Fig. 1 illustrates the general reaction scheme of the ester crosslinking, including an example of an undesired side-reaction for illustration purpose.

Fig. 2 shows particles according to the invention. On the right, a particle without pores is illustrated. On the left, a particle with pores is illustrated. Both pictures were taken using a confocal microscope.

Fig. 3 shows H&E-colored histology sections of the subcutaneous tissue area where the biomaterial of the invention was injected, at the time-point 3 weeks post-injection. It can be observed that the biomaterial is not inducing encapsulation and that it is being degraded.

Fig. 4 shows a quantification of the different types of areas observed throughout degradation in histology and the evolution of implant composition over time

Fig. 5 shows a quantification of the area where implanted biomaterial can still be identified in different degradation stages as compared to overall implant area.

Fig. 6 shows a kit for mixing biomaterial with an additional fluid prior to implantation.

Fig. 7 schematically illustrates particles in the form of spiculae.

Fig. 8 shows an embodiment where dehydration is achieved during 3D printing by means of a selective membrane element.

Fig. 9 shows an embodiment where dehydration is achieved during deposition into a liquid forming an aqueous two-phase system.

Fig. 10 shows an embodiment where dehydration is achieved through the use of water-binding porogens.

Ester crosslinking density

The ester crosslinking density is the amount of ester groups present in a given amount of material. Ester density is expressed in terms of moles of ester groups present per mass unit of dry material. Ester density can be measured by titration (consumption of base during ester hydrolysis) or spectroscopic techniques (e.g FTIR). For titration, the amount of base required to induce hydrolysis of all the ester groups present in a given amount of material is determined by addition of an excess of base, and titration of the base remaining after complete hydrolysis, as detailed in the Methods section. Regarding FTIR, ester groups have a characteristic absorption band around 1730cm^-1, and with the necessary precautions and calibration, it is possible for some substances to use this band for semi-quantitative estimation of the ester crosslinking density.

Wall

In porous materials, the wall is the solid material between the pores. In the hydrated state, this material is typically a water-swollen hydrogel, whereas in the dry state, it is typically solid dry polymeric material. The walls are generally well visible in phase contrast microscopy, but can also be specifically visualized by fluorescent or colorimetric staining in microscopy for quantification purposes.

Wall thickness

The wall thickness is the local thickness of solid, typically water-swollen material between pores, i.e. it is the local thickness of the walls. Wall thickness is analyzed on aqueous suspensions of the gel-like biomaterial. For wall thickness analysis, confocal fluorescence images are acquired using either autofluorescence or fluorescence from a fluorescent dye with an affinity for the solid material, such as rhodamine 6G at a concentration of 5 microgram/mL, permitting to distinguish solid material and pore fluid by the contrast in fluorescence intensity. On images representing confocal planes, the wall thickness can be evaluated by random ray tracing as the average length of the segments defined by the individual intersections between the rays and the walls.

Pore diameter

The pore diameter is the length from wall to wall across pores in the material. Pore diameter is analyzed on aqueous suspensions of the gel-like biomaterial. For pore diameter analysis, confocal fluorescence images are acquired using either autofluorescence or fluorescence from a fluorescent dye with an affinity for the solid material, such as rhodamine 6G at a concentration of 5 microgram/mL, permitting to distinguish solid material and pore fluid by the contrast in fluorescence intensity. On images representing confocal planes, the pore diameter can be evaluated by random ray tracing as the average length of the segments defined by the individual intersections between the rays and the pores.

Settling volume

The settling volume is the volume occupied by spontaneous swelling in deionized water by a given amount of material. The settling volume is measured by suspending a given amount of material, characterized by its dry weight, in an excess of water, and letting the suspended material sediment to its equilibrium volume. The equilibrium volume can for instance be read on a graduated cylinder. The settling volume is reported in g of water that can be absorbed per gram of dry material [g/g]. Exogenous crosslinkers

Exogeneous crosslinkers are crosslinkers that add additional atoms to the molecular structure of the polysaccharides that they are crosslinking. Part of their mass remains within the crosslinked product, and this remaining mass is linked to their crosslinking action.

Endogenous crosslinks

Endogenous crosslinks are formed from atoms already present in the original polysaccharide molecules to be crosslinked. Activation agents may be used in some instances, but they not add mass to the chemical crosslinking structures (there may on occasions be individual atom replacement seen in isotopic studies).

Molar amount of ester groups

This is the number of molecular R-COO-R groups present in a sample, divided by Avogadro’s number to make moles.

Reversible compressibility

A reversibly compressible material is a material that can be compressed by preferably more than 50%, more preferrable more than 80%, and most preferable more than 90%, and essentially recover its original properties such as volume, dimensions, Young’s and shear modulus after relaxation to its original state before compression.

Protrusions

Protrusions are irregularities or asperities extending beyond the general biomaterial or biomaterial particles surface. As such, they define and reach the convex hull of the biomaterial or biomaterial particles, and are separated from each other by areas not reaching the convex. Protrusions can for example be defined on a digital representation acquired by fluorescent staining and confocal microscopy, by scanning electron microscopy, by micro-computed tomography, or by other suitable high-resolution imaging techniques. On digital representations, the convex hull can be found algorithmically in either 2D image planes or 3D stacks, and protrusions identified as reaching the convex hull.

Spicula

Particles can have an internal structure in the manner of spiculae. Particles with an internal structure organized in the manner of spiculae are porous, exclusively or mostly with walls resembling struts. Interconnectivity

Interconnectivity is a property of the pore space of a particle or scaffold. Pores can either be closed and isolated, or connected to the outside world, be it directly or through other pores. The ensemble of pores connected directly or indirectly to the outside of a biomaterial bulk or biomaterial particle defines the interconnected pore space. Interconnectivity is the fraction of the total volume of a particle or scaffold, or ensemble of particles or scaffolds, which is occupied by interconnected pores. Interconnectivity can be assessed digitally from high-resolution stacks or imaging planes by numerically detecting connectivity to the outside. Alternatively, interconnectivity can be gauged by the wicking test, where a biomaterial, biomaterial particles, or biomaterial particles assembly is placed on a wiping paper and the loss of weight due to spontaneous pore fluid withdrawal by the capillary effect of the wiping paper is measured. For this assay, no additional mechanical force should be applied onto the samples in order to avoid damaging the structure of the biomaterial.

Young’s modulus of the biomaterial

The Young's modulus is a well-known quantity in mechanical engineering. It is defined as the linear coefficient relating stress (force per area) to strain (deformation relative to the original length) as measured in uniaxial compression, typically for small strains (20% compression or less). It is thus the slope of the initial proportion of the stress-strain curve. The Young moduli as used here are measured under laterally unconstrained conditions, which in the case of porous materials includes the possibility of extrusion of pore fluid during the compression. The Young’s modulus of the biomaterial refers to the Young modulus measured on macroscopic samples, including hydration fluid and possibly pore fluid. For precision, the Young modulus is reported under reference conditions, that is, suspended in phosphate buffered saline at neutral pH (potassium chloride (KCI) 2.6mM, potassium phosphate monobasic (KH2PO4): 1.5mM, sodium chloride (NaCI) 138mM, sodium phosphate dibasic (Na2HPO4): 8.1 mM in deionized water).

Young’s modulus of the walls of the biomaterial

In the case of porous materials, one can distinguish the Young’s modulus of the biomaterial as defined above, from the local Young’s modulus of the biomaterial making up the walls. If the biomaterial making up the walls can be produced in homogeneous form, the Young’s modulus of the wall material can be assessed by macroscopic uniaxial compression. In cases where only the porous material is available, the Young's modulus of the wall biomaterial can be evaluated by microindentation techniques, directly on structured materials, as for instance described in Welzel et al., Adv Healthc Mater 2014, or Beduer at al,, Advanced Healthcare materials 2015. 4(2): p. 301-12. As above, the Young’s modulus of the wall biomaterial is reported under reference conditions.

Polymer/polysaccharide concentration in the walls of the biomaterial

The polymer/polysaccharide concentration in the walls of a porous biomaterial can be evaluated by relating the dry weight as obtained by drying to constant weight (at 70°C at ambient pressure, or at room temperature in vacuum better than 5mbar) to the weight of the wall phase measured in a hydrated state, but without the pore fluid. In practice, the weight of the wall phase is obtained by suspending the material to be measured in reference fluid (phosphate buffered saline as for the measurement of the Young’s modulus of biomaterial), followed by forced removal of the pore fluid by using a paper towel and gentle manual force to remove all free liquid, followed by weighing of the remaining solid hydrated material. The dry weight can then be determined after washing with deionized water to remove possible traces of phosphate buffered saline.

Particles

Particles in the sense of this invention are entities of solid or gel-like biomaterial that are contiguous. The presence of distinct particles can be evidenced by diluting the material in an excess of solvent (for instance, deionized water) and observing separated entities floating in the solution. The method can be combined with microscopic observation techniques and/or fluorescent or visible color staining for example with rhodamine 6G or methylene blue. The biomaterial can consist of one, a few, or many particles, and the particles may be porous or not. In some embodiments, only a fraction of the particles is porous or only a fraction of the biomaterial is composed of particles. The biomaterial can consist in a molecular solution expanding to arbitrarily large volumes of dilution, or in a mixture of one or several particles and molecular solution.

