and scaffolds thereof

* * *

FIELD OF THE INVENTION

The present invention concerns a novel method for manufacturing porous scaffolds for biomedical uses and scaffolds thereof.

BACKGROUND OF THE INVENTION

In tissue engineering scaffolds play a crucial role for cell sustain and guide to address 3D tissue growth and regeneration.

In the scaffold design the first factor to consider is the material choice, which is the first constituting interface to the biological environment. A suitable material should have at least the following properties: it should promote cell attachment and function and not provoke any unwanted tissue response from the cells (i.e. it has to be biocompatible) and should degrade in non-toxic products, leaving a scaffold-free living cell construct (i.e. it has to biodegradable).

Since now a wide range of materials have been proposed, both inorganic and organic, and, among them, both from synthetic and natural origin. Hydroxyapatite, bioactive glasses, calcium phosphate ceramics (inorganic); polyglycolic acid, polylactic acid, ε-polycaprolactone (synthetic organic); collagens, glycosaminoglycan, starch, chitin, chitosan, silk fibroin, hyaluronic acid (natural polymers) are just some examples.

Together with the choice of the material, another issue in the design of scaffolds for tissue engineering is to address the overall physical properties, starting from morphology at micro and sub-micrometric level. The presence of porosity has been proved to be fundamental for tissue regeneration because it can induce cell migration and proliferation inside the scaffold, leading to its optimal population. In this way the scaffold can substitute the temporary unavailable external cell matrix, providing mechanical support and biochemical signaling to the cell construct.

This is the reason why researcher are investigating on the possibility to optimize scaffolds in terms of mechanical properties, porosity, and degradation time together with release of bioactive factors such as drugs, hormones or any biochemical factor able to stimulate the cell signaling pathways.

There are many ways to fabricate porous scaffolds or foams. Some of the most used techniques are fiber felts, fiber bonding, phase separation (and sublimation), solvent casting and particulate leaching, membrane lamination, melt molding, polymer/ceramic fiber composite foam (with polymer phase dissolution), high-pressure processing (i.e. supercritical C0 ₂ ), hydrocarbon templating.

These techniques usually require multiple steps for realization and use of sacrificial materials to generate porosity.

It is therefore felt the need of improved processes for manufacturing porous, polymeric, biomedical scaffolds for use in regenerative medicine free of the drawbacks of the processes used until today. OBJECT AND SUMMARY OF THE INVENTION

The object of the present invention is to provide a method for preparing tridimensional, porous, biomedical scaffolds starting from a polymer in dispersion or melt state in a single manufacturing step. Right after preparation scaffolds are either ready to be used for cell culture or can undergo to different post processing steps with the aim of improving or modifying the scaffold properties.

According to the invention, the above object is achieved thanks to the method specified in the ensuing claims, which are understood as forming an integral part of the present description.

In an embodiment, the instant disclosure discloses a novel method for manufacturing a porous, polymeric, biomedical scaffold, the method comprising the following steps:

i) loading within an extrusion foaming apparatus at least one polymer in a dispersion or melt state at an initial temperature;

ii) introducing within the extrusion foaming apparatus a foaming agent at an initial pressure higher than atmospheric pressure, the foaming agent being soluble in the at least one polymer;

iii) allowing the foaming agent to dissolve within the at least one polymer to form at least one mixture including the least one polymer and the foaming agent; iv) extruding the at least one mixture through an extrusion die into an expansion region having an expansion pressure lower than the initial pressure such that the at least one mixture experiences a pressure drop and expands obtaining the porous, polymeric, biomedical scaffold,

wherein the foaming agent is selected among N ₂ 0, propane, butane and pentane, preferably the foaming agent is N ₂ 0, and

wherein the obtained scaffold is suitable for tissue engineering and regenerative medicine applications.

The method allows the production of tunable porous scaffolds constituted by a wide selection of natural and synthetic origin polymers optionally containing additives, such as i.a. drugs, inorganic compounds, bioactive factors, surfactants or emulsifiers.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described, by way of example only, with reference to the enclosed figures of drawing, wherein:

- Figure 1: Scheme of the foaming method.

