Improvements in 3D Printing
Field of the invention
This invention relates to a novel biocompatible ink for three-dimensional (3D) printing. In particular, though not exclusively, this invention relates to the preparation of a novel hybrid biocompatible ink for templated 3D cell printing (bioprinting) and a method of printing using the biocompatible ink.
Background to the invention
3D bioprinting is rapidly emerging as a key biofabrication strategy for engineering tissue structures with physiological form and complexity [1-4]. In practice, this process involves layer-by-layer deposition of a cell-laden biocompatible ink resulting in the additive manufacture of a patterned architecture with different cell types, growth factors or mechanical cues, which are positioned with far greater precision than can be achieved with conventional scaffold-based tissue engineering . While there have been significant advances in printing technology [6,7], progress has been limited by the rate of development of biocompatible inks that are compatible with both 3D printing and tissue engineering . These materials must be able to withstand extrusion, maintain structural fidelity for long time periods and permit adequate nutrient diffusion, all under cytocompatible conditions.
Due to their intrinsic porosity and capacity for high nutrient loading, hydrogels are the most promising candidate for biocompatible ink design , particularly when gelation can be externally triggered using chemical bonding , photoinduced crosslinking , thermal setting  or shear-thinning . However, integrating these factors into a system while maintaining printability, structural persistence and cell viability, is an enduring challenge . Poloxamers, a class of block copolymers of poly(ethylene oxide-b-propylene oxide-b- ethylene oxide) also known by trade names such as PluronicR™, SynperonicR™ and KolliphorR™, present a possible pathway to print gelation, as they undergo a sol-gel transition upon heating near physiological temperatures. Elevating the temperature of these non-ionic surfactants reduces the critical micelle concentration (CMC) and increases the micelle volume fraction (0m), which in turn exceeds the critical limit (0m > 0.53) resulting in micellar crystallisation and formation of a self-supporting gel phase . This phase
behaviour makes poloxamers ideal for patterning structures during 3D printing; however, their application as cell- supporting biocompatible inks is severely limited by the temperature and concentration dependence of the sol-gel transition, which results in rapid degradation of the printed structure upon cooling or immersion. Accordingly, single-component poloxamer gels are not used to print persistent cellularised structures, instead they have found application as cell-free "fugitive inks" for vascularised scaffold formation .
Conversely, chemically crosslinked gel systems offer excellent structural fidelity in aqueous solutions, but can be limited by poor compatibility with fibre extrusion and layer-by-layer printing. For example, the linear polysaccharide sodium alginate, found naturally in the cell walls of brown algae, can be rapidly crosslinked through chelation of divalent cations by the carboxylic acid groups founds on adjacent strands of the component β-D-mannuronate or a-L- guluronate epimers . Moreover, ionic crosslinking can be achieved using a wide range of readily available salts, for example Zn2+, Ni2+, Co2+, Ca2+, Ba2+ and Sr2+, and has been widely exploited to create persistent cell-laden gels for long-term culture and tissue engineering . Unfortunately, the rapid rate of crosslinking prevents effective interlayer adhesion during layer-by-layer immersion printing, and the limited shear thinning capacity during extrusion restricts formulations to low weight percentage sodium alginate gels that exhibit poor mechanical strength and non-viscoelastic rheological properties. Accordingly, the use of sodium alginate gels as biocompatible inks in 3D bioprinting has been limited to structurally simple objects with limited vertical size, which severely limits their application in tissue printing .
Previous work has provided hybrid multi-component biocompatible inks that integrate desirable physical properties from each constituent component. For instance, biodegradable polymers are commonly strengthened with osteoinductive ceramics, such as calcium phosphate , nanofibrous cellulose has been used to increase the shear thinning of alginate gels , while a mixture of poloxamer and acrylated poloxamer has been used to generate a synthetic gel that can be crosslinked using both temperature and ultraviolet irradiation . While these hybrid systems report printability and short-term cytocompatibility (4 to 14 days), they have not demonstrated practical applicability over a long-term, tissue engineering course.
WO 2016/073782, WO 2013/040087, WO 2013/158508, AU 2013/204780 and US 2015/0375435 all disclose various compositions comprising alginate or poloxamer.
Summary of the invention
The present disclosure relates to a multi-component biocompatible ink wherein the first component comprises poloxamer, a gel system that can undergo a sol-gel transition from liquid at cooler temperatures to gel at higher temperatures. The second component comprises an alginate, which is commonly used as a cell-supporting material due to its highly persistent crosslinking with many common multivalent ions .
According to a first aspect of the invention, there is provided a biocompatible ink which comprises a poloxamer in the range of from about 1 1 wt % to about 14 wt % and an alginate in the range of from about 5 wt % to about 7 wt %. This novel, multi-component biocompatible ink, comprising poloxamer and alginate, offers a versatile and new approach to generating cell-laden structures for tissue engineering. In particular, structures formed using this novel multi-component biocompatible ink have been found to exhibit favourable biomaterial properties compared to structures formed by cross-linked alginate, including increased shear thinning, compressive modulus and shear modulus. In particular, the structures exhibit increased porosity, providing for improved nutrient and oxygen diffusion, as well as increased compressive strength and elastic modulus. Significantly, this novel multi- component biocompatible ink exhibits greater shear thinning, which improves its printability compared to alginate alone.
Alternatively, the biocompatible ink may comprise a poloxamer in the range of from about 9 wt % to about 14 wt % and an alginate in the range of from about 5 wt % to about 7 wt %.
The hybrid nature of the biocompatible ink allows it to be used in a dynamic, two-step 3D printing and cross-linking approach, wherein a sacrificial poloxamer component templates both the macroscopic architecture but also creates large microscopic pores for effective nutrient diffusion. These templated structures can be used for 3D cell culture or tissue engineering, with good cell viability and retained structure for up to 35 days. This offers significant advantages over previous single and hybrid gel systems. For example, the inventors have advantageously found that a biocompatible ink comprising these weight percentages of poloxamer and alginate allows for the 3D printing of reliably smooth prints with reproducible geometries. Mixtures of poloxamer and alginate at different concentrations have been prepared previously [35, 36], but these compositions are not suitable for a biocompatible ink for 3D printing as described herein.
The biocompatible ink may comprise about 13 wt % of poloxamer, which may be poloxamer 407. The biocompatible ink may comprise about 6 wt % of alginate. The biocompatible ink may comprise about 13 wt % of poloxamer and about 6 wt% of alginate.
The biocompatible ink may comprise about 11 wt % of poloxamer and about 5 wt% of alginate, or about 11 wt % of poloxamer and about 6 wt% of alginate, or about 11 wt % of poloxamer and about 7 wt% of alginate. The poloxamer may be poloxamer 407.
The biocompatible ink may comprise about 12 wt % of poloxamer and about 5 wt% of alginate, or about 12 wt % of poloxamer and about 6 wt% of alginate, or about 12 wt % of poloxamer and about 7 wt% of alginate. The poloxamer may be poloxamer 407. The biocompatible ink may comprise about 13 wt % of poloxamer and about 5 wt% of alginate, or about 13 wt % of poloxamer and about 7 wt% of alginate. The poloxamer may be poloxamer 407.
The biocompatible ink may comprise about 14 wt % of poloxamer and about 5 wt% of alginate, or about 14 wt % of poloxamer and about 6 wt% of alginate, or about 14 wt % of poloxamer and about 7 wt% of alginate. The poloxamer may be poloxamer 407.
Poloxamers, also known by the trade name PluronicR™, are a class of non-ionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). Poloxamers have the general formula I below:
Examples of several members of the poloxamer class of compounds and details of their physical properties, chemical formulae and average molecular weights are shown in Table 1 below. The average numbers of ethylene oxide and propylene oxide units are calculated using the average molecular weights.
Table 1 - Exemplary poloxamer grades and their specifications.
The multi-component biocompatible ink of the invention may comprise a poloxamer that has a sol-gel transition temperature in the range of from 0 °C to 50 °C, for example at about 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C or about 40 °C. Any poloxamer that has a sol- gel transition temperature at a temperature of less than or equal to about 37 °C, at a concentration suitable to form an extrudable biocompatible ink, may be especially suitable. Poloxamer 407 is one example, in the concentration ranges described herein. Any of the wt % ranges or values specified above may be used for any of the poloxamer types specified in Table 1. For example, the biocompatible ink may comprise any of poloxamer 124, poloxamer 188, poloxamer 237, or poloxamer 338 at about 1 wt %, about 2 wt %, about 3 wt %, about 4 wt %, about 5 wt %, about 6 wt %, about 7 wt %, about 8 wt%, about 9 wt %, about 10 wt %, about 1 1 wt %, about 12 wt %, about 13 wt %, about 14 wt %, about 15 wt %, about 16 wt %, about 17 wt %, about 18 wt %, about 19 wt %, about 20 wt %, about 21 wt %, about 22 wt %, about 23 wt %, about 24 wt %, about 25 wt %, about 26 wt %, about 27 wt %, about 28 wt %, about 29 wt %, about 30 wt %, about 31 wt %, about 32 wt %, about 33 wt %, about 34 wt %, about 35 wt %, about 36 wt %, about 37 wt %, about 38 wt %, about 39 wt %, about 40 wt %, about 41 wt %, about 42 wt %, about 43 wt %, about 44 wt %, about 45 wt %, about 46 wt %, about 47 wt %, about 48 wt %, about 49 wt %, or about 50 wt %.