Particle diameter

The particle diameter characterizes the size of individual particles. The particle diameter can be obtained by digital analysis of microscopic images of highly dilute particle suspensions, where the particles are separated from each other. Such microscopic images can be obtained by fluorescence or by color-based staining. The particle diameter is defined as the diameter of a circle with the same area as the particle on the microscope image. The average particle diameter is defined as the weighted mean of the diameters of the particle observed, the areas being used as the weights for obtaining the mean. Chemical activator

A chemical activator is defined as an agent capable of inducing a chemical reaction. The chemical activator is consumed in the reaction. In one embodiment, the chemical activator enables to induce an ester-formation reaction. In one embodiment, the chemical activator transforms carboxylic acid or carboxylate anion groups into transient chemical species capable of forming an ester. In another embodiment, the chemical activator transforms hydroxyl groups into transient chemical species capable of forming an ester.

Incorporated chemical activator

An incorporated activator is an activator moiety that is irreversible bound to the biomaterial. Activator incorporation results from undesired side reactions and leaves the chemical activator essentially irreversible bound to biomaterial constituents, at least under usual conditions of use. Example of side reactions leading to such permanent incorporation are N-urea rearrangement in carbodiimides or reaction with hydroxyl instead of carboxyl groups for instance for uronium-type chemical activators.

Detailed description of the invention

Chemical activators of carboxylate groups such as carbodiimides can be used for ester synthesis. Figure 1 shows the reaction as applied to polysaccharide strands (1). Such ester formation reaction (2) in aqueous media are however plagued by low reaction efficiency and inadvertent covalent incorporation of activators (3). Carbodiimides are typically incorporated as urea derivatives. Surprisingly, we found that these problems can be addressed by carboxylic acid activation (for instance, with carbodiimide activation by using the water soluble carbodiimide EDC (N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide or its hydrochloride salt) under catalysis by aminopyridine catalysts (DMAP (4-Dimethylaminopyridine) and analogs), with an at least partial dehydration step.

Several methods for dehydration can be used. The choice of these methods is in part oriented by the desired target geometry of the biomaterial.

Non-porous biomaterials can advantageously be produced by the use of water-permeant, but salt impermeant membranes, known as reverse-osmosis membranes and commercially available. These membranes can be in tubular layouts, as illustrated on Figure 8. After extrusion through such dehydrating elements, the biomaterial may be incubated to permit the crosslinking reaction to complete, before further processing such as purification and sterilization. Alternatively, selective membranes can also be used for dialysis of fluid against osmotically highly active solutions such as concentrated polyvinylalcohol (PVA) or polyethyleneglycol (PEG) solutions. In other embodiments, the polysaccharide component itself is used in powder form to achieve dehydration. For this, polysaccharide powder is suspended in a small amount of non-solvent, for example acetone, to form dense slurries with paste-like or slightly liquid consistency. The slurry is the rapidly mixed with a reaction solution, which contains all the reactive ingredients, optionally including part of the polysaccharide in solution. The non-solvent permits rapid dispersal of the powdered polysaccharide in the reaction solution without formation of clumps. After this, the polysaccharide powder particles swell and partly or fully dissolves and create a dense reaction medium at polymer concentrations not amenable to facile mechanical mixing. In this dehydrated state, the reaction to form non-porous material completes at high efficiency.

Alternatively, or in combination, water can also be removed through the vapor phase. In one embodiment, reaction mix is distributed on a surface as thin film and dried under ambient air conditions, or optionally under air flow conditions or through the application of heat to accelerate the drying process. In another embodiment, reaction solution is dried by spray-drying in a spraydrying apparatus or by lyophilization. In such apparatuses, a water vapor trap in the form of chemical trap or a cold-trap, and the use of vacuum or heat can further accelerate the drying process.

The synthesis of porous materials typically requires the presence of a porogen in addition to the dehydration conditions. A first possibility is to use dehydration methods described above for non- porous biomaterial, while supplying the biomaterial reaction as a solution with a porogen insoluble or at least immiscible under reactions conditions, but that will later be dissolved or removed by the use of an appropriate solvent or possibly evaporated or otherwise removed. Such porogens can be poorly soluble salts, but also polymeric particles, gas bubbles or immiscible components forming an emulsion. In other embodiments, pore generation and dehydration are advantageously combined. In one embodiment, a poorly soluble anhydrous salt or a poorly soluble salts partly deficient in crystal water or hydration water as compared to their stable hydration state is supplied as both the porogen and the dehydration agent. An example for this is the addition of anhydrous calcium sulfate or calcium sulfate hemihydrate powder to the reaction solution. Such salts absorb water from the reaction solution (in the case of the calcium sulfate to form the dihydrate achieving the dehydration step), and can later be dissolved by solutions containing complexing agents such as ethylene-diamine-tetracetic acid EDTA or sodium citrate. Freezing can also be used as a means for forming porogens while dehydrating the solution. Lyophilization can remove water through the vapor phase while creating pores.

Aqueous emulsion systems can also be used for simultaneous dehydration and pore formation. For example, many carbohydrates including hyaluronic acid, carboxymethylcellulose, carboxymethylstarch and alginate form aqueous phases that separate from aqueous phases of polyethylene glycols. When reaction solutions of such carbohydrates are supplied with anhydrous polyethylene glycol in powder form, the polyethylene glycol dissolves by absorbing water from the reaction solution and simultaneously forms a non-miscible porogen phase. In this respect, many commercial polyethyleneglycols are hydroxyl-terminated and will, at least to some extent, be incorporated into the biomaterial by ester bond formation despite the phase separation. If desired, this can be completely prevented by the use of circular polyethyleneglycols. As an alternative to using powders, it is also possible to use mixtures of polyethyleneglycols and water with a high polyethyleneglycol concentration.

Numerous substances have been proposed as catalytic additives in reactions of chemically activated carboxylic acid groups. N-hydroxysuccinimide and derivatives, 1 -hydroxybenzotriazole hydrate and derivatives, phenol and derivatives, nitrophenol and derivatives, and many others have been proposed. Surprisingly, we found that among the many options, selected aminopyridine-type esterification catalysts permit to obtain high purities and ester densities in aqueous polysaccharides solutions at near neutral pH (pH 4 to 9, preferentially 5 to 8, and most preferentially 6 to 7.5) under dehydration conditions.

This discovery enables the production of high-purity ester-crosslinked hydrogels from biopolymers and combination of biopolymers presenting both carboxylate groups and hydroxyl groups. We have further discovered that such biopolymers, when synthesized at a specified ester crosslinking density between 0.01mol/kg and 1mol/kg, more preferentially 0.02 mol/kg and 0.7 mol/kg, most preferable between 0.05mol/kg and 0.5mol/kg and even more preferable between 0.07mol/kg and 0.4mol/kg can be readily processed and sterilized to produce implantable biomaterials. These biomaterials have a favorable local tissue response when implanted. The ester crosslinking density is measured according to the method described in the methods section.

The ester crosslinking density is determined by the reaction conditions (reactant concentrations, dehydration method, time, temperature). With the aid of the titration method, and examples disclosed here, it is possible to establish reaction conditions suitable for obtaining a desired ester crosslinking density.

In some instances, spectroscopic techniques can be used to detect the ester bonds. In Fourier Transform Infrared Spectroscopy FTIR, for instance, ester groups show an absorption band located typically around 1730cm^-1. Given that protonated carboxyl groups show a similar signal, it is often necessary to ensure complete deprotonation before proceeding with FTIR. Quantitative analysis further needs calibration with samples with known ester crosslinking density from titration since absorption coefficients in FTIR are not known generally known in advance. In addition to the yield in terms of ester crosslinking density, the aspect of purity is very important, as results from the below Table 1 (showing a quantification of the relation between encapsulation and purity) by the encapsulation thickness associated with different degrees of purity.

Table 1

Nuclear magnetic resonance, as well as chromatographic and mass spectroscopic techniques can be used to measure the amounts of chemical activator or catalysts covalently attached to the biomaterial. Details of these methods are given in the methods section. For instance, by NMR analysis, we find that incorporation of the chemical activator N-(3-Dimethylaminopropyl)-N'- ethylcarbodiimide (EDC) as an urea-derivative can be controlled to less than 0.4mol of chemical activator incorporated per mol of ester formed, preferentially less than 0.2mol of chemical activator incorporated per mol of ester formed, and even more preferentially less than 0.2mol of chemical activator incorporated per mol of ester formed, more preferentially less than 0.1 mol of chemical activator incorporated per mol of ester formed and most preferentially 0.05mol of chemical activator incorporated per mol of ester formed, and most preferentially 0.01 mol of chemical activator incorporated per mol of ester formed can be achieved.

Without the use of aminopyridine-type esterification catalyst(s) and dehydration step(s), for instance EDC is mainly incorporated as cationic urea as described in the literature and no stable gel is generally formed.