- Figure 2: FT-IR spectrum of a scaffold realized according to the method herein disclosed using a 5.4% silk fibroin dispersion by extrusion foaming through a standard nozzle (10 mm diameter nozzle), large and small diameter needles (4 mm and 2 mm, respectively), compared with the unprocessed 5.4% silk fibroin dispersion after freeze-dry. Amide I peak of the unprocessed dispersion is evidenced.

- Figure 3: SEM pictures of silk fibroin scaffolds realized according to the process herein disclosed after freeze-drying; the shown scaffolds have been realized using a standard nozzle (Figure 3A), a large needle (Figures 3B and 3C) and a small needle (Figures 3D and 3E) using different foaming pressures (0.55 or 1.1 MPa). Scale bars refer to distances of 100 μιη (pictures on the left, 200x magnification) and 20 μιη (pictures on the right, lOOOx magnification).

- Figure 4: SEM pictures of a scaffold realized according to the process herein disclosed from a 2% silk fibroin water dispersion by extrusion foaming through a small needle (2 mm diameter) at 1.1 MPa N ₂ 0 pressure. Scale bars refer to distances of 100 μιη (pictures on the left, 200x magnification) and 20 μιη (pictures on the right, lOOOx magnification). - Figure 5: SEM pictures of a scaffold realized according to the process herein disclosed using a 15% w/v gelatin dispersion after freeze-drying. Figure 5A is a SEM picture at 200x magnification (scale bar is 100 μιη); Figure 5B is a SEM picture at lOOOx magnification (scale bar is 20 μιη).

- Figure 6: FT-IR spectrum of a scaffold realized according to the process herein disclosed using a 15% w/v gelatin dispersion after freeze-drying.

- Figure 7: SEM pictures of a scaffold realized according to the process herein disclosed from a 4% w/v Fibroin/gelatin (1: 1 ratio) dispersion after freeze- drying. Figure 7A is a SEM picture at 200x magnification (scale bar is 100 μιη); Figure 7B is a SEM picture at lOOOx magnification (scale bar is 20 μιη).

- Figure 8: FT-IR spectrum of a scaffold realized according to the process herein disclosed from a 4% w/v silk fibroin-gelatin (1: 1 ratio) dispersion compared with those obtained from extrusion foaming a 2% w/v silk fibroin dispersion or a 2% w/v gelatin dispersion.

- Figure 9: SEM picture of a scaffold realized according to the process herein disclosed from a 40% w/v gelatin-hydroxyapatite (1: 1 ratio) dispersion after freeze-drying. Scale bars refer to distances of 100 μιη (picture on the left, 200x magnification) and 20 μιη (picture on the right, lOOOx magnification).

- Figure 10: SEM pictures of two scaffolds realized according to the process herein disclosed from 25% w/v and 50% w/v soy-lecithin dispersions

(Figures 10A and 10B, respectively). Scale bars refer to distances of 100 μιη (pictures on the left, 200x magnification) and 20 μιη (pictures on the right, lOOOx magnification).

- Figure 11: FT-IR spectrum of a scaffold realized according to the process herein disclosed from the soy-lecithin foam from 50% w/v dispersion compared with that obtained from the unprocessed 50% w/v soy-lecithin dispersion. No difference between the two described dispersions and the unprocessed dispersion after freeze dry was evidenced. DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail, by way of non limiting example, with reference to a method for functionalizing a decellularized biological material with at least one active principle, such as for example, a decellularized vascular vessel functionalized trough covalent bonds with an active principle.

In the following description, numerous specific details are given to provide a thorough understanding of embodiments. The embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the embodiments.

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

The headings provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

Within the instant description the term "dispersion" is intended to refer to a material state comprising more than one phase wherein at least one of the phases consists of finely divided phase domains, often in the colloidal size range, dispersed throughout a continuous phase (IUPAC Recommendations 2011, Terminology of polymers and polymerization processes in dispersed systems, Pure Appl. Chem., Vol. 83, No. 12, pp. 2229-2259, 2011. doi: 10.1351/PAC- REC-10-06-03). Among the different possible dispersions herein we refer to those obtainable mixing a solid or liquid dispersed phase (namely the polymer(s)) into a liquid continuous medium (namely the solvent(s)). This to include the following categories: solutions, sol, colloids, latexes, suspensions and emulsions.

