microenvironments (RDMEs) within each construct (300). In this configuration, diffusion continuity is preserved across construct boundaries, yet each embedded structure retains its internal gradient logic and physicochemical identity.

[0389] Composite materials (400) can be fabricated by several approaches, including:

[0390] • Sequential embedding, wherein preformed constructs (300) are immersed in or coated with a fresh precursor solution during a subsequent Phase I of the manufacturing method, followed by controlled crosslinking;

[0391] • Co-deposition, wherein amorphous pre-gel and macrostructured constructs are coextruded or layered within a mold or printer system prior to solidification; or

[0392] • Manual or robotic assembly, where constructs are spatially positioned within a mold and then encapsulated by an amorphous pre-gel solution. In certain optional embodiments, additional crosslinking is induced after deposition — for example, by ionic diffusion, photochemical activation, or enzymatic treatment — so that the macrostructured constructs (300) become partially anchored or fixed within the amorphous biomimetic material (200). This secondary crosslinking promotes the positional stability of each construct (300), preventing displacement or aggregation during handling or application while maintaining interfacial permeability and functional independence.

[0393] In some embodiments, the interface exhibits partial polymer interpenetration, producing a mechanically continuous yet functionally compartmentalized composite.

[0394] In certain embodiments, a plurality of macrostructured constructs (300) are fabricated with different configurations, material compositions, or regional distributions of biomimetic microenvironments (110).

[0395] Each construct may be independently tailored with distinct polymeric networks (A), crosslinking densities, ortypes of microenvironmental regions (e.g., shell (111), diffuser (112), or subtypes thereof such as 112-H, 112-N, or 112-B). These variations allow the combination of constructs optimized for specific diffusion profiles, degradation rates, microbial communities, or therapeutic functions within a single system.

[0396] In one representative embodiment, constructs with nutrient-rich diffuser regions are interspersed with constructs designed for protective or lag-phase stabilization, forming a heterogeneous composite assembly that delivers sequential or spatially differentiated biological responses.

[0397] Composite systems (400) offer several advantages over single-construct formats. They allow multi-regional functional integration, combining regions optimized for different purposes — for example, diffusion control, microbial activation, mechanical support, or controlled degradation — within one device.

[0398] They also provide greater mechanical robustness, extended release kinetics, and enhanced adaptability for applications such as bioactive scaffolds, multi-phase bioreactors, layered therapeutic implants, or mucosal-regenerative delivery systems. D. APPLICATIONS AND CONFIGURATIONS

[0399] The hydrogel-like biomimetic material (100) and its derived forms — amorphous material (200), macrostructured constructs (300), and composite materials (400) — are designed to function as modular platforms adaptable across multiple sectors.

[0400] The material’s combination of directed crosslinking, regionally distributed microenvironments (RDMEs), and dual-functional polymeric networks (A) enables a continuum of applications that span research, industrial bioprocessing, biomedical therapy, and personal-care technologies.

[0401] D.1 Research and Industrial Applications

[0402] D.1.1 Microbiological Research Tools

[0403] The biomimetic material (100) provides a reproducible, biomimetic substrate for microbiological studies, particularly those involving host-microbe and microbe-microbe interactions.

[0404] Unlike conventional culture plates or homogeneous hydrogels, this material reproduces native gradient conditions such as oxygen limitation, nutrient stratification, and mucin-associated adhesion.

[0405] Applications include:

[0406] • in-vitro simulation of gastrointestinal, cervico-vaginal, nasal, or environmental microbiomes;

[0407] • testing of antimicrobial, prebiotic, or probiotic agents under controlled oxygen and pH gradients;

[0408] • modeling of biofilm formation, dispersal, and microbial succession in complex communities

[0409] • microbiome mining; and

[0410] • observation of microbial metabolic shifts using fluorescence or sequencing-based assays.

[0411] The material supports both static and dynamic studies, and can be incorporated into microfluidic or high-throughput screening devices. D.1.1.1. Hydrogel-like Biomimetic Material (100) for microbiome mining

[0412] In different embodiments of the invention: A microbiome mining tool or kit

[0413] A microbiome mining tool comprising the biomimetic material (100), having a distribution (homogeneous or regional) of Bio-neutral (112-N) type of microenvironments (110), and sustaining metabolic activity of microorganisms and allowing to 3D sampling

[0414] A microbiome mining tool comprising the biomimetic material (100), having a distribution (homogeneous or regional) of Bio-holder (112-H) and / or Bio-booster (112-B) type of microenvironments (110), and selectively sustaining metabolic activity of microorganisms and allowing to 3D sampling

[0415] A microbiome mining tool comprising the biomimetic material (100), having a regional distribution of diffuser (112) type microenvironments (110), separated by shell (111) type microenvironments (110)

[0416] A microbiome mining tool comprising the biomimetic material (100), where the microbiome mining is from human donors. Included but not limited by human mucus samples (airways, cervicovaginal, gastrointestinal). In a preferred embodiment, from fecal samples.

[0417] A microbiome mining tool comprising the biomimetic material (100), having a distribution (homogeneous or regional) of Bio-holder (112-H) and / or Bio-booster (112-B) type of microenvironments (110), where the microbiome mining is from human donors, for boosting the growth of beneficial microbiota for humans.

[0418] A microbiome mining method is also provided, comprising the use of the biomimetic material (100) as a selective or enrichment matrix for capturing, growing, or isolating microorganisms from complex samples.

[0419] In one implementation, the microbiome mining is performed by placing a biological or environmental sample on top of the biomimetic material (100) and allowing passive diffusion of microorganisms and soluble components into the matrix.

[0420] In another implementation, the sample can be gently mixed or injected using syringes or pipettes to enhance penetration and contact between the sample and the internal microenvironments (110).

[0421] Optionally, the material (100) can be subjected to mild agitation, orbital shaking, or continuous flow, depending on the desired degree of oxygenation or nutrient exchange. In some embodiments, static incubation is preferred to favor the establishment of gradients and localized colonization, while in other embodiments periodic or continuous movement is applied to stimulate mass transfer and prevent surface overgrowth.

[0422] Following incubation, microbial colonies or enriched populations can be recovered by slicing, dissolving, or aspirating specific regions of the biomimetic material (100), enabling targeted microbiome isolation and mining under physiologically relevant conditions.

[0423] In different embodiments, it is possible to store the material (100) with the mined microorganisms for its preservation. For this it is possible to do a further implantation of the selected material into a new amorphous material (200) with Bio-holder (112-H) microenvironments.

[0424] In different embodiments, it is possible to continue to expand the material (100) with the mined microorganisms via selective boost. For this it is possible to do a further implantation of the selected material into a new amorphous material (200) with Bio-booster (112-B) microenvironments.

[0425] In different embodiments, the microbiome mining tool comprises the amorphous material (200) for the microbiome mining process. The amorphous material (200) being disposed in any container that allows the seeding of the samples and recovery of all or a section of the material (100).

[0426] In different embodiments, the microbiome mining tool comprises the Macrostructured Constructs (300) for the microbiome mining process. In different embodiments a plurality of Macrostructured Constructs (300) being dispersed or deposited on a solution or suspension containing the original sample to mine.

[0427] In different embodiments, the plurality of Macrostructured Constructs (300) share the same configuration of types of microenvironment (110) and same regional distribution of microenvironments within the Macrostructured Constructs (300).

[0428] In different embodiments, the plurality of Macrostructured Constructs (300) do not share the same configuration of types of microenvironment (110) or the same regional distribution of microenvironments within the Macrostructured Constructs (300). In this way it is possible to simultaneously select different microorganisms based on the selective probiotic properties of each macrostructured construct (300).

[0429] In different embodiments, the microbiome mining tool comprises the composite material (400). D.1.2 Industrial and Bioprocessing Uses

[0430] In industrial biotechnology, the constructs (300) serve as microbial carriers, immobilization matrices, or growth enhancers.

[0431] They may be integrated into bioreactors, fermentation systems, or wastewater treatment devices to improve mass transfer, stability, and productivity.

[0432] Key industrial embodiments include:

[0433] • Fermentation and metabolite production: constructs containing diffuser regions (112- B) enhance growth rates, reduce lag time, and increase biomass yield per unit volume.

[0434] • Probiotic manufacturing: beads or composite gels sustain high viability during storage and reactivation upon rehydration.

[0435] • Biofilm reactors and continuous bioprocesses: macrostructured systems allow reuse cycles with consistent mechanical stability.

[0436] • Agricultural bioinoculants: constructs containing beneficial bacteria (e.g., Bacillus, Pseudomonas, Azospirillum) serve as soil or plant inoculants with controlled hydration and nutrient release.

[0437] All embodiments can be tailored by adjusting the mechanical modulus, hydration level, and nutrient profile, providing both biological fidelity and industrial scalability.

[0438] D.1.2.1. Hydrogel-like Biomimetic Material (100) for boosting the growth of microorganisms for production of microorganisms for its subproducts.

[0439] In different embodiments, the invention allows it to surpass the difficulties of growing microorganisms in production settings, for R&D and for industrial settings. By controlling the lag and log targeted microorganisms, the invention allows a shorter time to peak production and to extend the viability of the microorganisms in time.

[0440] In different embodiments of the invention, the Macrostructured Constructs (300) are configured as boosters of targeted microbiota. In this presentation of the invention, a method of production of microorganisms includes supplementing Macrostructured Constructs (300) to a dynamic production of microorganisms. In a preferred embodiment, the Macrostructured Constructs (300) are supplemented in a volume of 1 % to 30% of the total of the system to be produced, 1% to 20%, more preferably, 1 to 10%. Then, periodic supplementation is provided for extending / sustaining the vitality of the dynamic production system. In a preferred embodiment, the production is done in a bioreactor and or a fermenter.

[0441] In different embodiments of the invention, the macrostructured constructs (300) are configured as single production units, where they comprise microenvironments (110) comprising viable microorganisms (C), which sustain metabolic activities, proliferate, and generate post biotics. As a result, each macrostructured construct (300) will act as a controlled factory and simultaneously a carrier of the microorganisms and its subproducts. In different embodiments, it can be dissolved for retrieving the desired products, or delivered as a whole as a carrier.

[0442] D.2 Lubricant and Personal-Care Applications

[0443] D.2.1 Biomedical Lubricants

[0444] The amorphous form (200) and thin films (300-F) of the biomimetic material (100) can function as biomedical lubricants for diagnostic and therapeutic procedures. Their viscoelastic behavior (G7G" ratio between 0.3 and 0.6), biocompatibility, and tunable hydration make them ideal for minimizing friction during the insertion of catheters, endoscopes, or surgical instruments.

[0445] Formulations can be adjusted to match local tissue pH by employing mucus mimicking material (100) as described above, and to maintain hydration and epithelial comfort during procedures such as colonoscopy, gynecological sampling, or device implantation.

[0446] D.2.2 Personal-Care and Sexual-Wellness Lubricants

[0447] The same principles can be adapted for personal-care lubricants, emphasizing physiological compatibility and microbial balance.

[0448] The inclusion of mucin or mucin-like proteins enhances tissue adherence and hydration retention.

[0449] Optional humectants (glycerol, xylitol), soothing agents (aloe vera, chamomile extract), or natural sensory enhancers can be incorporated. The lubricants may be sterile or probiotic-enriched, designed to preserve or restore mucosal microbiota.

[0450] Sustained-release formulations are possible, in which the gel gradually releases humectants, antioxidants, or prebiotic molecules over time.

[0451] These embodiments are fully compliant with ISO 10993 biocompatibility standards and exhibit favorable rheology, transparency, and shelf stability. D.3 Therapeutic Embodiments

[0452] The biomimetic material (100) also enables multiple therapeutic uses based on its mucosal mimicry, bioadhesion, and microbial modulation capabilities. Applications are grouped by target tissue and format (amorphous, macrostructured, or composite).

[0453] D.3.1 General Therapeutic Principles

[0454] The therapeutic embodiments exploit the material’s ability to:

[0455] • restore mucosal barrier function by forming a hydrated, adherent layer, and restructuration of the mucosal protein mesh;

[0456] • deliver beneficial microorganisms, postbiotics, or small molecules in a controlled manner;

[0457] • modulate local inflammation and oxidative stress through polymer degradation products; and

[0458] • support re-epithelialization and microbiota homeostasis.

[0459] Both bioactive-free (non-living) and viable microorganisms (C) versions are contemplated, depending on application.

[0460] D.3.2 Gastrointestinal Applications

[0461] Within the gastrointestinal tract, the biomimetic material (100) functions as:

[0462] • a delivery vehicle for probiotics, prebiotics, and postbiotics;

[0463] • a local therapeutic gel for ulcerative or dysbiotic conditions; or

[0464] • a protective mucosal coating that restores hydration and barrier integrity.

[0465] Preferred formats:

[0466] • amorphous gel (200) applied via oral, rectal, or endoscopic routes;

[0467] • macrostructured beads (300-B) or cylinders (300-C) used for oral delivery, optionally enteric-targeted through the controlled crosslinking of the outer shell (111);

[0468] • composite materials (400) combining multiple microenvironment types for staged release along the Gl tract. Microorganisms such as Lactobacillus, Bifidobacterium, Akkermansia, and Faecalibacterium can be incorporated in viable or lyophilized form, with protective shell layers ensuring viability through gastric passage. pH and oxygen gradients are tuned to match intestinal microenvironments by usage of mucus mimicking biomaterial (100).

[0469] D.3.3 Cervico-Vaginal Applications

[0470] In cervico-vaginal therapeutics, the invention supports local microbiota restoration, infection prevention, and mucosal repair.

[0471] Preferred formats include:

[0472] • oval or egg-shaped beads (300-B) inserted intravaginally;

[0473] • films (300-F) adhering to mucosal surfaces; and

[0474] • amorphous gels (200) dispensed via applicator.

[0475] The pH is adjusted to 3.8-4.5 and mucin or chitosan enhances mucoadhesion. Probiotic species (Lactobacillus crispatus, L. jensenii, L. gasseri) can be included for recolonization.

[0476] Active dopants such as lactic acid, hyaluronic acid, or plant-derived extracts may be incorporated to promote healing and hydration.

[0477] D.3.4 Airway and Nasal Applications

[0478] For the upper respiratory tract, the amorphous gel (200) and film (300-F) versions act as mucosal-protective formulations to maintain hydration, reduce irritation, and deliver bioactives or beneficial microbiota.

[0479] Targeted pH: 6.0-7.2; hydration: 80-95 %.

[0480] Applications include relief of dry-nose syndrome, post-infectious recovery, and adjunctive delivery of probiotics (Streptococcus salivarius, Corynebacterium accolens) or antimicrobial peptides.

[0481] D.3.5 Synergistic substrate for the delivery of therapeutic molecules

[0482] The biomimicking material is capable of retaining drugs, supplements or metabolizable molecules with therapeutic effect. These can be released upon adhesion of the material to any mucosal layer and delivered through the permeability of the epithelium. The macrostructured constructs in the shape of Beads (300-D) will be preferred for oral delivery with final target adhesion in the intestinal mucus layer for absorption of active components. Mucin in the construct will promote adhesion and increased release time while restoring permeability in the mucus.

[0483] Microstructured constructs in the shape of Films (300-F) or Disks (300D) can be selectively positioned and fixed in the mucus layers of different epithelial layers like the naso faringeal tract for nose-brain axis pathway delivery, gastrointestinal tract for intestinal absorption or cervicovaginal mucus layer for localized delivery of such molecules. The film will contain the active component that will be positioned in the targeted location.

[0484] None of the constructs mentioned above are stand alone delivery mechanisms and do not require an extra enteric coating for delivery.

[0485] D.3.5.1 Controlled release of electrolytes for hydration purposes

[0486] The biomimetic construct, in its capacity to retain an ionically balanced solution and salts, is able to deliver electrolytes through the gastrointestinal tract and in a proportional manner to the polymeric matrix degradation.