Aminopyridine-type esterification catalyst(s) are not incorporated. For instance, by NMR, no trace of incorporation of 4-dimethylaminopyridine (DMAP) could be detected under any condition. The reaction can also be carried out with different aminopyridines and nucleophilic catalyst analogs, such as 4-pyrrolidinopyridine, 1-Methyl-1,2,3,4-tetrahydro-1,6-naphthyridine, 1,4- Dimethyl-1 ,2,3,4-tetrahydropyrido[3,4-b]pyrazine, 1 ,4-Diethyl- 1 ,2,3,4-tetrahydropyrido[3,4- b]pyrazine, 9-Azajulolidine, 1 ,6-Dimethyl-2,3,5,6-tetrahydro-1 H,4H-1 ,3a,6,8-tetraazaphenalene, 1 ,6-diethyl-2,3,5,6-tetrahydro-1H,4H-1 ,3a,6,8-tetraazaphenalene, (Z)-N-Methyl-1-(4- pyridinyl)methanimine, 4-dimethylaminopyridine and can also be additionally enhanced with Lewis acid with further co-catalysts such as cerium(lll) chloride. Similarly, different chemical activators can be used, such as carbodiimides (e.g. N-(3-Dimethylaminopropyl)-N'- ethylcarbodiimide (EDC); Dicyclohexylcarbodiimide (DCC)) and also other carboxylic acid activation agents, to within the limits imposed by solubility in aqueous systems. Such carboxylic acid activation agents are widely known in the field of peptide coupling. Useful examples guanidine-based activation agents such as TSTU (N-[(Dimethylamino)[(2,5-dioxo-1- pyrrolidinyl)oxy]methylene]-N-methylmethanaminium Tetrafluoroborate(l-)), uronium-based coupling agents such as TNTU 2-(5-Norborene-2,3-dicarboximido)-1 ,1 ,3,3-tetramethyluronium tetrafluoroborate and HPTU 1,1,3,3-Tetramethyl-2-(2-oxopyridin-1(2H)-YL)isouronium hexafluorophosphate, phosphonium-based agents such as PyBOP (benzotriazol- 1- yloxytripyrrolidinophosphonium hexafluorophosphate), Castro’s reagent ((Benzotriazol-1- yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate) and BOMP (2-(benzotriazol-1- yloxy)-1 ,1-dimethyl-2-pyrrolidin-1-yl-1,3,2-diazaphospholidinium hexafluorophosphate), cyanuric chloride, 2-chloro-1-methylpyridinium iodide (Mukyiama’s reagent) and others. Some of these reagents can become incorporated through rearrangement reactions to stable ureas as for the carbodiimides, while others can react with hydroxyl group, leading again to their covalent incorporation onto the carbohydrate backbone. For selected polysaccharides such as pectin, alginate, carboxymethylcellulose and carboxymethylstarch, the activating agent can also be acidification, particularly if the reaction is carried out at high temperatures, preferentially above 100°C and below 200°C).

The combination of dehydration and catalysis with aminopyridine-types permits to obtain gel-like biomaterials for polysaccharide concentrations as low as 1%, preferentially 0.5%.

Ester bonds are known to be relatively facile to hydrolyse. Using ester bonds to crosslink polysaccharides such as alginate, hyaluronic acid and other carboxylated glycosaminoglycanes such as dermatan sulfate, chondroitin sulfate, and heparosan, carboxymethylstarch, carboxymethylcellulose, carboxymethylagarose and others, using the chemistry disclosed here, creates a strong contrast between the relatively stable chains and the more labile ester bonds. As a consequence, the degradation products become more predictable, they are anticipated to consist mostly in the original chains with limited chain shortening, which is near to impossible to achieve when crosslinking is performed through the use of external crosslinker, nor by the use of more resistant amide-groups. In some cases, short degradation fragments, with molecular weights below 70kDa, more preferentially below 50kDa and most preferentially below 40kDa are desired to facilitate renal clearance. In this case, the biomaterials can advantageously be synthesized using starting fragments with molecular weights below 70kDa, more preferentially below 50kDa and most preferentially below 40kDa. In other circumstances, and particularly for hyaluronic acid and other glycosaminoglycanes, higher molecular weights are desirable to avoid inflammatory effects associated with the smaller fragments. In this case, the biomaterial is advantageously synthesized with polymer chains with molecular weights above 100kDa, and more preferentially above 200kDa, and preferentially below 1 MDa to avoid excessive viscosity, even more preferentially below 700kDa. In some embodiments, small and large polymer fragments are combined to control the desired level of inflammation. Molecular weights of degradation fragments can be determined by techniques known in the art such as gel permeation chromatography after in-vitro degradation in basic or acidic environments calibrated such as to ensure complete degradation with a minimum of 95% of the ester bonds hydrolysed as determined by titration.

Some sterilization methods such as steam sterilization will decrease the ester crosslinking density by hydrolysis. The person skilled in the art can compensate for such hydrolysis by synthesizing by performing the titration on material samples before and after sterilization and increasing the original ester crosslinking density to compensate for the loss thus determined.

After synthesis, depending on the intended application, the biomaterial may be purified. This involves steps well-known in the art. Non-exclusive example methods include dialysis, washing of solids in excess aqueous or organic solutions, with or without mechanical agitation, as well as drying or evaporation steps. Ion exchange methods may also be used to provide appropriate counter-ions for the intended use. For example, for implantation, counter ions such as sodium or potassium cations and chloride, sulfate, phosphate or hydrogen carbonate anions are often used. Ion exchange is achieved by methods known in the art, which may in the simplest case include washing in excess electrolyte containing the desired counter-ions. In some embodiments, ion exchange medium and final biomaterial suspension medium are of identical composition, and may for example consist of physiological saline (0.9% NaCI) or phosphate buffered saline.

Mechanical properties such as the Young’s modulus of the biomaterial measured in compression and the elastic storage modulus G’ measured in shear rheology depend on many factors. The person skilled in the art can nevertheless adjust them to target values by noting that increasing the polymer concentration leads to an increase in these properties, permitting achieving a wide range of values between about 100Pa to about 100kPa for both moduli. At a polymer concentration, increased ester crosslinking density also leads to increased moduli, permitting compensatory adjustment to meet other target properties such as the polymer concentration in the walls.

For optimal mechanical and biological behavior in porous materials, we found out that a particularly desirable range of polymer concentration in the walls is between 20mg/g of hydrated wall material to 500mg/g of hydrated material, more preferentially from 75mg/g to 450mg/g, even more preferentially from 100mg/g to 400mg/g.

The settling volume describes the volume occupied by a biomaterial under free sedimentation. The settling volume for porous materials is larger than 10g/g, preferentially larger than 50g/g, and even more preferentially larger than 1OOg/g.

Both porous and non-porous biomaterials can be fragmented to obtain injectable formulations. For this, any means known in the art can be used, including sieving through grids, extrusion through narrow holes and mechanical mincing. Inversely, both porous and non-porous materials can be used as intact, monolithic biomaterials. In another embodiment, the crosslinking reaction is carried in molds imparting given shapes to the biomaterials, for instance to fabricate breast, buttock, nose, chin or other face implants or other body or organ implants with a pre-defined shape. In other embodiments, molding is achieved through compression with water loss through a selective membrane. Both porous and non-porous materials can also be molded in the form of individual particles, for example by carrying the crosslinking reaction in molds or in an emulsion.

In yet in another embodiment, after addition of chemical activator, but before crosslinking has proceeded substantially, the reaction mixture is 3D printed, as illustrated in Figure 8. The 3D printing can be concomitant with the dehydration step, for instance by using a reverse-osmosis tubular element for extrusion of the reaction mixture with simultaneous dehydration. An embodiment using a tubular dehydration element for 3D printing according to the invention is shown in Figure 8. Polysaccharides, buffer, catalyst in solution on the hand (12) and chemical activator (13) are mixed just prior to entry into the dehydration unit (14). Upon passing the dehydration unit, the reaction solution is concentrated beyond levels achievable by stirring, by at least partially selective water removal (15). Indeed, water is pressed through the water-selective (reverse osmosis) membrane (14) as a result of the pressure difference generated by the forces applied to maintain the reaction mass moving through the unit; optionally, a partial vacuum can also be applied at the water outlet (15). Typically, final solid concentrations larger than 10%, preferentially larger than 20%, and most preferentially larger than 30% are achieved. For 3D printing, the material is usually extruded through a nozzle (16) designed to form thin filaments; the nozzle can be a hollow needle or catheter available commercially (typically, but not exclusively, in the range of 22G to 27G). In 3D printing, the extruded thread-like material (17) is deposited onto a substrate (18) forming predefined patterns. By itself, the process forms a non- porous biomaterial in the form of non-porous tubular elements; depending on the design being printed, the end product is nevertheless porous, with pores resulting from the 3D design.

Alternatively, the reaction mix can be printed into a dehydrating highly concentrated, but immiscible solution such as a PEG solution in the case of dehydration in an aqueous two-phase system. This approach is illustrated in Figure 9. In this case, complete reaction solutions (20) consisting of polysaccharide(s), catalyst(s), activator(s), and optionally buffers and additives) is extruded through a nozzle (20) into a bath (23) consisting of a concentrated solution (more than 10%, preferentially more than 30%, more preferentially more than 50% of solute) of substances capable of forming aqueous 2-phase systems when mixed with aqueous solutions of polysaccharides. Upon extrusion, the extruded filament is immiscible with the surrounding bath solution, while water is attracted into the concentrated bath solution. In the process, a semi-dry filament (21) is formed. The viscosity and density of the bath maintain the filament in 3D-space, enabling generous designs with large pores. In one embodiment, carboxylated carbohydrate, catalyst, chemical activator and buffer form inner salts with absence or with minimal presence of bystander ions.

In one embodiment, the reaction mix is printed onto a cold substrate, in such a way as to form 3D printed structures after completion of the reaction in the cold and subsequent thawing, or for lyophilization. In this context, inks based on the ester crosslinking chemistry defined here can be combined with any other ink suitable in the same printing system, or to be added later or before printing the inks described here. In this way, biomaterials with locally and nearly arbitrarily variable mechanical properties or also locally and nearly arbitrarily variable degradation characteristics can be produced.

