Graft scaffold for cartilage repair and process for making same

Abstract

The present invention relates to a method of providing a graft scaffold for cartilage repair, particularly in a human patient. The method of the invention comprising the steps of providing particles and/or fibres; providing an aqueous solution of a gelling polysaccharide; providing mammalian cells; mixing said particles and/or fibres, said aqueous solution of a gelling polysaccharide and said mammalian cells to obtain a printing mix; and depositing said printing mix in a three-dimensional form. The invention further relates to graft scaffolds and grafts obtained by the method of the invention.

Claims

1. A graft scaffold comprising: 3% to 3.5% (w/v) of a gelling polysaccharide selected from the group consisting of gellan gum, acylated and sulfated gellan gum; mammalian cells; and alginate, wherein the graft scaffold is obtainable by a method comprising: providing an aqueous solution of the gelling polysaccharide and alginate; providing mammalian cells; mixing said aqueous solution of a gelling polysaccharide and alginate, and said mammalian cells to obtain a printing mix; and depositing said printing mix in a three-dimensional form, thereby obtaining the graft scaffold.

2. The graft scaffold of claim 1, wherein the alginate concentration is 2.5% or 3% (w/v).

3. The graft scaffold of claim 1, wherein said gelling polysaccharide is acylated gellan gum.

4. The graft scaffold of claim 1, wherein said aqueous solution of a gelling polysaccharide and alginate further comprises between 10 and 150 mmol/1 of divalent ions.

5. The graft scaffold of claim 1, wherein the graft scaffold further comprises particles selected from the group consisting of ECM, cartilage, hydroxyapatite (HA), polymer, biocompatible conductive material, and calcium phosphate; and/or fibres selected from the consisting of silk, elastin, and resilin.

6. The graft scaffold of claim 1, wherein the graft scaffold does not comprise particles selected from the group consisting of ECM, cartilage, hydroxyapatite (HA), polymer, biocompatible conductive material, and calcium phosphate or fibres selected from the consisting of silk, elastin, and resilin.

7. The graft scaffold of claim 1, wherein said aqueous solution of a gelling polysaccharide and alginate further comprises a monosaccharide sugar or disaccharide sugar at physiologic osmolarity.

8. The graft scaffold of claim 1, wherein the graft scaffold further comprises a growth factor and/or a mitogenic factor.

9. The graft scaffold of claim 8, wherein the growth factor or mitogenic factor is selected from the group consisting of BMP-2, BMP-7, TGF-β1, TGF-β2, TGF-β3, FGF-2, and IGF-1.

10. The graft scaffold of claim 8, wherein the concentration of growth factor and/or mitogenic factor is 0.1-5 ng/ml, 5-50 ng/ml or 50-500 ng/ml.

11. The graft scaffold of claim 1, wherein said mammalian cells are cartilage cells, cartilage stem cells, or cartilage precursor cells.

12. The graft scaffold of claim 1, wherein said mammalian cells are present at concentrations of 3×10.sup.6 cells/ml —50×10.sup.6 cells/ml.

13. The graft scaffold of claim 1, wherein the graft scaffold comprises 10 ng/ml TGF beta 3.

14. The graft scaffold of claim 1, wherein depositing said printing mix in a three-dimensional form is performed by deposition of lines of said printing mix, wherein each line has a width of 700 to 1100 μm and said lines overlap by 20% to 60%.

15. The graft scaffold of claim 1, wherein said depositing is performed by 3-D-printing methods.

16. The graft scaffold of claim 1, wherein said depositing is performed by additive manufacturing methods.

17. The graft scaffold of claim 16, wherein the additive manufacturing method is ink jet printing, bioprinting, extrusion printing or layer-by-layer method.

18. The graft scaffold of claim 1, wherein the 3-Dimensional form is generated based on a computer model of a contralateral organ.

Description

(1) The disclosure is further described with reference to the following figures and non-limiting examples, which depict particular embodiments.

(2) FIG. 1A is the three dimensional model created based on patient CT models and after internal support structure was added for better load bearing to the graft.

(3) FIG. 1B is a photograph of an intact tissue engineered ear construct.

(4) FIG. 1C is a photograph of internal support that could stabilize the ear structure for more natural like bending properties. Both images B and C were fabricated utilizing three-dimensional bioprinting and are composed of minced cartilage particles, gellan gum, and alginate.

(5) FIG. 2 shows graphs illustrating the rheological crosslinking kinetics and the final stiffness of the bioink with two compositions (Bioink, top panel; Bioink+HA, bottom panel).

(6) FIG. 3 illustrates the time dependency of the mechanical properties with the 20 mM strontium chloride solution where specimens (n=6) average ultimate stress at failure for each time point was measured in tension (left). Furthermore, the concentration of cations (black=calcium chloride and grey=strontium chloride) has similar effect on crosslinking despite the cationic source (right, top and bottom). It can be concluded that the mechanical properties are highly dependent on the cation concentration and crosslinking time.