[0487] In preferable presentations, the salts are selected from the set consisting of:

[0488] • Monovalent ions: Sodium (230-1150 mg / L), potassium (80-310 mg / L), (chloride at 350-2100 mg / L).

[0489] • Divalent ions: magnesium (7-36 mg / L), calcium (12-60 mg / L).

[0490] • Other salts or buffers: Phosphate at 15-60 mg / L, bicarbonate < 1 mg / mL (typically 300-900 mg / L)

[0491] D.4 Cross-Sectoral Functionalities

[0492] D.4.1 Bioadhesion and Targeted Retention

[0493] Mucin and chitosan components confer electrostatic and hydrogen-bond-mediated adhesion to epithelial or mucosal tissues, ensuring local retention and sustained activity. This property underlies the therapeutic performance of both probiotic and non-probiotic versions. D.4.2 Protective and Storage Functions

[0494] The outer shell (111) acts as a protective barrier that stabilizes encapsulated microorganisms or active molecules during storage and transport. Constructs (300) can preserve viability for extended periods (up to 12 months) under controlled humidity, then rehydrate to restore activity.

[0495] D.4.3 Compatibility with Devices

[0496] The material may be pre-loaded into capsules, syringes, applicators, or transdermal patches. It is compatible with coating technologies, allowing constructs or gels to be used in combination with medical devices for localized release or lubrication.

[0497] D.5 Environmental and Agricultural Embodiments

[0498] Although primarily designed for biomedical and microbiome-related applications, the same biomimetic logic applies to environmental and agricultural uses:

[0499] • Soil microbiota engineering: beads containing beneficial bacteria release microorganisms under moisture-triggered conditions.

[0500] • Bio-remediation matrices: gels act as scaffolds for pollutant-degrading microbes.

[0501] • Controlled nutrient delivery: composite constructs release fertilizers or signaling molecules in synchrony with microbial activation.

[0502] In selected embodiments for environmental and agricultural applications, within the biomimetic material (100), the water based suspension (B) may comprise molecules selected from the set: oxidoreductase enzymes, especially laccases, tyrosinases, lignin peroxidases, manganese peroxidases, and horseradish peroxidase, due to their high catalytic efficiency in degrading a broad spectrum of hazardous pollutants such as dyes, phenolic compounds, pharmaceuticals, and endocrine disruptors. While normal carriers offer many operational benefits, they are often limited by issues of enzyme leakage, diffusional inefficiency, stability inconsistency, and high cost or complexity of production, representing key barriers to efficient and sustainable biocatalysis in environmental applications.

[0503] In a more preferred embodiment, the invention is therapeutic for: Cervico- vaginal / Reproductive tract, genitourinary syndrome of menopause (atrophic vaginitis), vaginal dryness, thinning, loss of elasticity, altered microbiota (post-menopause); Bacterial vaginosis and recurrent yeast infections (Candida spp.); sexually transmitted infections (STIs): HPV, HSV, chlamydia, gonorrhea, syphilis; Radiotherapy, chemotherapy, or surgical treatments for cervical cancer; respiratory Tract (Nasal, Lung, Upper Airways); Chronic obstructive pulmonary disease (COPD); Cystic fibrosis (thick dysfunctional mucus, epithelial injury); Asthma (chronic inflammation); Respiratory infections: influenza, COVID-19, bacterial pneumonia; Chronic rhinosinusitis; Inhalation injuries (toxic gases, smoke, pollutants); Oral Cavity; Oral mucositis (chemo / radiotherapy); Periodontal disease; Oral candidiasis; Oral lichen planus; Ocular (Conjunctival I Corneal Surfaces); Dry eye disease (tear film instability, epithelial injury); Conjunctivitis (infectious or allergic); Chemical / thermal ocular burns; Stevens-Johnson syndrome / Toxic epidermal necrolysis (severe mucosal destruction); Systemic / Multi-Site Conditions; HIV / AIDS (broad mucosal vulnerability: gut, genital, oral); Graft-versus-host disease (post-transplant); Severe malnutrition (compromises gut mucosal barrier); Sjogren’s syndrome, systemic autoimmune dryness: oral, ocular, genital, respiratory mucosa; Inflammatory Bowel Syndrome, Chrons disease, Ulcerative Colitis, Celiac disease, Gut Dysbiosis and any other inflammatory disease in the intestinal region; colorectal cancer.

[0504] E. MANUFACTURING METHOD for Making the Material (100), Amorphous Material (200), Macrostructured Constructs (300), and Composite Material (400)

[0505] The invention further provides a method for manufacturing the hydrogel-like biomimetic material (100) and the macrostructured constructs (300) and composite systems (400) derived therefrom.

[0506] The method is based on the principle of directed crosslinking, enabling spatially controlled gelation and the generation of regionally distributed microenvironments (RDMEs). The method is modular and may include one or more iterative or parallel fabrication phases, allowing hierarchical embedding, multi-region formation, and scalable production.

[0507] E.1 General Process Structure

[0508] The process comprises three main phases: (1) Preparation of a hydrogel-like biomimetic material, (2) Shaping, and (3) Formation of composite or hybrid materials and an optional phase (4) for post processing.

[0509] Phase 0 - Precursor Preparation and Sterilization

[0510] All reagents and components are sterilized prior to mixing to ensure biocompatibility and asepsis. 1. Sterilization of polysaccharide polymers:

[0511] Expose to ultraviolet light for 30 minutes, followed by incubation in an oven at 50 °C for 30 minutes.

[0512] Autoclaving (121 °C, 15 min) may be applied when polymers are heat-stable.

[0513] 2. Sterilization of protein or mucin-based solutions: Mucin solutions are generally autoclaved to ensure sterility and viscosity retention and promote easy intake from the microbial population. Protein solutions are filter-sterilized (0.22 pm membrane) to prevent denaturation.

[0514] 3. Sterilization of powders and crosslinking modulators: Modulators such as CaCO3, GDL, or buffer salts are exposed to UV for 30 minutes followed by oven heating at 50 °C for 30 minutes.

[0515] 4. Sterilization of ionic-balancing solutions:

[0516] Aqueous solutions containing ionic salts are autoclaved and cooled prior to use.

[0517] This preparatory phase yields sterile polymeric and ionic solutions that are stable and ready for controlled gelation.

[0518] Phase 1 - Formation of amorphous biomimetic material (200)

[0519] This phase involves the creation of the amorphous hydrogel-like biomimetic matrix through the controlled initiation of crosslinking within a mixed polymeric precursor.

[0520] A polymeric network (A) is created to sustain a water-based suspension (B) and, optionally, viable microorganisms (C).

[0521] This phase involves three sequential steps: (1) preparation of a polymer mother solution, (2) addition of bioactive components, and (3) formation of the polymeric network by crosslinking.

[0522] Step 1 - Preparation of the Polymer Mother Solution

[0523] An ionic balancing solution is first prepared in water using ionic balancing molecules such as NaCI, KOI, CaCI2, Ca-gluconate, MgCI2, sodium phosphate, or HEPES.

[0524] In certain embodiments, the ionic solution is replaced by or supplemented with culture media including LB, Mueller-Hinton, TSB, MRS, BHI, RPMI, DMEM, M9, ROM, YCFA, or FAB.

[0525] The solution may be sterilized by autoclaving when required. Dissolution is carried out under magnetic agitation, ultrasonication, or manual stirring, at 100- 500 RPM, room temperature to 120 °C, for 3-15 minutes.

[0526] After dissolution, one or more polymers are added slowly while maintaining agitation between 100 and 320 RPM, temperature between room temperature and 50 °C, and time between 15 minutes and 8 hours.

[0527] If necessary, HCI 1 M or NaOH 1 M may be added to adjust pH and improve solubility.

[0528] Suitable polymers include:

[0529] • Anionic polysaccharides: sodium alginate, pectin, hyaluronic acid, and carboxymethyl cellulose.

[0530] • Neutral polysaccharides: dextran, pullulan, agarose, starch, guar, and locust bean gum.

[0531] • Cationic polysaccharides: chitosan.

[0532] • Proteins: mucin, gelatin, collagen, and silk fibroin.

[0533] • Synthetic polymers: polyethylene glycol (PEG), polyethylene glycol diacrylate (PEGDA), polyvinyl alcohol (PVA), polyacrylic acid (PAA), polyhydroxyethyl methacrylate (PHEMA), and Pluronics.

[0534] The polymer identity and concentration determine the mesh size, viscoelasticity, and degradation or release profile of the resulting material (100).

[0535] Step 2 - Addition Bioactive Components within Water-Based Suspension

[0536] A bioactive suspension is prepared in the same ionic solution, in water, or in a selected culture medium, containing one or more bioactive components, such as:

[0537] • Proteins or glycoproteins (e.g., mucin, enzymes, peptides);

[0538] • Small molecules (e.g., antibiotics, short-chain fatty acids, quorum modulators, metabolites);

[0539] • Particulates (e.g., nanoparticles, liposomes, microparticles); and / or

[0540] • Viable microorganisms (e.g., bacteria, yeasts, spores, bacteriophages).

[0541] • Agitation is adjusted according to the sensitivity of the components: - for protein-based elements, speeds remain below 100 RPM; for insoluble compounds, the suspension is vortexed prior to mixing. The bioactive suspension is then incorporated into the polymer mother solution under gentle agitation to maintain bioactivity.

[0542] This step imparts bio-incubating capability, mucoadhesiveness (through mucin inclusion), and targeted-delivery potential to the material.

[0543] Step 3 - Formation of Amorphous Biomimetic Material with ROME

[0544] 1. Initiation of directed crosslinking:

[0545] Directed crosslinking is initiated by introducing crosslinking agents in a spatially controlled manner: o In a preferred embodiment, ionic crosslinking is achieved using crosslinkers such as CaCI2, Ca-gluconate, BaCI2, or SrCI2, optionally combined with CaCO3+ GDL systems for delayed internal gelation. Agitation is maintained between 100 and 500 RPM to ensure uniform distribution.

[0546] Modulators such as mild chelators or pH adjusters can be added under 100- 250 RPM for 3-30 minutes to homogenize the reaction front. o In photo-crosslinking embodiments, polymers bearing photo-reactive groups (e.g., methacrylate or thiol-ene functionalities) are exposed to light (365-405 nm) in the presence of initiators such as Irgacure 2959 or LAP until the desired mechanical properties are reached. o In biologically induced crosslinking, polymerization occurs through enzymatic catalysis (e.g., transglutaminase, peroxidase / H2O2) or by microbial activity. The mixture is incubated at a temperature suitable for the embedded organism (typically 20-37 °C) for at least 12 hours, or until the intended network consistency is obtained. o Enzymatic crosslinking: addition of oxidases, transglutaminases, or peroxidases localized by microdispensing. o Field-assisted crosslinking: application of electric, magnetic, or electrochemical fields to direct ion migration or polymer alignment.

[0547] Reaction conditions (time, concentration, temperature, exposure geometry) define the crosslinking gradient and, consequently, the distribution of microenvironments (110). 2. Formation of microenvironments:

[0548] During crosslinking, differences in ion concentration, polymer density, or diffusivity generate discrete microenvironments with unique hydration and mechanical profiles. These are stabilized as regionally distributed microenvironments (RDMEs) within the forming gel.

[0549] The process can be designed to create specific configurations, such as concentric shells, asymmetric gradients, or random atomized distributions.

[0550] 3. Temperature and environmental control:

[0551] Ambient conditions are typically maintained between 20 °C and 40 °C for viable microorganisms, or higher (up to 60 °C) for sterile batches. pH is maintained between 4 and 9.

[0552] At the end of Phase 1 , the material corresponds to the biomimetic material in its amorphous state (200) or pre-structured form, ready for shaping or additional processing.

[0553] Phase 2 - Shaping and Structuring

[0554] Phase 2 imparts macrostructure and spatial hierarchy to the gel. The amorphous gels (200) can be shaped into macrostructured constructs (300)

[0555] Step 1 - Formation of Additional Polymeric Meshes

[0556] If the first crosslinking stage consumes all available reactive groups, a new polymer solution can be mixed with the pre-formed gel to create a superposed network. A fresh polymer solution is prepared as in Step 1 of Phase 1 , using either identical or different polymers, and mixed under low-shear agitation (20-80 RPM).

[0557] Step 2 - Formation of Defined Macrostructured Constructs (300) through crosslinking

[0558] Crosslinking is initiated by altering pH or ionic strength — e.g., immersing the gel in acidic / basic solutions or in concentrated salt or deionized water.

[0559] This procedure generates regionally distributed microenvironments (110) within the Macrostructured Constructs (300) enabling differential degradation, protective outer layers for microorganisms, and multi-polymer functional behavior. Additional crosslinking may occur during or after shaping to stabilize macrostructure. Controlled ion diffusion or localized UV exposure finalizes the geometry and establishes mechanical integrity.

[0560] Crosslinking may occur in one, two, or all directions to generate gradient, layered, or isotropic structures respectively.

[0561] Directional crosslinking and controlled molding provide three-dimensional geometries.

[0562] • Beads (300-B): crosslinker exposure from all directions without a mold yields shapes such as spherical, ellipsoidal, pear-like, bean-like, comet-like, drop-like, Prince Rupert’s drop-like, wobbly, or egg-like; diameters range 0.5-20 mm, preferably 1-10 mm, more preferably 2-7 mm, most preferably 3-5 mm.

[0563] • Disks (300-D): crosslinking from one or both sides of a gel in a mold having height- to-diameter ratio 1 :10-1 :1 ; thickness 2-30 mm, diameter 5-200 mm (preferably 3-10 mm thick, 10-150 mm diameter).

[0564] • Cylinders (300-C): lateral crosslinking within a mold of height-to-diameter 1 :1-100:1 , optionally by continuous extrusion through a crosslinking zone; top and base may later be sealed by brief exposure.

[0565] • Films (300-F): thin layers formed in molds with height-to-length or width ratio 1 :5- 1 :50, exposed from one side to the crosslinker.

[0566] • Tubes (300-T) : Crosslinking using a mold that maintains the hollow shape while crosslinking stabilizes. Crosslinkers may be applied from the lumen and / or the external surface of the tube.

[0567] Crosslinker may be applied after a pre-shaping step of pouring the amorphous gel in the mold or upon contact with a crosslinking molecule fixed in the mould.

[0568] Shaping techniques: o Casting or molding: the pre-gel is poured into molds defining desired geometries (e.g., bead, disk, cylinder, film). o Co-extrusion: two or more precursors are extruded simultaneously to generate layered or core-shell architectures. o 3D printing: extrusion or inkjet printing of gel precursors followed by localized crosslinking (UV or ionic). o Manual or robotic positioning: constructs can be manually or robotically arranged to form composite assemblies.

[0569] Phase 3 — Formation of Composite Materials (400) and Integration of Pre-Existing Constructs

[0570] In certain embodiments, pre-formed materials or macrostructures are combined with new polymeric solutions to obtain composite materials (400) featuring hierarchical or hybrid architectures.

[0571] Representative configurations include:

[0572] 1. Bead-in-gel composites, embedding beads (300-B) within a freshly forming hydrogel matrix. This new hydrogel matrix may be shaped into a new macrostructured construct.

[0573] 2. Laminated stacks of films (300-F) or disks (300-D) joined by interfacial crosslinking or mild swelling followed by gelation.

[0574] 3. Co-extruded core-shell fibers or rods, in which inner and outer polymer streams are crosslinked sequentially.

[0575] 4. Interpenetrating polymer networks (IPNs) formed by swelling an existing construct in a new polymer solution and initiating a secondary crosslinking reaction.

[0576] 5. Mosaic assemblies, obtained by arranging distinct macrostructures (e.g., beads, disks, films) inside a mold and backfilling the interspaces with polymer solution before crosslinking.

[0577] Mixing and assembly occur under 20-80 RPM agitation, 20-37 °C, and contact times between 5 and 180 minutes, conditions that preserve both the integrity of pre-existing meshes and the viability of any contained microorganisms.