It is further understood that mixtures containing porogens can also be 3D printed if the porogens are chosen to be sufficiently small or flexible to be able to pass through the extruding nozzle.

Some porous biomaterials are reversibly compressible to more than 50%, more preferrable more than 80%, and most preferably to more than 90%. Reversible compressibility arises in part through pore interconnectivity because the pore fluid is easily evacuated. Pore interconnectivity can be achieved with various porogens by the person skilled in the art. An important element is the choice of porogens that easily merge, as is for instance the case for porogens capable of growth such as through water-binding or crystallization, for example during ice formation. Gasbased foaming can also lead to interconnected pores, particularly for slow crosslinking and lower surfactant concentrations. Reversible compressibility is also linked to large pore diameter to wall thickness ratios. Reversibly compressible biomaterials can be injected or implanted in a dehydrated state, to gain volume in situ for instance by absorption of interstitial fluid, or by attracting liquid from blood vessels or lymphatic vessels by their expansive properties and ensuing alteration of the local filtration equilibrium. The additional liquid expands the pore space and provides additional space for example for tissue ingrowth. This can be advantageous for minimally invasive delivery of large intact implants through catheters or other narrow-bore tubing or instrumentation, a process that can be further aided by deliberate mechanical folding or rolling of the dehydrated material. In multiparticulate injectables, particles significantly larger than the diameter of the delivery catheter or needle can be delivery by transient compression during delivery, or by pre-existing dehydration before delivery. In situ, both in the case of injectable formulations and implants, the expansion process can help favoring tissue ingrowth by rapid uptake of fibrin, extracellular matrix proteins or cells during the expansion. Reversible compressibility can also be advantage in-vivo for recovery of the shape and volume of the implants after incidental compression, and to provide a soft, yet resilient natural feeling of the implants.

Non-porous and porous biomaterials can be used for various applications in-vivo, including prevention of adhesions, as a transitory implantable or injectable volumizing agent or as a drug or substance release depot, and also as an implant or injectable fulfilling more than one of these functions. In one embodiment, sieved, multiparticulate non-porous material is used as a dermal filler in the face, for example for volumizing nasolabial folds, lips, cheeks, temples or the like. In another embodiment, non-porous material is used for the volumizing the back of the hand. In another embodiment, lidocaine or another anaesthetic agent is added for the use as a dermal filler. In yet another embodiment, an antiseptic agent such is povidone iodine at a final concentration between 0.1mg/mL of iodine to 20mg/mL of iodine, more preferentially between 0.5mg/mL of iodine and 5mg/mL of iodine, is mixed with a suspension of non-porous particles for use as a dermal filler.

In another embodiment, porous and non-porous biomaterials can be used for various applications in-vivo, including as a transitory implantable or injectable volumizing agent or as a drug or substance release depot, and also as an implant or injectable fulfilling more than one of these functions. Tissue ingrowth can be controlled by pore size and pore fraction, with larger pore size and pore fraction being more conducive to tissue ingrowth as known in the literature. In one embodiment, the biomaterial is used as a dermal filler in the face, for example for volumizing nasolabial folds, lips, cheeks, chin, temples or the like. In another embodiment, the biomaterial is used for the volumizing the back of the hand. In another embodiment, the biomaterial is used for volumizing buttocks, breasts, hips and other body areas. In another embodiment, the biomaterial is used to provide volume and smoothen the surface of the skin, such as for the treatment or correction of cellulitis. In another embodiment, it is used in combination with energy-based devices. In yet another embodiment, the biomaterial is used as a hepatic regeneration template, as a muscle regeneration template, as a urethral implant against incontinence. In some embodiments, the biomaterial is used in injectable from, in others, it is used as a scaffold with predefined shape. In some embodiments, the biomaterial is delivered minimally invasively in a compressed state through catheters or other tubular elements to regain its volume after placement. In another embodiment, lidocaine or another anaesthetic agent is added for the use as a dermal filler. In yet another embodiment, an antiseptic agent such is povidone iodine at a final concentration between 0.1mg/mL of iodine to 20mg/mL of iodine, more preferentially between 0.5mg/mL of iodine and 5mg/mL of iodine, is mixed with a suspension of non-porous particles for use as a dermal filler.

In biomaterials supplied either as pre-shaped single item or as multiparticulate injectable bulking agent, geometric particle parameters are important as well. Spherical particles, for example produced in emulsion polymerization, provide materials that tolerate little deformation before permanent deformation. Such materials may be used when purely volumetric correction with internal redistribution of the material is required. Irregular particles form materials with high resistance to deformation, and are preferentially used to impart shape to injected implants, through massaging shortly after application. The effect can be further amplified with irregular particles with significant protrusions (expanding 10%, preferentially 20%, and even more preferentially 30% or more above the general particle surface). Such particles form particularly cohesive implants and can for instance be obtained by mechanical fragmentation of porous bulk material or 3D printing of individual particles. A possible application is shaping after implant injection, by external massaging into the desired shape or by application of an external mold. Porosity and ensuing ingrowth ensure anchorage to surrounding tissues for the creating of localized implants without migration. Porous material, in particulate or bulk form, with average pore diameters from 10 micrometers to lOmillimeters, more preferentially from 20 micrometers to 2mm, and most preferentially from 50 micrometers to 1mm are conducive to tissue ingrowth and vascularization. Pores can be templated by ice crystals formed during freezing, with pore size control enabled for instance through the use of different mold materials. Porogens such as gas bubbles or salt crystals are also typically used for introduction of controlled porosity. Porosity as characterized by pore volume as compared to total biomaterial volume is also an important parameter regarding both degradation kinetics and material colonization. A large pore fraction (more than 30%, preferentially more than 50%, and even more preferentially more than 80%) is useful for enhancing tissue ingrowth. To deliver intact sponge-like particles with large porosity through narrow-bore tubing, catheters, or needles, particles with reversible compressibility are needed when the particles are larger than the bore. Reversibly compressible particles recover their mechanical and geometrical properties following recovery from large scale compression (up to 50% of the volume, more preferentially up to 70% of the volume, and even more preferentially 80% or 90% of the volume). Reversible compressibility is obtained for instance through particle design, with large pore diameter to wall thickness ratios (more than 0.5, preferentially more than 1 , more preferentially more than 2, and most preferentially more than 3). Reversible compressibility is also advantageous in-vivo to create geometrically stable implants supporting vascularization.

In one embodiment, the biomaterial is provided in a compressed or dry or partially dry state and will be hydrated before, during, or after the implantation step. The hydration step can be performed by adding cell suspension, tissue suspension, a solution or suspense of proteins, live or dead cells, exosomes, agents... The hydration step can also happen through absorption of liquid at the implantation site from the surrounding tissue or the surrounding blood and lymphatic vessels.

The biomaterial can be made by ester crosslinking from various carboxylated polysaccharides such hyaluronic acid, dermatan sulfate, chondroitin sulfate, heparosan, heparin and other glycosaminoglycanes, carboxymethylated polysaccharides such as carboxymethylstarch, carboxymethyldextrin and carboxymethylcellulose, and other natively carboxylated polysaccharides such as alginates, pectins and xanthans. Binary or multi-component mixtures of such polysaccharides can also be used, and neutral polysaccharides or non-carboxylated polysaccharides such as starch and maltodextrins, cellodextrin, chitosan, glucomannan, carrageenans and others can also be admixed for crosslinking via their hydroxylgroups. Carboxylated polymers such as polyacrylic acid, polymaleic acid, polyaspartic acid, polyglutamic acid, polyvinylbenzoic acid and others, copolymers with carboxylated monomers, and mixtures of such polymers can also be admixed for co-crosslinking. Likewise, hydroxylated synthetic and semi-synthetic polymers can be admixed and will be incorporated through ester formation. Some examples include polyvinylalcohol, hydroxyethyl- and hydroxylpropylcellulose, hydroxyethylmethacrylate, linear and branched polyglycerol, phenol-formaldehyde resins, epoxide self-polymerisates such as the self-polymerized polymer of butanedioldiglycidylether and diglycidylether, silicone-polyols such as dimethicone-co-polyol, hydroxy- or carboxyl-terminated polyethylene glycols, including polymerization grades of about M_n=500, M_n=5000, M_n=50 000; and others. Small molecules with hydroxyl or carboxyl functionalities can also be incorporated by ester formation during polymerization. In light of tissue reconstruction, metabolic carbon sources are of special interest, namely saturated and unsaturated fatty acids such as palmitic, oleic and linoleic acid, glycerol, phospholipids, mono- and diglycerides, hexoses and pentoses such as glucose, fructose, ribose and desoxyribose and ascorbic acid. For the purpose of favoring adipogenesis, adipogenic pharmacologically active agents such as hydroxylated glitazones such as troglitazone or hydroxypioglitazone can covalently incorporated through ester formation and released intact during degradation. For the purpose of favoring muscle fiber generation and regeneration, omega-3 poly-unsaturated fatty acids can likewise by added for incorporation, but also prostaglandin E-2, prostaglandin F2alpha, testosterone and analogs, as well as betamimetics and particularly beta-2-mimetics such as clenbuterol or salbutamol. The small molecules listed here can not only be incorporated, but also admixed without covalent coupling, or supplied as combination of both forms. Generally, covalently coupled molecules will be released more slowly, through material degradation, while admixed molecules will be released more rapidly. Combinations of covalent incorporation and admixing can be used to tailor release profiles, with a burst release (preferable, more than 50% of the admixed amount released during the first 1 h, during the first 2h, during the first 12h, the first 24h or the first week) due to admixing and sustained release (preferably, more than 50% retained up to 1 week, up to 2 weeks, up to 1 month, up to 2 month or up to 6 months). Different release profiles can be chosen for different molecules, for example rapid release of inductors of differentiation and slower release of maturation factors.