(7) FIG. 4 is a graph illustrating the metabolic activity of the chondrocytes embedded into the printing mix material in printing process. Metabolic activity assay (Promega MTS one solution assay was performed in several time points analyzed with a plate reader (Synergy H1, Biotek). Positive control was alginate 1% (light gray), printing mix material corresponds to printing mix material without tissue particles (gray) and printing mix material+ECM (dark gray) consists of cartilage extra cellular matrix particles <100 μm in diameter. All conditions were analyzed in triplicates.

(8) FIGS. 5A and 5B illustrate the co-deposited support structure providing initial crosslinking molecules such as cations from any source, enzyme, protein or other activating molecule that initiates the crosslinking cascade (FIG. 5A) and the final construct with overhanging features after elution of the support (FIG. 5B).

(9) FIG. 6 is a photograph of an intact native size tissue engineered nose construct composed of particles, gellan gum, and alginate. Construct was fabricated utilizing three-dimensional bioprinting in less than 17 minutes. Space between the lines represents 1 mm.

(10) FIG. 7 is a bright field microscopy image showing 10% PMMA fiber orientation in 3% gellan gum before shear (left), after uniaxial shear in two directions corner to corner (middle) and after uniaxial shear vertically. Scale bar is 50 microns.

(11) FIGS. 8A and 8B illustrate the gellan gum composition of high acylated gellan (FIG. 8A) and non acylated gellan (FIG. 8B).

(12) FIG. 9 illustrates the conversion of patient specific three dimensional model during the printing process into tissue engineered nasal graft from left to right. Space between the lines represents 1 mm.

(13) FIGS. 10A and 10B show the Fourier transform infrared spectroscopy (FTIR) results of several degrees of sulfation in polymer backbones of alginate (FIG. 10A) and gellan gum (FIG. 10B). Arrow in 1300 cm.sup.−1 marks the peak of sulfation. In higher degrees of sulfation in polymer the growth factor binding is increased leading to better delivery of molecules.

(14) FIGS. 11A-11D show the result of a rheological characterization of the bioink compositions with and without particles. Shear thinning was measured in rotation (FIG. 11A), shear recovery in oscillation after shear of 1 second (100.sup.−8 shear rate) for two cycles (FIG. 11B), Bioink alone was ionically crosslinked with several cation conditions (FIG. 11C), and maximum storage modulus G′ of the samples crosslinked for 30 minutes with 20 mM SrCl.sub.2 (FIG. 11D). Error bars represent standard deviation.

(15) FIGS. 12A-12D show the result of a determination of the tensile and swelling properties of the printed constructs. Tensile testing was performed on printed dumbbell specimens where the nozzle path is shown by the black lines and the printed structure is shown after swelling (FIG. 12A). Representative stress-strain curves where failure occurred in the central region of the specimen (FIG. 12B). Swelling behavior of the bioink compositions based on equation (2) and (3) to evaluate total water retention (FIG. 12C) and water retention after crosslinking (FIG. 12D) respectively. The smallest divisions on the ruler are 1 mm and error bars represent standard deviation.

(16) FIGS. 13A-13C show the result of a determination of cell viability of printed constructs and the cell proliferation assay. Viability after printing one layer thick discs was evaluated with live dead staining (FIGS. 13A and 13B) where 80% viability was observed 3h after printing, which recovered to 97% by day 4. To assess viability in a large structure, a young adult size nose was printed and the viability was evaluated from a central slice (diffusion distance ˜5 mm) evaluated by live dead staining. A cell viability of 60% was observed. Scale bar 5 mm (FIG. 13B, left), and 50 μm (FIG. 13B, right). Additionally, cell number in casted disks were evaluated with DNA quantification (FIG. 13C) where a statistically significant increase in DNA from day 1 to day 21 was observed with Bioink+Cartilage particles and both TGF-β3 supplemented compositions. Error bars represent standard deviation and level of significance was (p<0.05).

DETAILED DESCRIPTION

(17) Material

(18) The bioink material comprises at least one cytocompatible polymer and at least one of particles and cells, the crosslinking being provided by spontaneous or externally triggered reaction of reactive groups and molecules, at least one of these types being present on at least one of the polymer, minced tissue and cells. The cytocompatible polymers (hereinafter referred to as “the polymers”) for use in this method may be any suitable polymers with the necessary cytocompatibility, that is, their presence is not harmful to cells. They may be natural (biopolymers) or synthetic materials, or combinations of these. The necessary reactive groups allowing the crosslinking may be already present on the polymers, or the polymers may be modified to include such groups. Typical non-limiting examples of natural polymers include alginate, alginate sulfate, heparin, fibrin, heparin sulfate, elastin, tropoelastin, chondroitin sulfate, dermatan sulfate, hyaluronic acid, hyaluronan sulfate, cellulose, dextran, dextran sulfate, poly-l-lysine, chitosan, gelatin, gellan gum of varying acylation degree, gellan sulfate, guar gum, cassia gum, konjac gum, Arabic gum, ghatti gum, locust bean gum, xanthan gum, xanthan gum sulfate, carrageen, carrageen sulfate, silk and collagen of varying type. All sulfated versions of these polymers are included.