[0578] These hybrid processes enable seamless integration of old and new structures, creating continuous or discontinuous gradient, layered, or compartmentalized microenvironments within a single construct. In other embodiments, two or more pre-gel formulations can be generated in parallel, each with different compositions or crosslinking parameters. These are later combined in Phase 2 to produce spatially distinct regions within the same construct, ensuring voluntary geographic deposition and defined RDMEs.

[0579] Phase 4 - Post-Processing and Drying

[0580] Post-processing adjusts the final physical state and storage stability of the material.

[0581] 1. Drying and dehydration:

[0582] The material can be air-dried, solvent-cast, or lyophilized. For lyophilization, water removal exceeds 90 %, yielding a rehydratable matrix that maintains structural fidelity.

[0583] 2. Solvent casting:

[0584] Gel precursors are cast into thin films and dried under laminar airflow or controlled humidity to form flexible sheets.

[0585] 3. Partial dehydration:

[0586] For constructs intended for immediate use, partial water removal (30-70 %) provides handling strength while retaining rehydration capacity.

[0587] 4. Conditioning:

[0588] Constructs are equilibrated under controlled humidity (40-70 % RH) and temperature (4-25 °C) to stabilize physical and biological properties.

[0589] 5. Sterile packaging:

[0590] Post-processed materials are packaged under sterile or aseptic conditions in sealed containers, optionally under inert gas, ready for rehydration or direct application.

[0591] E.2 Directed Crosslinking and Gradient Control

[0592] Directed crosslinking underlies the reproducibility and complexity of the invention.

[0593] 1. Ionic systems:

[0594] Preferred crosslinkers include calcium salts such as CaCI2, CaCO3+ gluconolactone (GDL), or Ca-lactate in concentrations from 0.01 M to 2 M. Diffusion time can range from 3 minutes to 20 hours depending on desired gradient. 2. Photocrosslinking:

[0595] Achieved by exposure to controlled UV intensity (e.g., 10-200 mW / cm2) through masks or patterned illumination.

[0596] Particularly suited to 3D printing applications.

[0597] 3. Enzymatic and redox systems:

[0598] Provide mild, cell-compatible crosslinking without chemical initiators. Examples: horseradish peroxidase + H2O2, laccase, or glucose oxidase systems.

[0599] 4. Secondary crosslinking:

[0600] Microorganisms (C) may generate local pH or ionic changes post-fabrication, creating new microenvironments dynamically.

[0601] 5. Gradient measurement and reproducibility:

[0602] Oxygen and ion gradients are measurable and reproducible using microelectrodes, ensuring manufacturing control for regulatory compliance.

[0603] E.3 Hierarchical and Iterative Fabrication

[0604] The method allows re-entry of intermediate products into earlier stages of the process, enabling iterative design and structural hierarchy.

[0605] 1. Construct embedding:

[0606] A bead (300-B) may be embedded in an amorphous gel (200) or within another bead to create nested geometries.

[0607] This process may be repeated multiple times, producing multiple hierarchical levels.

[0608] 2. Parallel formation and fusion:

[0609] Independent gels produced in parallel can be positioned adjacently or fused by partial crosslinking to form heterogeneous composite materials.

[0610] 3. Controlled regional deposition:

[0611] Multiple dispenser heads, extrusion nozzles, or robotic arms can deposit distinct pregel formulations at specific coordinates, achieving planned RDME distributions.

[0612] 4. Automation and scale-up:

[0613] Robotic and mechanical dispensing systems can automate mixing, extrusion, and layering, ensuring reproducibility and throughput for industrial production. Microfluidic devices enable continuous fabrication of uniform beads or layered fibers. E.4 Process Parameters and Conditions

[0614] To ensure reproducibility, key process parameters are defined as follows:

[0615] Section F - Examples 1) Tailoring the biomimetic material in different regions of the gastrointestinal tract

[0616] Through the gastrointestinal tract, the characteristics of the gut mucus change in terms of pH and viscoelastic properties. The formulation and production protocols were adapted as follows:

[0617] 1. Development of material for mimicking homogenous colon mucus. 2. Development of material for mimicking gradient colon mucus.

[0618] 3. Development of material for mimicking flow colon mucus.

[0619] 4. Development of material for mimicking the small intestine mucus.

[0620] See Fig. 11.

[0621] To achieve a biomimetic material replicating the composition, structure and barrier properties of a homogeneous healthy human colon mucus a polymeric network with embedded bioactive materials was prepared following the next steps. Solution with NaCI concentration of 0.57 mg / mL and mucin from porcine gastric origin to a final concentration of 29 mg / mL were autoclaved. Alginate powder, calcium carbonate powder and delta-gluco lactone powder were sterilized through UV exposure for 30 min and subsequent 50 °C exposure for further 30 min. Alginate powder was added to the mixture to a final concentration of 11 mg / mL at a velocity of 220 rpm. Following homogenization, calcium carbonate powder was ultrasonicated for 1 minute, vortexed for 30 seconds and incorporated into the mixture at a final concentration of 1 mg / mL at a velocity of 220 rpm. After additional homogenization the delta-gluco lactone powder was vortexed for 30 seconds and subsequently added to a final concentration of 10 mg / mL at a velocity of 400 rpm. The obtained viscoelastic properties are the ones shown in Fig. 12. The pH of this formulation was 6.0.

[0622] With this information and considering the Maxwell model, the mesh size was calculated to be 23 nm.

[0623] A directional crosslinking step was done to provide the material with a gradient microenvironmental distribution, to recreate an uneven O2 distribution through the material for it to be suitable to culture anaerobic species and to provide the material with a biomimetic permeability to the gut membrane. Upon homogenization of the polymeric network, the mixture was deposited onto a surface with previous sterilized and dried calcium gluconate at a concentration of 20 mg / mL. See Fig. 13.

[0624] This second crosslinking step reduced the mesh size to 18 nm.

[0625] To use this biomimetic material in a microfluidic device, viscoelastic properties were altered by modifying the proportions of the used components: Solution with NaCI concentration of 0.57 mg / mL and mucin from porcine gastric origin to a final concentration of 28.5 mg / mL were autoclaved. Alginate powder, calcium carbonate powder and delta-gluco lactone powder were sterilized through UV exposure for 30 min and subsequent 50 °C exposure for further 30 min. Alginate powder was added to the mixture to a final concentration of 3.0 mg / mL at a velocity of 220 rpm. Following homogenization, calcium carbonate powder was ultrasonicated for 1 minute, vortexed for 30 seconds and incorporated into the mixture at a final concentration of 0.6 mg / mL at a velocity of 220 rpm. After additional homogenization the delta-gluco lactone powder was vortexed for 30 seconds and subsequently added to a final concentration of 7 mg / mL at a velocity of 400 rpm. See Fig. 14.

[0626] The formulation was then adjusted to mimic the healthy human small intestine mucus. Solution with NaCI concentration of 0.57 mg / mL and mucin from porcine gastric origin to a final concentration of 14.3 mg / mL were autoclaved. Alginate powder, calcium carbonate powder and delta-gluco lactone powder were sterilized through UV exposure for 30 min and subsequent 50 °C exposure for further 30 min. Alginate powder was added to the mixture to a final concentration of 7 mg / mL at a velocity of 220 rpm. Following homogenization, calcium carbonate powder was ultrasonicated for 1 minute, vortexed for 30 seconds and incorporated into the mixture at a final concentration of 0.8 mg / mL at a velocity of 220 rpm. After additional homogenization the delta-gluco lactone powder was vortexed for 30 seconds and subsequently added to a final concentration of 1 .4 mg / mL at a velocity of 400 rpm. With this new formulation, pH was raised to 6.2 and viscoelastic properties were reduced. See Fig. 15.

[0627] 2) Tailoring the biomimetic material for the different conditions in the airway

[0628] In different conditions the characteristics of the airway mucus change in terms of pH and viscoelastic properties. The formulation and production protocol were adapted as follows:

[0629] 1. Development of material for mimicking the physiologic airway mucus.

[0630] 2. Development of material for mimicking the homogeneous cystic fibrosis airway mucus.

[0631] 3. Development of material for mimicking the gradient cystic fibrosis airway mucus.

[0632] To achieve a biomimetic material replicating the composition and viscosity of the healthy human airway mucus the following protocol was followed:

[0633] A solution composed of NaCI at a concentration of 4.78 mg / mL and mucin from porcine gastric origin at a final concentration of 20 mg / mL was autoclaved. Alginate powder, calcium carbonate powder and delta-gluco lactone powder were sterilized through UV exposure for 30 min followed by a 30 minutes exposure at 50 °C. Alginate powder was added to the mixture at a final concentration of 3 mg / mL at a velocity of 220 rpm. Following homogenization, calcium carbonate powder was ultrasonicated for 1 minute, vortexed for 30 seconds and added to the mixture at a final concentration of 1 mg / mL at a velocity of 220 rpm. After additional homogenization the delta-gluco lactone powder was vortexed for 30 seconds and subsequently added at a final concentration of 4 mg / mL at a velocity of 400 rpm. The pH of the resulting solution was 6.0. Viscoelastic properties were measured through rheology using a frequency sweep. See Fig. 16.

[0634] Crosslinking density and mesh size were calculated based on molar ratios of used calcium and alginate and maxwell model from viscoelastic properties. Mesh size is approximately 108 nm.

[0635] To mimic the more viscous mucus and higher salinity of cystic fibrosis, the manufacturing protocol and formulation were changed as follows. This new formulation was intended to be used to recreate an infection model, this required the inclusion of Muller Hinton Broth as a bioactive agent within the ionic balancing solution.

[0636] A solution composed of Muller Hinton Broth and mucin from porcine gastric origin at a final concentration of 25 mg / mL was autoclaved. Alginate powder was sterilized through UV exposure for 30 min followed by 30 min exposure at 50 °C. The powder was then added to the mixture at a final concentration of 25 mg / mL at a velocity of 220 rpm. After homogenization the mixture was deposited onto a surface with previous sterilized and dried calcium gluconate at a concentration of 20 mg / mL. See Figs 17a and 17b.

[0637] These formulation changes maintained a similar crosslinking density but reduced the mesh size to 20 nm. 3) Tailoring of biomimetic material inspired by the cervicovaginal mucus at the different moments of the menstrual cycle

[0638] Throughout the menstrual cycle, the characteristics of the cervicovaginal mucus change in terms of pH and viscoelastic properties. The formulation and production protocol were adapted as follows:

[0639] 1. Development of material for mimicking homogeneous cervicovaginal mucus in nonovulatory conditions

[0640] 2. Development of material for mimicking gradient cervicovaginal mucus in non-ovulatory conditions

[0641] 3. Development of material for mimicking homogeneous cervicovaginal mucus in non- ovulatory conditions in MRS

[0642] 4. Development of material for mimicking gradient cervicovaginal mucus in non-ovulatory conditions in MRS

[0643] 5. Development of material for mimicking homogeneous cervicovaginal mucus in ovulatory conditions

[0644] 6. Development of material for mimicking gradient cervicovaginal mucus in ovulatory conditions

[0645] 7. Development of material for mimicking the cervicovaginal mucus during pregnancy. See Fig. 18.

[0646] To achieve a biomimetic material replicating the average features of cervicovaginal microenvironment, a polymeric network with embedded bioactive materials was prepared following the next steps.

[0647] A solution composed of NaCI at a concentration of 0.07 mg / mL, mucin from porcine gastric origin at a final concentration of 1.5 mg / mL and mucin from submaxillary gland at a final concentration of 1.5 mg / mL was autoclaved. Pectin powder, calcium carbonate powder and delta-gluco lactone powder were sterilized through UV exposure for 30 min followed by 30 min exposure at 50 °C. Pectin powder was added to the mixture to a final concentration of 5 mg / mL at a velocity of 220 rpm. Following homogenization, calcium carbonate powder was ultrasonicated for 1 minute, vortexed for 30 seconds and added to the mixture at a final concentration of 2 mg / mL at a velocity of 220 rpm. After additional homogenization the delta- gluco lactone powder was vortexed for 30 seconds and subsequently added to a final concentration of 0.36 mg / mL at a velocity of 400 rpm. The obtained viscoelastic properties are the ones shown in Fig. 19. The pH of this formulation was 6.0.

[0648] Considering these viscoelastic properties, the Maxwell model was used to calculate the mesh size, obtaining a value of 27 nm.

[0649] According to molar relationships between pectin and calcium in the mixture and tailored pH, the crosslinking density can vary from 5.5 to 30%.

[0650] A directional crosslinking step was done to provide the material with a gradient microenvironmental distribution, to recreate an uneven O2distribution through the material for it to be suitable to culture anaerobic species and to provide the material with a biomimetic permeability to the gut membrane. Upon homogenization of the polymeric network, the mixture was deposited onto a surface with previous sterilized and dried calcium gluconate at a concentration of 20 mg / mL. See Fig. 20.

[0651] This second crosslinking step reduced the mesh size to 21 nm.

[0652] To promote the growth of Lactobacillus the formulation was then adjusted to include MRS as a bioactive agent within the ionic balancing solution. A solution composed of acidic NaCI at a concentration of 0.07 mg / mL, mucin from porcine gastric origin at a final concentration of 1.5 mg / mL and mucin from submaxillary gland at a final concentration of 1.5 mg / mL was autoclaved. Pectin powder, calcium carbonate powder and delta-gluco lactone powder were sterilized through UV exposure for 30 min followed by 30 min exposure at 50°C. Pectin powder was added to the mixture to a final concentration of 7 mg / mL at a velocity of 220 rpm. Following homogenization, calcium carbonate powder was ultrasonicated for 1 minute in previous autoclaved acidic MRS, vortexed for 30 seconds and incorporated into the mixture at a final concentration of 1.5 mg / mL at a velocity of 220 rpm. After additional homogenization the delta-gluco lactone powder was mixed in previous autoclaved acidic MRS, vortexed for 30 seconds and subsequently added to a final concentration of 1.5 mg / mL at a velocity of 400 rpm. With this new formulation, pH was reduced to 5 and viscoelastic properties are the ones shown in Fig. 21.

[0653] A directional crosslinking step was done to provide the material with a gradient microenvironmental distribution, to recreate an uneven O2distribution through the material for it to be suitable to culture anaerobic species and to provide the material with a biomimetic permeability to the gut membrane. Upon homogenization of the polymeric network, the mixture was deposited onto a surface with previous sterilized and dried calcium gluconate at a concentration of 20 mg / mL. See Fig. 22.

[0654] The formulation was then adjusted to mimic the hydrated, low viscosity of cervicovaginal mucus in ovulatory conditions.

[0655] A solution composed of NaCI at a concentration of 0.07 mg / mL, mucin from porcine gastric origin at a final concentration of 1.2 mg / mL and mucin from submaxillary gland at a final concentration of 1.2 mg / mL was autoclaved. Pectin powder, calcium carbonate powder and delta-gluco lactone powder were sterilized through UV exposure for 30 min followed by 30 min exposure at 50 °C exposure. Pectin powder was added to the mixture to a final concentration of 2.5 mg / mL at a velocity of 220 rpm. Following homogenization, calcium carbonate powder was ultrasonicated for 1 minute, vortexed for 30 seconds and incorporated into the mixture at a final concentration of 0.9 mg / mL at a velocity of 220 rpm. After additional homogenization the delta-gluco lactone powder was vortexed for 30 seconds and subsequently added to a final concentration of 0.36 mg / mL at a velocity of 400 rpm. The obtained viscoelastic properties are the ones shown below. The pH of this formulation was 7.0. Considering these viscoelastic properties the obtained mesh size is 59 nm. See Fig. 23.