Additional agents can also be admixed with the formulations. Such agents include the caine family of anaesthetic agents and particlulary Lidocaine and its salts such as lidocaine hydrochloride. Other anaesthetic agents include, without limitation, ambucaine, amolanone, amylocalne, benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine , butacaine, butamben, butanilicaine, butethamine, butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine, dibucaine, dimethysoquin, dimethocaine, diperodon, dycyclonine, ecgonidine, ecgonine, ethyl chloride, etidocaine, beta-eucaine, euprocin, fenalcomine, formocaine, hexylcaine, hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate, levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methyl chloride, myrtecaine, naepaine, octacaine, orthocaine, oxethazaine, parethoxycaine, phenacaine, phenol, piperocaine, piridocaine, polidocanol, pramoxine, prilocaine, procaine, propanocaine, proparacaine, propipocaine, propoxycaine, psuedococaine, pyrrocaine, ropivacaine, salicyl alcohol, tetracaine, tolycaine, trimecaine, zolamine, procaine, chloroprocaine, cocaine, cyclomethycaine, cimethocaine (larocaine), propoxycaine, procaine (novocaine), proparacaine, tetracaine (amethocaine). Non-limiting examples of aminoamide local anesthetics include articaine, bupivacaine, cinchocaine (dibucaine), etidocaine, lidocaine (lignocaine), mepivacaine, piperocaine, prilocaine, ropivacaine, and trimecaine. A composition disclosed herein may comprise a single anesthetic agent or a plurality of anesthetic agents, with individual agents being present as such, or as salts. A non-limiting example of a combination local anesthetic is lidocaine/prilocaine (EMLA). Other agents include adrenaline, antimicrobials, antifungals, antibiotics, antivirals, antiparasitics, growth factors such as FGF-1 , FGF-2, EGF, TGF-beta PDGF and others, DNA, RNA, including gene expression vectors, synthetic nucleic acids, nanoparticles, superparamagnetic nanoparticles, ferromagnetic nanoparticles, carbon nanotubes, graphene sheets, cytostatics, extracellular matrix molecules, nucleic acids, neurotransmitters, adjuvants, exosomes, liposomes, viral particles, exosomes, transfection agents, immunological adjuvants, anti-inflammatory agents, cytostatic agents, antibodies, immune checkpoint inhibitors such as anti-CTLA4 or anti-PD1 , radioisotopes, radiological contrast agents, colorants, tattooing pigments. The biomaterial can also be coated on the internal and external surfaces, or bulk adsorbed or chemically coupled with extracellular matrix proteins such as collagen I, collagen III, collagen IV and other collagens, laminin 511 and other laminins, Matrigel, fibrin, fibrinogen, fibronectin, vitronectin, entactin, elastin, but also immune signalling molecules such as interleukins (il-1 , il6, il-12, il-18 and others), interferon-gamma, mcp1 , mcp2 and other CCL-type of chemokines, sdfla, cxcll , cxcl3, cxcl8, and other CXCL-type of chemokines, but also peptides consisting of or containing motives such as RGA, RGD, RADA, IKVAV, LRE, YIGSR, CLPFFD, VGVAPG, YNAAGGHNA and others. The biomaterial can also be coated, impregnated, and covalently or non-covalently modified with autologous, heterologous orxenologous decellularized material, for example decellularized adipose tissue, decellularized skin, decellularized heart tissue, decellularized muscle tissue, decellularized bladder tissue. For these modifications, reversibly compressible biomaterials can advantageously be rapidly mixed with a given solution or a sequence of solution through repeated compression and rehydration cycles; other biomaterials can be modified through perfusion or sufficiently long diffusion times.

The biomaterials described here can also be used as cell- or tissue-delivery vehicles. To enhance survival of adherent cells, cell-adhesion motives can be provided by covalent or non-covalent biomaterial coating or modification with extracellular matrix proteins, and particularly collagens such as collagen I, III, and IV, laminins, fibrin and fibronectin, and adhesion peptides such as RGA, RGD, RADA are useful. Porous, and particularly porous reversibly compressible biomaterials can be efficiently loaded with cell or tissue suspensions by transient compression, followed by mechanical aspiration of the suspensions. The cells may include stem cells such as embryonic stem cells, embryonic stem cells having undergone directed differentiation to various stem cell or terminal cell types, induced pluripotent stem cells and induced pluripotent stem cells having undergone directed differentiation, stem cells or differentiated cells obtained by transdifferentiation, stromal, mesenchymal or hemapoietic stem cells isolated or mobilized from the bone marrow, stromal, mesenchymal or adipous stem cells isolated from adipous tissue or lipoaspirate, neural or glial stem or progenitor cells isolated from peripheral or central nervous tissue, organ-specific stem cells such as cardiac stem cells, pancreatic stem cells, satellite cells, endothelial stem and progenitor cells, keratinocyte stem cells, hair follicle stem cells, urothelial stem cells, intestinal epithelium stem cells, hematopoietic and side-population stem cells. Somatic cells include a multitude of cells, including osteoblasts, chondrocytes, fibroblasts, endothelial and outgrowth endothelial cells, smooth muscle cells, dermal fibroblasts, myofibroblasts, myoblasts, cardiac muscle cells, keratinocytes, hepatocytes, intestinal epithelial cells, adipocytes, chromaffine cells, sweat gland epithelial cells, sebaceous gland epithelial cells, mammary epithelial cells, adipous progenitor cells, lymphatic endothelial cells, dendritic cells, macrophages, polarized macrophages, naive, activated or regulatory B- and T-cells, T-cells with selected or modified t-cell receptors, B-cells producing specific antibodies, hormone producing cells such as beta-cells, alpha-cells, thyrotropic, lactotropic, corticotropic, gonadotropic or somatotropic pituitary cells, mucosal epithelial cells, pulmonary epithelial cells, thyroid and parathyroid cells and others, as well as combination of different cell types. Tissue and tissue extracts include lipoaspirate, blood plasma, platelet-rich plasma, whole blood, serum, excised and mince subcutaneous tissue, fat tissue, muscle tissue, liver tissue, of autologous, heterologous or xenologous origin. Tissue and tissue extracts may or may not be treated or preconditioned, such as by enzymatic digestion, centrifuging or incubating. For combining the biomaterial with cell or tissue extracts reversibly compressible biomaterials can advantageously be rapidly mixed with a given solution or suspension or a sequence of solution through repeated compression and rehydration cycles; other biomaterials can be modified through perfusion or sufficiently long diffusion times. Multiple cell, tissues, agents and other modifications may be freely combined.

The biomaterials described here can be implanted in-vivo either as intact, pre-shaped scaffolds or in injectable form. Porous biomaterials are typically found with substantial ingrowth of host tissue after a few weeks to months, and are nearly completely replaced by host tissues after 1 month to 5 years, preferentially two months to 1 year. Upon degradation, materials freed can in some cases be hydrolyzed by endogeneous enzymes (hyaluronic acid, dermatan sulfate, keratan sulfate, heparosan, heparin and other endogeneous polysaccharides). In most cases, enzymatically and non-enzymatically degraded fragments are taken up transitorily by tissue macrophages before excretion or terminal metabolization. Non-porous implants are less amenable to tissue ingrowth and act more typically as transitory bulking agent.

The degradation time can be tuned by tuning the ester crosslinking density and the polymer concentration in the walls. Tissue ingrowth and particularly vascularization depend on the pore size, larger pore sizes being more favorable to vascularization than smaller ones, with a threshold of about 50 micrometer average pore diameter favoring larger blood vessels and capillaries, growth of nerves, growth of lymphatic vessels and capillaries.

In one embodiment, the biomaterial is reversibly compressible and porous. When injected subcutaneously in-vivo, the biomaterial lifts the surrounding tissues and the porous space is progressively occupied by extracellular matrix proteins, different types of cells including stem cells, fibroblasts, adipocytes, progenitor cells and macrophages, and is vascularized. In another embodiment, the cells include adipocytes. In another embodiment, the cells include lymphatic endothelial cells. In another embodiment, the cells include neurons. In another embodiment, the cells include hematopoietic stem cells. In one embodiment, the tissue occupying the pore space includes nerves, lymphatic vessels, blood vessels or combinations thereof. In one embodiment, the tissue occupying the pore space includes a lymph node.

Figure 3, 4 and 5 illustrate results obtained upon implantation of an ester-crosslinked biomaterial according to the invention in mice. Figure 3 shows that at 3 weeks, some biomaterial still subsists without major tissue ingrowth in the center of the subcutaneously injected area (4). In this particular example, at 3 weeks, most of the material is partially degraded and has been absorbed by phagocytic cells (5) with some tissue ingrowth. No capsule is seen, the material here has been injected into a septum (6) and no dense layer is seen at the interface.

Fig. 4 quantifies the implant evolution over the following months, with increasing tissue ingrowth and decrease contribution of the phagocytic cells. Figure 5 shows that by 6 months, most material has been cleared including from the phagocytic cells.