The biocompatible ink may comprise any of poloxamer 124, poloxamer 188, poloxamer 237, or poloxamer 338 in the range of from about 1 wt % to about 5 wt %, in the range of from about 5 wt % to about 10 wt %, in the range of from about 15 wt % to about 20 wt %, in the range of from about 20 wt % to about 25 wt %, in the range of from about 25 wt % to about
30 wt %, in the range of from about 30 wt % to about 35 wt %, in the range of from about 35 wt % to about 40 wt %, in the range of from about 40 wt % to about 45 wt %, or in the range of from about 45 wt % to about 50 wt %.
In any of the above embodiments, the biocompatible ink may comprise alginate at about 3 wt %, about 4 wt %, about 5 wt %, about 6 wt %, about 7 wt %, about 8 wt%, about 9 wt %, or about 10 wt %
The sol-gel transition temperature is defined as the temperature at which the solution of poloxamer undergoes a sol-gel transition from a liquid phase to gel phase. A sol-gel transition (also known as gelation) is generally defined as the point at which the solution switches from a liquid phase, whereby the poloxamer is free to diffuse, to a gel, whereby the poloxamer entraps the solvent . The sol-gel transition temperature of the biocompatible ink described herein may differ from the sol-gel transition of the poloxamer component alone. For example, the sol-gel transition temperature of the formed biocompatible ink may be higher, or may be lower, than the sol-gel transition temperature of a poloxamer component alone, when not included as a component of the ink.
In an embodiment of the invention, the poloxamer comprises a triblock copolymer of the formula I:
Formula I wherein x = 80 to 120, y = 40 to 90, and z = 80 to 120, or more preferably wherein x = 90 to 110, y = 50 to 80, and z = 90 to 110. For example, the poloxamer may be poloxamer 407.
Alginates are a family of non-branched binary copolymers of 1-4 glycosidically linked β-D- mannuronic acid (M) and a-L-guluronic acid (G) residues. The relative amount of the two uronic acid residues and their sequential arrangement along the polymer chain vary widely, depending on the origin of the alginate. Alginate is the structural polymer in marine brown algae such as Laminaria hyperborea, Macrocystis pyrifera, Lessonia nigrescens and Ascophyllum nodosum. Alginate is also produced by certain bacteria such as Pseudomonas aeruginosa, Azotobacter vinelandii and Pseudomonas fluorescens.
Alginate gels are produced when a multivalent cation forms ionic bonds with the negatively charged group from a G residue from each of two different alginate polymers, thereby cross- linking the two polymers. The formation of multiple cross-linkages among numerous alginate polymers results in the matrix that is the alginate gel structure. The alginate may comprise alginic acid, an ester of alginic acid, a salt of alginic acid or a combination thereof. An ester of alginic acid may for example comprise ethylene glycol alginate or propylene glycol alginate. A salt of alginic acid may, for example, comprise a quaternary ammonium or phosphonium salt of alginic acid, such as ammonium alginate.
Alternatively, a salt of alginic acid may for example comprise an alkali or alkaline earth metal salt of alginic acid, such as sodium alginate, potassium alginate, calcium alginate, magnesium alginate, barium alginate, strontium alginate, or a combination thereof. In a preferred embodiment, the alginate comprises sodium alginate.
The alginate may optionally be functionalised with a functionalising moiety. Functionalising the alginate with a functionalising moiety involves bonding the alginate backbone to the functionalising moiety, for example, with one or more covalent bonds. The term "functionalised" and related words indicates that the alginate is modified to enable interaction with components such as molecules, reagents and/or cells. A "functionalising moiety", therefore, is any moiety which enables the interaction of a molecule, reagent or cell (for example) with the alginate, for example by binding or other interaction. The functionalising moiety may be a natural polymer, such as a carbohydrate, protein, nucleic acid, or lipid. The functionalising moiety may be a growth factor. The functionalising moiety may be a synthetic polymer. Advantageously, the functionalising moiety may comprise a peptide or carbohydrate for the purpose of cell recognition and/or adhesion. The peptide may, for example, comprise an amino acid sequence, for example, selected from RGD, VGVAPG, GEFYFDLRLKGDK, YIGSR, WQPPRARI, or a combination thereof. In a preferred embodiment, the peptide is RGD. The carbohydrate may, for example, comprise hyaluronic acid (HA), chondroitin sulphate, dermatan sulphate, keratan sulphate, and/or heparan sulphate, heparin, agarose, dextran, cellulose, derivatives of any of these carbohydrates, or a mixture thereof.
Alternatively, the functionalising moiety may be a growth factor such as a protein or steroid hormone for stimulating cell growth and/or promoting cell proliferation. The growth factor
may, for example, comprise platelet-derived growth factor (PDGF), insulin-binding growth factor- 1 (IGF-1), insulin-binding growth factor-2 (IGF-2), epidermal growth factor (EGF), bFGF, aFGF, FGF-10, transforming growth factor-a (TGF-a), transforming growth factor-β (TGF-β) I through III including the TGF-β superfamily, platelet factor 4 (PF-4), osteogenin and other bone growth factors, collagen growth factors, heparin binding growth factor- 1 (FIBGF-1), heparin binding growth factor-2 (FIBGF-2), a BMP molecule selected from BMPl, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9, BMP10, BMPl l or BMP 12, a GDF molecule selected from GDF1, GDF2, GDF3, GDF4, GDF5, GDF6, GDF7, GDF8, GDF9, GDF 10, GDF 11 or GDF 12, dpp, 60A, BIP, OF, IGF-1, KGF, TGF^3, TRX, VEGF, copper peptide, acetyl hexapeptide, palmitoyl pentapeptide, CPP, and UDN glycoprotein, derivatives of any these growth factors, or a mixture thereof. Any single, or any combination, of the functionalising moieties mentioned herein may be used to functionalise the alginate.
The mixture of alginate and poloxamer forming the ink may be prepared in an aqueous solution, for example, in a cell growth medium suitable for a given cell type, such as (but not limited to) Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), RPMI medium, TheraPeakR™ medium, nutrient broth, lysogeny broth, terrific broth, super optimal broth, super optimal broth with catabolite repression, yeast extract nutrient broth, or yeast extract peptone dextrose. Selection of a suitable aqueous solution is within the routine ability of the skilled person.
The biocompatible ink may further comprise at least one cell. There are no limitations on the types of cell which might be included, so the cell may be a prokaryotic or a eukaryotic cell. For example, the cell may be a bacterial, algal, diatom, fungal, yeast, plant, avian, fish, amphibian, reptile or mammalian cell. Particular examples of suitable cells include (but, as mentioned, are not limited to) chondrocytes, connective tissue fibroblasts, tendon fibroblasts, bone marrow reticular tissue fibroblasts, non-epithelial fibroblasts, pericytes, osteoprogenitor cells, osteoblasts, osteoclasts, keratinocytes, hair root cells, hair shaft cells, hair matrix cells, exocrine secretory epithelial cells, hormone secreting cells, epithelial cells, neuronal cells, neural or sensory cells, photoreceptor cells, muscle cells, extracellular matrix cells, blood cells, cardiovascular cells, endothelial cells, kidney cells, hepatic cells, pancreatic cells, immune cells, stem cells, germ cells, nurse cells, interstitial cells, stellate cells, progenitors, and a combination thereof.
The cell may be a mesenchymal stem cell. As mentioned, cells may, for example, be mammalian or plant cells, for example mouse, rat, human, canine, bovine, porcine or feline cells or a mixture thereof. The cell may be one which has not been obtained via a method which involved destruction of an embryo, for example, a human embryo. The cell may also be a microorganism such as a eukaryotic microorganism, a prokaryotic microorganism, or an archaeal cell. The microbial cell may be from yeast such as Saccharomyces cerevisiae. The microbial cell may be from algae genera such as Chlorella or Dunaliella. The cell may also be a bacterial cell, such as gram positive or gram negative bacteria. The bacterial cell may be Escherichia coli or Lactobacillus acidophilus. The cell may be one which may be isolated from commensal flora from the gastrointestinal tract, respiratory tract, oral cavity, urinary tract, or skin. Where a cell is mentioned in this specification regarding any of the aspects of the invention, any of the aforementioned cell types, or mixtures thereof, may be contemplated.
The biocompatible ink may also comprise a component of extracellular matrix, a cellular material, a cellular component, a growth factor, a peptide, a protein, a lipid, a natural polymer, an inorganic particle, a nanoparticle, a synthetic molecule, a synthetic polymer, or a combination thereof. As used throughout this specification, the term "an additive" may indicate any one or more of these components, although the term need not be limited to these.
According to a second aspect of the invention, there is provided a method for forming a biocompatible ink according to the first aspect of the invention comprising a step of combining a poloxamer and an alginate.