[0656] A directional crosslinking step was done to provide the material with a gradient microenvironmental distribution, to recreate an uneven O2distribution through the material for it to be suitable to culture anaerobic species and to provide the material with a biomimetic permeability to the gut membrane. Upon homogenization of the polymeric network, the mixture was deposited onto a surface with previous sterilized and dried calcium gluconate at a concentration of 20 mg / mL. This reduced the mesh size up to 32 nm. See Fig. 24.

[0657] The formulation was then adjusted to mimic a thicker biomimetic material for the cervicovaginal mucus during pregnancy, with presence of small proteins that mimic the presence of antibodies in the pregnancy mucus. A solution composed of NaCI at a concentration of 0.07mg / mL, mucin from porcine gastric origin at a final concentration of 1.5 mg / mL, and mucin from submaxillary gland at a final concentration of 1.5 mg / mL was autoclaved. Pectin powder, calcium carbonate powder and delta-gluco lactone powder were sterilized through UV exposure for 30 min followed by 30 min exposure at 50 °C exposure. Pectin powder was added to the mixture to a final concentration of 5 mg / mL at a velocity of 220 rpm. Following homogenization, albumin was filtered through a 0.22 pm syringe filter and added to the mixture to a final concentration of 5 mg / mL. After homogenization, calcium carbonate powder was ultrasonicated for 1 minute, vortexed for 30 seconds and incorporated into the mixture at a final concentration of 2 mg / mL at a velocity of 220 rpm. After additional homogenization the delta-gluco lactone powder was vortexed for 30 seconds and subsequently added to a final concentration of 0.36 mg / mL at a velocity of 400 rpm. With this new formulation, pH was raised to 8.0 and viscoelastic properties were reduced. The mesh size from this hydrogel is 40 nm. See Fig. 25.

[0658] 4) Mesh size changes through microbiota interactions

[0659] Microbiota from faecal matter isolation was cultured in biomimetic material for up to 72 hours. At each time point the viscoelastic properties were measured and mesh size was calculated through Maxwell Model using room temperature and Boltzmann constant. = ( / c_B T / G')A(l / 3)

[0660] See Fig. 26.

[0661] The biomimetic material proved to maintain structure over time with reduced changes on mesh size. However, this maintenance of mesh size over time needs to be analysed in relation to the remodelling of the structure caused by the increase in abundance of exopolysaccharide producers like Lactobacillus and Bifidobacterium. See Fig. 27

[0662] This was also verified by the confirmation of the activity of these microorganisms by quantification of butyrate over time. See Fig. 28.

[0663] The changes in microbial relative abundance or butyrate concentration are much more substantial than mesh size changes. See Fig. 29. 5) Capacity of biomimetic material to selectively favour microbial growth and its application for microbiome mining

[0664] Microbiota isolates from faecal matter from two donors (D4 and D6) were cultured in the colonlike biomimetic material with 3D gradual microenvironmental structure. This material was fabricated at a final mucin concentration of 254 mg / mL, NaCI 0.57mg / mL, by directional crosslinking from the bottom up using calcium gluconate and BHI as ionic balancing solutions. The resulting viscoelastic properties were of G’ of 634 Pa and 63 Pa.

[0665] After an initial 24-hour stabilization of the microbiota in the biomimetic material with three- dimensional structure, the material was dissolved, and the microbial composition of the sample was assessed through sequencing. This sample was then inoculated into a new sterile biomimetic material with three-dimensional structure and incubated at 37 °C under normoxia for 24 and 48 hours. Following the incubation periods, the biomimetic hydrogels were dissolved, and the microbial composition of the samples was assessed through 16S rRNA amplicon sequencing (V3-V4 region).

[0666] The following two figures show microbial relative abundances at the family level for the microbial community after 24 hours of stabilization in the colon-like microenvironment and then after 24 and 48 hours of subculturing in a new sterile colon-like biomimetic hydrogel.

[0667] For both donors, Lactobacillaceae displayed the most substantial increase in relative abundance. This result suggests the biomimetic hydrogel can selectively favour the growth of exopolysaccharide-producing bacteria in detriment to other generally dominant microbiota species in the gut community like Ruminococaceae.

[0668] This capacity of the biomimetic hydrogel promotes the boosting of probiotic-like species that would contribute to promote health benefits.

[0669] A second finding is related to the increase of Enteroccaceae and Enterobacteriaceae. Both families include species that are classified as opportunistic pathogens in the gut environment and could be considered a health threat if the biomimetic hydrogel was used in the therapeutic context. However, the observed increase of these species could be derived from sample handling and incubation in normoxia, which can be permissive to the growth of aerobic species. It would be expected that when delivered to the gastrointestinal tract in a highly anaerobic environment those species would be reduced in abundance as seen in the stabilization period in both donors. Furthermore, this trait could potentially be helpful when considering in vitro diagnosis aid, in which difficult to detect strains may be amplified in aerobic culture within the biomimetic hydrogel.

[0670] The results obtained clearly show that the tailored microenvironmental gradients created in the biomimetic colon-like hydrogel selectively boost some species from the donor sample. See Figs. 30 and 31.

[0671] In fact, the alpha diversity of the sample within the subcultured gel was reduced with culture time to less than half the initial Hill-Shannon index. This reduction suggests that the new substrate favoured the growth of a narrower subset of species that could efficiently adapt to the new conditions, while many other members of the original community either declined in abundance or were lost entirely. For donor 4, for example, the community shifted from being composed of approximately 60 equally abundant species to the equivalent of only 20, reflecting a dominance of fewer taxa. Such a decrease in effective species number implies that the new substrate imposed selective pressures that reduced community evenness and richness. See Fig. 32.

[0672] In a following experiment, after shaping this biomimetic hydrogel into a 3D sphere like geometry (bead) by extrusion over a 10%w / v CaCI2 solution they were added to a planktonic culture of faecal matter isolates in NaCI 0.09% solution. The samples were then maintained in incubation at 37°C under agitation in a closed system. Limiting the access to 02 from the environment.

[0673] After V3-V4 sequencing and bioinformatic analysis the results show that after 24, 48 and 72h of incubation the most prevalent species adhered to the biomimetic bead were from the Ligilactobacillus, and Limoscilactobacillus genera. Compared to the normoxia cultured held in the three- dimensional substrate with unidirectional shaping crosslinking, the Enterobacteriaceae relative abundance was maintained suggesting that the favourability of the growth of this type of species is related more to the culturing conditions rather than the substrate itself.

[0674] This capacity of the beads to promote adherence of probiotic-like species could be exploited for mining desired species to be then later delivered for a therapeutic or food supplement application or for fundamental research. See Fig. 33.

[0675] 6) Therapeutic use of colon-like biomimetic hydrogel Tailored composition and viscosities from the gastrointestinal mucus-like biomimetic hydrogel may be used for the treatment and recovery of mucosal lesions along the intestinal tract. Such lesions include the characteristics of ulcerative colitis and Crohn’s disease, which are associated with localized or diffuse tissue damage and impaired mucus barrier function. The biomimetic hydrogel described herein is designed to reproduce the rheological, biochemical, and microstructural properties of native colonic mucus, providing a protective and restorative scaffold that promotes epithelial regeneration and local homeostasis.

[0676] The hydrogel composition consists primarily of mucin, alginic salts, and controlled ionic crosslinking systems (e.g., calcium chloride or calcium carbonate with glucono-b-lactone) that yield tunable viscoelastic properties. The mechanical parameters (storage modulus G' and loss modulus G") are modulated through the concentration of mucin and the degree of crosslinking, allowing the hydrogel to adapt to distinct delivery routes and lesion localizations throughout the gastrointestinal tract. The formulations can be prepared as sterile, ready-to- use hydrogels suitable for direct clinical application.

[0677] The material can be delivered as a high-viscosity gel, a spreadable or flowable formulation, or an enema-type viscous solution. Each configuration is optimized for compatibility with standard clinical dosage volumes, injection pressures, and endoscopic or rectal administration devices. The hydrogel’s viscoelasticity ensures that it adheres to mucosal surfaces and remains in place long enough to protect and hydrate the lesion area while releasing mucin and other bioactive components that support the regeneration of the native mucus layer and epithelial repair.

[0678] The following compositions illustrate representative formulations and their corresponding usecases across different intestinal regions:

[0679] 7) Fabrication of stable colonic mucus models in the shape of beads for dynamic studies in faecal matter and for boosting the growth of Akkermansia muciniphila

[0680] A gut-mimicking biomimetic hydrogel bead system (Gut3Beads™) is provided to replicate the physicochemical properties of the colonic mucus layer. This embodiment demonstrates the capacity of the beads to maintain structural stability, reproduce physiological gradients of pH and oxygen, and support the selective growth of mucin-dependent bacterial species such as Akkermansia muciniphila. The system is designed to be compatible with dynamic studies involving faecal matter and to serve as a test platform for nutraceutical or microbiome-related formulations.

[0681] Fabrication of mucus mimicking beads

[0682] A NaCI solution (0.57 mg / mL) was prepared in deionized water. Alginic salt was weighed to a final concentration of 80 mg / mL, sterilized by UV exposure (30 min) and oven drying (50°C, 30 min), and dissolved in the NaCI solution. A mucin solution (28.5 mg / mL in NaCI) was prepared and autoclaved. Both solutions were mixed and homogenized.

[0683] Calcium carbonate was sterilized (UV + oven drying) and weighed to a final concentration of 8.57 mg / mL. Glucono delta-lactone (GDL) was weighed to a final concentration of 14.28 mg / mL. Both powders were dissolved in NaCI (0.57 mg / mL) before addition. The calcium carbonate solution was added to the polymer solution under magnetic stirring for 5 min, followed by addition of the GDL solution with continued stirring for 20 min.

[0684] Calcium chloride solutions (4% w / v and 10% w / v) were prepared in deionized water and autoclaved. After homogenization, the gel was transferred to an 80 mL syringe fitted with a 5.5 mm tip and dispensed via syringe pump into the 4% calcium bath under agitation. After overnight crosslinking, the bath was replaced with 10% calcium chloride and incubated until the following day. The beads were washed with NaCI (0.57 mg / mL), excess liquid was removed, and the beads were stored at 4 °C until use

[0685] Characterization of the beads.

[0686] Given the future use of the beads to propel the growth of selective species within the intestinal microbiota the beads were tested in their capacity to sustain the microenvironment during the testing times.

[0687] 1. Shell stability and thickness

[0688] Different shaping crosslinking protocols were tested to achieve the most homogeneous resistant microenvironment to prevent release of mucin or other molecules from the bead to the external environment. Calcium chloride was stained with Alizarin Red 50mM to characterize the diffusion of the Calcium towards the internal alginate-mucin environment. Later beads were submitted to micrometric oxygen tension measurement to characterize the homogenous

[0689] As shown in the pictures in Fig. 34, a double subsequent crosslinking ensured a more evenly distribution of calcium within the bead and guaranteed even distribution of oxygen and other molecules within the bead.

[0690] 2. Regionally distributed Gut3Beads™ characterization

[0691] Upon fabrication, biomimetic beads were submitted to Unisense micro-measurement of oxygen and pH to confirm homogeneity within the microenvironments within the bead a low oxygen permeability (representative of small mesh size and high crosslinking density). See Fig. 35.

[0692] Average pH across the bead is 6.5 while the oxygen profile decreases from 240 pM / mL to 180 pM / mL linearly through the depth of the bead. This linear decrease is relatable to a homogenous crosslinked structure from the shell to the inner core of the beads.

[0693] 3. Stability in faecal matter

[0694] One of the applications of these beads is to sustain mucin dependent microorganisms while in vitro testing with faecal matter isolates for the development of nutraceuticals or any other biotics. Beads immersed in fecal matter solution under agitation for 48 hours exhibited only ~20% mass loss, confirming their suitability for extended incubations (>24 hours) in dynamic assays involving complex biological matrices. See Fig. 36. Growth boosting of Akkermansia muciniphila

[0695] Cultures of A muciniphila grown in PYG + mucin medium supplemented with Gut3Beads™ demonstrated enhanced viability and biomass accumulation compared to controls without beads. Within one week of incubation, DNA concentration and MTT assay results both indicated higher cell density and metabolic activity in bead-supplemented conditions. The biomimetic beads provided a stable mucin reservoir and microaerophilic gradient that sustained bacterial growth over prolonged culture periods. See Fig. 36.

[0696] Gut3Beads™ serve as a colonic-mimicking platform for microbial culture, stability testing, and functional evaluation of microbiome-targeted products. Potential applications include:

[0697] Dynamic faecal matter studies, enabling long-term in vitro evaluation of nutraceuticals, probiotics, and postbiotics under physiologically relevant conditions.

[0698] Selective cultivation of mucin-dependent species, supporting growth of fastidious bacteria such as Akkermansia muciniphila and other mucus-associated commensals. Bioreactor and production optimization, improving yield and viability of mucinophilic strains for live biotherapeutic or probiotic production.

[0699] Microbiota model development, recreating intestinal gradients of oxygen, pH, and nutrient diffusion for research and industrial applications.

[0700] Nutraceutical and drug screening, assessing effects of bioactives on mucus-dwelling microbiota within a controlled, native-like microenvironment.

[0701] Predicted advantages include long-term stability in complex media, tuneable oxygen gradients, reproducibility, and compatibility with both laboratory-scale and industrial-scale microbiome culture systems.

[0702] 8) Cervicovaginal mimicking beads for selective mining from cervicovaginal isolate

[0703] A biomimetic material is provided in the form of cervicovaginal mucus-mimicking beads designed to selectively promote the growth of beneficial bacterial species present in cervicovaginal microbiota isolates. This embodiment demonstrates the capacity of the biomimetic material to recreate key physicochemical properties of the cervicovaginal environment — such as pH, mucin content, and ionic composition — thereby favouring the enrichment of Lactobacillus species from mixed microbial populations. To validate the capacity of the biomimetic material to selectively promote the growth of desired bacterial species. A suspension of isolated microbiota from exudates from the cervicovaginal canal was cultured planktonically with the supplementation of cervicovaginal-like biomimetic material in the shape of a bead. For this purpose, the biomimetic material for the cervicovaginal mucus was directionally crosslinked from the outside by extrusion into a CaCI2bath.

[0704] In summary, 10% volume of the culture was added in the shape of beads to an MRS planktonic culture containing isolate collected through a menstrual cup. Upon cultivation, the biomimetic beads were collected and dissolved for posterior DNA extraction. This was followed by 16S rRNA amplicon sequencing (V3-V4 region) after incubation, after 24, 48 and 72 h.

[0705] The initial microbial population within the cervicovaginal isolate exhibited broad taxonomic diversity, consistent with a heterogeneous community typical of the vaginal microbiome. Following incubation in the presence of cervicovaginal-like beads, a marked enrichment of Lactobacillus species was observed within the hydrogel-associated fraction.

[0706] Lactobacillus crispatus emerged as the dominant species after 72 hours, indicating that the biomimetic environment selectively favoured its proliferation. This selective enrichment suggests that the pH, mucin concentration, and diffusion-limited microenvironment of the beads recreated the physicochemical conditions characteristic of the healthy cervicovaginal niche.

[0707] These results support the hypothesis that the biomimetic beads act not only as a culture matrix but also as a selective mining tool, isolating beneficial Lactobacillus species from mixed cervicovaginal microbial communities. See Fig. 37.

[0708] The cervicovaginal mimicking beads can be used as a platform for:

[0709] Selective isolation and enrichment of Lactobacillus species and other beneficial microorganisms from complex cervicovaginal microbiota samples.

[0710] In vitro modelling of the cervicovaginal environment, enabling the study of microbial interactions and host-microbe dynamics under physiologically relevant conditions. Development of next-generation vaginal probiotics, leveraging the selective growth properties of the biomimetic matrix to cultivate and stabilize health-associated bacterial strains. Microbiome mining and strain discovery, facilitating the identification and propagation of candidate probiotic species for therapeutic and industrial use.

[0711] Production applications, supporting the cultivation and yield optimization of vaginal probiotic strains for large-scale production and commercialization.