The present description concerns a method for manufacturing a porous, polymeric, biomedical scaffold comprising the following steps:

i) loading within an extrusion foaming apparatus at least one polymer in a dispersion or melt state at an initial temperature;

ii) introducing within the extrusion foaming apparatus a foaming agent at an initial pressure higher than atmospheric pressure, the foaming agent being soluble in the at least one polymer;

iii) allowing the foaming agent to dissolve within the at least one polymer to form at least one mixture including the least one polymer and the foaming agent;

iv) extruding the at least one mixture through an extrusion die into an expansion region having an expansion pressure lower than the initial pressure such that the at least one mixture experiences a pressure drop and expands obtaining the porous, polymeric, biomedical scaffold,

wherein the foaming agent is selected among N ₂ 0, propane, butane and pentane, preferably the foaming agent is N ₂ 0.

The method is extremely simple, inexpensive and tunable in term of physical properties (namely, degree of porosity and pore dimensions) of the scaffolds.

Porous, polymeric, biomedical scaffolds can be realized from either biodegradable and not degradable polymers in a dispersion or melt state using high pressure foaming agents.

The biodegradable polymers suitable to be used for the manufacturing of a porous, biomedical scaffold can be selected from proteins, polysaccharides or lipids (e.g. albumin, collagens, glycosaminoglycan, chitosan, phospholipids, starch, chitin, chitosan, silk fibroin, hyaluronic acid, alginate), biodegradable synthetic polymers (e.g. polyethylene glycol derivates, polyglycolic acid, polylactic acid, poly-DL-lactic acid), or mixtures thereof.

When the at least one polymer is employed in the method object of the instant application in a dispersion state, the preferred solvent is water, either pure, or having at least one salt dissolved therein (such as a saline buffer solution, like PBS), or having a non-neutral pH (in the range of 2 to 10). The use of water or water-based solution avoids removal of the solvent from the scaffold before use. The use of other solvents is possible, but solvent removal is necessary after foaming. Both polar (e.g. dichloromethane, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide, propylene carbonate, formic acid, n-butanol, isopropanol, n-propanol, ethanol, methanol, acetic acid, nitromethane) and non-polar solvents (e.g. diethyl ether, pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, 1,4-dioxane, chloroform) can be successfully employed.

The process is particularly efficient in the case of aqueous proteins dispersions wherein proteins present both hydrophilic and hydrophobic sites. Usually these proteins fold and assemble in water to minimize the hydrophobic sites exposed at the water phase. Due to the high solubility of N ₂ 0 (i.e. a foaming agent) in the hydrophobic phase, during the gas expansion the bubbles can drive the self-assembly of the proteins to form a stable interface at the gas/water interface. Moreover, in view of the tendency of proteins to generate relatively strong intermolecular bonds, the stability of the scaffold itself can be increased.

Typical concentrations of the polymer dispersions are between 1% and 50% w/v of polymer, preferably 1 to 20% w/v; lower or higher concentrations may determine the reduction of the porous scaffold mechanical structure for example determining the self-collapsing of the foam or an excessive increase of the viscosity, which hinders the expansion of the dissolved foaming agent, limiting the foam expansion. In case of high viscosities polymer dispersions extrusion foaming can be realized by increasing the process temperature (i.e. the initial temperature at which the foaming agent is introduced within the extrusion foaming apparatus). Molten state thermoplastic polymers can be processed (e.g. ε- polycaprolactone) according to the instant description by loading them into the extrusion foaming apparatus and pressurizing with the foaming agent.

The foaming process occurs thanks to the fact that the foaming agent is capable to dissolve into the at least one polymer, allowing its expansion when pressure is released and the at least one polymer/foaming agent mixture is extruded from the extrusion foaming apparatus.

The foaming agent can be selected among N ₂ 0, pentane, propane, and n- butane, preferably N ₂ 0.