The method may comprise the steps of: (a) sterilising the poloxamer and (b) sterilising the alginate, wherein steps (a) and (b) may be sequential or concurrent, and the order of the steps may be reversed, and wherein steps (a) and (b) are completed prior to combining the poloxamer and alginate. In an alternative embodiment, the poloxamer and an alginate are combined to form a biocompatible ink, which is subsequently sterilised.
Sterilisation refers to any process that effectively kills or eliminates transmissible agents such as fungi, bacteria, viruses, prions and spore forms. Sterilisation can be achieved through the application of heat, chemicals, irradiation, or high pressure, or by filtration. Heat sterilisation includes autoclaving (using steam at high temperatures). Radiation sterilisation can include using X-rays, gamma rays, UV light and subatomic particles. Chemical sterilisation can
include treating with ethylene oxide gas, ozone, chlorine bleach, glutaraldehyde, formaldehyde, ortho phthalaldehyde, hydrogen peroxide or peracetic acid, or any other chemical sterilisation agent known in the art.
In one embodiment, the poloxamer may be sterilised by autoclaving. The alginate may be sterilised by use of UV light.
The mixture of poloxamer and alginate may be cooled to a temperature in the range of from 0 °C to 10 °C, or in the range of from 3 to 5 °C for up to about an hour or for at least about an hour, for example for about 1-2 hours, optionally also being stirred. The mixture of poloxamer and alginate may be cooled to a temperature of about 4 °C and stirred for at least about an hour. Without wishing to be bound by theory, it is thought that this step aids with solubilising the poloxamer.
After cooling the mixture of poloxamer and alginate as described above, the mixture may be warmed to about room temperature (for example, about 20 °C, 21 °C, 22 °C, 23 °C, 24 °C or about 25 °C) at least about 1 minute or about 5 minutes or at least about 10 minutes, for example, for up to about 10 minutes, or up to about 20 minutes, or up to about 30 minutes, optionally also being stirred. Without wishing to be bound by theory, it is thought that this step aids with re-solubilising any alginate precipitated during the cooling step.
The term "stirred" as used herein may refer to any appropriate agitation method, such as (but not limited to) stirring, shaking or swirling. At least one cell may be added to the mixture of poloxamer and alginate. At least one cell may be added to the poloxamer prior to it being mixed with the alginate. Alternatively or additionally, at least one cell is added to the alginate prior to it being mixed with the poloxamer. Advantageously, the at least one cell is added to the mixture of poloxamer and alginate, or to the alginate, or to the poloxamer, after sterilisation. Examples of suitable cells may be any as described above, in relation to the first aspect of the invention.
An additive as defined above may, alternatively or additionally, be added to the mixture of poloxamer and alginate, or may be added to the poloxamer prior to it being mixed with the alginate, or may be added to the alginate prior to it being mixed with the poloxamer.
The additive may be added after the completion of any sterilisation steps.
According to a third aspect of the invention, there is provided a method for forming an alginate gel structure using a biocompatible ink according to the first aspect of the invention, which may optionally be prepared to according to the second aspect of the invention. The method may comprise extruding (which term may encompass other methods of depositing) the biocompatible ink onto a temperature controlled surface to form the alginate gel structure. The method may additionally, or optionally, comprise a three-dimensional (3D) printing process.
The method may comprise the steps of:
(i) extruding (or otherwise depositing) the biocompatible ink onto a temperature controlled surface to form a printed structure, and
(ii) cross-linking the printed structure to form an alginate gel structure.
The method may further comprise a step before (i), comprising preparing a biocompatible ink comprising a poloxamer and an alginate using a method according to the second aspect of the invention.
The biocompatible ink may be extruded onto the temperature controlled surface using any suitable extrusion means, for example such as a funnel, nozzle, needle, tube or pipe. The extrusion means may in particular be a syringe. Alternatively, the extrusion means may suitably be the printhead nozzle of a 3D printer.
The biocompatible ink may be extruded/deposited onto the temperature controlled surface to form a single layer of printed structure. Advantageously, the biocompatible ink may be extruded/deposited onto the temperature controlled surface to form a plurality of layers of printed structure.
In order to initiate the sol-gel transition of the poloxamer component of the biocompatible ink, the temperature controlled surface may be maintained at a temperature of between 10 °C and 50 °C, or between 25 °C and 50 °C, or between 35 °C and 40 °C, or between 36 °C and 38 °C. In an embodiment, the temperature controlled surface is maintained at a temperature of about 35 °C, or about 36 °C, or about 37 °C, or about 38 °C, or about 39 °C, or about 40 °C. The temperature controlled surface may be maintained at a lower temperature if, for example, the biocompatible ink is extruded from or via a cooled source, for example, via a
cooled nozzle or syringe. For example, a nozzle cooled to about 10 °C could extrude a biocompatible ink with a sol-gel transition of about 12 °C onto a surface maintained at a temperature of about 14 °C.
The term "temperature controlled" in this context indicates that the surface is maintained at a generally constant temperature, for example within about 5 °C above or below a set temperature, ideally within about 1 °C, 2 °C, 3 °C or about 4 °C. In some instances, this may require heating of the surface to a temperature greater than the temperature of the surrounding atmosphere, so that the temperature controlled surface may be termed a heated surface. The set temperature of the temperature controlled surface may be set at a temperature appropriate to initiate the sol-gel transition of the poloxamer component of the biocompatible ink. Therefore, the temperatures and ranges provided in the preceding paragraph may be the set temperature of the temperature controlled surface. Determining a suitable set temperature is within the routine abilities of the skilled person.
The temperature controlled surface may also be located in an environment where the ambient temperature results in the surface reaching and maintaining a suitable temperature, which may also be termed a set temperature. That is, maintaining the surface at a suitable temperature may not require any specific or active intervention, but may be the result of the environment in which the surface is located.
In step (ii) of the method, the printed structure may be cross-linked by immersing the structure in an ionic salt solution, thereby forming the alginate gel structure. The ionic salt, as mentioned throughout this specification, may comprise, for example, a halide salt such as chloride, fluoride, or iodide, or a combination thereof. Alternatively, the ionic salt may comprise a carbonate, phosphate, sulphate, acetate, citrate or nitrate salt. The ionic salt may comprise zinc ions, nickel ions, cobalt ions, calcium ions, barium ions, strontium ions, or a combination thereof. The ionic salt may be calcium chloride. The ionic salt may comprise any combination of salt types as mentioned herein.
The printed structure may be immersed in an ionic salt solution for at least about 5 minutes, or more preferably for at least about 10 minutes. The printed structure may be immersed in an ionic salt solution having a cationic concentration of at least about 1 mM, or at least about 5 mM, or at least about 10 mM, or at least about 20 mM, or at least about 50 mM, or at least about 100 mM. The printed structure may be immersed in an ionic salt solution with a
cationic concentration in the range of from 5 mM to 500 mM, more preferably 5 mM to 200 mM. The printed structure may be immersed in an ionic salt solution with a cationic concentration of about 5 to 100 mM, for example about 5 mM calcium chloride or about 100 mM calcium chloride. Immersion may be conducted at about room temperature (for example, about 20 °C, 21 °C, 22 °C, 23 °C, 24 °C or about 25 °C).
The term "immersed" may indicate that the printed structure is partially, substantially or wholly surrounded by and contacted with the ionic salt solution, or may encompass the printed structure being washed with the solution or otherwise contacted with the solution. The printed structure may be wholly or substantially surrounded by and contacted with the ionic salt solution. Without wishing to be bound by theory, it is believed that this enables the ions to diffuse into the printed alginate gel structure from a substantial number of different angles, resulting in the formation of a cross-linked alginate gel with an anisotropic mesh structure. Optionally, one side or surface of the printed structure may be contacted with the ionic salt solution. Without wishing to be bound by theory, it is believed that this enables the ions to diffuse into the printed alginate gel structure from a substantially single direction, so that diffusion of the ions into the printed gel structure is substantially unidirectional. This results in the formation of a cross-linked alginate gel with an isotropic mesh structure, comprising a plurality of microchannels that are substantially aligned, perpendicular to the side or surface of the alginate gel structure that was contacted with the ionic salt solution. The surface may be an upper surface (i.e., the surface furthest away from the temperature controlled surface) so that ions diffuse into the printed structure in a substantially single downward direction (i.e., in a direction perpendicular to the upper surface). This results in the formation of a plurality of microchannels that are substantially aligned perpendicular to the upper surface of the alginate gel structure.
At least about 50% of the total weight of poloxamer initially present in the printed structure after step (i) may be expelled from the structure in step (ii) when forming the cross-linked alginate gel structure. That is, after the completion of step (i), at least about 50% of the initial total weight of poloxamer is no longer present in the formed and cross-linked alginate gel structure. Alternatively, at least about 75%, 80%, 85%, 90% or at least about 95% of the total
weight of poloxamer present in the printed structure after step (i) may be expelled from the structure in step (ii). At least about 95%, 96%, 97%, 98% or at least about 99% of the total weight of poloxamer present in the printed structure after step (i) may be expelled from the structure in step (ii). Substantially all of the total weight of poloxamer present in the printed structure after step (i) may be expelled from the structure in step (ii).