[0712] Predicted advantages include selective growth of beneficial Lactobacillus species, compatibility with microbial consortia, and the ability to replicate natural mucosal environments for both research and manufacturing purposes.

[0713] 9) Use of colonic mimicking material in the shape of a bead to propel the growth of Veillonella parvula

[0714] A colonic-mimicking biomimetic hydrogel is provided in the form of spherical beads that replicate the physicochemical properties of the human colon mucus. This embodiment demonstrates that the biomimetic beads can support the growth of anaerobic and slow- growing microorganisms, such as Veillonella parvula, by providing a structured, nutrientcompatible, and microaerophilic microenvironment.

[0715] Fabrication of colon mucus biomimetic material in the shape of a bead

[0716] A NaCI solution (0.57 mg / mL) was prepared in deionized water. Alginic salt was weighed, sterilized by UV exposure (30 min) and oven drying (50 °C, 30 min), and dissolved in the NaCI solution to a final concentration of 80 mg / mL. A mucin solution (28.5 mg / mL in NaCI) was prepared and autoclaved. Both solutions were mixed and homogenized.

[0717] Calcium carbonate was sterilized (UV + oven drying) and weighed to a final concentration of 8.57 mg / mL. Glucono delta-lactone (GDL) was weighed to a final concentration of 14.28 mg / mL. Both powders were dissolved in NaCI (0.57 mg / mL) before addition. The calcium carbonate solution was added to the polymer solution under magnetic stirring for 5 min, followed by addition of the GDL solution with continued stirring for 20 min.

[0718] Calcium chloride solutions (4% w / v and 10% w / v) were prepared in deionized water and autoclaved. After homogenization, the gel was transferred to an 80 mL syringe fitted with a 5.5 mm tip and dispensed via syringe pump into the 4% calcium bath under agitation. After overnight crosslinking, the bath was replaced with 10% calcium chloride and incubated until the following day. The beads were washed with NaCI (0.57 mg / mL), excess liquid was removed, and the beads were stored at 4 °C until use

[0719] Use of beads as additive to propel the growth of Veillonella parvula

[0720] Veillonella parvula was grown in PyG medium with and without beads supplementation. Briefly, 10% volume of beads were added to a planktonic culture of V. parvula and put in incubation under mild agitation. At determined timepoints, metabolic activity was measured, and the DNA concentration was quantified.

[0721] Cultures of V. parvula supplemented with the biomimetic beads showed a pronounced increase in both DNA concentration and metabolic activity compared to controls. After 72 hours, bacterial growth in the Beads group surpassed that of the control, reaching a peak DNA concentration of approximately 285 ng / pL after one week — whereas the control cultures remained below 41 ng / pL. Elevated DNA levels were maintained after two weeks, indicating sustained bacterial growth within the bead-associated environment. See Fig. 38.

[0722] MTT assay data further confirmed enhanced cell viability in the Beads group, aligning with the DNA quantification trends. At both the one- and two-week timepoints, viability in the Beads group was significantly higher than in controls, demonstrating the ability of the biomimetic beads to maintain bacterial metabolic activity over time. See Fig. 39.

[0723] Macroscopic inspection showed only slight surface degradation of the beads after prolonged incubation, indicating mechanical and structural resilience under culture conditions. These results collectively confirm that the biomimetic colonic hydrogel beads create a favorable environment for V. parvula proliferation and sustained viability. See Fig. 40.

[0724] This biomimetic system provides a tuneable and physiologically relevant platform for the cultivation of mucin-dependent or anaerobic microorganisms that are otherwise challenging to grow in standard media. Potential applications include:

[0725] In vitro microbiota modelling, supporting the growth of fastidious commensals under colon-like conditions

[0726] Microbial bioproduction or screening, enabling cultivation of species involved in short-chain fatty acid and metabolite production

[0727] Microbiome therapeutic development, serving as a culture or delivery matrix for live biotherapeutic products (LBPs) Prebiotic research and microbiome mining, facilitating the enrichment and isolation of beneficial but difficult-to-culture gut species

[0728] Boosting production and yield of hard-to-culture probiotic bacteria within producers, improving process scalability and strain recovery efficiency.

[0729] Expansion of commercially available probiotic strains, enabling producers to broaden their product portfolios by cultivating novel, previously non-culturable or low-yield bacterial species.

[0730] Predicted advantages include prolonged bacterial viability, maintenance of physiological oxygen gradients, and structural stability compatible with extended culture periods. This embodiment demonstrates that colonic-mimicking biomimetic beads can sustain and enhance the growth of Veillonella parvula, validating their use as functional microbial culture and delivery platforms.

[0731] 10) Probiotic effect of biomimetic material: Delivery of mucin and polysaccharides for mucosa restoration and microbiota re-balance. Pre-digestion protocol and probiotic effect evaluation

[0732] A gut-mimicking biomimetic material bead system is provided for the delivery of mucin and polysaccharides to the intestinal environment. This embodiment demonstrates that the biomimetic material can withstand gastrointestinal digestion conditions, adhere to intestinal mucus layers, and modulate microbial community composition in vitro. The hydrogel beads are designed to act both as delivery vehicles and as prebiotics capable of supporting microbiota rebalance and mucosal restoration.

[0733] Fabrication of Beads with gut mucus mimicking characteristics

[0734] A NaCI solution (0.57 mg / mL) was prepared in deionized water. Alginic salt was weighed to a final concentration of 80 mg / mL, sterilized by UV exposure (30 min) and oven drying (50 °C, 30 min), and dissolved in the NaCI solution. Separately, a mucin solution (28.5 mg / mL in NaCI) was prepared and autoclaved. Both solutions were mixed and homogenized.

[0735] Calcium carbonate was sterilized (UV + oven drying) and weighed to a final concentration of 8.57 mg / mL. Glucono delta-lactone (GDL) was weighed to a final concentration of 14.28 mg / mL. Both powders were dissolved in NaCI (0.57 mg / mL) before addition. The calcium carbonate solution was added to the polymer solution under magnetic stirring for 5 min, followed by addition of the GDL solution with continued stirring for 20 min. Calcium chloride solutions (4% w / v and 10% w / v) were prepared in deionized water and autoclaved. After homogenization, the gel was transferred to an 80 mL syringe fitted with a 5.5 mm tip and dispensed via syringe pump into the 4% calcium bath under agitation. After overnight crosslinking, the bath was replaced with 10% calcium chloride and incubated until the following day.

[0736] The beads were washed with NaCI (0.57 mg / mL), excess liquid was removed, and stored at 4 °C until use

[0737] Characterization of delivery to the gut and persistence in the mucosal layer

[0738] A predigestion protocol was conducted over a batch of gut-mimicking biomimetic beads. Briefly, a known number of Biomimetic beads, weighted and characterized through Unisense for uniformity and microenvironmental conditions, was immersed in enzyme solutions that mimic the different regions of the gastrointestinal tract in a serial manner like this:

[0739] 1. Amylase (75 U / mL) pH 7 for 0.5 h

[0740] 2. Pepsin (2000 U / mL) pH 2 for 0.5 h

[0741] 3. Pepsin (2000 U / mL) pH 7 for 2 h

[0742] 4. Pancreatin (100 U / mL) pH 7 for 2 h

[0743] Solutions were prepared in an isotonic sodium chloride solution to the beads and the full predigestion protocol was conducted under sterility and incubation was performed at 37°C.

[0744] The biomimetic beads remained structurally stable throughout the simulated digestion sequence, with no significant mass loss observed. Minor weight fluctuations were attributed to reversible hydration and swelling. See Fig. 41.

[0745] Microsensor profiling revealed that both pH and oxygen diffusion gradients were preserved, confirming maintenance of internal microenvironmental structure. See Fig. 42.

[0746] This can be confirmed by the oxygen profiling through the beads. It is evident that despite the treatment, the linear profile of oxygen tension is maintained which indicates that through the depth of the bead there has not been significant disruption. See Fig. 43.

[0747] Later, beads were tested in their capacity to be adhered to the mucus layers of the intestine. Mucin-containing beads were placed on porcine intestinal sheets for 6 h at 37 °C, followed by washing with NaCI solution using a controlled flow rate to evaluate bead adhesion to mucus surfaces. After confirming the resistance to NaCI solution washing, the intestine sheet was turned upside down inside the NaCI solution. See Fig. 44.

[0748] The Biomimetic beads (Gut3Beads™) demonstrated strong mucoadhesion to intestinal tissue, remaining attached even after inversion and washing, indicating robust mucus-layer affinity. Gut3Beads™ can potentially adhere to the mucus layers of the human gut for the delivery of bioactive molecules and have prebiotic action.

[0749] Characterization of the changes in the community of bacteria in-vitro upon treatment with Beads

[0750] Microbiota isolates from two donors (donor 6 - D6; and donor 11 - D11) were cultivated in gut mimicking hydrogel tailored to have physicochemical characteristics that resemble those from the human colon. After 24 hours of stabilization in the hydrogel, these microbial communities were supplemented with Gut3Beads™ in a 10% v / v ratio with BHI media to determine their prebiotic activity.

[0751] After 24, 48 and 72 hours the microbial composition in the gut mimicking model was assessed using 16S rRNA amplicon sequencing (V3 - V4 region).

[0752] Principal Coordinates Analysis (PCoA) based on weighted UniFrac distances (based on abundance and phylogeny) reveals a clear separation of microbial communities by donor. The addition of beads produced moderate shifts in community composition, which were more evident for donor 6. Additionally, the presence of beads resulted in more pronounced community composition shifts for donor 6. Within each condition, temporal progression from 0 to 72 hours indicated gradual community changes, particularly in samples without beads supplementation. See Fig. 45.

[0753] Cultivation of donor 11 (D11) intestinal microbiota in the gut mimicking model resulted in increased relative abundance of Lactobacillaceae species in the samples supplemented with beads. Species from the Lactobacillaceae family are known for their ability to produce lactate and antimicrobials that control the proliferation of other microbes and increase butyrate levels. Butyrate is a product that serves as an essential nutrient for the metabolic processes of mucus remodelling microorganisms that depend on its availability for the successful completion of metabolic function. Concomitantly, the selective growth of Lactobacillaceae promotes the reduction of Mycobacteriaceae levels. This family of bacteria includes opportunistic pathogens with pro-inflammatory activity commonly present in immunosuppressed populations. When supplemented with beads, the microbiota from donor 6 displayed an increase in the relative abundance of Bifidobacterium, Lactobacillus, Ligilactobacillus, and Limosilactobacillus. These bacterial genera include probiotic species that thrive with the supplementation of fibers to produce fermentation products that modulate inflammation, stabilize the mucosal barrier and metabolites that serve as nutrients to other species of microorganisms that also have health promoting properties. Additionally, Ligilactobacillus species, such as L. salivarius, support gut health by strengthening the intestinal barrier, by producing antimicrobial compounds that inhibit pathogens, and by modulating immune responses to reduce inflammation. As for Limosilactobacillus species, L. reuteri and L. fermentum are particularly well known for producing reuterin, a broad-spectrum antimicrobial, as well as for promoting microbiota balance, improving metabolic health, and supporting women’s vaginal health. Together, these genera contribute to maintaining a resilient gut ecosystem and protecting against infections while also offering systemic immunomodulatory benefits.

[0754] For donor 6 (D6), an extra experimental group was included to control for the structural effect of the beads. In this case, the intestinal microbiota was supplemented with 10 % v / v mucin. In contrast to beads supplementation, mucin did not promote an increase in relative abundance of Bifidobacterium, and Lactobacillaceae species, suggesting that the beads’ structural and chemical properties promote the proliferation of probiotic species. Consequently, a potential application of the 3D structured gut-like hydrogel is microbiome mining. This is also supported by the strong enrichment of Ligilactobacillus and Limosilactobacillus species that migrated from the microbiota seeded in the biomimetic material towards the beads, as verified through sequencing (Beads_D6). See Fig. 46.

[0755] The results mentioned above provide evidence of the potential use of this 3D structure as a prebiotic supplementation device with therapeutic or supplementation potential. Indeed, the gut-mimicking biomimetic beads can serve as prebiotic capable of promoting microbiota balance and mucosal regeneration. Potential uses include:

[0756] - Therapeutic mucosal restoration, enhancing epithelial barrier repair and mucus layer renewal

[0757] Prebiotic supplementation, fostering growth of beneficial bacterial species while suppressing opportunistic pathogens

[0758] Microbiome mining and biotherapeutic discovery, enabling selection of probiotic or commensal strains under mucus-like conditions Controlled delivery of bioactive molecules and probiotics within intestinal environments Retention or boost of mucus-associated microbiota in in vitro dynamic gut simulators. Predicted advantages include resistance to digestion, strong mucoadhesion, biocompatibility, and selective stimulation of beneficial microbiota. These results collectively support the use of mucus-mimicking hydrogel beads as next-generation probiotic and prebiotic delivery systems with therapeutic potential.

[0759] 11) Biomimetic Bead as Delivery system for Spermidine

[0760] A biomimetic material shaped as bead is provided for the encapsulation and controlled delivery of bioactive or nutraceutical molecules such as spermidine. This embodiment demonstrates the ability of the biomimetic material to create a stable, nutrient-rich microenvironment suitable for both supporting microbial growth and enabling the gradual release of bioactive compounds.

[0761] The system is designed to emulate gut-like physicochemical conditions, providing a compatible habitat for mucin-dependent and slow-growing microorganisms while simultaneously acting as a carrier for postbiotic or prebiotic molecules.

[0762] Fabrication of Biomimetic Bead with encapsulated Spermidine

[0763] In Phase I, a homogeneous aqueous formulation containing mucin (50 mg / mL), sodium alginate (80 mg / mL), and calcium carbonate (60 mg / mL) was prepared. A solution of the postbiotic compound spermidine (commercially available as Sprevive®) was incorporated into this mixture at a final concentration of approximately 35 mg / mL. Internal crosslinking was initiated through gradual acidification by adding glucono-5-lactone (GDL) at 100 mg / mL.

[0764] In Phase II, this mixture was shaped into spherical beads by droplet extrusion into a 4% (w / v) calcium chloride solution, establishing a stable bead geometry with a defined microenvironmental gradient.

[0765] To enhance stability and modulate the diffusion profile of spermidine, a Phase III reinforcement step was added by immersing the preformed beads in a 10% (w / v) calcium chloride solution, increasing crosslinking density at the periphery and generating a multilayered matrix with regionally distributed microenvironments.

[0766] Validation of Gut3Beads™ with encapsulated spermidine After washing with isotonic saline, the resulting biomimetic beads were used as growth supplements in cultures of common and hard-to-grow bacterial species, including Lactobacillus paracasei and Akkermansia muciniphila. Both supplementation with Biomimetic beads and spermidine-loaded Biomimetic beads increased metabolic activity of the cultured bacteria over time.

[0767] Both standard (non-supplemented) and spermidine-loaded biomimetic beads successfully supported microbial culture. The inclusion of spermidine within the biomimetic material did not interfere with bead formation or structural stability.

[0768] Cultures supplemented with either type of bead exhibited a gradual increase in metabolic activity over time compared to control conditions without beads, as indicated by visible metabolic activity.

[0769] These results confirm that the biomimetic bead system can stably encapsulate spermidine and provide it to microbial cultures in a controlled manner, maintaining both molecular integrity and bioactivity within the 3D matrix. See Fig. 47.