In one embodiment, the biomaterial is degradable through the action of macrophages. In another embodiment, the biomaterials are degradable through the action of endogeneous enzymes such as hyaluronidase, esterases, lipases and amidases present in the implant environment. In one embodiment, the molecular weight of the polysaccharide composing the backbone of the biomaterial is chosen to achieve a specific degradation time and a specific time of residence in macrophages involved in the degradation process. In another embodiment, the molecular weight of the polysaccharide composing the backbone of the biomaterial is chosen to achieve renal or hepatic excretion with minimal residency in macrophages.

In some embodiments, the biomaterial is packaged sterile in a sterile syringe. In some embodiments, the sterile syringe containing the sterile biomaterial is further packaged into a sterile pouch bag. In other embodiments, one or several sterile syringes are assembled with accessory elements to form a kit, such as the one shown in Figure 6, which consists of a sterile pouch bag (7), a sterile syringe containing biomaterial (8), a sterile connector for mixing (9) and a further sterile syringe with medium (10). Such a kit can for example be used to prepare and mix cell suspensions with the biomaterial or to prepare and mix tissue suspensions with the biomaterial. Alternatively, the provision as a kit permits to store the biomaterial in conditions preventing ester hydrolysis, for instance by adaption of pH and salinity to minimize hydrolysis, with mixing or reconstitution of a physiologically suitable injectable immediately prior to use by mixing the reconstitution medium provided in the kit with the biomaterial. Such mixing under is conveniently carried out under sterile conditions by using a sterile connector (9) which can also be provided in the kit. This permits to join the syringes as shown in (11), and allows the components to be mixed via back-and-forth movements applied to the plungers.

The kit may also contain delivery catheters, delivery needles, tubing, vials and other components. Delivery catheters and needles may be supplied separately but within the kit, or may already be screwed to the biomaterial syringe. Catheters and needles may for example, and non-exclusively, conform to 16G, 19G, 22G, 25G, 27G, 30G or 31G standards, and they may or may not conform to thin wall standards.

In one embodiment, the kit comprises a device for purifying and sorting cells from a tissue extract, such as a closed system for isolating Stromal vascular fraction from lipoaspirated tissues. In another embodiment, the kit comprises a centrifuge tube. In another embodiment, the biomaterial is supplied in a centrifuge tube or in a filtration device or filtration bag. In another embodiment, the biomaterial is provided in a closed system for isolating cells, or tissue fractions, such as stromal vascular fraction.

Method for Ester density analysis by infrared spectroscopy (Fourier Transform Infrared Spectroscopy FTIR)

If components known to interfere with infrared spectroscopy are present in the material to examined, it is first washed free of such contaminants, for example by suspension in a 50% isopropanol : 50% water mixture for polysaccharides insoluble in this mixture, in the presence of neutral salts such as sodium chloride if additionally, ion exchange is required.

The material of interest, if necessary freed of interfering impurities, is next neutralized to pH 7.0 in suspension. This can be done directly in an aqueous suspension by addition of dilute sodium hydroxide or hydrochloric acid while monitoring the pH to achieve pH 7.0. The material is then dried at 80°C or under vacuum. The dried material is then analyzed either directly by attenuated internal reflection FTIR or after compaction into a potassium bromide pellet at a concentration of 1 % to 3% by transmission FTIR.

FTIR is a relative method and ester crosslinking density needs to be identified on the basis of known samples established through the titration method, by identifying a signature peak of the ester bonds (typically around 1730cm^-1) and a constant peak elsewhere in the spectrum. In many cases, FTIR is suitable only for the detection of high ester crosslinking density. Method for Ester density analysis by titration

The ester crosslinking density is determined by the consumption of base during ester hydrolysis. The amount of base consumed is determined by back-titration after addition of a known excess of base to induce complete hydrolysis.

In cases where the material is suspended in solutions containing additional salts, the material is first washed free of contaminating salts prior to further processing to obtain the dry weight of the polymeric content.

The biomaterial sample is neutralized to pH 7.0. This is done in an aqueous suspension by addition of dilute sodium hydroxide or hydrochloric acid. The material is then dried at 80°C or under vacuum and the weight is measured. The dry material is suspended in deionized water, and the pH verified and possibly re-adjusted to be neutral. An exactly known amount molar amount of nNaon of sodium hydroxide is then added. The amount of sodium hydroxide added should exceed the molar amount of ester bonds anticipated in the sample. The resulting solution is incubated in an airtight container at 80°C for a minimum of 2h, or twice the time required for all visible biomaterial to dissolve, whichever is longer.

After hydrolysis, the solution is left to cool down to room temperature within the airtight container. Assessing that indeed base was added in sufficient amounts (pH>10.5), following this, the solution is titrated back to pH 7.0 (+/- 0.2) using dilute acid solution and the molar amount of acid required for neutralization is recorded. The molar amount of ester groups present in the sample is the difference between the excess base nNaon added initially and the molar amount of nnci required for back-titration. The ester crosslinking density is then reported as the molar concentration of ester groups as detected by the base consumption in the dry solid of mass m. It is found from (nNaon - n_Hci)/m. Controls are typically added to the titration as is well known in the art, particularly in the presence of additional hydrolysable groups, which need to be either determined separately or by controls as their hydrolysis may also consume base.

Some polysaccharides present particularities that make it necessary to adjust the ester crosslinking density quantification method described here. These adjustments are related to particularities of the polysaccharides which are generally known in the art. For instance, if base- hydrolysable groups are present, the consumption of base will appear greater depending on the additional hydrolysis occurring during incubation. The person skilled in the art will proceed to adjust the amount of base consumed by the ester hydrolysis compared to suitable controls without ester bonds, or based on separate evaluation of the additional groups being hydrolysed, with titrimetric or spectroscopic techniques. Such correction is for example necessary when sensitive amides are present. Method for determination of the amount of chemical activator incorporated in the biomaterial

Nuclear magnetic resonance NMR can be used to quantify the amount of reactants incorporated into the ester-crosslinked materials. The typical chemical constitution of the polysaccharides leads to a majority of the protons being associated with a chemical shift in a band from about 3.0ppm to 5.0ppm relative to the tetramethylsilane standard, whereas many activators and catalysts are easily identifiable and quantifiable protons outside this band.

Other techniques can also be used and may be necessary for certain cases. For example, chromatographic techniques such as high-performance liquid chromatograph or gas chromatography coupled to mass spectroscopy can be used to identify fragments of incorporated activators or catalysts with high precision. For quantitative assessment, these techniques need calibration with known compounds at known concentrations, as known in the art.

Method for determination of the settling volume

A known amount of material (determined by weighing to constant mass) is washed with deionized water and then suspended in an excess amount of deionized water in a graduated cylinder. The material is left to sediment overnight, and then the volume of the sediment is assessed by using the graduations. If the sediment occupies the entire volume, the experiment is repeated with more deionized water until a separation into sediment and supernatant is observed. The settling volume can be reported in terms of mass of liquid absorbed in relation to the dry mass originally present, giving rise to a g/g ratio.

Examples

Example 1

A solution with 2% sodium hyaluronate, 20mM 4-pyrrolidinopyridine,15mM PIPES and 10mM TPTLI ([dimethylamino-(2-oxopyridin-1-yl)oxymethylidene]-dimethylazanium) was adjusted to pH 6.5. The solution was extruded, using a 25G needle, into of 50% w/w of polyethylene glycol with a molecular weight of 10kDa. This yielded a biomaterial with a Young modulus of 500Pa, and an ester crosslinking density of 0.075mol/kg. A biomaterial consisting of long, irregular particles was produced through manual fragmentation and sterilization with 70% ethanol and extensive washing with physiological saline.

Example 2

5mL of a solution containing 30mg/mL sodium alginate, HEPES buffer ((4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid) 50mM at pH 7.0, 50mM DMAP-HCI (dimethylaminopyridinehydrochloric acid salt) and 30mM of EDC-HCI (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloric acid salt) was prepared. The solution was frozen in a Falcon tube and left to incubate for one month in the cold. After thawing, the biomaterial was washed extensively with physiological saline (0.9% sodium chloride NaCI). The biomaterial was measured to have a Young modulus of 2500 Pa and an ester crosslinking density of 0.18mol/kg and less than 0.05mol of EDC was incorporated per mol of ester. The biomaterial was fragmented using an electric mincer to produce irregular porous particles according to the invention and sterilized during 20 minutes at 121°C using a standard autoclave. The settling volume of the resulting biomaterial was 150g/g. The pore fraction was 85%, for an interconnected pore fraction of 75%. The mean pore diameter was 55.6 micrometers, with a pore-to-wall ratio of 2.4

Example 3

A solution with 2% sodium carboxymethylstarch, 20mM 4-pyrrolidinopyridine,15mM PIPES and 10mM TPTLI O-[2-Oxo-1(2H)-pyridyl]-N,N,N',N'-tetramethyluronium-tetrafluoroborate) was adjusted to pH 7.0 and spray-dried at 40°C and 50mmHg of pressure. After 3 days of incubation, this yielded a powder, which upon hydration to 3% of reticulated mass by weight had a Young modulus 900Pa, and an ester crosslinking density of 0.35mol/kg as indicated by titration. The biomaterial was washed extensively in physiological saline, sterilized at 121°C using a standard autoclave.

After degradation at pH 1 and 80°C, NMR indicated that 0.12% of the residues of the carboxymethylstarch chains were modified with an TPTU-derived urea, which is less than 0.015 mol of incorporated activator per mol of ester formed.