(19) Typical non-limiting examples of synthetic polymers include, but are not limited to, polymers, or polymers derived from, polyethylene glycol, polypropylene glycol, polaxomers, poly oxazolines, polyethylenimine, polyvinyl alcohol, polyvinyl acetate, polymethylvinyl ether-co-maleic anhydride, polylactide, poly N-isopropylacrylamide, polyglycolic acid, poly methylmethacrylate, polyacrylamide, polyacrylic acid, and polyallylamine.

(20) By “at least one” of the groups being present on at least one of the polymer, particles and cells, is meant that the added reactive groups may be present on all or any of these entities.

(21) Particles incorporated to polymer solution can consist of but are not limited to extracellular matrix tissue particles, loaded or unloaded beads and fibers in size range between 5-500 microns.

(22) Crosslinking

(23) The formation of hydrogel based on the material combination, particles and cells can be initiated by many factors or agents, including but not limited to mono-, di-, trivalent cations, enzymes and radical initiators. Additionally, physical and physical-chemical methods may be employed, for example, treatment in low or high pH solution and different temperature regions during the manufacturing process.

(24) In certain embodiments, either one of said printing mix and said polymer scaffold comprises reactive groups covalently attached thereto, particularly reactive groups facilitating linking of said printing mix, or its constituent components, to said particles, by crosslinking by spontaneous or externally triggered reaction, wherein reactive groups are present on at least one of the polymer, minced tissue and cells to reconstruct functional and native cartilage like tissue grafts.

(25) Particles

(26) The size of the minced tissue to be used may be any suitable size, but in a particular embodiment, it is from 5 microns-500 microns, so that it can be extruded without clogging the dispensing unit such as needle or valve. The minced tissue for use in the method may be any suitable tissue, but it is advantageously tissue of a similar or identical nature to that of the cartilage. Exemplary and non-limiting examples of suitable tissue include articular cartilage, nucleus pulposus, meniscus, trachea, nasal cartilage, rib cartilage, ear cartilage, synovial fluid, tracheal cartilage, vitreous humor, brain, liver, spinal cord, muscle, connective tissues and subcutaneous fat, intrapatellar fat pad, small intestinal submucosa. A particular example is tissue with high content of elastin and glycosaminoglycan, particular examples being any type of cartilage, nucleus pulposus and meniscus. The tissue may be minced by any suitable method, exemplary and non-limiting methods including homogenizing, cryomilling, dry milling, cutting, chopping, crushing and slicing. The tissue may be subject to decellularization to remove epitopes which can cause acute inflammatory responses and pathogens including HIV. Recently, decellularized tissues, that is, tissue in which the cells have been killed and their remnants removed, have attracted interest as scaffold material alternatives to simpler approaches where the scaffold is composed of a single material (Hoshiba et al. “Decellularized matrices for tissue engineering”. Expert Opinion on Biological Therapy. 2010; 10:1717-28). Tissue decellularization results in a scaffold of extracellular matrix ideally suited for regenerating injured or diseased tissue since it retains the high resolution architecture and biological cues necessary for recapitulation of function. Decellularization may be done, for example, by using detergents, hydrogen peroxide, sodium hydroxide and enzymes, RNase and DNase. Particles can be manufactured by methods such as but not limited to colloid formation by hydrophilic/hydrophobic interactions, two phase emulsions and in oil interfaces. Fibers can be manufactured by methods such as but not limited to electrospinning, fiber extrusion and fiber pulling. Particles and fibers of any kind may be minced by any suitable method, exemplary and non-limiting methods including homogenizing, cryomilling, dry milling, cutting, chopping, crushing and slicing. These additive tissue pieces, particles and fibers may be further modified with functional groups binding to carrier polymer or combination of these materials or treated to expose reactive groups for crosslinking. Furthermore growth factors, antioxidants and drug molecules may be loaded in or on the added polymers, tissue pieces, particles and fibers.

(27) Cells

(28) The use of the term “cells” in this description encompasses not only individual cells, particularly mammalian cells, more particularly human cells, most particularly autologous human cells, but also encompasses agglomerations of the described cells which form spheroids, pellets, and microtissues, which are well known to and commonly used by the art. The cells for use in the method are advantageously cells of a similar type as those present on the cartilage tissue. Typical non-limiting examples of suitable cell types include primary autologous chondrocytes, primary allogenic chondrocytes, chondroprogenitor cells, chondroblasts, mesenchymal stem cells, induced pluripotent stem cells and adipose-derived stem cells, neural crest derived stem cells.