According to a fourth aspect of the invention, there is provided an alginate gel structure formed using the method according to the third aspect of the invention. In a fifth aspect, there is provided a tissue culture scaffold formed by an alginate gel structure which comprises a plurality of microchannels. The scaffold may be formed using the biocompatible ink according to the first aspect of the invention.
In both aspects, the plurality of microchannels in the gel structure or scaffold may be substantially aligned. Advantageously, the plurality of aligned microchannels may be aligned substantially perpendicular to an upper surface of the alginate gel structure. The microchannels may possess a mean pore diameter of from 1 to 15 μπι, for example, from between 2 to 10 μπι or from between 5 to 8 μπι. The microchannels may possess a mean pore diameter of about 5 μπι, about 6 μπι, about 7 μπι or about 8 μπι.
The alginate gel structure or tissue culture scaffold may comprise at least one type of cell, which may be any as described in accordance with the first aspect of the invention. In an embodiment, the alginate gel structure or tissue culture scaffold comprises tissue selected from one or more of skin, cartilage, bone, bone marrow, skeletal muscle, smooth muscle, cardiac muscle, fat, nerves, brain, eye, pancreas, spleen, thyroid, adipose, sinus, oesophagus, kidney, heart, lung, intestine, stomach, colon, rectum, breast, ovary, uterus, cervix, prostate, bladder, liver or a combination thereof. In an embodiment, the alginate gel structure or tissue culture scaffold comprises a plurality of tissue layers and/or types of tissue. The alginate gel structure or tissue culture scaffold may comprise a non-mammalian cell such as a bacterial, algal, diatom, fungal, yeast, plant, avian, fish, amphibian or reptile cell. The cell could be a cell which is capable of forming a biofilm, such as a bacterial or a fungal cell.
The alginate gel structure or tissue culture scaffold may additionally or alternatively comprise a component of extracellular matrix, a cellular material, a cellular component, a growth factor, a peptide, a protein, a lipid, a natural polymer, an inorganic particle, a nanoparticle, a synthetic molecule, a synthetic polymer, or a combination thereof.
According to a sixth aspect of the invention, there is provided an apparatus for three- dimensional (3D) printing, comprising a controller, a printhead and a syringe or other container containing a biocompatible ink according to the first aspect of the invention.
According to a seventh aspect of the invention, there is provided a method of culturing cells, wherein an alginate gel structure according to the fourth aspect of the invention, or a scaffold according to a fifth aspect of the invention, in either case comprising at least one cell, is supplemented, immersed, washed or otherwise contacted (all of which terms being mutually interchangeable) with a cell culture growth medium. It is within the routine ability of the skilled person to select a cell culture growth medium suitable for the cell type or types to be cultured. For example, Dulbecco's Modified Eagle's Medium (DMEM), MEM, RPMI TheraPeakR™, nutrient broth, lysogeny broth, terrific broth, super optimal broth, super optimal broth with catabolite repression, yeast extract nutrient broth, or yeast extract peptone dextrose may be used.
The cell culture growth medium may comprise glucose, at least one inorganic salt, at least one amino acid, at least one vitamin, at least one growth factor, at least one antibiotic or any combination thereof. The cell culture growth medium may comprise insulin, penicillin, streptomycin, sodium pyruvate, dexamethasone, insulin-transferrin-selenium, ascorbic acid 2- phosphate, β-glycerophosphate, recombinant human bone morphogenetic protein 2, or combinations thereof. The growth medium may also comprise further cells, of a type the same as or different to those contained within the alginate gel structure or scaffold.
The growth medium may be supplemented with an ionic salt solution which may, for example, be calcium chloride or another ionic salt as mentioned above. The growth medium may comprise ionic salt at a cationic concentration of at least about lmM, or at least about 5 mM, or at least about 10 mM, or at least about 20 mM, or at least about 50 mM, or at least about 100 mM, for example calcium chloride at any of these concentrations. The growth medium may be supplemented with ionic salt at a cationic concentration in the range of from 1 mM to 100 mM, or from 2 mM to 60 mM, more preferably from 3 to 30 mM, for example, about 5mM, for example calcium chloride at any of these concentrations.
The alginate gel structure or scaffold comprising at least one cell and supplemented with a cell culture growth medium may be incubated at a temperature between 25 °C and 50 °C, or between 35 °C and 40 °C, or between 36 °C and 38 °C, for example, a temperature of about
35 °C, or about 36 °C, or about 37 °C, or about 38 °C, or about 39 °C, or about 40 °C.
The alginate gel structure or scaffold comprising at least one cell and supplemented with a cell culture growth medium may be incubated for at least about 1 day, or for at least about 2 days, or for at least about 3 days, or for at least about 5 days, or for at least about 10 days, for example, for at least 12-30 hours, for example for at least about 24 hours. Incubation periods of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or about 10 weeks are also contemplated.
The alginate gel structure comprising at least one cell and supplemented with a cell culture growth medium may be incubated at about 37 °C for about 5 days.
The cell culture growth medium may be partially or wholly changed during the incubation period, for example every 1, 2, 3, 4 or 5 days, for example 2, 3, 4, 5 or 6 times per week. The composition of the cell culture growth medium during the incubation period may be constant, or may vary when the medium is changed.
Other routine cell culture conditions, adjustable according to the routine abilities of the skilled person, may also be used, such as oxygen concentration, carbon concentration, pH, temperature, light exposure or other atmospheric conditions, pressure conditions, reagent supplementation and any other variable.
According to an eighth aspect of the invention, there is provided a kit comprising a biocompatible ink according to the first aspect of the invention, which may optionally be prepared by the method according to the second aspect of the invention. The kit may further include co-ordinates, or programming or other instructions for use to enable or to instruct a 3D printing apparatus to generate a desired 3D printed structure using the biocompatible ink contained in the kit.
Alternatively or additionally, the kit may further comprise at least one cell for mixing with the biocompatible ink. Examples of suitable cells may be as described above, in relation to the first aspect of the invention.
The at least one cell may be provided as a suspension in a sterile medium. Alternatively, the at least one cell may be provided in an immobilised or freeze-dried form. The at least one cell may be cryopreserved.
In a related aspect, there is provided a kit comprising an alginate gel structure and/or tissue culture scaffold according the invention. The kit may further comprise at least one cell culture medium suitable for use in a cell culture method according to the invention.
Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", mean "including but not limited to", and do not exclude other moieties, additives, components, integers or steps. Moreover the singular encompasses the plural unless the context otherwise requires: in particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Other features of the invention will become apparent from the following examples. Generally speaking the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims and drawings). Thus features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. Moreover unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.
Where upper and lower limits are quoted for a property then a range of values defined by a combination of any of the upper limits with any of the lower limits may also be implied.
In this specification, unless stated otherwise, properties are measured under standard temperature and pressure. Throughout the specification, the weight percentage (wt %) of a given component in a composition or structure defines the weight of that component as a percentage of the overall weight of the composition or structure.
Brief description of the figures
The invention will now be described, by way of example only, with reference to the following Figures 1 to 18 in which: Figure 1 shows the effect of poloxamer 407 and sodium alginate concentration on hybrid gel
Figure 2 shows the effect of sodium alginate concentration on the structural fidelity of printed structures;
Figure 3 shows the effect of incubating the printed structures in cell media supplemented with varying quantities of CaCl2;
Figure 4 is a comparison of the FTIR spectra obtained from 13 wt% poloxamer 407 / 6 wt% sodium alginate hybrid gel prior to and after crosslinking with CaCl2;
Figure 5 shows optical microscope image of a safranin O stained 10 μπι section of a paraffin- embedded printed square after crosslinking with CaCl2; Figure 6 shows scanning electron microscopy (SEM) micrographs used to investigate the effect of poloxamer 407 expulsion on the micro- and nanostructure of the hybrid gel;
Figure 7 is a bar chart of pore diameters from the SEM images in Figure 6;
Figure 8 shows rheological and mechanical testing of 13 wt% poloxamer 407 / 6 wt% sodium alginate hybrid gel, 6 wt% sodium alginate gel and 13 wt% poloxamer 407; Figure 9 shows the print geometries and input parameters for various structures printed with 13 wt% poloxamer 407 / 6 wt% sodium alginate biocompatible ink;
Figure 10 demonstrates the macroscale and microscale printing capability of the 13 wt% poloxamer 407 / 6 wt% sodium alginate hybrid gel;
Figure 11 shows the effect of encapsulated hMSCs on structural retention of structures printed with 13 wt% poloxamer 407 / 6 wt% sodium alginate biocompatible ink;
Figure 12 shows image obtained from the confocal fluorescence microscopy of hMSCs within a structure printed with 13 wt% poloxamer 407 / 6 wt% sodium alginate biocompatible ink;
Figure 13 is a bar chart of cell viability of hMSCs within a structure printed with 13 wt% poloxamer 407 / 6 wt% sodium alginate biocompatible ink over time;
Figure 14 shows the results of live-cell imaging performed on 3D printed structures of
hMSC-laden 13 wt% poloxamer 407 / 6 wt% sodium alginate biocompatible ink;
Figure 15 shows a confocal microscopy image of human kidney glomerular endothelial cells (GEnCs) and human podocytes encapsulated in a structure printed with 13 wt% poloxamer 407 / 6 wt% sodium alginate biocompatible ink, stained with calcein (white) and CellTrackerR™ Deep Red (black);
Figure 16 shows a phase contrast microscopy image of bacterial cell-aggregate encapsulated in a structure printed with 13 wt% poloxamer 407 / 6 wt% sodium alginate biocompatible ink;
Figure 17 shows a SEM micrograph of Thalaisiosira pseudonan encapsulated in a structure printed with 13 wt% poloxamer 407 / 6 wt% sodium alginate biocompatible ink; and
Figure 18 shows a SEM micrograph of Phaeodactylum tricornutum encapsulated in a structure printed with 13 wt% poloxamer 407 / 6 wt% sodium alginate biocompatible ink.