Example 4

A solution containing 10.5mg/mL of protonated pectin was prepared; it had a pH of 4.1. The solution was frozen at -40°C and extracted in cold (-40°C) isopropanol until complete dissolution of the ice. After warming to room temperature, isopropanol was replaced with ethyl acetate and the porous biomaterial dried at room temperature. After drying, it was exposed to 160°C in an oven for 30 minutes. The resulting biomaterial was suspended in physiological saline and fragmented using a bowl mortar to produce particles.

The ester crosslinking density was found to be 0.98mol/kg, for a Young modulus of 0.5kPa. The measured pore fraction was 89.4+/-4%, whereas the interconnected pore fraction was 87.5+/-6%. The mean pore diameter was 90.6micrometers, with a pore to wall diameter ratio of 5.1. The particles were irregular with protrusions.

Example 5

A mixture of pullulan (20%) and sodium alginate (3%), buffered by 50mM PIPES buffer (piperazine-N,N'-bis(2-ethanesulfonic acid), pH 6.5), containing 20mM DMAP-HCI and 40mM EDC-HCI was frozen at -20°C and dried under vacuum. The product obtained was an elastic, slightly porous biomaterial with an ester crosslinking density of 0.18mol/kg, with an EDC incorporation rate of 0.03mol of EDC per mol of ester formed.

Example 6

2.5mL of a reaction solution containing 5% hydroxyethylcellulose and 3% sodium carboxymethylstarch, 20mM 2-(5-Norbornene-2,3-dicarboxamido)-1 ,1 ,3,3-tetramethyluronium Tetrafluoroborate (TNTLI), HEPES buffer ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) 30mM, and 20mM 9-Azajulolidine hydrochloride was mixed with 5g of anhydrous calcium sulfate. The mix, in the form of a humid paste, was incubated for 3days at ambient temperature, before dissolution of the calcium sulfate with 100mM NasEDTA solution for 2 weeks. This formed a transparent, finely porous gel with an ester density of 0.08mol/kg.

Example 7

5mL of a reaction solution containing 1% of polyvinylalcohol, 6% hydroxypropylcellulose and 4% sodium alginate, 25mM PyBOP (benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate), 50mM MES buffer (2-N morpholinoethansulfonic acid), pH 5, and 20mM 4-pyrrolidinopyridine hydrochloride was mixed with 1mL of polyethyleneglycol solution with a molecular weight of 10kDa and a mass concentration of 50%. The mix was well stirred, yielding a gel-like emulsion which was incubated for 3days at ambient temperature, before dissolution in an excess of water and fragmentation by a magnetic stirbar to irregular, porous particles.

Example 8

1 mL of a solution containing 30mg/mL sodium alginate, HEPES buffer ((4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid) 50mM at pH 7.5, 50mM DMAP-HCI (dimethylaminopyridinehydrochloric acid salt) and 20mM of EDC-HCI (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloric acid salt) and 5mM of sodium oleate was prepared. The solution was spread on a glass surface and dried under a moderate vacuum of 20mbar. After 1 day of incubation, a transparent solid membrane with a wax-like touch was recovered. This membrane was hydrated to a flexible sheet-like hydrogel with a total ester density of 0.4mol/kg and an incorporation of the chemical activator of less than 0.02mol/mol of ester.

Example 9

Reversibly compressible, porous biomaterial consisting of irregular particles with protrusions is injected into the subcutaneous tissues of the hands, 5mL per hand for hand rejuvenation. Example 10

To prepare an injectable implant from a solution containing cells in suspension, contained in a syringe, a kit alike to the one shown in Figure 6 can be used. Such a kit may consist of a sterile pouch bag (7), a sterile syringe containing biomaterial (8), a sterile connector for mixing (9) and a further sterile syringe with medium (10), and possibly additional syringes or accessories. A cell suspension, for example for gene therapy based on previously prepared autologous cells is available, may be available in a third syringe external to the kit. In this case, the mixing connector (9) can be used first to mix medium and cells from the unrelated syringe by connecting the syringes via the connector, and applying back-and-forth movements of the plungers. Similarly, mixing with the biomaterial can take place through by back and forth movements of the plungers after connecting medium and biomaterial syringe (11). If the cell suspension is available in a recipient, it can be aspirated into the medium syringe provided in the kit, if necessary by discarding part of the medium to accommodate for the volume of the cell suspension.

Example 11

Human adipose tissue is harvested by lipoaspiration from a suitable anatomical location such as the abdominal wall or the hip reaction. Optionally, the lipoaspirate is processed by collagenase digestion, centrifugation, sedimentation, sieving or other techniques known in the art. Optionally, the stromal vascular fraction is isolated from the lipoaspirate by a mechanical process. The lipoaspirate may also be more concentrated or more dilute as compared to the original harvest, depending on the processing, and may optionally be complemented with additional components such as stem cell suspensions, stem cell fractions, plasma, physiological saline, platelet-rich plasma, serum, pharmacological agents such as adrenalin, or aneasthetic agents such as lidocaine and others. The biological component thus obtained is then combined with biomaterial through the use of elements of a kit. The kit can for example contain biomaterial at a concentration higher than the final concentration for injection, for example about 1.1 to 5x higher, more preferentially about 1.5x to 3x higher. The kit may provide a connector for connecting a with biomaterial under sterile conditions to a syringe containing a suitable amount of biological component or pharmaceutical/chemical component to reach an acceptable biomaterial concentration upon mixing. A composite living biomaterial is then created by mixing the biological component with the biomaterial, using a series of back-and-forth movements of the pistons to achieve acceptable homogenization. The composite living biomaterial can then be implanted through minimally invasive delivery.

Example 12

Human tumor tissue is harvested by means of a biopsy, and combined with biomaterial through the use of mixing kit as described above. The resulting mixed biomaterial is used in immunocompetent or immunocomprised laboratory rodents, preferentially but not necessarily mice, to establish a patient-derived xenograft model for the study of anticancerous drugs suitable to treat the cancer in a personalized manner.

Example 13

Liver tissue is harvested by means of a needle biopsy from an immune-matched donor. The liver biopsy material is liquefied by means of mechanical shearing and mixed with the biomaterial by a mixing kit as described above. The assembled, living biomaterial is implanted minimally invasively in a recipient, for example for liver regeneration in terminal cirrhosis.

Example 14

The biomaterial according to the invention is supplied with 1%-5% lidocaine or other anaesthetic agent content for local anaesthesia concomitant with injection.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the scope of the appended claims.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Claims

CLAIMS:

1. A biomaterial comprising a polysaccharide having both hydroxyl and carboxyl groups crosslinked by internal ester crosslinks formed essentially from these hydroxyl and carboxyl groups, characterized in that the ester crosslinking density of the biomaterial is between 0.01 mol/kg and 1 mol/kg.

2. The biomaterial according to claim 1 , characterized in that the ester crosslinking density is between 0.02 mol/kg and 0.7 mol/kg, preferably between 0.05 mol/kg and 0.5 mol/kg and even more preferably between 0.07 mol/kg and 0.4 mol/kg.

3. The biomaterial according to one of the claims 1 to 2, characterized in that the ester crosslinking density is larger than 0.07 mol/kg and preferably larger than 0.1 mol/kg.

4. The biomaterial according to one of the claims 1 to 3, characterized in that it is in the solid state.

5. The biomaterial according to one of the claims 1 to 4, characterized in that it is in a hydrated, gel-like state.

6. The biomaterial according to one of the claims 1 to 5, characterized in that its pH is between 4 and 9, more preferentially between 5 and 8.

7. The biomaterial according to one of the claims 1 to 6, characterized in that non-esterified carboxyl groups are essentially supplied as sodium, potassium, calcium or magnesium salts.

8. The biomaterial according to one of the claims 1 to 7, characterized in that it has an amount of chemical activator moieties, irreversibly bound to the biomaterial during formation of the ester crosslinking which is less than 0.4 mol per mol of ester formed, preferably less than 0.2mol of chemical activator incorporated per mol of ester formed.

9. The biomaterial according to claim 8, characterized in that it has an amount of chemical activator moieties of less than 0.1 mol of chemical activator incorporated per mol of ester formed, more preferentially less than 0.05 mol of chemical activator and most preferentially less than 0.01 mol of chemical activator.

10. The biomaterial according to claim 8 or 9, characterized in that the chemical activator is a carbodiimide, preferentially a water-soluble carbodiimide, and most preferentially 1-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC).

11. The biomaterial according to claim 10, characterized in that less than 0.4 mol of carbodiimide is incorporated per mol of ester formed, preferentially less than 0.2 mol of carbodiimide incorporated per mol of ester formed, and even more preferentially less than 0.2 mol of carbodiimide incorporated per mol of ester formed, more preferentially less than 0.1 mol of carbodiimide incorporated per mol of ester formed, more preferentially 0.05 mol of carbodiimide and most preferentially less than 0.01 mol.

12. The biomaterial according to one of the claims 1 to 11, characterized in that it has the form of irregular particles.

13. The biomaterial according to claim 12, characterized in that the particles comprise protrusions at their surfaces.

14. The biomaterial according to claim 12 or 13, characterized in that the particles are internally built in the manner of spicula.

15. The biomaterial according to one of the claims 12 to 14, characterized in that it consists of a multitude of particles with an average diameter of 0.05 mm to 5 mm, preferentially 0.075 mm to 1 mm.

16. The biomaterial according to one of the claims 1 to 15, characterized in that it has a porous structure.

17. The biomaterial according to claim 16, characterized in that the pores have an average diameter between 5 micrometers and 5 mm, more preferentially between 10 micrometers and 1 mm, and most preferentially between 20 micrometers and 500 micrometers.