N ₂ 0 is the preferred foaming agent since it is a non-flammable gas, is highly soluble in hydrophobic polymers and does not induce acidification of the polymer water dispersion (as for example C0 ₂ does), leading to unwanted side effects, like protein denaturation, accelerated hydrolysis or additives degradation. The use of N ₂ 0 positively influences the preservation of the integrity of the pressurized dispersions in the time, without causing dispersion instability, polymer degradation or loss of sterility, and reduces the fire risks in the case of use in surgery room or special environments.

There is no specific limitation on the initial pressure used during introduction of the foaming agent within the extrusion foaming apparatus, which is actually limited by the extrusion foaming apparatus mechanical properties and the foam expansion limits. The initial pressure of the foaming agent used in the instant method is about 0.2 to 30 MPa, preferably 0.5 MPa to 2 MPA, more preferably 0.55 to 1.1 MPa. An initial pressure of about 0.55 to 1.1 MPa provided good results in terms of pores dimensions (considering the desired application) and can be considered a reasonable reference range.

The porosity of the obtained porous scaffold depends on the pressure difference between the initial addition pressure of the foaming agent inside the extrusion foaming apparatus and the pressure of the expansion region, wherein higher initial addition pressure determines bigger pore size and pore density (as shown by way of example in Figure 3). Mean pore size of the scaffold obtained according to the method herein disclosed lies typically in the range of 10 to 1,000 μιη, but, being pressure related, it can be tuned by increasing/decreasing the initial addition pressure under which the foaming agent is loaded within the extrusion foaming apparatus.

The obtained scaffold can be post processed i.a. to reduce solubility in solvents or to remove the solvent used to realize the polymer dispersion (e.g. by freeze drying, exposure to chemical agents such as methanol, ethanol or water vapor, crosslinking for example by UV exposure or by chemical reaction).

The use of different diameter extrusion dies provides the possibility to produce differently shaped foams: from cake-shaped to wire-shaped structures.

The possibility to realize porous scaffolds by means of a small needle allows the realization of porous wire-like scaffolds that, in some cases, may determine functional changes in the scaffolds themselves making them suitable for specific applications, for example, in combination with a microfabrication apparatus.

Microfabrication is a process of fabrication of miniature structures of micrometer scales and smaller. A microfabrication apparatus is typically constituted by an extrusion foaming apparatus provided with a terminal extrusion dies (i.e. a dispensing needle or nozzle) coupled to a 3 axes computer-aided positioning system. This makes it a suitable technique for the realization of meshes, networks or multi-layer 3D structures.

A further advantage of the method herein disclosed lies in the possibility of direct injection of the extruded scaffold in a body site as filler (e.g. in a critical bone defect) for tissue regeneration support, if the polymer dispersion is biocompatible and sterile.

Another advantage of the method herein disclosed lies in the possibility of direct injection of the extruded scaffold in a shape mold to obtain scaffolds of any desired shape (foam injection molding). The disclosed method allows realization of porous, biomedical scaffolds in a single step and with a high degree of reproducibility obtaining the desired degree of porosity and/or pore dimensions necessary for allowing cell migration and proliferation inside the scaffold itself.

Moreover, the method herein disclosed is successful in extrusion foaming polymers particularly difficult to process with other techniques. In particular, among these, relevant results according to the instant description have been obtained with silk fibroin water dispersions obtaining compact, highly porous, water stable scaffolds, not obtained with the other known techniques.

The method for manufacturing a biomedical, porous, polymeric scaffold herein disclosed allows also use of additives in order to improve the biological and/or the physical/mechanical properties of the scaffold itself.