(29) Printing Mix Material

(30) The term “printing mix” in the context of the present specification refers to an extruded mass comprising the key constituent components: Particles made of natural (optionally: dried) tissue or fibre, or made of biocompatible, optionally bioresorbable, polymer, or both polymer and natural tissue/fibre, An aqueous solution of a gelling polysaccharide, particularly gellan gum or a derivative thereof, and Mammalian cells.

(31) The composition of the printing mix material may be varied across a wide range, depending on the nature of the materials and the end-use. The polymers are typically present in a weight proportion of from 0.5-20%. When minced tissue, particles or fibers are present, they are typically present at a weight proportion of from 10-40% of dry polymers or equally 1-20% in total weight. When cells are present, they are typically used at concentrations of 3×10.sup.6 cells/ml-50×10.sup.6 cells/ml.

(32) In addition to the major components hereinabove described, the crosslinkable material may include other materials, present to confer particular properties on the material. One particular example is elastin, which is abundant in auricular and nasal ECM to provide the elasticity of the tissue and other examples include growth factors, cytokines, drugs, biologics, siRNA, DNA, antioxidants such as polyphenols into the polymeric solutions, which could augment regeneration of the tissues. Added growth factors could be bound to sulfated polymers or unmodified polymer for enhanced delivery and effectivity in the proximity of the cells residing in the printing mix.

(33) The printing mix material in its ready-to-use form is a readily thermally gelled state that can easily be applied to take desired shape in the manufacturing process. Powders of the molecules and lyophilized minced tissue, particles and fibers can be stored and sterilized separately. All the components can be combined before packaging or rehydrated just prior to use thus preserving the growth factors and proteins for long periods of time.

(34) Shape

(35) Patient specific tissue grafts are tailored for each patient or certain model catalogs can be created for situations where patient imaging is not desired or not possible. The three-dimensional model obtained from external ear and nose scans can be modified to contain internal support structures, gradient of polymers for versatile mechanical properties and porosity for enhanced cell survival in large constructs. Furthermore region could be tuned in terms of stiffness, growth factor cocktail and concentration, for example, to induce regional variations in cell proliferation. For example the periphery of the cartilage graft could be more porous or softer allowing more nutrient flow into the deep structure. Also the regional specificity and tissue types are found in these constructs, for example, in the lobe of the ear fat is the main tissue and is responsible for the mechanical properties. The regional properties and specified structures can be easily built in a layer-by-layer manner. In such a layered approach, the crosslinking mechanism would take place not only within individual layers, but also between adjacent layers, thus forming a completely integrated continuous structure. This can be achieved by initiating crosslinking in the periphery of the construct in contact with support structure containing the reactive molecule reservoir.

(36) Support

(37) Support structure can be co-deposited with the printing mix material to support overhanging structures, to initiate crosslinking or to prevent drying of the material during deposition. Support material can contain crosslinking factors including but not limited to mono-, di-, trivalent cations, enzymes and radical initiators. Additionally, physical and physical-chemical methods may be employed by support material interactions to modify the pH and molecule concentration. After the construct manufacturing the support structure can be eluted. Elution can be due to but not limited to temperature change, pH change or degrading molecules.

(38) The result is a cartilage repair that is quick, effective and long-lasting. The longevity is an important factor in the graft to preserve the mechanical properties until sufficient ECM production of the cells has been achieved to produce a native cartilage-like structure.

(39) Typical examples of the use to which the method of this disclosure may be put include: Reconstruction of craniofacial defects; Filling and reconstruction partial tissue loss and integrating them with native tissue; Reconstruction of trachea (windpipe), meniscus or costal cartilage with patient specific grafts; Filling of osteochondral defects

(40) The method of the invention is characterized by the following advantages: Possibility to produce patient specific tissue grafts for craniofacial and orthopaedic applications such as but not limiting to: ear, nose, articular cartilage. Possibility to tune the bending properties to match the scaffold with physiological parameters and specific regions of the native tissue. Possibility to include functional load bearing regions of more compact polymers and reinforced structures to tune the mechanical properties of the graft. Provides better patient satisfaction and decreased pain levels due to elimination of the need for cartilage harvest. Utilizes autologous, allogenic or xenogenic native tissue which already contains the complex array of tissue-specific extracellular matrix components in physiologically accurate proportions. These particles are mainly responsible of the proliferation cues stimulating the chondrocytes. Tissue fragments from any possible ECM particles can be incorporated into hydrogel blend for additive manufacturing purposes to produce any desired geometry without compromising its biochemical composition thus rising prospects for organ bioprinting. Possibility to incorporate therapeutic factors within the scaffold including, but not limited to: pharmaceutical compounds, growth factors, peptides, proteins, carbohydrates, and gene therapy vectors. Additionally, homing molecules can be included that would induce host cell migration into the scaffold. Possibility to achieve zonal organization of tissue architecture by layering various tissues/compositions using additive manufacturing techniques.

