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WO2018053565A1 - Appareil et procédé de biofabrication manuelle de forme libre - Google Patents

Appareil et procédé de biofabrication manuelle de forme libre Download PDF

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Publication number
WO2018053565A1
WO2018053565A1 PCT/AU2016/050886 AU2016050886W WO2018053565A1 WO 2018053565 A1 WO2018053565 A1 WO 2018053565A1 AU 2016050886 W AU2016050886 W AU 2016050886W WO 2018053565 A1 WO2018053565 A1 WO 2018053565A1
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WO
WIPO (PCT)
Prior art keywords
bioink
extrusion
nozzle
chambers
cells
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Application number
PCT/AU2016/050886
Other languages
English (en)
Inventor
Peter CHOONG
Gordon George Wallace
Claudia DI BELLA
Stephen Thomas Beirne
Christopher John RICHARDS
Adam Christopher TAYLOR
Zhilian Yue
Fletcher William THOMPSON
Original Assignee
St Vincent's Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by St Vincent's Hospital filed Critical St Vincent's Hospital
Priority to PCT/AU2016/050886 priority Critical patent/WO2018053565A1/fr
Publication of WO2018053565A1 publication Critical patent/WO2018053565A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/16—Macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/222—Gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A61L2300/414—Growth factors

Definitions

  • the present invention relates to apparatus and methods for handheld, free-form biofabrication.
  • Osteoarthritis is a leading cause of disability in developed countries and in the United States it is second only to cardiovascular disease as the most common cause of disability, affecting 80% of people over the age of 65.
  • Early physical symptoms such as the development of chondral lesions on the articular surface of the knee, are typically not detected by the individual as articular cartilage is aneural and these defects cannot self- repair.
  • Current available treatments for chondral defects include autologous chondrocyte implantation, mosaicplasty, and microfracture. While healing of the repair has been documented, an inferior tissue, fibrocartilage, is the legacy of this treatment. To date, no clinical strategies are as yet able to consistently reproduce normal hyaline cartilage which has the cellular and mechanical characteristics to sustain the everyday demands of shear and compression.
  • tissue engineering has been explored as a potentially viable alternative to treat chondral injuries, for example through the implantation of prefabricated bioprinted biomaterial constructs to fill in the defect and encourage native tissue formation.
  • Bioprinting techniques involving the creation of organised 3D tissue constructs through layer-by-layer assembly, have come to the fore given the depth- dependent composition of the cartilage structure to be recapitulated.
  • a major advantage of the bioprinting approach is the ability to tailor implants to the anatomy of the defect and/or specific lesion by using medical imaging data to inform implant design.
  • surgical approaches to chondral injury repair nominally require an initial debridement step to remove excess fibrous tissue around the defect. This means the size and shape of the final defect to be filled may not be accurately known prior to surgery.
  • the resulting graft/host mismatch of the laboratory prefabricated scaffold may be one of the reasons that might impede good integration of the scaffold.
  • a device for free-form biofabrication comprising: a plurality of extrusion chambers that are hand-holdable and individually actuatable; an extrusion nozzle having a plurality of openings that are closely adjacent one another and respectively fluidly connected to the plurality of extrusion chambers; wherein one of the extrusion chambers contains a bioink that comprises living cells which, in use, are controllably deliverable in situ with a structural biomaterial to form a biofabricated structure.
  • the nozzle openings may be separate from each other such that no mixing of content between chambers occurs prior to extrusion.
  • the nozzle openings may be in a collinear side-by-side arrangement.
  • One nozzle opening may be positioned within another opening in a co-axial arrangement.
  • a largest width of each nozzle opening at a tip of the nozzle may be between about 0.1 mm and 1 .5 mm.
  • the device may further comprise a chassis configured to removably receive the chambers and/or the extrusion nozzle.
  • the device may further comprise a radiation source for curing extrudate.
  • Independent extrusion from each chamber may be pneumatically or mechanically actuated.
  • One chamber may comprise a structural biomaterial, one or more growth factors, a photoinitiator, a curing agent, a biocompatible adhesive, a biocompatible sealant, or combinations thereof
  • a maximum shear stress applied to the bioink during extrusion through the nozzle is preferably insufficient to adversely affect the viability of the living cells.
  • the bioink may be a shear-thinning fluid, such that the viscosity of the bioink decreases as the bioink is extruded through the nozzle.
  • the bioink may comprise a biocompatible hydrogel of GelMa and HAMa.
  • the bioink may comprise a biocompatible hydrogel of about 10 wt% GelMa and about 2 wt% HAMa.
  • the bioink may comprise TGFp3, BMP6, hEGF, hFGF-2, or combinations thereof.
  • the bioink may comprise about about 0.5 wt% 2,2'-Azobis [2-methyl-N-(2 hydro-xyethyl) propionamide] (VA-086).
  • the bioink may comprise adipose stem cells at a concentration of at least about 2 10 6 cells/ml.
  • a bioink for biofabrication comprising a biocompatible hydrogel comprising gelatin- methacrylamide (GelMa) and hyaluronic acid-methacrylate (HAMa).