MATERIALS AND METHODS Human Mesenchymal Stem Cell Culture
Human mesenchymal stem cells (hMSCs) were harvested from the proximal femur bone marrow of osteoarthritic patients undergoing total hip replacement surgery, in full accordance with Bristol Southmead Hospital Research Ethics Committee guidelines (reference #078/01) and having received informed consent from all patients. hMSCs were cultured at 37 °C and 5% carbon dioxide in an "expansion medium" using low glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 100 units mL"1 penicillin / 100 mg mL"1 streptomycin (Sigma Aldrich, UK), 2 mM GlutaMAX (Invitrogen, USA), 10% (v/v) foetal bovine serum and 5 ng mL"1 freshly supplemented basic human fibroblast growth factor (Peprotech, USA). Cells were harvested using Dulbecco's phosphate buffered saline (Sigma Aldrich, UK) and trypsin / ethylenediaminetetraacetic acid (EDTA) solution (Sigma Aldrich, UK) and centrifuged into a pellet. This was re-suspended in a small (<50 μΕ) volume of phenol-free DMEM, and then counted, ready for addition to the biocompatible ink.
Human glomerular endothelial cell and podocyte culture
Conditionally immortalised glomerular endothelial cells (GEnCs) and podocytes were
cultured in monolayers using T175 flasks in humidified incubators at 5% C02. 20 mL of culture medium was used. Podocytes were cultured in RMPI-1640 medium (ThermoFisher, Scientific, UK) supplemented with 1% insulin-transferrin-selenium (ITS) (Sigma Aldrich, UK) and 10% (v/v) FBS. GEnCs were cultured in EBM-2 Endothelial Growth Basal Medium (Lonza) supplemented with an EGMRTM-2-MV BulletKitR™ (Lonza) containing 5% (v/v) FBS, hydrocortisone, R3-insulin like growth factor (R3-IGF-1), human fibroblast growth factor (hFGF-B), human endothelial growth factor (hEGF) and ascorbic acid. The cells were cultured at a permissive temperature of 34 °C, which allowed them to proliferate as they were de-differentiated and exhibited a cobblestone-like morphology. For co-culture, GEnC medium was used as this was also suitable for podocyte growth and differentiation. The cells were expanded until they reached confluency, which was monitored using bright field microscopy. The cells were then passaged by incubating with 5 mL of trypsin/EDTA solution for 5 minutes, before 5 mL of GEnC or podocyte medium was added and the cell suspension was centrifuged for five minutes at 524 χ g. The resulting cell pellet was resuspended in a small (<50 μΐ) amount of fresh medium, and then counted, ready for addition to the biocompatible ink.
Bacterial Cell Culture
BL21 Escherichia coli (New England Biolabs, US) transformed with heterologous plasmid were cultured in terrific broth at 37°C in a shaking incubator until cells reached an optical density at 600 nm of > 0.5 arbitrary units. The cells were then centrifuged at 5000 x g for 10 minutes, and the supernatant discarded. The pellet was then resuspended in a small (<50 μΐ) volume of terrific broth.
Diatom Cell Culture
Thalassiosira pseudonana and Phaeodactylum tricornutum were isolated from Culture Collection of Algae and Protozoa (CCAP) strains 1085/12 and 1052/1 A, respectively. They were cultured at 16 °C with 12-hour light/dark cycles under fluorescent cool-white bulbs, in f/2 Guillards media (Sigma Aldrich, UK). The culture flasks were inverted twice a week to resuspend the cells. Prior to printing, the cells were centrifuged into a pellet, and resuspended in a small (< 50 μΐ) volume of Guillards media, counted, and added to the biocompatible ink. Biocompatible ink Preparation
A 40 wt% stock solution of poloxamer 407 (PluronicR™ F127, Sigma Aldrich, UK) in low glucose, phenol-free Dulbecco's Modified Eagle's Medium (DMEM, Sigma Aldrich, UK)
was autoclaved to 121 °C for 40 minutes, cooled to 4 °C and used as a sterile solution for 3-4 weeks. A fresh 10% stock solution of sodium alginate (Sigma Aldrich, UK) in DMEM was mixed for 30 minutes at room temperature using a BDC250 overhead stirrer (Caframo, Canada), and then sterilised under UV irradiation for 20 minutes. These solutions were used to prepare working gel formulations; for instance, a 13 wt% poloxamer 407 / 6 wt% sodium alginate biocompatible ink was made by mixing 1.2 g of stock poloxamer 407, 0.65 g stock sodium alginate and 0.15 g phenol-free DMEM for 1-2 hours at 4°C and then 10 minutes at room temperature. When prepared for cell printing, the DMEM was excluded at this stage to allow the hMSC suspension to be added after the mixing steps. The cell-loaded or cell-free gels were then transferred to a sterile 5-mL syringe (Terumo Corporation, Japan), which was capped with a Mono-Ject tip caps (covered with parafilm) and then pulse centrifuged to 3000 x g. A micropipette was used to create a small air channel in the gel, allowing the syringe plunger to be inserted, ready for printing. 3D Printing
A MendelMax 2.0 desktop 3D printer (Maker's Tool Works, USA) was assembled and used to print a syringe-based universal paste extruder, designed by Richard Home and available online under a creative commons licence (Thingiverse #20733). Polyvinyl alcohol was used for the extruder body and acrylonitrile butadiene styrene for the gears. Non-printed parts included a timing belt and pulley (2.5 mm pitch), and a NEMA17 high-torque stepper motor. The paste extruder was mounted on the MendelMax printer, replacing the plastic extruder, and then calibrated in x, y, and z directions and extruder step size. For extrusion, the printer was transferred to a tissue culture hood, wiped clean with 70% ethanol and then sterilised under UV light for one hour. Commercial hairspray was used to adhere coverslips to the print bed, which was then heated to 37 °C. The gel-loaded syringes were equipped with a cut pipette tip of 0.5 mm internal diameter (Starlab, UK), fitted to the plastic extruder and secured with the extrusion belt. Structures were printed at a rate of 600 mm min"1 (extrusion rate of 0.7 mL min"1), immersed for 10 minutes in phenol-free DMEM supplemented with 100 mM CaCl2, washed with phosphate buffered saline (PBS) and then maintained under standard conditions, until needed. For higher resolution structures, a reduced printing rate of 200 mm min"1 (extrusion rate of 0.02 mL min"1), and either flat-tip needles (25-gauge or 30- gauge, RS Components, UK) or 23 -gauge hypodermic needles (Terumo Corporation, Japan) shortened with a Dremel, were used to print structures that were crosslinked and then maintained in 5 mM CaCl2. In certain cases, a 0.1% (v/v) doping of 10 mg mL"1 RITC
(Sigma Aldrich, UK) was used to visualise extruded fibres using widefield fluorescence microscopy. Photographs were taken using a Canon 1200D DSLR with an 18-55 mm lens. All 3D models in the STL file format were processed into G-code for layer-by-layer printing using Slic3r software (Open Source, http://slic3r.org). Cell Viability Studies
Biocompatible ink containing hMSCs (3 x 106 cells mL"1) was extruded into square prints or single lines, which were crosslinked with 100 mM CaCl2 and cultured in expansion medium containing 5 mM CaCl2. For imaging, the prints were transferred to a 35-mm diameter Petri dish with glass substrate (MatTek, USA) in phenol-free media supplemented with a commercial live/dead stain (Life Technologies, UK) and 20 mM HEPES buffer (Sigma Aldrich, UK). These samples were imaged on either an SP8 confocal fluorescence microscope (Leica, UK) using a 10X objective lens, or a DMI3000 inverted widefield fluorescence microscope (Leica, UK) using a 2.5X objective lens and an excitation filter of 450-490 nm. Cell counting image analysis was performed on confocal fluorescence microscopy images using ImageJ software (NIH, USA).