EXAMPLES

Example 1a: Bioprinting of Patient Specified Tissue Wafts

(41) Clinical computed tomography imaging was performed and the resulting computational three-dimensional object (FIG. 1) was obtained. The patient specific external ear model was then mirrored for the contralateral side and a new 3D model was generated. Together with the new model the external support structure model was generated to support the ear structure especially in the overhanging regions during the printing. Support structure was designed to be in contact with the ink in the strategically important places to initiate the crosslinking and to support the overhanging features (FIG. 5). The co-extrusion of the support material was shown to preserve horizontal bioink lines without sagging and the printed shape accurately after elution of the support. Furthermore the internal support structure of more dense polymers was prepared to allow better force distribution in internal structure (FIG. 2, 3). All models were converted into machine code in STL-converter (RegenHU) and transferred into the bioprinter (BioFactory, RegenHU) for the printing process.

(42) Using the same technique the inventors have demonstrated the printing of several cartilaginous structures including meniscus, intervertebral discs and nose. Two-component intervertebral disc grafts could be printed with two bioink compositions mimicking the nucleus pulposus and the annulus fibrosus.

Example 1b: Production of Cartilage Particles for Three-Dimensional Printing Purposes

(43) Cartilage was harvested from the fresh bovine articular or auricular cartilage by removing thin layers of cartilage into a petri dish containing PBS and penicillin-streptomycin 1%. The harvested cartilage was transferred into cryomill (Retsch) and milled for three cycles in 30 Hz intensity. Milled cartilage was collected and lyophilized to obtain dry powder that could be sieved into the desired particle size range. These particles can be further loaded with growth factors or other molecules to enhance the proliferation and other cell responses. After loading the particles were lyophilyzed and cryopreserved to maximize the biomolecule availability for prolonged shelf life.

Example 1c: Printing Mix Material Preparation and the Printings Process

(44) Printing mix material (“Bio-Ink”) was produced by combining gellan gum in 3.5% concentration with the alginate 3%. Gellan gum was dialyzed against ultrapure water to minimize the cation residues in the material. Dialysis was performed over three days in 70-80° C. ultrapure water changing the water one to two times a day. Gellan was further lyophilized to obtain a dry powder. Purified gellan gum was dissolved into glucose containing deionized water making it more cell compatible and alginate solution was added to obtain final concentration of polymers. The polymer blend was mixed with ECM particles and 6×10.sup.6 cells/ml to obtain the final printing mix material. This printing mix material stimulated the cell proliferation significantly compared to positive control (FIG. 4). Cartilage extracellular matrix production was evaluated with histology and immunostaining after 8 weeks in culture for Bioink alone and Bioink+ECM with and without growth factor TGF-β3. The Bioink+ECM without growth factors stimulated cell proliferation above Bioink alone which was clearly visible in H&E staining. The Bioink+Cartilage particles showed a slight increase in Alcian blue staining and a slight collagen II staining was observed suggesting the need for additional growth factor stimulation. Cells were often seen proliferating around the particles without growth factor whereas the Bioink+Cartilage particles with TGF-β3 had no site-specific proliferation which suggests that the particles are a source of mitogenic growth factors. After 8 weeks, the gross appearance of the scaffolds suggested that the growth factor stimulation had a clear effect on cartilage matrix production as seen in the size and opaque appearance of TGF-β3 supplemented samples. Both supplemented bioink compositions showed a significant increase in cartilage ECM components and had areas which began to resemble the cell density and GAG content of native cartilage. Furthermore, collagen II deposition was strong throughout the graft in the growth factor supplemented conditions while only pericellular staining was seen in the samples cultured without TGF-β3. Collagen type I and alizarin red staining were performed to determine the fibrocartilage production and calcification. Collagen I was found in Bioink+Cartilage particles and in both TGF-β3 supplemented conditions suggesting some fibrocartilage production, perhaps due to the passaging of the cells. In all the conditions calcification was absent suggesting the cartilage phenotype of the chondrocytes was stable.

(45) The printing mix material was printed onto a substrate and the support polymer Pluronic F127 was co-extruded to fill subsequent layer. Pluronic contained 20 mM of SrCl.sub.2 to initiate the bioink crosslinking upon contact with the ink. Cations diffused into the printing mix material due to osmotic balance and electrostatic forces which initiated the crosslinking. Structures were generated with 410 μm needles and 800 mm/minute feedrate. Pressure applied to extrusion syringe varied between 1.2-1.4 bar. After layer-by-layer deposition of the material into desired form, the sacrificial support Pluronic was eluted in a 20 mM SrCl.sub.2 bath for few minutes before the construct was transferred to 37° C. cell culture medium. FIG. 1 illustrates the ear cartilage internal support structure and FIG. 6 shows the nose grafts generated by this technique. FIG. 2 illustrates the initial storage modulus being 100 kPa after crosslinking which is comparable to high stiffness hydrogels.