Additives which may be added to the polymer in dispersion or melt state are represented by:

- salts, bases or acids (e.g. HC1, NaOH, NaCl) that can improve polymer dispersion stability;

- drugs (e.g. anti-inflammatory agents or other drugs) that being released by the scaffold are able to exert their pharmacological effect in the surrounding tissues;

- ligands (e.g. surfactants or emulsifiers) that can improve the porous scaffold stability or improve the polymer dispersion stability;

- cross-linkers, that by chemically binding the polymeric chains improve stability and mechanical properties of the scaffold (e.g. genipin in the case of silk fibroin),

- not degradable synthetic polymers, that incorporated in the scaffold permit to address special physical properties, such as electricity conduction by using conductive polymers (e.g. poly-pyrrole, poly-aniline, polythiophenes, Poly(3,4-ethylenedioxythiophene) Polystyrene sulfonate (PEDOT:PSS),- biomolecules (e.g. antioxidants, hormones, growth factors, etc.), that being released by the scaffold are able to exert their biological function in the surrounding tissues; and/or

- inorganic compounds, preferably inorganic nanoparticles, nanorods, nanowires (e.g. graphene, carbon nanotubes, magnetic nanoparticles, hydroxyapatite or calcium phosphate nanoparticles) that are able to modify the mechanical properties of the scaffold, and to induce some specific biological response. As an example hydroxyapatite or bio-glass particles added to polymeric scaffolds are reported to improve osteoconductivity and are commonly proposed as filler for scaffolds in bone defect repair.

The main features controlling the method herein disclosed are temperature, pressure and polymer composition as well as composition of the polymer dispersion; such features allow to tune the polymer (either in a dispersion or melt state) viscosity, influencing the expansion kinetic and the stability and morphology of the obtained porous scaffold.

For what concerns the polymer dispersions the initial concentration determines its viscosity, which influences the expansion rate and the final scaffold porosity porous. Polymer dispersions concentration ranges between 1% and 50% w/v in water are here reported, but foaming can occur from low concentrations (0.1 % w/v) up to high concentrations (95% w/v). Longer polymer chains provide better chances to produce stable foams at low concentrations, due to the increase of the number of chain-to-chain interactions.

Low concentration dispersions require lower pressures for successful foaming, while high concentration dispersions require higher pressures. For molten polymer foaming the highest pressures are required because of the higher polymer viscosity.

Excessive pressure usually leads to foam collapsing upon its weight.

Temperature can favor or contrast the foam expansion and its stability after foaming. Increase of temperature from the environmental condition determines progressive reduction in polymer dispersion viscosity, permitting a faster and higher expansion at constant pressure (at least for dispersions having direct proportionality between viscosity and temperature increase). Fast reduction of temperature after extrusion foaming can block the expansion process, permitting to obtain the desired result in term of bubble size. The process can be tuned to exploit physical transitions of the dispersion, like the case of gelatin which presents a sol/gel transition at about 35°C. In this case the dispersion is foamed at temperature higher than 35°C and temperature dropdown following the expansion allow the gel transition and stabilize the final foam structure.

Similar consideration can be done in the case of molten polymers, where the foaming temperature is set higher than the polymer melting temperature to address the wanted polymer viscosity.

Depending on the requested porosity, the foaming dispersion temperatures allowed are those at which the dispersion is in liquid state. While in the case of the molten polymer, i.e. when no solvent is required, the practical limit for the process is the temperature at which the polymer chains start degrading.

Typical values of the initial temperature in case of foaming polymer(s) in a dispersion state are in the range -20 to 120 °C, preferably 20 to 60 °C.

Typical values of the initial temperature in case of foaming polymer(s) in a melt state are in the range 30 to 250 °C, preferably 40 to 200°C.

In cases when the extrusion phase (iv) is carried out at an extrusion temperature that is lower than the initial temperature, typical values of the extrusion temperature are in the range -200 to 37 °C.

The porous scaffold manufactured according to the method herein disclosed can then be subjected to post-processing, which may allow i.a. to reduce solubility of the scaffold itself in solvents or to remove the solvent still present in the porous scaffold in case the polymer was loaded inside the extrusion foaming apparatus in dispersion state. Such post-processing can be any of freeze drying, exposure to chemical agents such as methanol, ethanol or water vapor, crosslinking for example by UV exposure or by chemical reaction.

The method herein disclosed was successfully tested with different polymer dispersions containing silk fibroin, soy-lecithin, gelatin and alginate and with ε-polycaprolactone (PCL) in melt state.