Biocompatible ink containing hMSCs (6 x 106 cells mL"1) was printed (square geometry, trachea), crosslinked with 100 mM CaCl2 and cultured in a differentiation medium supplemented with 5 mM CaCl2. Media and growth factor stocks were prepared as follows: chondrogenic basal medium was prepared using high glucose DMEM (Sigma Aldrich, UK) supplemented with 100 units mL"1 penicillin / 100 mg mL"1 streptomycin, 2 mM GlutaMAX (Invitrogen, USA), 1% (v/v) sodium pyruvate (Sigma Aldrich, UK) and 1% (v/v) insulin- transferrin-selenium (Sigma Aldrich, UK); osteogenic basal medium was prepared using a- MEM (Sigma Aldrich, UK) supplemented with 100 units mL"1 penicillin / 100 mg mL"1 streptomycin, 2 mM GlutaMAX (Invitrogen, USA) and 2% FBS; dexamethasone (Sigma Aldrich, UK) was dissolved in ethanol as a 100X solution, then diluted in chondrogenic basal medium to yield a 100 μΜ stock solution that was filter sterilised; 80 mM ascorbic acid 2- phosphate (Sigma Aldrich, UK) in deionised water was prepared and filter sterilised; 10 mg mL"1 insulin (Sigma Aldrich, UK) in acetic acid (pH 2.0, Sigma Aldrich, UK) was prepared and filter sterilised; 1 M β-glycerophosphate disodium salt hydrate (Sigma Aldrich, UK) in deionised water was prepared and filter sterilised; recombinant human transforming growth factor β3 (TGF β3, R&D Systems, UK) was dissolved in a filter-sterilised 1 mg mL"1 solution
of bovine serum albumin (BSA, Sigma Aldrich, UK) in 4 mM hydrochloric acid (Sigma Aldrich, UK) to yield a 100 μg mL"1 stock solution; recombinant human bone morphogenetic protein 2 (BMP 2, Peprotech, UK) was dissolved in a filter-sterilised 1 mg mL"1 solution of BSA in 4 mM hydrochloric acid (Sigma Aldrich, UK) to yield a 100 μg mL"1 stock solution. For bone tissue engineering, prints were cultured in osteogenic basal medium freshly supplemented with 0.1 μL mL"1 dexamethasone, 2.5 μL mL"1 ascorbic acid 2-phosphate, 10 μL mL"1 β-glycerophosphate and 0.25 μL mL"1 BMP 2, with media changes 3 times a week for three weeks. For cartilage engineering, prints were cultured in chondrogenic basal medium freshly supplemented with 1 μL mL"1 dexamethasone, 1 μL· mL"1 ascorbic acid 2- phosphate and 1 μL· mL"1 TGF β3 for 5 weeks, with media changes 3 times a week, with the addition of 1 μL· mL"1 insulin for the final 4 weeks of tissue engineering.
The printed tissue was harvested, photographed and then fixed in Fixation Buffer (BioLegend, USA) for 2 hours, transferred to a 70% (v/v) ethanol solution and then submitted to Histology Services Unit (University of Bristol), where they were embedded in paraffin, cut into 10-μπι sections and affixed to Polysine microscope slides (VWR, UK). Samples were rehydrated using 2-minute immersions in xylene, 100% ethanol, 70% (v/v) ethanol and then deionised water. For calcium staining, slides were immersed in 2% Alizarin Red S (Sigma Aldrich, UK) for 5 minutes, before excess stain was cleared by dipping the samples 20 times in acetone, in 50:50 acetone :xylene, and then in xylene. For Von Kossa phosphate staining, slides were immersed for 90 minutes in a 5% solution of silver nitrate (Sigma Aldrich, UK), with the container covered in foil and illuminated from above using a table lamp. The slides were then washed 4 times with deionised water, immersed for 3 minutes in 5% sodium thiosulphate (Sigma Aldrich, UK), washed twice with deionised water, and then dehydrated using 2 minute immersions in 90% (v/v) ethanol and 100% ethanol. For collagen staining, slides were immersed in 0.1% Sirius Red (Sigma Aldrich, UK) for 1 hour at room temperature, briefly washed in 2 changes of 0.5% (v/v) acetic acid and then dehydrated using 2 minute immersions in 70 (v/v) ethanol, 90% (v/v) ethanol and 100% ethanol. For glycosaminoglycan staining, slides were briefly washed with acetic acid (pH 2.3), stained for 2 minutes using 0.1% Safranin O (pH 2.3), dipped in 95% (v/v) twenty times, 100% ethanol ten times and then immersed for two minutes in xylene. Sodium alginate staining used a similar protocol, using less acidic solutions of acetic acid and safranin O (both pH 2.9) to allow deprotonation of the carboxylic acid chains. Stained sections were mounted in DPX
(Fisher Scientific, UK) and imaged using a DMI300 inverted bright field microscope (Leica, UK) with a 100X oil immersion objective lens.
Fourier Transform Infrared Spectroscopy
Lyophilised samples (crosslinked and uncrosslinked 13 wt% poloxamer 407 / 6 wt% sodium alginate hybrid gel) were analysed using a Spectrum One FTIR spectrometer (Perkin Elmer, USA). Data were collected in transmission mode, scanned across a wavenumber range of 600-4200 cm"1, with a blank background subtracted from the measured data.
Small amplitude oscillatory measurements were performed on the gels at 37°C using a Kinexsus Pro+ Rheometer (Malvern Instruments, UK). Crosslinked or un-crosslinked samples were added to the rheometer plate, the test geometry (diameter = 20 mm, 4° angle) was lowered to a gap height of 500 μπι and any excess hydrogel was discarded. A strain sweep from 0.05-10% was carried out at an oscillation frequency of 1 Hz to determine the linear viscoelastic (LVE) region. From this, an optimised LVE strain of 0.1% was used during a frequency sweep performed from 0.1-10 Hz. The linear region of the frequency sweep was then used to determine the elastic modulus (G) and the viscous modulus (G"). Steady state shear measurements were performed at a temperature of 25°C and a shear rate range of 0.1-10 s"1. Non-linear regression analysis was performed using the Ostwald-de Waele relationship to calculate the flow index. Compression Testing
The mechanical behaviour of the gels was evaluated using unconfined compression testing under displacement control. Samples of sodium alginate or hybrid gel were washed in 100 mM CaCl2 to crosslink the chains and, in the latter case, expel any poloxamer 407. The diameter and thickness were measured, and the sample was positioned between impermeable plates on an Instron 3343 1 kN single column universal testing machine (Instron, UK), fitted with a 10 N load cell. The test rig was lowered to make contact with the gel, and the load and displacement readings were set to zero. The samples were compressed at a rate of 1 mm min"1 until either the maximum displacement of 5 mm was reached or the maximum load of 10 N was exceeded. The load and displacement data were recorded in real time, allowing the compressive modulus to be determined from the linear region of the compression phase.
Scanning Electron Microscopy
Small sections of either the 13 wt% poloxamer 407 / 6 wt% sodium alginate hybrid gel or the 6 wt% sodium alginate gel were dehydrated using critical point drying, mounted on stubs and sputter coated with silver using a High Resolution Sputter Coater (Agar Scientific, UK). These samples were images using a JSM IT300 Scanning Electron Microscope (Jeol Ltd., Japan). For each gel, pore diameters were evaluated using line and measurement functions in Image J software. 50 pores were sampled across each micrograph, and were used to calculate the mean average and the standard deviation. Unidirectionally-crosslinked samples were prepared by adding the CaCl2 on top of the gel in a microcentrifuge tube (Eppendorf, UK). Calcium Depletion Measurements
A square print, crosslinked for 10 minutes in 100 mM CaCl2, was immersed in 5 mL phenol- free DMEM. A calcium combined ion selective electrode (VWR, UK), calibrated over the range 0-10 mM was used to measure the solution concentration of calcium in this solution, with measurements taken every 2 minutes for 24 hours. RESULTS AND DISCUSSION
A cellularised biocompatible ink was formulated using sodium alginate and poloxamer 407, which involved developing a new methodology to meet the conflicting conditions required to solubilise the separate gel components, retain cell viability and maintain sterility. Briefly, autoclaved 40 wt% poloxamer 407 and UV-sterilised 10 wt% sodium alginate were mixed at 4°C and then at 25°C, to create a homogenous fluid which could be loaded with hMSCs. A MendelMax 2.0 3D printer retro-fitted with a syringe pump was used to extrude the pre- gelled fluid onto a heated stage set to 37°C. The elevated temperature instigated a spontaneous sol-gel transition mediated by the poloxamer 407 component of the printed layers, allowing the generation of self-supporting 3D geometries, which were further stabilised by crosslinking the alginate chains with a CaCl2 wash. Hollow square-based rectangular prisms with outer dimensions of 10 x 10 x 2.4 mm and a wall thickness of 1.6 mm were printed from six 400-μπι thick layers, and this structural template was used to enable high-throughput printing and facile assessment of print quality and structural fidelity.
The 3D printing compatibility of the biocompatible ink was tested over a range of poloxamer 407 and sodium alginate concentrations. Figure 1 shows the effect of poloxamer 407 and sodium alginate concentration on hybrid gel printing. Photographs were taken immediately
after crosslinking 1 cm x 1 cm structures printed using 5 to 8 wt% sodium alginate solution supplemented with poloxamer 407 at a concentration of 11 to 15 wt%.