Example 2: Bioink Composition Optimized for its Mechanical Properties and Growth Factor Retention

(46) Bioink preparation: Gellan was added to D-glucose (300 mM) containing ultra-pure water at 90° C. to achieve a 3.5% solution, of which 85% was low-acyl gellan gum and 25% was high-acyl gellan gum. Alginate was added to the mixture to achieve 2.5% solution. The boiling flask was kept at 90° C. with agitation until the solution was homogeneous, typically for one hour. The homogeneous solution was cooled down to 30° C. prior the cell mixing. Briefly, the bovine chondrocytes (4×10.sup.6 cells/a) were mixed in the DMEM solution and added to the bioink in culture medium in 1:10 volume ratio to pre-crosslink the bioink. Mixing was performed until the solution reached room temperature and the printing syringes were loaded.

(47) Gellan gum high acyl (GG-HA) and low acyl (GG-LA) compositions (FIG. 8) contribute to the stiffness and the elasticity of the final bioink. By varying the ratio between the acylation forms the materials can crosslink tighter yielding stiffer matrix whereas by disrupting the tight packing of the polymer chains in crosslinking more elastic matrix can be produced. These parameters were optimal for craniofacial applications in 85% GG-LA, 25% GG-HA composition which provides tunable crosslinking properties up to 230 kPa ultimate stress and an average strain of 68% at failure (FIG. 3). To further optimize the growth factor retention in the bioink a concentration of 2% of sulfated gellan gum (GG-3%) (FIG. 8) was added to the bioink. This composition was superior in retaining the loaded growth factors, in this case TGF-β3 and FGF-2, in the bioink compared to the non-sulfated bioink.

Example 3: Optional Printing Material and Crosslinking Process with Support Material

(48) Base polymer gellan 3% with additive hyaluronan conjugated with tyramine 3% were mixed together to generate enzymatically crosslinkable hydrogel in the presence of horseradisch peroxidase (HRP) and hydrogen peroxide. Materials were dissolved into deionized water in presence of monosaccharide glucose in physiologic osmolarity, specifically 300 mM. Hydroxyapatite particles in concentration of 4% (w/v) were added to the polymer mixture. This bio-ink composition was further bioprinted in the presence of HRP and hydrogen peroxide when HRP was mixed either to the bio-ink in 1 unit/ml concentration or to the pluronic F127 30% mixture together with the hydrogen peroxide in 0.0012% concentration. Layer-by-layer constructed scaffold crosslinked immediately upon contact with the support structure. The support structure was eluted in the cold medium to decrease the negative effects of hydrogen peroxide in the presence of the cells. The structure was washed several times after to minimize the amount of hydrogen peroxide residues.

Example 4: Fiber Reinforced Materials for Bioprinting

(49) Gellan 3% was dissolved in deionized water and 10% (w/v) polymethyl metacrylate (PMMA) fibers were added as chopped eletrospun fibers into the gellan solution. Fibers were imaged with scanning electron microscopy to determine the fiber diameter to be approximately 2 micron. The fiber reinforced gellan was imaged before the shear (FIG. 7 left) and after two different shear orientation for 2 minutes (FIG. 7 middle and right). Fiber orientation was greatly increased in uniaxially shear already after 2 minutes. Furthermore, the uniaxial but not unidirectional shear oriented the fiber shorter than 50 microns to shear direction, thus allowing us to form heterogeneous load bearing structures in the matrix. However, fibers longer than 50 microns were not able to keep the orientation in shear when direction of shear was altered. During the extrusion printing the shear pattern is uniaxial and unidirectional in the nozzle, thus orientating the fibers to flow direction. Upon fast cessation of flow we can keep the fiber orientation uniaxial which will affect the load bearing capability of the structures. These structures are nature inspired and for example collagen II fibers in articular cartilage are changing the orientation in different cartilage layers.

Example 4a Bioink Crosslinking

(50) An exemplary bioink is a blend of gellan and alginate mixed with human micronized Cartilage particles or HA particles (≤40 μm size). Upon addition of mono-, di- or trivalent cations, gelation (sol-gel transition) occurs as the helices aggregate into junction zones which are linked into a three dimensional network via the coiled part of the molecule. The printing process is divided into three stages namely bioink pre-printing, printing process and post-printing crosslinking. Initially the bioink was loaded into a syringe and the support polymer into a second syringe. At this stage, a small amount of cations were present in the bioink to increase viscosity and enhance printing properties. During the printing process the co-extruded of the support, cations diffused to the periphery of the printed structures to initiate the crosslinking. After the final structure is completed, the support can be eluted in 4° C. cation-supplemented medium.