[0770] This multilayer biomimetic bead design demonstrates the feasibility of incorporating nutraceutical molecules into mucus-mimicking hydrogels for microbiome-related applications. Potential uses include:

[0771] Microbiome nutrition studies to evaluate postbiotic effects on mucin-dependent or slow-growing species

[0772] Targeted delivery of bioactive compounds to microbial ecosystems in vitro or in vivo

[0773] Co-encapsulation platforms for combined microbial and molecular delivery under physiologically relevant conditions

[0774] Predicted advantages include tuneable release kinetics, compatibility with diverse bioactive additives, structural robustness during handling, and maintenance of microbial viability. This embodiment highlights the use of mucin-alginate matrices as smart delivery systems bridging microbial support and controlled nutraceutical release. 12) Encapsulation of Akkermancia muciniphila

[0775] A three-dimensional biomimetic material is provided for the encapsulation of Akkermansia muciniphila, a mucin-dependent bacterium associated with anti-inflammatory and mucus- regenerative pathways in the gastrointestinal tract. This embodiment addresses the challenges of cultivating and maintaining A. muciniphila, whose viability strongly depends on mucin availability and tightly regulated microenvironmental conditions such as oxygen tension, pH, and ionic composition.

[0776] By embedding A. muciniphila within a gut-like biomimetic matrix containing mucin and polysaccharide components, it is expected that the hydrogel will provide a supportive microenvironment that mimics the native mucus layer, thereby sustaining bacterial metabolism and viability over extended periods.

[0777] Production of biomimetic material encapsulating A. muciniphila

[0778] An inoculum of Akkermansia muciniphila was prepared by activating the strain in PYG + 0.01 % medium for 48 hours. After this time, the inoculum was centrifuged and resuspended to a final concentration of 109cells / ml in a mucin solution (28.5 mg / mL) prepared in sterile saline (0.57 mg / mL). To this bacterial-mucin suspension, an alginic salt solution was added to reach a final alginate concentration of approximately 11.6 mg / mL. After homogenization, calcium carbonate (8.57 mg / mL) was incorporated, followed by the addition of glucono-5- lactone (12.28 mg / mL). The mixture was then left to crosslink for 5 minutes (Phase I).

[0779] In Phase II, the shaping step was performed by creating droplets into 4% CaCI solution and left crosslinking for 3 minutes, after which they were collected and washed in an isotonic saline solution to remove unbound ions.

[0780] The resulting biomimetic beads maintained their structure and the viability of encapsulated A. muciniphila in a mucin-rich microenvironment that promotes metabolic stability and prevents rapid decline due to storage or environmental stress.

[0781] This encapsulation strategy provides a foundation for stabilizing A. muciniphila in both research and therapeutic contexts. The encapsulated form is envisioned for use in: microbiome research models, where it can simulate mucus-microbe interactions; probiotic and biotherapeutic formulations, offering a viable platform for oral or colonic delivery of A. muciniphila and other mucus-associated microorganisms, while protecting cells from oxygen exposure and gastrointestinal stress; cryopreservation and transport, leveraging the mucinrich hydrogel as a protective carrier for maintaining bacterial viability during storage and shipment.

[0782] Predicted advantages include sustained bacterial viability, controlled release in gut-like environments, and preservation of the functional mucin-microbe interface. This embodiment demonstrates the feasibility of using mucin-based biomimetic hydrogels to host and stabilize mucinophilic microorganisms that are otherwise difficult to culture and handle under standard laboratory conditions.

[0783] 13) Encapsulated Lactobacillus plantarum and stability in pectin-based biomimetic material

[0784] A biomimetic three-dimensional hydrogel structure is provided for the encapsulation and stabilization of Lactobacillus plantarum within a cervico-vaginal-like biomimetic material in the shape of a bead. The formulation aims to preserve microbial viability during storage and simulate mucosal conditions relevant to probiotic delivery and microbiota modulation.

[0785] Manufacturing of cervicovaginal-like biomimetic mucus model encapsulating L. plantarum

[0786] Briefly, an inoculum of L. plantarum was activated in MRS medium. The next day, this inoculum was diluted to a final concentration of 107 CFU / mL and mixed within a biomimetic gel formulation. This mixing was performed by mixing a pectin solution at a concentration of 10 mg / mL and resuspended lactobacillus in acidic saline solution. For bead shaping this gel structure was immersed in droplets into a CaCI solution at a 10% w / v concentration. After 3 minutes such beads containing alive microorganisms were washed with NaCI solution 0.07mg / mL and left for incubation at room temperature and at 37°C.

[0787] An inoculum of Lactobacillus plantarum was activated overnight in MRS culture medium. The following day, the culture was diluted to a final concentration of 1 x 107CFU / mL and mixed into a pectin-based biomimetic gel formulation.

[0788] The biomimetic material was prepared by dissolving pectin at a concentration of 10 mg / mL in an acidic saline solution, followed by incorporation of the bacterial suspension (Phase I). For shaping the biomimetic material in the bead form, the mixture was immersed in droplets into a 10 % (w / v) calcium chloride solution. The mixture was then extruded dropwise into a 10 % (w / v) calcium chloride crosslinking bath for approximately three minutes to induce gelation and form spherical hydrogel beads encapsulating viable microorganisms (Phase II).

[0789] After gelation, the beads were washed with 0.07 mg / mL sodium chloride solution and transferred to incubation media (MRS). The quantity of viable microorganisms both in planktonic conditions and encapsulated was measured through CFU plating. Encapsulated L. plantarum maintained comparable or higher viability than planktonic cultures, confirming the protective effect of the biomimetic matrix. See Fig. 48.

[0790] Vitality verification after storage and incubation

[0791] The encapsulation of this microorganism within the biomimetic material conferred protection and vitality was expanded over prolonged periods of time, supporting the bio-holding capacity to sustain life over shelf life. For this purpose, long-term vitality assays demonstrated sustained microbial survival after storage at room temperature and at 4 °C for periods of 24h, and after 1 week and 3 weeks. DNA quantification and metabolic activity indicated that the encapsulation of L. plantarum in the biomimetic material shaped as beads preserved both viability and metabolic activity over time. See Fig. 49.

[0792] Evaluation of the protection capacity of the microorganisms against the gastrointestinal tract

[0793] When exposed to simulated gastrointestinal conditions, including pancreatin and fecal matter solutions, the encapsulated microorganisms are expected to exhibit markedly higher survival rates compared to free cells. Live / dead staining and fluorescence microscopy confirmed that pectin-based biomimetic beads provided an effective barrier against digestive stress, maintaining bacterial integrity and viability. See Fig. 50.

[0794] This embodiment can be used for the development of probiotic delivery systems designed to protect beneficial bacteria during storage and transit through the gastrointestinal tract or other mucosal environments.

[0795] Predicted benefits include enhanced microbial stability under variable temperature conditions; protection of viability during gastrointestinal transit; controlled release of viable bacteria at target mucosal sites; applicability to vaginal, oral, or intestinal probiotic formulations This example demonstrates the potential of pectin-based biomimetic beads as stable carriers for live microorganisms in therapeutic and microbiome-modulating applications.

[0796] 14) Encapsulation of active therapeutic molecules within three-dimensional biomimetic material

[0797] A three-dimensional biomimetic material is provided in the form of beads for the controlled release of therapeutic molecules in microbial environments. The biomimetic material reproduces the physical and chemical composition of mucus and allows the localized and sustained release of antibiotic or bioactive compounds.

[0798] The biomimetic beads were produced using a mucin-alginate-based hydrogel formulation, like the colon mucus-like biomimetic material. The active compound — such as indomethacin, an antibiotic known to inhibit Escherichia coli growth — was incorporated directly into the waterbased suspension that is added mixed with the polymeric solution to form the mother solution for the construction of the biomimetic material (Phase I).

[0799] In Phase II, the resulting suspension was submitted to directional crosslinking by exposure to calcium chloride solution for 3 minutes, promoting ionic gelation and formation of spherical beads. The beads were then washed in an isotonic balancing solution to remove unbound surface molecules and equilibrate osmotic conditions.

[0800] After fabrication, the biomimetic beads were immersed in an isotonic balancing solution to initiate the diffusion and release of the antibiotic into the surrounding medium. Samples of the release medium were collected at predetermined time intervals and stored at 4 °C for subsequent analysis.

[0801] Following 24 hours of stabilization of Escherichia coli planktonic cultures, aliquots of the collected release samples were added to the cultures to evaluate the inhibitory effect of the released antibiotic on bacterial growth. See Fig. 51.

[0802] The release of the antibiotic from the biomimetic beads was detectable after approximately 3 hours and reached its maximum concentration around 20 hours. This release profile was reflected in the biological response of Escherichia coli cultures, where a reduction in colonyforming units (CFU) was observed, indicating an inhibitory effect attributable to the antibiotic diffusing from the beads. At this stage, the concentration of the released antibiotic was sufficient to suppress bacterial growth relative to the expected proliferation observed in untreated culture media.

[0803] This biomimetic delivery platform can be used for studying drug-microbiota and hostmicrobiome interactions under physiologically relevant mucus-like environments; screening and optimizing controlled-release formulations for antibiotics, peptides, and small molecules; developing in vitro models for infection dynamics, antimicrobial resistance testing, or microbiota-targeted therapies.

[0804] 15) Modification of biomimetic 3D structure to include microenvironment stabilizer for cryopreservation

[0805] A biomimetic three-dimensional biomimetic material is provided in the form of beads capable of maintaining the viability of encapsulated microorganisms during cryogenic storage. The embodiment includes a cryoprotective additive integrated into the biomimetic material to stabilize the internal microenvironment and prevent damage associated with ice crystal formation and osmotic stress.

[0806] A modified formulation was produced with adding 10% glycerol to the standard biomimetic formulation based on alginic salt, calcium carbonate and gluco-delta-lactone. The biomimetic material was then shaped in the form of beads.

[0807] Upon manufacturing, the glycerol-containing biomimetic beads were submitted to different preservation conditions overnight at 4°C, -20° C and -80°C and both their shape and oxygen distribution was determined.

[0808] After storage under the different temperature conditions, the glycerol-containing biomimetic beads exhibited stable spherical morphology across all tested temperatures, with no visible cracking or surface collapse. See Fig. 52.

[0809] Unisense microsensor readings showed that oxygen tension across the thickness of glycerolcontaining biomimetic beads remained constant throughout all tested temperatures ranging from approximately 253 to 235 pmol / L. This was also observed for biomimetic beads stored at 4°C in the absence of glycerol, though lower temperatures affected their integrity showcasing the advantage of including glycerol or other cryoprotectants on the biomimetic beads formulations. See Fig. 53 Storage at 4°C, Fig. 54 Storage at -20°C and Fig. 55 Storage at -80°C. The cryostabilized biomimetic beads may be used as carriers for microbial storage, transport, and biotechnological inoculation. Potential applications include long-term preservation of probiotics, anaerobic consortia, or engineered microbial systems for research, pharmaceutical, or agricultural purposes.

[0810] Predicted benefits include improved viability and recovery of microorganisms after thawing; maintenance of oxygen and ionic gradients within the bead microenvironment; prevention of matrix cracking and internal ice formation; compatibility with scalable fabrication and automated cryostorage workflows.

[0811] This embodiment demonstrates a simple and effective modification of a biomimetic material that enables robust cryopreservation without compromising its biological performance or structural fidelity.

[0812] 16) Alginate-based hydrogel beads for controlled release of nanoparticles and nanofertilizers

[0813] An alginate-based three-dimensional carrier is provided for the encapsulation and controlled release of nanoparticles and nano-fertilizers. The carrier will be formed as spherical hydrogel beads with probiotic and nutrient-delivery functionality. This embodiment is intended to improve nutrient bioavailability and reduce nutrient losses in agricultural applications while maintaining soil microbial balance.

[0814] The hydrogel beads will be fabricated through a two-phase process.

[0815] In Phase I of the manufacturing process, a sodium alginate solution will be prepared, and bioactive nanoparticles will be dispersed uniformly in the water-based suspension using continuous stirring. In Phase II, the nanoparticle-alginate mixture will then be extruded dropwise into a calcium chloride bath for multi directional crosslinking, forming spherical hydrogel beads via ionic crosslinking.

[0816] After curing and washing, the beads will be dried at 40 °C or freeze-dried for storage stability.

[0817] These beads will achieve encapsulation efficiencies of 75-90% and exhibit swelling in soil moisture. Nutrient release will follow concentration gradient diffusion initially and matrix erosion at later stages, maintaining nutrient availability longer than the traditional fertilizers. When applied to soil, the beads will gradually release encapsulated nutrients, such as Zn, Fe, or N, enhancing root uptake efficiency and reducing nutrient losses compared to conventional fertilizers.

[0818] The system will be adaptable to various nanoformulations including nano-urea for nitrogen delivery, Fe3O4nanoparticles for iron supplementation, and SiO2nanoparticles for stress resistance.

[0819] Furthermore, in a multiple tailored micro-environmental design, the beads will co-encapsulate beneficial microorganisms like Rhizobium and Azospirillum, creating bio-nano hybrid fertilizers that will enhance soil fertility and microbiome stability. Modifications to the alginate matrix, such as the inclusion of polyvinyl alcohol (PVA), humic acid, or starch, will adjust mechanical strength and release profiles. The release rate will be tuneable by varying alginate concentration, calcium crosslinking density, or by adding sodium and potassium ions to emulate natural soil ionic conditions.

[0820] Predicted advantages will include sustained nutrient release, minimized nanoparticle toxicity, prevention of aggregation, and complete biodegradability, reducing environmental impact. The proposed manufacturing process will be simple, scalable, and cost-effective, making it suitable for industrial production and large-scale agricultural use.

[0821] The resulting alginate-based nanocarriers will promote efficient nutrient absorption, maintain soil microbial health, and safeguard groundwater quality through controlled, eco-friendly release mechanisms. Their tuneable design will allow adaptation to diverse crops, soils, and climatic conditions, establishing alginate hydrogels as next-generation smart fertilizer platforms.

[0822] 17) Three-dimensional gastrointestinal-like biomimetic material in bead shape for mining microbiota from fecal matter, that can be cultured and reimplanted in the donor for microbiota rebalancing

[0823] A three-dimensional gastrointestinal-like biomimetic material is provided for the selective capture, enrichment, and culture of commensal microorganisms derived from faecal matter. The material is designed to simulate the biochemical and structural characteristics of the intestinal mucosa, thereby promoting the adhesion and proliferation of beneficial microbial species. The enriched microbiota can subsequently be reintroduced into the original donor to restore microbial balance and enhance mucosal health. The three-dimensional gastrointestinal-like biomimetic material was prepared following the traditional method considering 25 mg / mL mucin and 0.57 mg / mL ionic balancing solution (Phase I). To sustain the harsh conditions of faecal matter incubation, the mixture was subjected to directional dual crosslinking in a calcium chloride bath for 12 hours per cycle to produce spherical beads of approximately 2-3 mm in diameter. Following crosslinking, the beads were washed three times with sterile physiological saline and stored at 4°C prior to use.

[0824] Faecal matter was collected under sterile conditions according to standard procedures. Immediately following defecation, a ratio of 30 g of faecal matter per 150 mL of sterile 0.9% NaCI solution was homogenised using a blender. The homogenate was filtered through a sterile mesh to remove coarse particulates and aliquoted into 50 mL conical tubes.

[0825] The biomimetic material in bead shape was added to the faecal suspension at a concentration of 10% v / v, and the mixture was incubated at 37°C. After 24, 48 and 72h the microbiota inside of the beads was sequenced to determine the evolution of the community within and the capacity of the beads to attract the desired species and promote the growth of commensal species. See Fig. 56.

[0826] Microbial communities residing within the beads were analysed by 16S rRNA gene sequencing and compared to the free-living communities in the surrounding medium. The results demonstrated a selective enrichment of bacterial species belonging to the genera Ligilactobacillus and Limoscilactobacillus within the beads.

[0827] In therapeutic use, the microbiota-enriched beads may be rectally delivered into the donor as an autologous microbiota restoration treatment. Because the microbial species are native to the host, engraftment efficiency is expected to be higher, reducing immune rejection and promoting long-term microbiota stability.