18. The biomaterial according to claim 16 or 17, characterized in that the polysaccharide density in the walls of the porous structure in the hydrated state is 20 mg/g of hydrated wall material to 500 mg/g of hydrated material, more preferentially from 40 mg/g to 400mg/g, even more preferentially from 50 mg/g to 300 mg/g, more preferentially from 75 mg/g to 250 mg/g.

19. The biomaterial according to one of the claims 16 to 18, characterized in that it has a spongelike structure with a porosity, preferably larger than 50 %.

20. The biomaterial according to claim 19, characterized in that the porosity is larger than 70%, preferably larger than 90%.

21. The biomaterial according to one of the claims 16 to 20, characterized in that it has a ratio of mean pore diameter to mean wall thickness of at least 1 , preferentially 2, and most preferentially 3 or larger.

22. The biomaterial according to one of the claims 1 to 21, characterized in that the polysaccharide is chosen from the group of:

- hyaluronic acid;

- dermatan sulfate;

- chondroitin sulfate;

- heparin

- heparosan;

- alginates;

- carboxymethylcellulose; and

- copolymers thereof.

23. The biomaterial according to one of the claims 1 to 22, characterized in that is synthesized from a polysaccharide with a molecular weight in the range of 1 kDa to 10 MDa, more preferentially 10 kDa to 1 MDa, most preferentially 100 kDa to 500 kDa size.

24. The biomaterial according to one of the claims 1 to 22, characterized in that is synthesized from a polysaccharide with a molecular weight in the range of 1kDa to 60kDa, more preferentially 10kDa to 50kDa.

25. The biomaterial according to one of the claims 1 to 24, characterized in that upon essentially complete hydrolysis of the ester bonds of the biomaterial frees soluble fragments with a molecular weight in the range of 1 kDa to 10 MDa, more preferentially 10 kDa to 1 MDa, and most preferentially 100 kDa to 500 kDa.

26. The biomaterial according to one of the claims 1 to 24, characterized in that upon essentially complete hydrolysis of the ester bonds of the biomaterial frees soluble fragments with a molecular weight in the range of 1kDa to 60kDa, more preferentially 10kDa to 50kDa.

27. The biomaterial according to one of the claims 1 to 26, characterized in that the settling volume is larger than 10 g/g, preferentially larger than 50 g/g, and most preferentially 100 g/g.

28. The biomaterial according to one of the claims 1 to 27, characterized in that it has a Young modulus in the range of 100 Pa to 50 kPa, more preferentially 200 Pa to 10 kPa and most preferentially between 500 Pa to 5 kPa.

29. The biomaterial according to one of the claims 1 to 28, characterized in that it is in sterile form.

30. The biomaterial according to one of the claims 1 to 29, characterized in that it has a shear modulus G’ at 1 Hz of oscillatory shear rheology and a strain smaller than 1% in the range of 100 Pa to 50 kPa, more preferentially 200 Pa to 10 kPa and most preferentially between 500 Pa to 5 kPa.

31. The biomaterial according to one of the claims 1 to 30, characterized in that the biomaterial contains an anesthetic agent, an antibiotic, and/or a vasoactive agent.

32. The biomaterial according to one of the claims 1 to 31 , characterized in that the biomaterial when subjected to hydrolysis in a pH range between 2 and 13 and a time between 5 min and 1 year, more than 80% of the ester bonds are hydrolysed while the molecular weight of the polysaccharide decreases by no more than 50%.

33. The biomaterial according to one of the claims 5 to 32, where the material is injectable with a peak force of less than 50N, preferentially less than 30N, and even more preferentially less than 20N, through a needle or catheter of 22G, 25G or 27G.

34. A kit comprising a syringe loaded with the biomaterial according to one of the claims 1 to 33 in its hydrated or dry state.

35. A ready to use kit comprising:

(i) one or several sterile syringes according to claims 34;

(ii) one or several sterile syringes or vials containing a reconstituting solution, or an additive, or several additives, such as physiological saline, buffer, cell suspension, tissue suspension, anesthetic product, antibiotic product, osmotically active substance, cell culture medium, implantation medium, vasoactive product and the like; and

(iii) one or several sterile connectors or septums.

36. Method of manufacturing the biomaterial according to one of the claims 1 to 33, characterized by combining a dehydration process with a catalysis process for the ester bond formation by means of a chemical activator capable of transforming carboxylic acid groups or carboxylate anion groups into more reactive chemical species.

37. Method according to claim 36, characterized in that the chemical activator leads to an endogeneous crosslinking.

38. Method for according to claims 36 or 37, characterized by the following steps e) preparing an aqueous solution of a salt of a polysaccharide or mixture of polysaccharides having both hydroxyl and carboxyl groups, each one of the groups present at at least 0.01 mol/kg of dry mass of the polysaccharide or mixture of the polysaccharides; f) adding a pH buffer and an activator of carboxylic acids, in particular a carbodiimide, and catalytically active amounts of dimethylaminopyridine or related aminopyridine, leading to solution with an initial pH between 4 and 7.5, more preferentially between 6 and 7.3; g) subjecting said solution to a dehydration; and h) incubating the solution obtained in step c) until the the ester bond formation has substantially be completed.

39. Method according to claim 36 or 37, characterized by the following steps: a) preparing an aqueous solution of a salt of a polysaccharide or mixture of polysaccharides having both hydroxyl and carboxyl groups, each one of the groups present at at least 0.01 mol/kg of dry mass of the polysaccharide or mixture of the polysaccharides; b) adding a pH buffer and an activator of carboxylic acids, such as a carbodiimide, and catalytically active amounts of dimethylaminopyridine, leading to solution with an initial pH between 4 and 7.5, more preferentially between 6 and 7.3; and c) freezing the solution to a subzero temperature in the range of -2° to -273.15° for a time of 1 h to 1 month, preferentially from 4 h to 2 weeks, and most preferentially from 8 hours to 1 week.

40. Method according to the claim 39, characterized in that the cryoincubation temperature in step c) is in the range of -2°C to -273.15°C, preferably -5°C to -80°C, and more preferable between - 5°C and -40°C.

41. Method according to claim 39 or 40, characterized in that the polysaccharide concentrations are 3% or below, 1 % or below, or 0.5% or below.

42. Method according to one of the claims 36 - 38, where the dehydration is performed through the use of a hygroscopic salt such as anhydrous calcium sulfate

43. Method according to claim 42, where the hygroscopic salt is dissolved by addition of a suitable solvent, such as solution containing EDTA for the calcium sulfate

44. Method according to one of the claims 36 - 38, where the dehydration is performed through air drying.

45. Method according to one of the claims 36 - 38, where the dehydration is performed through lyophilization.

46. Method for manufacturing the biomaterial according to claim 1 to 33, characterized by the following steps: a) preparing an acidic solution of a salt of a polysaccharide having both hydroxyl and carboxyl groups; b) freezing the solution to a subzero temperature in the range of -2° to -273.15°; c) extracting the water ice in a cold organic solvent with high water solubility or miscibility with water such as isopropanol at a temperature between -2° and the freezing point of the solvent, more preferentially between -5°C and -40°C; d) evaporating the solvent; and e) thermally treating the resulting sponge-like substance at 100°C to 200°C, more preferentially 120°C to 180°C.

47. The biomaterial according to one of the claims 1 to 33 for use as a replacement tissue and regeneration of original tissue in patients.

48. The biomaterial according to one of the claims 1 to 33 for use as an implantable tissue engineering material, preferably a soft tissue engineering material.

49. The biomaterial according to one of the claims 1 to 33 for use as a shapeable tissue or organ body implant.

50. The biomaterial according to one of the claims 1 to 33 for treating tissue defects, in particular tissue defects caused by severe trauma or cancer ablation.

51. The biomaterial according to one of the claims 1 to 33 for use in a method of breast reconstruction.

52. The biomaterial according to one of the claims 1 to 33 for use in lip reconstruction or augmentation.

53. The biomaterial according to one of the claims 1 to 33 for use

54. The biomaterial according to one of the claims 1 to 33 for use in a method of: cell delivery into body tissues or, body organs, or body fluids; cell culture, differentiation, preparation, with or without subsequent in-vivo delivery; in vivo cell culture for the production or consumption of differentiating factors, antibodies, hormones, cells, genetic vectors, vessels, red blood cells, white blood cells, stem cells, exosomes, lipids, energy, heat or light; lifting or expanding tissues, in particular skin tissues, breast tissues or supporting sphincters; enhancement of soft tissue volumes; create synthetic cellular organizations, in particular in the ovarian environment and in dentistry; drug delivery, coating, retaining, delivering molecules (drugs, proteins, nucleic acids, viruses, differentiation factors, growth factors, carbohydrate, adjuvants, fatty acids, triglycerides, cholesterol, with loading before, during, or after delivery; and immunoengineering through the effect of ester crosslinking density.

55. Composition comprising: a) a multitude of irregular porous particles obtained from the biomaterial according to one of the claims 1 to 33 and b) a physiologically acceptable fluid.

56. Composition according to claim 55, characterized in that the amount of fluid is such that the particles are only partially hydrated.

57. Method of manufacturing the biomaterial according to one of the claims 1 to 33, characterized by the use of ester crosslinks from endogenous groups of the backbone polymer to create a stable polymer-based hydrogel without addition of exogenous modifications.

58. Method of manufacturing the biomaterial according to one of the claims 1 to 33, characterized in that the crosslinking is based only on endogeneous carboxylate and hydroxyl groups.