  • the hydrogel may comprise between about 5 wt% and about 20 wt% GelMa, or between about 7.5 wt% and about 17.5 wt% GelMa, or between about 10% and about 15% GelMa.
  • the hydrogel may comprise between about 1 wt% and about 4 wt% HAMa, or between about 1 .5 wt% and about 3.5 wt% HAMa, or between about 2 wt% and about 3 wt% HAMa.
  • the hydrogel comprises about 10 wt% GelMa and about 2 wt% HAMa.
  • the bioink may further comprise a photoinitiator.
  • the photoinitiator may be VA- 086.
  • the bioink comprises less than about 1 .5 wt% VA-086, or less than about 1 wt% VA-086, or less than about 0.5 wt% VA-086, or less than about 0.25 wt% VA-086.
  • the bioink may further comprise adipose stem cells.
  • the stem cells may comprise human infrapatellar fat pad derived stem cells.
  • the bioink may comprise stem cells at a concentration of at least about 2 10 6 cells/ml, or at least about 4 10 6 cells/ml, or at least about 6 ⁇ 10 6 cells/ml, or at least about 10 ⁇ 10 6 cells/ml, or at least about 12 ⁇ 10 6 cells/ml.
  • the bioink may be deposited using the device described.
  • a biofabricated structure formed using the device described and/or the method described and/or the bioink described.
  • the biofabricated structure may have free-formed compositional variations and/or gradients in two-dimensions and/or three-dimensions.
  • Figure 1 is a perspective view of a device for free-form biofabrication according to an embodiment
  • Figure 2 is a photograph of the device during handheld operation
  • Figure 3 is a schematic illustration of the device
  • Figure 4 is a cross-sectional view of a nozzle for the device according to an embodiment
  • Figure 5 is a cross-sectional view across the tip of the nozzle
  • Figure 6 is a cross-sectional view across the tip of a nozzle according to another embodiment
  • Figure 7 shows results from a cell viability test for cells in a bioink according to an embodiment and printed using the device of an embodiment
  • Figure 8a and 8b show the shear-thinning behaviour and temperature-dependent viscosity of a bioink according to an embodiment
  • FIGS. 9a to 9d show the ultraviolet (UV) absorbance and UV-dependent rheology of components of a bioink according to an embodiment
  • FIGS. 10a to 10d illustrate the pressure-dependent extrusion performance of the device
  • Figures 1 1 a to 1 1 c illustrate the pressure-dependence of extruded lines deposited by the device
  • Figures 12a to 12e demonstrate various printing capabilities of the device
  • Figures 13a to 13d illustrate the in vivo test procedure of Example 4.
  • Figures 14a and 14b illustrate macroscopic test results from Example 4.
  • FIGS 15a to 15e illustrate biomechanical test results from Example 4.
  • Figures 16a and 16b illustrate histological assessment results from Example 4.
  • a device 10 for free-form biofabrication comprises two chambers 2a, 2b, each ending in an extrusion nozzle 4. Deposition or extrusion of content from each chamber is independently actuatable or controllable by the user, ie the user can extrude from chamber 2a or chamber 2b or both chambers simultaneously.
  • Each chamber 2a, 2b is in fluid connection with a separate nozzle opening 6a, 6b such that no mixing of content between the chambers occurs prior to extrusion.
  • At least one of the chambers 2a, 2b contains a bioink comprising living cells for free-form biofabrication.
  • the device 10 is sized and configured to fit in a user's hand for handheld operation. The small form factor of the device 10 allows for an ergonomic fit to the user's hand while the lightweight body maximises printing control.
  • the nozzle openings 6a, 6b may be arranged in a collinear, side-by-side arrangement.
  • Collinear deposition together with independent extrusion from each chamber, allows for the creation of compositional gradients (and therefore functional gradients) in two-dimensions, ie within a layer, and/or in three- dimensions, ie between layers, when each chamber is filled with a different material or formulation. This permits the delivery of different layers and cell types making it ideally placed to tackle cyto-matriceal variations often found in complex tissues such as cartilage.
  • the separate nozzle openings 6a, 6b maintain separation of the contents of each chamber while in the device 10 and prior to extrusion, but are preferably arranged closely adjacent each other (at least at the tip 8 of the nozzle) such that the deposited materials converge, contact and/or mix with each other.
  • the largest width of each of the collinear nozzle openings 6a, 6b at the tip 8 of the nozzle is between about 0.7 mm and 1 .0 mm.
  • the cross-sectional geometry of each nozzle opening 6a, 6b within circular nozzle 4 is semi-circular, however other alternative shapes may be provided, eg circular, elliptical, etc.
  • the nozzle is preferably made of a material capable of manufacture to high tolerances, such as a metal or metal alloy.
  • the nozzle is three- dimensionally (3D) printed in titanium 6AI4V alloy. The small size of the nozzle tip allows the user to print directly within small spaces in fine resolution, for example, directly on or inside a wound.
  • Figure 6 illustrates a nozzle 4 with co-axial nozzle openings 6a, 6b.