Optimum printing performance was observed using 13 wt% poloxamer 407 with 6 wt% sodium alginate, which produced reliably smooth prints with reproducible geometries. For 5 and 6 wt% concentrations of sodium alginate, the 13 wt% hybrid gel produced the most well- defined structure, in contrast to the deformation observed with the hybrid gel containing 11 wt% poloxamer 407, or the "castled" structure with accumulated material at the corners that occurred when using the hybrid gel containing 15 wt%. When higher concentrations of 7 and 8 wt% sodium alginate were used, a "castled" structure was also observed at 11 wt% poloxamer 407, and above 12 wt% the gel could no longer be printed. 4 wt% sodium alginate gels were either too liquid to print or disintegrated over 5 days incubation.
The alginate constituent allowed post-printing calcium crosslinking, which prolonged stability of the printed structure in cell culture media. Figure 2 shows the effect of alginate concentration on the structural fidelity of the printed structures (scale bars = 2 mm). Photographs were taken of squares printed using 13 wt% poloxamer 407 supplemented with sodium alginate at a loading of: (a) 2 wt%, (b) 4 wt% and (c) 6 wt%. The structures were crosslinked in cell medium supplemented with 100 mM CaCl2 for 10 minutes and then incubated in un-supplemented cell medium at 37°C for 120 hours.
At 48 hours, the hybrid gel containing 2 wt% alginate had dissolved into solution, while at 120 hours the hybrid gel containing 4 wt% alginate had disintegrated. In contrast, the hybrid gel structure containing 6 wt% alginate exhibited a retained geometry up to 120 hours, after which the printed structure deformed, lost definition, and disintegrated.
Taken together, these results indicated that the optimum concentration range for the poloxamer is 11-14 wt% and for the alginate is 5-7 wt%. It was found that by incubating the printed structures in cell media supplemented with millimolar quantities of CaCl2, it is possible to retain the structural fidelity of the printed structures for up to 240 hours. Figure 3 shows photographs of printed structures containing 13 wt% poloxamer 407 / 6 wt% sodium alginate crosslinked in 100 mM CaCl2 and subsequently cultured in cell media supplemented with CaCl2 at a concentration of: (a) 0 mM (un-supplemented media), (b) 1 mM, (c) 3 mM, (d) 5 mM, (e) 10 mM.
The loss of structural fidelity of the printed constructs after 5 days appeared to be largely independent of the CaCl2 concentration used during crosslinking (between 5 and 100 mM). Rather, disintegration was attributed to the higher ionic strength within the gel compared to the surrounding cell medium, which led to osmotic swelling of the printed structure and the displacement of the chelated calcium ions . This hypothesis was supported by an observed increase in the concentration of calcium ions (1 mM over 4 hours) in cell media containing a single crosslinked structure.
To counter the effect of calcium efflux, the printed structures were incubated in cell media supplemented with millimolar quantities of CaCl2. Incubating the printed structures in cell media supplemented with low levels of CaCl2 (<3 mM) failed to preserve the structure of the prints after 240 hours (Figure 3a-c), however, supplementing the media with 5 mM or 10 mM CaCl2 gave excellent structural fidelity after 240 hours (Figure 3d, e).
In summary, the biocompatible ink optimisation experiments led to a standard protocol whereby 13 wt% poloxamer 407 / 6 wt% alginate was crosslinked in 100 mM CaCl2 for 10 minutes, with a 5 mM CaCl2 media supplement used to maintain the structure during long term culture.
After printing, calcium crosslinking and washing steps were performed at room temperature to allow the poloxamer 407 to dissolve and diffuse out of the printed gel structure. This dynamic process was monitored using Fourier transform infrared (FTIR) spectroscopy, which was used to track the changes in the transmission bands associated with the mannuronate and guluronate monomers of alginate (for instance, carboxylate stretches at 1411 and 1604 cm"1) and the repeat ether moiety in poloxamer 407 (prominent stretches at 1103 and 2885 cm"1).
Figure 4 shows the FTIR spectra obtained from (a) the 13 wt% poloxamer 407 / 6 wt% sodium alginate hybrid gel prior to calcium crosslinking and after crosslinking, along with (b) reference spectra of poloxamer 407 and sodium alginate. Significantly, the un-crosslinked hybrid gel possessed a composite spectrum with transmission peaks originating from both components, while the FTIR trace from the post-crosslinked structure was identical to sodium alginate alone. This was consistent with complete expulsion of the poloxamer 407 component from the hybrid gel leaving behind a macroscopically templated alginate print.
Optical microscopy was performed on an alginate print section stained with the carbohydrate dye Safranin O. Figure 5 shows Safranin O staining performed on a 10 μπι section of a
paraffin-embedded 1 cm x 1 cm printed square, which revealed homogenous staining throughout the printed structure.
The effect of poloxamer 407 expulsion on the micro- and nanostructure of the hybrid gel was investigated by scanning electron microscopy (SEM). Crosslinked gel samples were prepared using critical point drying, an established dehydration technique that preserves the integrity of hydrogels for high-resolution imaging .
Figure 6 shows SEM micrographs of: (a) 13 wt% poloxamer 407 / 6 wt% sodium alginate hybrid gel and (b) 6 wt% sodium alginate gel, in each case, with unidirectional crosslinking using 100 mM CaCl2 (scale bar in all SEM images = 20 μπι). Significantly, the SEM micrographs revealed an extensive porous architecture within the crosslinked gel, with a mean pore diameter (d) of 6.4 ± 0.6 μπι that was much larger than those observed in alginate alone (d = 0.76 ± 0.06 μπι). Furthermore, in the hybrid gel, aligned pores were observed, whereas the sodium alginate gel possessed an isotropic mesh.
Additionally, Figure 6 shows SEM micrographs for 6 wt% sodium alginate hybrid gel with varying poloxamer 407 concentration: (c) 9 wt% poloxamer 407, (d) 1 1 wt% poloxamer 407, (e) 13 wt% poloxamer 407, (f) 15% poloxamer 407. Significant differences in microstructure of the poloxamer 407 / sodium alginate hybrid gels were observed, with a trend towards larger pore structures in the hybrid gels with more poloxamer 407. Figure 7 is a bar chart of pore diameters from the SEM micrographs, showing a systematic increase of pore size with increasing concentration of poloxamer 407.
This indicated that the poloxamer 407 not only provided a physical template for macroscopic structure formation, but also acted as a microscopic template where the amphiphilic nature of the surfactant molecule stabilised the formation of the large macropores. Porosity is a critical feature in tissue engineering structures, with large pores shown to increase elastic moduli, enhance nutrient mass transport and provide interstitial space for extracellular matrix (ECM) deposition [24, 25]. Moreover, unidirectional calcium diffusion into the hybrid gel generated aligned channels oriented perpendicular to the gel surface (Figure 6a).
This behaviour has previously been observed in alginate hybrid gels [26, 27] and, without being bound by theory, is thought to occur when hydrodynamic flow arises from friction between the contracting alginate chains and the bulk solution . Such patterned architectures are highly desirable for tissue engineering applications; for example, anisotropic
gel environments are used to guide cellular alignment and microchannel architectures are exploited for enhanced nutrient mass transport . Conversely, under the same experimental conditions, unidirectional calcium diffusion resulted in alginate gels with an isotropic mesh structure (Figure 6b) which suggests that poloxamer 407 acts as an interfacial stabiliser during the formation of both large pores and aligned channels . This is a significant advantage obtained by the use of the biocompatible ink described herein.
Rheology and compression testing revealed that the hybrid gel, before and after calcium crosslinking, had physical properties significantly different to the equivalent neat alginate and poloxamer 407 systems. Figure 8 shows the results of testing 13 wt% poloxamer 407 / 6 wt% sodium alginate hybrid gel (circular data points), 6 wt% sodium alginate gel (square data points) and 13 wt% poloxamer 407 (triangular data points).
A shear rate ramp performed on un-crosslinked hybrid gels at printing temperature (T = 25°C) showed a significant decrease in viscosity with increased shear, producing a flow index (n) of 0.126 ± 0.001, which is indicative of a shear thinning material (n < 1) (Figure 8a). The Ostwald-de Waele model was used to estimate the flow index. This behaviour was far less pronounced for the 6 wt% alginate system (n = 0.629 ± 0.003), while the 13 wt% poloxamer 407 showed significant shear thinning, which together suggest that poloxamer 407 enhances the shear thinning behaviour of the hybrid gel.
Frequency sweeps were used to measure the elastic component of the shear modulus (G') for the uncrosslinked gel (light grey data points) and crosslinked gel (black data points), and also the viscous components of the shear modulus (G") for the uncrosslinked gel (white data points) and crosslinked gel (dark grey data points), all at 37 °C . These revealed that the uncrosslinked hybrid gel was viscoelastic (G' > G , slope in G), in contrast to the uncrosslinked 6 wt% alginate (G' < G") (Figure 8b, c). Calcium ion crosslinking of the hybrid gel resulted in an order of magnitude increase in the shear modulus, which was attributed to the increased rigidity conferred by the inter-chain chelation of calcium ions. Indeed, no crossover between G' and was observed at low frequencies, which is consistent with the formation of a persistent rigid gel network. Significantly, the crosslinked hybrid gel exhibited a shear modulus approximately twice that of crosslinked 6 wt% alginate.