Porous biomedical scaffolds can be produced starting from high concentration up to low concentration protein dispersion, namely 50% w/v lecithin and 2% w/v silk fibroin water dispersions. The present inventors did not verify specific limitation about the concentrations other than the increase of viscosity that hinders the expansion of the dissolved foaming agent or the reduction of the structural properties, which can lead to self-collapsing scaffolds.

The extrusion foaming process through a small diameter needle of low concentration fibroin dispersions (2% w/v, see Figures 3D and 3E) allows the realization of porous biomedical scaffolds, which resulted to keep stability if immersed and kept in water after more than 30 days, without the use of any further treatment. This is reasonably due to the combined contribution of the self- assembly mechanism at the bubbles interface and the shear stress derived by the dispersion flowing through the needle when expelled at high pressure.

The FTIR spectra obtained using gelatin, silk fibroin-gelatin and soy- lecithin dispersions did not show particular difference in the structures of the spectra with respect of the respective unprocessed dispersion after freeze dry (Figures 6, 8 and 11, respectively). The absence of bands shift in the spectra indicates that the process does not (deeply) modify the inter- or intra- molecular interactions between the polymer chains.

In the example disclosed in Figure 9 the high concentration of hydroxyapatite within the gelatin dispersion allowed the thermal post treatment of the obtained porous scaffold in order remove by pyrolysis the polymeric phase (gelatin), obtaining an inorganic porous scaffold usable for example as bio- absorbable bone graft substitute.

The extrusion foaming method was also successfully tested with alginate dispersion to assess the possibility to foam polysaccharides, which generally are prevalently hydrophilic and have high dissolution rates in water. Due to the fact that alginate turns to gel if exposed to divalent cations (which causes ionic crosslink of the macromolecule chains) the foam has been produced in a CaCl solution. In this case the diffusion rate of Ca ⁺⁺ ions and the N ₂ 0 expansion are in contrast to each other. This leads to lower homogeneous porosity foams.

The possibility of extrusion foaming polymers in a melt state was also tested. In particular poly ε-polycaprolactone (PCL) at 90°C was successfully extrusion foamed by means of pressurized N ₂ 0 at 1.1 MPa. The foaming was realized directly into a dewar flask containing liquid nitrogen in order to suppress bubbles coalescence and to quench the material expansion by fast cooling the foam right after production. This procedure leads to the realization of high-density foams, because of the polymer concentration. Also in this case the foaming process can be tuned by acting on pressure and temperature.

Materials and methods

Foaming dispersion

The polymers tested herein were constituted by dispersion in water of silk fibroin, pig-skin gelatin, soy-lecithin and alginate.

Silk fibroin was obtained from white cocoons produced by polyhybrid

Bombyx mori (Cooperativa Sociolario, Como, Italy). Following silk degumming with a 4.24 g/1 Na ₂ C0 ₃ water solution at 98°C for 1 h and repetitive rinsing, fibroin was dissolved in 9.3 M LiBr solution (Fluka, Switzerland) at 65°C for 2 h, yielding a 20% (w/v) solution. This solution was dialyzed against deionized water using 3 to 12 ml Slide-A-Lyzer dialysis cassettes (molecular weight cut-off (MWCO) 3500 g/mol, Pierce) for 4 days with periodic changes of deionized water, obtaining 5.4% w/v final concentration. 2.7% and 2% concentrations were obtained by dilution.

Soy lecithin, pig-skin gelatin and alginate were provided by Sigma Aldrich and suspended in warm water (40°C) and gentle stirring to improve dispersion. Tested concentrations for the various dispersions were between 2% and 30% w/v, but there is no specific limitation other than the increase of viscosity that hinders the expansion of the dissolved foaming agent.

Raw ε-polycaprolactone (PCL, Fluka) at 90°C in melt state was also tested (Sigma Aldrich, molecular weight 80000 Da).

Foaming agents

The foaming agent used is nitrous oxide.

Other foaming agents have been successfully tested, such as propane, n- butane, pentane.