Example 4b: Rheological Analysis

(51) The cation related viscosity enhancement and crosslinking properties can be investigated with rheology and mechanical testing. Rheological properties of the Bioink, Bioink+HA, and Bioink+Cartilage Particles were measured with an Anton Paar MCR 301 (Anton Paar, Zofingen, Switzerland) rheometer to determine the shear behavior and shear recovery. All of the bioink compositions showed shear thinning behavior which is critical for extrusion (FIG. 11a). Furthermore, all the compositions had a yield point (weak gel formation) prior to extrusion which is important in preventing particle and cell sedimentation in the syringe (Table 1).

(52) TABLE-US-00001 TABLE 1 Summary of the rheological measurements. The yield points were calculated using the Herschel/Bulkley equation. Bioink + Cartilage Bioink Bioink + HA Particles Yield point 15.6 Pa ± 17.7 Pa ± 122 Pa ± 0.7 Pa 6.5 Pa 22 Pa Cessation 21% 90% 98% in 10 s* Maximum 152 kPa ± 110 kPa ± 96 kPa ± G′ 3.0 kPa 2.0 kPa 1.0 kPa *Shear recovery at 10 s after the 2nd shear sequence.

(53) The shear recovery curves (FIG. 11b) illustrate the recovery of the bioink structure after the printing process. Shear recovery after the second shear sequence was 98% in Bioink+Cartilage Particles and 90% in Bioink+HA after ten seconds. At the same time the Bioink alone recovered to only 21% of the original modulus. FIG. 11c illustrates the storage modulus G′ after cation-induced crosslinking of Bioink alone where the cation concentration and source had a clear influence. FIG. 11d illustrates the final storage modulus for the three bioink compositions. The Bioink alone had the highest final storage modulus (152 kPa±3 kPa) compared to Bioink+Cartilage Particles (96 kPa±1 kPa) and Bioink+HA (110 kPa±2 kPa), suggesting that crosslinking is somewhat hindered by the particles irrespective of their source.

Example 4c: Mechanical Properties and Swelling Behavior

(54) Mechanical properties of the bioprinted cartilaginous structures were assessed in tension. Tensile dumbbell specimens were printed using Bioink+HA particles with or without cells. The nozzle path (printing direction) in the gage section of the specimen was chosen to be parallel to the direction of tension (FIG. 12a). Young's modulus was significantly higher in acellular constructs (E=230 kPa±7.0 kPa) compared to cellular ones (E=116 kPa±6.8 kPa) (p<0.001), suggesting that the cells increase the compliance of the construct and/or inhibit the crosslinking. There was no difference in failure strain between the acellular (37%±6.4%) and cellular (34%±2.1%) (p=0.54) constructs.

(55) Swelling of the bioink with and without particles was quantified to assess the total water retention and the water retention after gel crosslinking (FIG. 12c-d). Swelling at 37° C. up to 48 hours increased the hydrogel weight between 2000-3800% of the dry weight of the sample which is typical of hydrogels and between 26% and 54% of the crosslinking weight of the hydrogels. Fully hydrated state was achieved after 24 hours and in more specific the Bioink and Bioink+Cartilage particles were fully hydrated after 5 hours suggesting faster swelling kinetics. Comparison between swelling ratios of the Bioink alone and the particle containing compositions after 48 hours suggested dependency on the particle type.

Example 4d: Bioink Compatibility

(56) Cellular bioprinting process was investigated with Bioink+HA to exclude all the interactions and proliferation cues between particles and cells. One layer thick discs were printed to assess the cell viability after printing (FIG. 13a) which was compared to the initial viability of the cells prior to mixing. To investigate cell viability in large structures, a young adult sized nose (3.1 cm, 2.6 cm and 1.5 cm) was printed and kept in static culture until the cell viability in the middle of the construct was evaluated from a central slice (minimum diffusion distance of 5 mm). Bioprinting with the particles showed an 80% viability three hours after printing, however, after four days the cell viability recovered to 97% where it remained until the end of the experiment. The young adult sized nose graft had decreased viability in the center of the scaffold (60% viable cells at day 7) compared to 96% viability in the periphery (FIG. 13b). This suggests the need for incorporating internal porosity or channels to enhance nutrition transport. By introducing interconnected porosity into 1.5 cm high cubes the viability in the center of the structure was as high as in the periphery. Such nutrition channels .sup.[or engineered porosity can be incorporated into the bioprinted structures by extruding the support polymer within the grafts, which could later be cleared in subsequent washing/crosslinking steps. With this technique a complex 3D interconnected porous network can be created that is used to perfuse the grafts with nutrient-rich medium. To further enhance mass transport of nutrients, grafts can also be pre-conditioned in dynamic bioreactors.