Furthermore, strain sweeps were used to measure the elastic component of the shear modulus (G') for the uncrosslinked gel (light grey data points) and crosslinked gel (black data points),
and also the viscous components of the shear modulus (G") for the uncrosslinked gel (white data points) and crosslinked gel (dark grey data points), all at 37 °C (Figure 8d, e). The strain sweep of the crosslinked hybrid gel showed a linear viscoelastic (LVE) region extending to approximately 0.7% of the complex shear strain at 37°C, which was an order of magnitude larger than the limit measured for crosslinked 6 wt% alginate (-0.06%).
Finally, unconfined compression testing at 37°C gave a Young's modulus (E) of 45 ± 4 kPa for the crosslinked hybrid gel (Figure 8f), a value that was 50% higher than crosslinked 6 wt% alginate (E = 30 ± 5 kPa) and similar to soft tissues such as articular surface hyaline cartilage (E = 79 ± 39 kPa, as measured in bovine tissue) . Taken together, it was evident that the presence of poloxamer 407 in the initial mixture enhanced the shear thinning and rheological characteristics of the uncrosslinked biocompatible ink (n, G G ), while the structural templating significantly improved the rheological and mechanical properties of the final crosslinked structure (E, G G and LVE region).
In light of the favourable mechanical properties and high porosity of the hybrid gel, an array of anatomical structures were printed. Figure 9 shows the print geometries and input parameters (not to scale), including the maximum length (L), width (W), feature width (T) and height (H) of: (a) nose with L = 46.2 mm, W = 29.2 mm, H = 17.2 mm, (b) ear with L = 46.8 mm, W = 32.5 mm, H = 6.4 mm, (c) square test print with L = 10.0 mm, T = 1.6 mm, H = 2.4 mm, (d) tracheal cartilage ring with L = 18.5 mm, W = 13.5 mm, T = 2.2 mm and H = 3.2 mm, and (e) Crosshatch with a line width of 0.3 mm separated by a gap of 0.3 mm.
To demonstrate the macroscale printing capability of the hybrid 13% poloxamer 407 / 6% alginate gel, a full size ear and nose, with a heights of 0.64 and 1.72 cm respectively were printed (Figure 10, scale bars = 1 cm). The geometry of these printed structures was faithful to the input design, moreover, the final print height of the nose (17.2 mm) demonstrated that the thermally induced sol-gel transition could be propagated effectively through the structure over a large distance from the heated bed.
In addition to these macroscopic structures, the hybrid gel was used to print microscopic structures, which were observed using fluorescence spectroscopy (Figures lOc-f). The hybrid gel was used to print a fine crosshatched mesh with a mean voxel area of 0.17 ± 0.03 mm2 (Figure 10c). This demonstrated how the resolution could be modified simply by tuning the internal diameter (0) of the extrusion tip. For example, a mean line width of 0.69 ± 0.04 mm
was achieved using a standard micropipette tip (0 = 0.50 mm) (Figure lOd), which reduced to 0.41 ± 0.05 mm in the Crosshatch pattern by using a 25-gauge syringe needle (0 = 0.26 mm), and a mean line width of 0.19 ± 0.01 mm was attained using a 30-gauge syringe needle (0 = 0.16 mm) (Figure lOe). Taking the average diameter of an adult hMSC to be approximately 10-20 μπι, these results demonstrate the potential of the hybrid gel for high resolution cell patterning. Moreover, high magnification fluorescence imaging of extruded biocompatible ink revealed alignment of the gel fibres parallel to the print vector, which was attributed to shear flow alignment during extrusion (Figure lOf).
A time course study was performed to analyse whether cell loading affected the printing process or structural fidelity of the hybrid gel construct. Figure 1 1 shows the effect of encapsulated hMSCs on structural retention of the printed structures. Three constructs were printed in 3 wt% poloxamer 407 / 6 wt% sodium alginate hybrid gel either (a) cell-free, or (b) with 3 x 106 hMSCs mL-1 (scale bar = 2 mm). These constructs were crosslinked using 100 mM CaCl2 and then cultured in cell media supplemented with 5 mM CaCl2 for five days, with one construct in each group photographed after 0, 48 and 120 hours. Significantly, the addition of 3 x 106 cells mL-1 hMSCs to the hybrid gel prior to printing produced no observable effect on extrusion efficacy or structural retention of square prints over the course of 5 days in culture with cell medium.
The viability of the encapsulated hMSCs was assessed over a one-week period using live / dead staining, with confocal fluorescence microscopy. Figure 12 shows confocal fluorescence microscopy of hMSCs within a printed 13 wt% poloxamer 407 / 6 wt% sodium alginate hybrid gel, with the cells stained using calcein-AM (live cells = white) and propidium iodide (dead cells = black). Images were taken (a) immediately after printing, and after (b) day 1, (c) day 2, (d) day 3, (e) day 4 and (f) day 7 (scale bars = 100 μπι). Image analysis demonstrated a cell viability of 87 ± 4% (immediately after printing), 79 ± 8% (1 day), 76 ± 3% (2 days), 88 ± 2% (3 days), 81 ± 6% (4 days) and 83 ± 6% (7 days).
Figure 13 is a bar chart of cell viability of hMSCs within a printed 13 wt% poloxamer 407 / 6 wt% sodium alginate biocompatible ink over time. Image analysis revealed high post-print cell viability (87 ± 4%), which remained in excess of 75% throughout the one-week culture period.
Figure 14 shows live-cell imaging performed on 3D printed structures of hMSC-laden 13
wt% poloxamer 407 / 6 wt% sodium alginate hybrid gel. Live-dead staining using calcein AM (live cells = white) and propidium iodide (dead cells = black) revealed high post-print viability in (a) fine fibres printed with 23-gauge needles (scale bar = 500 μπι), and throughout a printed square (b) immediately after printing and (c) after ten days of culture (scale bar = 1 mm).
Figure 14a shows that cell death from flow-induced shear forces was not evident even at high-resolution prints (23-gauge needle, 0 = 0.34 mm), which gave a post-print cell viability of 83%. Low magnification widefield fluorescence microscopy performed on cell-laden square prints allowed clear visualisation of the hMSC distribution, with negligible cytotoxicity observed either post-printing or after 10 days in culture in serum-containing cell media (Figure 14b, c). Previous studies have shown cytotoxic effects when poloxamer 407 is used as an encapsulating gel (60-70% viability for 15.6 wt% poloxamer 407), but not when used as a liquid additive (80-90% viability for 10% poloxamer 407), with more severe effects at higher weight percentage and longer exposure time (quoted figures for HepG2 cells, t = 1 day) . These values correlate well with the results from the present study, where hMSCs are only exposed to 13 wt% poloxamer 407 during mixing and printing (typically 30-60 minutes), before poloxamer 407 is expelled from the hybrid gel. In general, hMSCs encapsulated within the hybrid gel adopted a spherical cell morphology, an equilibrium geometry assumed in the absence of microscale features or integrin binding sites, and typical of nanofibrous gels, such as alginate . Although cells such as chondrocytes and osteoblasts are naturally rounded  an adherent analogue would benefit cells with fibroblastic or neuronal morphology and enable methodological flexibility in printing complex, multi-responsive structures. This could be achieved by introducing cell-binding motifs; for instance, enhanced cell adhesion and spreading has been observed when alginate is covalently tagged with arginylglycylaspartic acid (RGD), a tripeptide sequence present in the cellular adhesion protein fibronectin.
Figure 15 shows that it is possible to print structures encapsulating two complementary human cell types with 13 wt% poloxamer 407 / 6 wt% sodium alginate biocompatible ink.
Figure 16 shows that it is possible to print structures encapsulating bacterial cells with 13 wt% poloxamer 407 / 6 wt% sodium alginate biocompatible ink.
Figures 17 and 18 show that it is possible to print structures encapsulating diatoms with 13
wt% poloxamer 407 / 6 wt% sodium alginate biocompatible ink.
In conclusion, the new biocompatible ink described herein offers a multitude of advantages for bioprinting, compared to single-component gel systems. The inventors have shown that combining separate specialised, functional components can produce a smart soft biomaterial that can be extruded at high-resolution and effectively crosslinked to produce cytocompatible structures with long-term structural fidelity. Moreover, by using poloxamer 407 as a sacrificial guest, it is possible to template both the macroscopic and microscopic structure, producing a porous alginate framework with upgraded mechanical properties and enhanced rheological characteristics. This provided a platform for tissue engineering using cell-laden prints, which resulted in widespread matrix production within a confined geometry, a result that opens up new opportunities for printing tissue structures with complex physiological structure and represents a significant advance towards the ultimate goal of recapitulating physiological tissue structures in vitro.
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