Nitrous oxide has high solubility in the hydrophobic phase of the used polymers, does not induce dispersion acidification and is not flammable, which makes it a preferable foaming agent.

0.55 and 1.1 MPa N ₂ 0 pressures were used.

Additives

Additives can be either soluble or not soluble, or partially soluble (like surfactants or emulsifiers) in the polymer in its dispersion or melt state, this determining how the additives will be available in the obtained biomedical scaffold or interfere with the biomedical scaffold structuring.

In the present disclosure hydroxyhapatite nanoparticles (Sigma Aldrich) were loaded into a gelatin dispersion. Hydroxyhapatite powder was added to the gelatin dispersion at 50°C under gentle stirring until complete dispersion.

Extrusion foaming apparatus

A stainless steel 0.5 liters (ICO, Whip it) siphon was used as extrusion foaming apparatus.

The purging valve was implemented with different extrusion dies to obtain different biomedical scaffold physical structure. In particular, a standard nozzle having 50 mm length and 10 mm inner diameter, as well as two different needles, both 100 mm long and having 5 mm inner diameter (large needle), or 2 mm inner diameter (small needle) were used.

Foaming method The method consist in four main steps (as shown in Figure 1):

i. An extrusion foaming apparatus is loaded with a polymer (in the form of a polymer liquid dispersion or melt material) with the residual volume occupied by a low solubility aeriform (typically air);

ii. The extrusion foaming apparatus is pressurized with the foaming agent at a predetermined pressure, typically between 0.5 and 2.0 MPa;

iii. The polymer is allowed to absorb the foaming agent obtaining a polymer/foaming agent mixture (gas dissolution can be speed up by shaking or stirring);

iv. The polymer/foaming agent mixture is expelled out of the extrusion foaming apparatus by releasing the pressure inside the extrusion foaming apparatus by opening a valve. Foaming occurs as polymer/foaming agent mixture flows outside the apparatus through a nozzle or a needle because of the foaming agent expansion.

To obtain a dry porous scaffold after foaming, the obtained scaffold underwent to freeze drying.

The obtained porous scaffolds were analyzed by optical imaging and electron microscopy (after freeze drying). FT-IR spectra were collected to assay molecular rearrangement.

Results

Silk fibroin, gelatin, lecithin, alginate dispersions were tested at different concentrations and different foaming conditions (needle/nozzle diameter, foaming agent pressure) to assess the limits of the disclosed method.

Here below the obtained remarkable results are reported.

Fibroin dispersion

5.4% fibroin dispersion was extruded from standard 10 mm diameter nozzle, 4 mm and 2 mm diameter needles (100 mm long).

0.55 and 1.1 MPa N ₂ 0 pressure were used.

The obtained porous scaffolds have been analyzed by FT-IR (Figure 2) to check different protein structuring, compared with that obtained from the unprocessed dispersion. The spectrum reveals a slight shift of the Amide I peak towards 1500 cm ^"1 , which is addressable to a β-sheet organization of the protein structure, conferring to the obtained foams higher stability to water.

To assess the effects of foam extrusion through thin needles a 2% (w/v) silk fibroin dispersion was extruded from a 10 mm diameter nozzle in a petri dish and 2 mm diameter needle directly in water, to probe water stability.

While silk fibroin dispersion extruded through a large and short nozzle (Figure 3A) does not maintain stability and re-dissolve in water if not post- processed (i.e. exposure to methanol water solution to induce β-sheet formation inside silk fibroin), the use of small and large needles (Figures 3 B to E) induce stability to the obtained scaffold which results to be not soluble in water, even after 30 days.

The resulting scaffold reveals evident fiber-like structures, aligned along the extrusion direction (Figure 4), thus maintaining a wide porosity, with pores size ranging between 10 and 100 μιη. The organization in fibrils or fiber-like structures is evidence of a higher molecular organization, which prevents the foam to solubilize in water, even after long time.

Gelatin dispersion

15% gelatin dispersion in water was foamed using a standard nozzle.

The dispersion was heated at 50°C to liquefy gelatin inside the extrusion foaming apparatus.