[0828] 18) Controlled Release in multiple microenvironment substrate (Beads within Disks)

[0829] A biomimetic material is provided that enables controlled release of bioactive agents through the integration of multiple microenvironments within a single continuous structure. This configuration allows localized modulation of molecular diffusion and mimics the physicochemical gradients present in biological mucus layers. Fabrication of Multiple Microenvironments Biomimetic Device for Controlled Molecule Release

[0830] The biomimetic substrate comprises a polymeric network formed from one or more biocompatible hydrogel-forming polymers, such as alginic salts, gelatin, collagen, or pectin, dispersed within an aqueous medium containing mucin at concentrations between 10 mg / mL and 30 mg / mL. The polymeric components are selected and combined to achieve controlled hydration, ionic balance, and elastic mechanical properties compatible with mucosal tissues.

[0831] The microenvironmental differentiation within the substrate is achieved through spatially controlled crosslinking, producing discrete yet interconnected zones that differ in ionic strength, crosslink density, or polymer composition. This can be accomplished through sequential or gradient deposition of crosslinking agents, including calcium ions (for ionic crosslinking) and gluco-5-lactone (for controlled internal activation).

[0832] Each region thus created exhibits a distinct permeability coefficient, allowing region-specific release rates of encapsulated molecules while maintaining overall structural continuity of the hydrogel network.

[0833] In this example, a biomimetic material (Biomimetic material A) was prepared by mixing alginate solution (40 mg / mL), calcium carbonate (0.25 mg / mL) and gluco-delta-lactone (2.5 mg / mL) with the addition of food colouring (Phase I). It was later shaped into beads by second phase crosslinking through immersion of droplets in calcium chloride 10% w / v for 3 minutes (Phase II).

[0834] In parallel a biomimetic material was prepared mixing alginic salt solution to a final concentration of 32 mg / mL, 0.3 mg / mL calcium carbonate and 3mg / mL gluco-delta-lactone and let homogenize for 3 minutes (Biomimetic material B).

[0835] Then the device was assembled by preparing a mold with a base of 100 pL of calcium chloride 10% solution, pouring 1 mL of the second biomimetic material (B), manually dispersing 3 units of beads, covering with a second layer extra 1 mL of biomimetic material B. Upon which extra 100 pL of calcium chloride 10% was added on top. For a final conformation of an encapsulated number of Biomimetic material A in the shape of beads within a Biomimetic material B.

[0836] Incorporation and Release of the Active Component

[0837] The bioactive compound or model therapeutic molecule is uniformly distributed or selectively localized within one or more regions of the substrate prior to or during crosslinking. Encapsulation can occur either during Phase I network formation (bulk incorporation) or Phase II directional structuring (layer-specific inclusion).

[0838] Using a USB microscope, a picture was taken every hour to evaluate the diffusion of the red coloured molecule contained in Biomimetic material A towards the external environment (Biomimetic material B). See Fig. 57.

[0839] The higher crosslinking density on the shell from the Biomimetic material B contains the diffused molecule while it is being released through time from the Biomimetic material A.

[0840] These pictures were then analysed to quantify the amount of red dye released from the Biomimetic material A to the Biomimetic material B. A histogram of intensity for red colour was created for a region outside of the beads (specifically in the Biomimetic material B). See Fig. 58.

[0841] The evolution of the curve with steeper peak in the pure red (255 from RGD colour) demonstrated the change in the contained molecules within the polymeric network of material A. See Fig. 59.

[0842] As it is seen from the mean values in the figures analysed, the amount of red colour is increased through time, but more importantly the standard deviation of such colour is reduced. Meaning that the diffused red dye was homogeneously distributed in the polymeric matrix A by the 24h of experiment.

[0843] Furthermore, it was noted during the experiment, and it can be seen in the figures, that the red dye released from Biomimetic material A was retained by the Biomimetic material B, without diffusing to the external container.

[0844] Applications

[0845] The regionally distributed microenvironments within the biomimetic material render it adaptable for a wide range of therapeutic, pharmaceutical, and biotechnological applications.

[0846] In certain embodiments, the material is configured for controlled release of drugs, peptides, proteins, nucleic acids, or other bioactive agents at mucosal or epithelial surfaces, enabling localized and sustained therapeutic delivery. In additional embodiments, the biomimetic composition facilitates encapsulation and protection of living microorganisms, enzymes, or biologically active cells, maintaining their viability under storage and physiological conditions.

[0847] The system may further be utilized for the simultaneous delivery of living microorganisms in combination with prebiotic substrates, wherein the incorporated prebiotic component supports the viability and metabolic activity of the encapsulated microorganisms or of resident commensal microbiota.

[0848] 19) Biomimetic hydrogel-like material provides a microenvironment that promotes the growth of commensal microorganisms while limiting the growth of opportunistic pathogens like E. coli

[0849] A biomimetic hydrogel-like material with regionally distributed microenvironments is provided that recreates the microenvironment of the gut mucosa, promoting the growth of commensal bacteria while preventing the proliferation of opportunistic pathogens, such as Escherichia coli (E. coli).

[0850] The biomimetic material was prepared to a final concentration of mucin 25mg / mL, alginic salt 11 g / mL; crosslinked internally through activation of calcium carbonate 1 mg / mL with Gluco- Delta-Lactone 10mg / mL (Phase I). Upon homogenization, the biomimetic material was shaped unidirectionally from the bottom up to mimic the gradual mesh gradient characteristic of the mucus layer in the lower gastrointestinal tract.

[0851] A Phase II shaping step was performed with calcium chloride 20 mg / mL deposited in the bottom of a mold to induce directional ionic crosslinking, establishing a structural and ionic gradient along the vertical axis of the material.

[0852] Microbiota Culture and Community Evolution

[0853] Microbiota samples derived from human faecal matter isolates were inoculated in the biomimetic hydrogel-like material with regionally distributed microenvironments and cultured for up to 72 h. Following incubation, the DNA was extracted and sequenced in the V3-V4 regions of the 16S rRNA gene to characterize microbial community evolution within the biomimetic material.

[0854] The results indicate that the biomimetic material supported the proliferation of probiotic and commensal taxa, including members of Bacteroidota and Actinobacteriota, as well as extreme anaerobes that participate in metabolic processes like Firmicutes. In contrast, the relative abundance of Proteobacteria, including opportunistic pathogens like E. coli, decreased over time during culture in the biomimetic material. See Fig. 60.

[0855] These results are also accompanied by the evidenced relationship between the growth of Lactobacillus and Bifidobacterium species with the changes in the polymeric network of the biomimetic material, indicating an intrinsic capacity of the biomimetic material to promote the maintenance and rebalancing of the mucus layer. See Fig. 61.

[0856] Metabolite analysis indicated that microbial catabolism within the material led to an increase in short-chain fatty acid (SCFA) production, compounds known to exert anti-inflammatory and gut-health-promoting effects. See Fig. 62.

[0857] Microbiome mining potential of the biomimetic material

[0858] In a further embodiment, the biomimetic material demonstrated capacity for microbiome mining, particularly for the identification and harvesting of bioactive molecules secreted by cultured microbiota.

[0859] In this example, faecal microbiota isolates from two individual donors were independently cultured within the biomimetic hydrogel material under identical conditions. The resulting culture supernatants were collected after 24 h and 48 h of incubation, filtered through sterile syringe filtration, and subsequently supplemented into E. coli cultures grown in Luria-Bertani (LB) medium.

[0860] The results showed that in both donor-derived cultures, the supernatants from microbiota grown in the biomimetic material exhibited inhibitory activity toward E. coli growth, confirming the generation of antimicrobial metabolites within the system. Notably, in one donor, the native microbiota also displayed inherent antimicrobial capacity even without prior culture in the biomimetic material, whereas in the second donor, the activity was enhanced only after culture within the biomimetic environment.

[0861] These findings indicate that the biomimetic hydrogel composition is capable of selectively promoting microbial metabolic interactions that lead to the production of bioactive and antimicrobial compounds. Accordingly, the system may be further exploited as a functional platform for microbiome mining, enabling the discovery, isolation, or enrichment of therapeutically relevant or value-added metabolites produced by host-associated microbial communities. See Figs. 63a and 63b. 20) Mucus adherent film (300-F) for the delivery of therapeutic agent in the nose to brain pathway

[0862] The nose to brain pathway is a demonstrated effective pathway in surpassing the blood brain barrier for central nervous system therapeutic molecules.

[0863] The biomimetic muco-adherent film is fabricated by crosslinking a polymeric mesh that contains embedded mucin-like proteins at a concentration representative of the nasal mucus (10-20 mg / mL). This polymeric network is tailored to maintain a desired ionic balance compatible with nasal mucus, therefore enabling a controlled release of the encapsulated therapeutic molecule.

[0864] The polymeric components comprise alginic salts and pectin, where the mesh size and release rate would be tailored through adjustment of polymer concentration and the ratio of ionic crosslinker. The initial preparation step (Phase I) involves mixing the therapeutic molecule within the bioactive agents in the water-based solution, ensuring homogeneous distribution throughout the hydrogel matrix prior to crosslinking.

[0865] To form the sheet-like structure, the biomimetic hydrogel is deposited into a mould containing an ionic crosslinking agent (calcium chloride, calcium gluconate) within its base. Upon contact with the mold, directional crosslinking occurs, forming a thin, cohesive shell structure with the desired mechanical stability and mucoadhesive properties.

[0866] The final biomimetic mucus-adherent film (300-F) would be fabricated with a thickness of 1- 3 mm and squared or rectangular shape having side dimensions of 3 to 5 mm. These dimensions are selected to ensure optimal placement and retention within the nasal cavity while maintaining patient comfort and effective diffusion characteristics.

[0867] For administration, the film is delivered using a nasal probe insertion device and positioned in the upper region of the nasal cavity, adjacent to the olfactory mucosa. Upon placement, the film would adhere to the mucus layer, forming a temporary mucoadhesive interface.

[0868] Following adherence, the film gradually would release the therapeutic molecule into the nasal mucosa, allowing sustained diffusion along the olfactory pathway and subsequent transport to the central nervous system.

[0869] 21) Stent-like hydrogel for application in highly permeable regions of the colon Mucin-containing biomimetic material is provided for the creation of a stent-like tubular structure intended for localized placement within regions of the colon exhibiting increased permeability or damage of the mucus barrier.

[0870] For that purpose, a polymeric network solution is mixed with a water-based suspension with high mucin concentration (greater than 10 mg / mL) within an ionically balanced solution compatible with the endothelial environment, such as NaCI 0.9%. This solution may be composed of more than one polymer to maximise elasticity and reduce fragility during delivery, including but not limited to alginate, collagen and combinations thereof.

[0871] An initial crosslinking step (Phase I) is carried out using a slow-crosslinking molecule, such as calcium gluconate, to reach low crosslinking density suitable for subsequent shaping. This controlled crosslinking permits subsequent directional crosslinking of the manufacturing process (Phase II) for customized geometry formation.

[0872] During Phase II, the hydrogel is introduced into a cylindrical mould configured to produce a tube-like geometry. The mold permits sequential or regional crosslinking of individual layers of the polymeric network, while selectively masking the internal lumen or the external surface, thereby allowing precise control over wall thickness and structural distribution.

[0873] The process may include the regional deposition of different biomimetic materials with distinct compositions generated during Phase I, enabling spatial differentiation of the tubular hydrogel. This allows regional tuning of microenvironmental features designed for compatibility with the changing geometry, for example, variations in elasticity, permeability, or degradation rate - adapted to the differing diameter, thickness (100-300pm) and composition of the mucus layer through the intestinal tract.

[0874] After finishing the Shaping step (Phase I and II), the material may be submitted to lyophilization for long-term storage prior to clinical use or maintained at 4°C until implantation. It is recommended to rehydrate and allow swelling of the tube-like biomimetic material with 0.9% NaCI before use.

[0875] The tube-like biomimetic material is configured for delivery through a catheter equipped with an inflatable balloon, enabling placement via colonoscopy at the targeted region of the colon. Upon reaching the desired site, balloon inflation expands the tubular hydrogel into contact with the intestinal mucosa. The biomimetic material is adapted to adhere to the epithelial surface, functioning as a temporary stent-like barrier that prevents direct contact between luminal contents and the injured mucosal surface. See Fig. 64.

[0876] The tube-like biomimetic material thereby reduces local inflammation, prevents microbial invasion, and serves as a temporary bio-scaffold. Over time, the tube-like biomimetic material is degraded by the resident microbiota within the colon. In certain embodiments, degradation occurs over a period of approximately 72 hours, during which the microbiota metabolize the hydrogel components and initiate the formation of a new exopolysaccharide matrix that promotes restoration of the native mucus barrier.

[0877] 22) Lyophilization of the biomimetic materials

[0878] For long term stability and maintenance of the viability of encapsulated microorganisms, low- hydration microenvironments (<35%) and low nutrient availability (2-3 mg / ml in 72h) are preferred. These will create bio-holder-like structures that will modulate microbial metabolism, enable stable storage and reduce metabolic activity.

[0879] For this purpose, after structure forming and polymeric network mesh creation around the bioactive components, the biomimetic material can be submitted to drying through air drying at temperatures below 50°C or lyophilization.

[0880] Lyophilization is the preferred method to remove moisture when microorganisms are encapsulated within the material, as the rapid freezing at very low temperatures effectively halts metabolic activity of the bacteria while minimizing cell death.

[0881] Furthermore, the combination between lyophilization and the protective polymeric mesh from the biomimetic material act synergistically to provide less harm for the microorganisms owing to gentle moisture removal.

[0882] Biomimetic structures both in bead-shape and disk-shape were lyophilized following a -80°C freezing curve under vacuum. The final materials successfully maintained their structural integrity after lyophilization and recovered their initial microenvironmental profile upon rehydration. See Fig. 65.

[0883] 23) Fabrication and characterization of biomimetic materials in different formats

[0884] A series of hydrogel-like biomimetic materials were produced in multiple macrostructured configurations — beads, disks, tubes, and films — to demonstrate the versatility of the manufacturing method and the capacity of the biomimetic material (100) to maintain its regional physicochemical characteristics across different geometries and scales. All formats were fabricated according to Phases I and II of the manufacturing method, using a standard biomimetic formulation composed of alginate (8 mg / mL), mucin (25 mg / mL), calcium carbonate (1 mg / mL), and glucono-5-lactone (10 mg / mL) within an ionic balancing solution (Phase I). Directed ionic crosslinking was induced by exposure to calcium chloride solutions (Phase II).

[0885] (a) Beads (300-B) of Different Sizes

[0886] Spherical and ellipsoidal biomimetic beads were obtained with adaptable diameter (2 mm, 4 mm, 5 mm and 6 mm). See Fig. 66.

[0887] (b) Disks (300-D) of Different Sizes

[0888] Disk-shaped biomimetic materials were prepared by casting the same pre-gel formulation into molds of 0.5 cm, 1 .5 cm, 2.0 cm and 3.2 mm diameter with a thickness of 0.4 cm. Though all these features may be adjusted.

[0889] Directional crosslinking from both bottom and top surfaces created asymmetric oxygen and hydration profiles. The smaller disks were optimized for in-vitro colon or cervico-vaginal models, while larger disks serve as macro platforms for co-culture or biofilm studies as well as oral intake. See Fig. 67.

[0890] (c) Tubes (300-T)

[0891] Tubular biomimetic materials were obtained by injecting the pre-gel into a cylindrical mold fitted with a removable inner rod, followed by external and internal exposure to calcium chloride to produce dual-sided crosslinking.

[0892] The resulting tubes had outer diameters of 6 mm, inner diameters of 4 mm, and lengths between 40 mm and 100 mm. See Fig. 68.

[0893] (d) Films (300-F) With and Without Reservoir

[0894] Thin films were fabricated by mold crosslinking in square geometries (30 x 30 mm x 2 mm thick).

[0895] For films without a reservoir, a homogeneous crosslinking was applied to create a continuous diffusion barrier. See Fig. 69a. For films with a central reservoir, a shallow cavity was formed before final crosslinking, in which a softer biomimetic material is included. This configuration enables controlled release of encapsulated contents through the surrounding diffusion layers, mimicking mucus turnover or topical drug-delivery dynamics. See Fig. 69b.