(57) The effect of cartilage particles and growth factor, in this case TGF-β3, supplementation on cell proliferation was evaluated in casted gels cultured for 21 days. The Bioink alone did not stimulate cell proliferation; in fact there was a loss in DNA at day 7 which slowly recovered. Bioink+Cartilage particles, on the other hand, stimulated proliferation and caused a statistically significant increase (p<0.001) in DNA over 21 days. With TGF-β3 supplementation, there was a statistically significant increase in DNA in the Cartilage particles containing samples at day 7 (p<0.001). By day 21, both bioinks showed increases in DNA, which were not statistically significantly from each other.

Example 4e: Extracellular Matrix Production and Cartilage Formation

(58) Cartilage extracellular matrix production was evaluated in bioink alone and bioink+Cartilage particles with histology and immunostaining after 3 and 8 weeks in culture. Histological evaluation after 3 weeks revealed a clear increase in cell number, GAG synthesis and collagen II production in both bioink compositions supplemented with TGF-β3 (10 ng/ml). Furthermore, Bioink+Cartilage particles without growth factors stimulated cell proliferation above Bioink alone which was clearly visible with 3 and 8 week H&E staining. At both time points the Bioink+Cartilage particles showed a slight increase in Alcian blue staining and at the 8 week time point a slight collagen II staining was observed suggesting the need for additional growth factor stimulation. Cells were often seen proliferating around the particles without the growth factor supplementation which suggests that cell-particle adhesion and/or growth factors in the particles are important. However, because in the Bioink+Cartilage particles with TGF-β3 samples, no site-specific proliferation was observed, the results suggest rather the particles are a source of mitogenic growth factors and not specific cell-matrix adhesive cues. After 8 weeks, the gross appearance of the scaffolds suggested growth factor stimulation had a clear effect on cartilage matrix production as opaque appearance and increase in size was observed. At 8 weeks, both supplemented bioink compositions showed a significant increase in cartilage ECM components and had areas which began to resemble the cell density and GAG content of native cartilage. Furthermore, collagen II deposition was strong throughout the graft in the growth factor supplemented conditions while only pericellular staining was seen in the samples cultured without TGF-β3. Collagen I was found in Bioink+Cartilage particles and in both TGF-β3 supplemented conditions suggesting some fibrocartilage production, perhaps due to the passaging of the cells. In all the conditions calcification was absent suggesting the cartilage phenotype of the chondrocytes was stable.

Example 4f: Magnetic Resonance Imaging

(59) To assess the shape retention of the printed structures several MRI techniques were evaluated. The printed nose was kept in PBS for 2 weeks to assure complete swelling prior T2-weighted MR imaging. These images were thresholded and converted into a .STL file and compared to the original model used for printing and to the cartilaginous graft immediately after printing. Comparison of the original model and the printed graft illustrates precise material extrusion and detailed structures. However, slightly thicker nostril walls were observed in comparison to the original model. Furthermore, when comparing the printed structure to the MRI model after 2 weeks swelling, a slight thickening of the nostril walls were observed, however, no sign of degradation or deterioration of the shape was detected.

Example 5: Bioprinting Process Parameters

(60) One important factor of the reproducible printing process is the connectivity of the consecutive lines. In order to assess the effect of line spacing an optimization of line thickness must be conducted. Printing parameters such as pressure, feed rate and needle diameter were tested to standardize the line thickness to 900 μm±53 μm. After the determination of the average line thickness the effective line-line adhesion was investigated by printing a series of tensile testing dumbbells having different line spacing. The dumbbells were tensile tested until failure and the data illustrates that by increasing the line spacing the possibility of defects in the structure increased suggesting that in order to provide reproducible mechanical properties for printed structures the lines should overlap approximately 40-50%. The data suggested that the variance of the ultimate stress at failure did not differ in the tested samples with amount of overlapping lines down to 20% whereas the number of samples that were not stabile enough for testing increased with increasing line spacing. According to the data the optimal line spacing is affected by the bioink in question however by increasing the overlapping the probability of internal printing process related defects decreases. Furthermore the line thickness can be freely chosen by changing the process parameters such as pressure, printing speed and needle diameter.

(61) Several mechanical testing measurements were performed for the newly designed bioink to investigate the parameters affecting the reproducibility of the structural and mechanical properties. The tensile evaluation of specimens printed with varying printing directions and with cell laden bioink revealed that the youngs modulus, ultimate stress and the failure strain are not altered by adding of the cells in the seeding density of 4×10.sup.6 illustrating that the volume fraction of cells (˜1% approx.) is compensated by the strong surrounding matrix. Furthermore, dumbbell specimens were printed in varying printing directions with respect to the tension, namely parallel to tension (0°), perpendicular to tension(90°) and in 45° angle to the tension (45°). The printing direction did not show any statistically significant differences between the groups suggesting that the bioprinted structures can be designed based on the printing and process related parameters rather than based on the estimated mechanical loading of the final structures.