The extruded polymer/foaming agent mixture was deposited on a metal plate floating on a liquid nitrogen bath to suppress bubbles coalescence permitting gelatin to structure and become solid. Similar results can be obtained by putting the metal plate into a refrigerator right after the foaming process.

Figure 5 shows the SEM structure of the gelatin scaffold after freeze- drying, which presents a closed porosity in the range of 50 to 250 μιη. Pores result homogeneously distributed and present a roughly spherical shape inherited by the foaming gas bubbles expansion.

The FT-IR spectrum of the foamed gelatin (Figure 6) reveals no difference with the unprocessed gelatin dispersion.

Silk fibroin/Gelatin dispersion

4% total protein concentration obtained by mixing equal parts of silk fibroin and gelatin in water was extruded using a standard nozzle to assay the possibility to make scaffold consisting of polymers blends. The obtained scaffolds are uniform and homogeneously mixed.

Results are shown in Figure 7. The produced foams are compact and stable. The internal structure from the SEM pictures reveals an open porosity ranging from 50 to 250 μιη. A second order, lamellar structure porosity (measuring few microns) is also present and fills the gaps between the bigger pores. This is addressable to the freeze-drying process and is supposed to widely swell once the foam is rehydrated.

The FT-IR spectrum of the foamed gelatin/fibroin blends are reported in Figure 8 and is a combination of the spectra obtained from the spectra of silk fibroin and gelatin alone. No molecular structuring is evidenced.

Gelatin dispersion and hydroxy apatite nanometric powder

To assess the possibility to introduce large quantities of additives to the polymeric dispersion, a 40% water dispersion obtained by mixing equal parts of gelatin and hydroxy apatite nanometric particles (particle size < 200 nm) was extruded using a 6 mm diameter nozzle. Dispersion (and extrusion foaming apparatus) temperature was set at 50°C to keep the gelatin liquid, 0.55 MPa N ₂ 0 pressure was used. The polymer/foaming agent mixture was extruded on an aluminum plate floating on a liquid nitrogen bath, in order to quick cool the obtained porous scaffold, to preserve the porous structure by accelerating the gelatin sol to gel transition.

An example of the resulting foam is shown in Figure 9 where the porosity is appreciable and ranges from about 50 to 250 μιη, with a combination of closed and open cells structure.

Lecithin dispersion

To assess the possibility to extrude highly concentrated soy lecithin dispersions (25% and 50% w/v of soy lecithin) were extruded using a standard nozzle (10 mm diameter) and 0.55 MPa N ₂ 0 pressure (Figure 10).

The resulting foam appears relatively compact, but, even at so high protein concentration, the foaming occurs thus indicating that better results could be obtained by increasing the dissolved foaming agent volume (and increasing the pressure). The resulting material present a closed cells porosity structure, with a pore size ranging from few microns to about 150 μιη. From the higher magnification images it is possible to evidence the presence of micrometric and sub-micrometric cavities, reasonably due to the expansion limited by the dispersion viscosity.

The FT-IR spectrum of the foamed soy lecithin (50% w/v in Figure 11) reveals no difference with the unprocessed gelatin dispersion.

Alginate dispersion

10% alginate dispersion was extruded through a thin needle in 2% CaCl water solution (0.55 MPa N ₂ 0 pressure). Ca ions induce physical cross-link of the alginate polymeric chains during the N ₂ 0 expansion, structuring the scaffold in a semi-transparent alginate foamed wire.

Melt PCL

To assess the foaming of melt state polymers, ε-polycaprolactone (80000 MW) was loaded into the pressurized extrusion foaming apparatus, filled with N ₂ 0 (1.1 MPa) and heated at 90°C. After complete melting and foaming agent dissolution inside the polymer, the polymer/foaming agent mixture was extruded using a standard nozzle (10mm inner diameter) into a dewar flask containing liquid nitrogen. The contact with the cold liquid permits to stop the expansion, quickly solidify the foam and thus to obtain the desired solid porous scaffold.

Naturally, while the principle of the invention remains the same, the details of construction and the embodiments may widely vary with respect to what has been described and illustrated purely by way of example, without departing from the scope of the present invention.