[0896] These results confirm that the hydrogel-like biomimetic material (100) maintains its internal physicochemical gradients and viscoelastic identity across multiple macro-formats, each providing a distinct functional architecture adaptable to research, industrial, or therapeutic use.

[0897] 24) Mechanisms of use of the Vag3Gel™as a therapeutic dressing for promoting rebalance of the cervicovaginal mucosa environment, promoting adequate microbiota colonization, re-establishing acidic pH and mucosal barrier integrity while providing hydration for the cervicovaginal canal.

[0898] IFU: The gel is indicated for people with diagnosed permeable gut, Crohn’s disease, IBD or microbiota dysbiosis.

[0899] Draft Protocol: - Cervicovaginal Therapeutic Administration

[0900] Type: Mucoadhesive, bioactive hydrogel (mucin-based)

[0901] Delivery Form: Pre-filled sterile syringe with rectal applicator

[0902] Volume per Dose: 4 mL

[0903] Intended Use:

[0904] 1. Target Patient Population

[0905] Patients with:

[0906] • Recurrent Bacterial Vaginosis, Aerobic Vaginosis, or Candida Recurrent Infections, Lactobacillus-Depleted Vaginal Microbiota

[0907] • Evidence of mucosal barrier dysfunction: like vaginal atrophy from postmenopausal stages, or breast cancer survivors in which Estrogen levels are reduced, or damaged mucosal associated to STI like chlamydia, gonorrhea or Trichomoniasis

[0908] • Post antibiotic recovery

[0909] • Preparation or recovery from vaginal procedures . Materials Required

[0910] • Cervicovaginal biomimetic mucus 4 mL pre-filled sterile applicator

[0911] • Disposable gloves

[0912] • Protective hygiene pad or towel

[0913] • Antiseptic wipes (optional) . Administration Instructions

[0914] 1. Wash your hands with soap and water.

[0915] 2. Open one applicator (or fill the syringe with the premeasured 4 mL gel if reusable).

[0916] 3. Lie down on your back with knees slightly bent, or stand with one foot on a chair — whichever is more comfortable.

[0917] 4. Gently insert the applicator tip into the vagina, about 5 cm deep (not painful).

[0918] 5. Press the plunger slowly until all the gel is released.

[0919] 6. Remove the applicator and discard it if single-use.

[0920] 7. Remain lying down for a few minutes to allow the gel to spread. . Dosage schedule

[0921] • Induction phase: Apply one full applicator (= 4 mL) once daily, preferably at bedtime, for 7 days in a row.

[0922] • Maintenance phase: Apply 2-3 times per week as advised by your doctor. . Dosage & Frequency

[0923] Phase Frequency Duration

[0924] Induction phase 1 dose daily 14 consecutive days

[0925] Maintenance phase 1 dose every 2-3 Up to 8 weeks, based on clinician days assessment

[0926] • Administration preferably in the evening before bed.

[0927] • Adjust frequency based on: o OLE follow-up o Mucosal healing markers o Patient tolerance and response . Storage Conditions

[0928] • Store at 2-25 °C (room temperature or refrigerated) Protect from direct sunlight

[0929] Do not freeze

[0930] 6. Precautions & Contraindications

[0931] • Not to be used during menstruation

[0932] • Avoid in patients with known allergy to any of the biomimetic mucus components (mucin, pectin, salts)

[0933] • Not intended for systemic absorption

[0934] • Safe for use alongside biologies or oral therapies

[0935] 25 Cultivation of Candida albicans in a Cervico-Vaginal-Like Biomimetic Material (Vag3Gel™)

[0936] A cervico-vaginal-like biomimetic material, herein designated Vag3Gel, was fabricated to reproduce the physicochemical conditions of non-ovulatory cervico-vaginal mucus and to evaluate its suitability for culturing fungal species such as Candida albicans

[0937] Preparation of the Vag3Gel™Formulation

[0938] The formulation was prepared following the Phase I and II. An aqueous solution of NaCI (0.07 mg / mL) was prepared and supplemented with mucin from porcine gastric origin (1 .5 mg / mL) and mucin from bovine submaxillary gland (1.5 mg / mL). The solution was autoclaved to ensure sterility and homogeneity.

[0939] Pectin powder was sterilized by UV exposure for 30 minutes followed by incubation at 50 °C for 30 minutes and then added to the mucin-containing solution to a final concentration of 5 mg / mL under constant stirring at 220 rpm for 15 minutes. Calcium carbonate (2 mg / mL) and glucono-5-lactone (0.36 mg / mL) were then introduced sequentially to induce internal ionic crosslinking. The resulting mixture was allowed to homogenize to form the biomimetic material. The pre-gel was subsequently molded into disks (300-D). Inoculation and Culture of Candida albicans

[0940] An overnight culture of C. albicans was prepared in Luria Bretania (LB) broth at 37 °C under agitation (200 rpm). After overnight inoculation, the yeast suspension was diluted to a final concentration of 1 x 106CFU / mL.

[0941] Each Vag3Gel™disk was placed in a 24-well plate and inoculated with 700 pL of the C. albicans suspension, ensuring full surface contact. Samples were incubated at 37 °C for 24, 48, 72 and 144 hours.

[0942] Assessment of Fungal Growth and Viability

[0943] At each timepoint, viable cells were quantified by counting colony-forming units (CFU). Results demonstrated progressive colonization of Vag3Gel™with CFU increasing in the first 24 h followed by stationary state. See Fig. 70

[0944] These results validate Vag3Gel™as a physiologically relevant substrate for studying fungal- mucus interactions, biofilm initiation, and therapeutic testing under cervico-vaginal conditions.

[0945] Potential applications include:

[0946] • In-vitro models for Candida colonization, antifungal susceptibility, or probiotic competition studies;

[0947] • T esting of therapeutic or probiotic formulations aimed at rebalancing the vaginal microbiota; and

[0948] • Screening of mucoadhesive or anti-biofilm materials under physiologically controlled pH and oxygen gradients.

[0949] The Vag3Gel™ formulation successfully mimics the physical and chemical environment of the cervico-vaginal mucus, sustaining the adhesion, morphogenesis, and proliferation of Candida albicans while maintaining quantifiable physicochemical gradients. This confirms the adaptability of the biomimetic material (100) for modeling host-microbe interactions in mucosal ecosystems.

Claims

CLAIMS1. A hydrogel-like biomimetic material (100) having a three-dimensional distribution of biomimetic microenvironments (110), wherein each biomimetic microenvironment (110) comprises:(a) a polymeric network (A) that simultaneously provides structural support and functional activity; and(b) a water-based suspension (B); wherein the water-based suspension (B) is sustained by the polymeric network (A).

2. The biomimetic material (100) according to claim 1 , wherein the polymeric network (A) comprises at least one polymer selected from natural polysaccharides, glycopolysaccharides, proteins, glycoproteins, synthetic polymers, or combinations thereof.

3. The biomimetic material (100) according to any of the preceding claims, wherein the polymeric network (A) is configured to:(i) form and maintain the three-dimensional structure of each biomimetic microenvironment (110);(ii) retain the water-based suspension (B) and any viable microorganisms (C); and(iii) upon degradation, release both the components of the polymeric network (A) and the contents retained within the biomimetic microenvironment (110).

4. The biomimetic material (100) according to claim 3, wherein the polymeric network (A) protects the contents of the biomimetic microenvironment (110) against environmental stress during transport, storage, or delivery, and wherein the degradation products of the polymeric network (A) provide a functional biological and / or physicochemical activity.

5. The biomimetic material (100) according to any of claims 1 to 4, wherein the polymeric network (A) comprises one or more crosslinking modulators selected from calcium carbonate (CaCO3), glucono b-lactone (GDL), calcium chloride (CaCI2), or calcium gluconate, optionally in combination with a biological or photo-crosslinking step.

6. The biomimetic material (100) according to any of claims 1 to 5, wherein the waterbased suspension (B) comprises water, ionic balancing molecules, and optionally one or more bioactives or biological modulators selected from short-chain fatty acids, prebiotics, postbiotics, vitamins, peptides, proteins, mucin or mucin-like proteins, culture media, or environmental stabilizers.

7. The biomimetic material (100) according to any of claims 1 to 6, further comprising viable microorganisms (C) in a structural manner, wherein the viable microorganisms (C) are suspended within the water-based suspension (B) and / or adhered to the polymeric network (A).

8. The biomimetic material (100) according to claim 7, wherein the viable microorganisms (C) are selected from bacteria, fungi, or viruses, either as single species or as consortia.

9. The biomimetic material (100) according to any of the preceding claims, wherein the biomimetic microenvironments (110) are distributed homogeneously or regionally throughout the material (100).

10. The biomimetic material (100) according to claim 9, wherein the regional distribution defines regionally distributed microenvironments (RDMEs) exhibiting discrete physicochemical parameters and, optionally, internal gradients of hydration, ion concentration, oxygen tension, or crosslinking density.

11. The biomimetic material (100) according to any of the preceding claims, wherein each biomimetic microenvironment (110) is of a type selected from diffuser, shell, bio-holder, bio-neutral, or bio-booster.

12. The biomimetic material (100) according to claim 11 , wherein at least two types of biomimetic microenvironments (110) coexist within the same biomimetic material (100), defining a macrostructure comprising external shell-type microenvironments surrounding internal diffuser-type microenvironments.

13. The biomimetic material (100) according to any of claims 1 to 12, wherein each biomimetic microenvironment (110) exhibits:(a) a hydration level between 5 % and 95 % of its total mass;(b) a mesh size between 18 nm and 110 nm;(c) a crosslinking density between 34 % and 100 % of the moles of polymer of the polymeric network (A);(d) a storage modulus G' between 1 .5 Pa and 3 000 Pa and a loss modulus G" between 0.3 Pa and 500 Pa for shear-strain frequencies of 0.01 Hz to 100 Hz; and(e) a pH between 4 and 9.

14. The biomimetic material (100) according to claim 13, wherein said ranges are selected to reproduce the viscoelastic and diffusive properties of natural mucus from human mucosal tissues.

15. The biomimetic material (100) according to any of the preceding claims, presented as an amorphous gel (200).

16. The biomimetic material (100) according to any of claims 1 to 14, presented as a macrostructured construct (300) shaped as a bead, disk, cylinder, or film, each comprising regionally distributed biomimetic microenvironments (110).

17. The biomimetic material (100) according to claim 16, wherein the macrostructured construct (300) comprises at least one external region with shell-type microenvironments (111) surrounding one or more internal regions of diffuser-type microenvironments (112).

18. The biomimetic material (100) according to any of claims 1 to 17, presented as a composite material (400) comprising a dispersion of macrostructured constructs (300) within an amorphous biomimetic material (200).

19. The biomimetic material (100) according to any of claims 15 to 18, wherein the macrostructured construct (300) comprises hierarchical embedding of one or more constructs (300), forming nested or multi-scale architectures selected from beads- within-beads, disks-within-beads, disks-within-cylinders, or combinations thereof.

20. The biomimetic material (100) according to any of the preceding claims, wherein the material is in a dehydrated or rehydratable state obtained by lyophilization, solvent casting, or air-drying.

21. The biomimetic material (100) according to any of claims 13 to 20, wherein the amorphous material (200) is configured as a biocompatible lubricant for application on mucosal or epithelial surfaces.

22. The biomimetic material (100) according to claim 21 , wherein the lubricant is formulated for use in sexual activity, for insertion of medical or diagnostic devices, or to facilitate endoscopic or colonoscopic procedures.

23. The biomimetic material (100) according to any of claims 21 to 22, wherein the amorphous material (200) exhibits:(a) a pH between 3.8 and 7.2;(b) viscoelasticity compatible with natural mucus; and(c) non-irritant, biocompatible, and non-cytotoxic properties.

24. The biomimetic material (100) according to any one of the preceding claims for use in the recovery of mucosal layers.

25. The biomimetic material (100) for use according to claim 24, wherein the mucosal layer is selected from gastrointestinal, cervico-vaginal, or respiratory mucosa.

26. The biomimetic material (100) for use according to claim 25, wherein the material (100) or its amorphous form (200) is administered rectally, intravaginally, or nasally.

27. The biomimetic material (100) for use according to any of claims 24 to 26, wherein the material is presented as one or more macrostructured constructs (300) configured for therapeutic delivery of bioactive agents, viable microorganisms, or both.

28. The biomimetic material (100) for use according to claim 27, wherein the macrostructured constructs (300) are shaped as beads, disks, cylinders, or films, and are administered orally, rectally, intravaginally, or topically to a mucosal surface.

29. The biomimetic material (100) for use according to any of claims 24 to 28, wherein the material promotes rehydration of the mucosal surface, restoration of mucus viscoelasticity, and rebalancing of the native microbiota composition.

30. A method for treating a mucosal disorder or damage in a subject in need thereof, comprising administering to the subject an effective amount of the biomimetic material (100) according to any of claims 1 to 23.31 . The method according to claim 30, wherein the mucosal disorder affects gastrointestinal, cervico-vaginal, or respiratory mucosa.

32. The method according to any of claims 30 to 31 , wherein the biomimetic material (100) is provided in the form of macrostructured constructs (300) configured for therapeutic delivery of bioactive agents, viable microorganisms, or both.

33. The method according to claim 32, wherein the macrostructured constructs (300) are shaped as beads, disks, cylinders, or films, and are administered orally, rectally, intravaginally, or topically to a mucosal surface.

34. The method according to any of claims 30 to 33, wherein administration of the biomimetic material (100) restores mucosal hydration, re-establishes mucus barrier function, and rebalances the mucosal microbiota.

35. A method for manufacturing a hydrogel-like biomimetic material (100) and macrostructured constructs (300) derived therefrom, comprising:Phase 1 — preparation: providing an aqueous mixture containing one or more polymers forming the polymeric network (A); incorporating a water-based suspension (B) containing ionic-balancing molecules and optionally bioactives or viable microorganisms (C); and crosslinking the mixture to obtain a polymeric network (A) that sustains the water-based suspension (B);Phase 2 — shaping: subjecting the biomimetic material (100) obtained in Phase 1 to directional or sequential crosslinking to form regionally distributed biomimetic microenvironments (110), thereby obtaining macrostructured constructs (300) comprising distinct internal and external microenvironmental regions.

36. The method according to claim 35, wherein the crosslinking in Phase 1 is selected from ionic, photo-induced, or biologically induced crosslinking.

37. The method according to claim 36, wherein the ionic crosslinking employs a system of calcium carbonate (CaCO3) and glucono 5-lactone (GDL) to modulate crosslinking kinetics.

38. The method according to claim 35, wherein the directional or sequential crosslinking of Phase 2 generates regional distributions (RDMEs) and, optionally, physicochemical gradients of hydration, crosslinking density, or oxygen tension.

39. The method according to any of claims 35 to 38, wherein the polymers forming the polymeric network (A) are selected and crosslinked such that, upon degradation, thenetwork progressively releases its polymer components and the contents retained within, thereby imparting structural and functional activity to the resulting biomimetic material (100).

40. The method according to any of claims 35 to 39, wherein the shaping is carried out by extrusion, moulding, or droplet formation.41 . The method according to any of claims 35 to 40, wherein the biomimetic material (100) obtained after Phase 1 and / or the macrostructured construct (300) obtained after Phase 2 is reused as an input material for a subsequent repetition of Phase 1 , thereby producing hierarchically embedded constructs (300) within one another.

42. The method according to any of claims 35 to 41 , wherein two or more Phase 1 preparations are performed in parallel, and the resulting biomimetic materials (100) are selectively deposited during Phase 2 to achieve regional or geographically discrete distributions of biomimetic microenvironments (110) within the macrostructured construct (300).

43. The method according to any of claims 35 to 42, further comprising a dehydration step performed by lyophilization, solvent casting, or air-drying to produce a rehydratable biomimetic material (100).

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