  • the coaxial arrangement may advantageously allow for deposition of an outer 'shell' structure that can better confine cells encapsulated in an inner 'core' structure.
  • the diameter of the inner opening 6a at the tip 8 of the nozzle is between about 0.2 and 0.4 mm, and the diameter of the outer opening 6b at the tip 8 of the nozzle is between about 0.8 and 1 .1 mm.
  • the device 10 may comprise modular components to allow for facile and rapid switching of materials, nozzle designs, printing geometries, etc.
  • the nozzle 4 and/or the chambers 2a, 2b are selectively removable and interchangeable.
  • the components may be removed for sterilisation, or may be disposed and replaced with sterile components after each use.
  • This feature, together with its small form factor allows the device 10 to be easily brought into and out of the surgical field. Further, this allows for the ability to 3D print multiple layers in substantially real-time using different biomaterials and/or cells to reconstitute different tissues, simply by swapping the chambers.
  • the device 10 may comprise a chassis 12 that is configured to removably receive the chambers 2a, 2b and/or the deposition nozzle 4.
  • the size of the chassis 12 is similar to a pen, and is therefore configured to fit in a user's hand for handheld operation in a direct-write fashion.
  • the chassis 12 is 3D printed in a thermoplastic polymer such as acrylonitrile butadiene styrene (ABS).
  • ABS acrylonitrile butadiene styrene
  • the modular components may be detachably mounted to each other via any suitable fastening means, eg snap fit, friction fit, screw threads, bayonet mounts, etc.
  • the device 10 may comprise a source of radiation 14, such as a UV lamp or a light emitting diode (LED), for photocuring of a photopolymer deposited by the device.
  • a source of radiation 14 such as a UV lamp or a light emitting diode (LED)
  • UV LED 14 is directed towards the extruder nozzle 4, such that the focal distance of the LED lens substantially coincides with the tip 8 of the nozzle, and the UV beam may be focused on the extrudate 20 during or after deposition from the nozzle.
  • the user activates the radiation source 14 using a foot pedal.
  • Figure 2 shows the process of handheld printing and simultaneous photocuring.
  • the radiation source 14 may be movable or rotatable on the device, to allow the user to adjust the distance and/or angle of radiation.
  • extrudate may be cured by reacting with a curing agent.
  • the control afforded by the collinear deposition system is especially amenable to reactive printing mechanisms whereby a curing agent (such as divalent cations for ionic crosslinking) is delivered along with the bioink to cure it in situ.
  • the curing agent may be provided in one chamber, such that it is not mixed with the bioink in the other chamber until both are selectively extruded.
  • independent extrusion from each chamber 2a, 2b may be pneumatically actuated.
  • the user can extrude from chamber 2a or chamber 2b or both chambers simultaneously.
  • the extrusion pressure and rate may be controlled via pneumatic regulator 16.
  • Sensors such as flowmeters or rotameters may additionally be provided to monitor and regulate extrusion flow rate.
  • Extrusion from each chamber may alternatively be actuated via a mechanical mechanism, eg piston or screw-based extruders.
  • Mechanical extrusion may allow for a constant and controllable volumetric dispensation irrespective of temperature fluctuations in the chambers 2a, 2b and the resultant change in viscosity of the content of the chambers.
  • the device 10 may comprise a thermally controlled chassis 12, eg thermally insulated, to reduce or control heat transfer from the user's hand to the contents of the chambers during operation.
  • the ability to selectively deposit multiple materials opens up a range of strategies for biofabrication of cartilage and other tissues.
  • the two chambers 2a, 2b could be loaded with two different materials (e.g. different stiffness) to help build up the required 3D gradient of mechanical properties;
  • the two chambers 2a, 2b could be loaded with cell-laden and non-cell-laden bioinks to enable control of cell distribution within a construct;
  • loading one or other chamber with morphogens such as growth factors could allow 2D and 3D gradients to be set up which could influence depth- dependent differentiation and phenotype of bioprinted cells or tissues,
  • one chamber could contain a curing agent that is selectively deposited to cure the extrudate from the other chamber.
  • the device 10 may be used to build up high mm to cm scale 3D structures, with compositional gradients in 3D.
  • the device 10 may be combined with image guided or robotic surgical systems to allow precise deposition of biomaterials, for example in keyhole surgery, where the degree of biomaterial extrusion from the nozzle can be assisted or controlled by intraoperative detection devices. Detection devices can be incorporated into the hand-held device 10 and the data fed into image guided computers available intraopertively.
  • the bioink contained in at least one of the chambers 2a, 2b comprises a biocompatible matrix that supports cellular activity, in order to allow for biofabrication of biological structures using the device 10.
  • the matrix may be biologically-derived or artificial.
  • the matrix is a hydrogel.
  • a range of requirements must be considered when selecting the matrix components, including compressive modulus, bonding to the osteochondral interface, cell viability and proliferation, hydrogel pore-size and diffusivity, degradability and the capability for chondrocytes to remodel the environment with native cartilage components.
  • the chosen bioink must also serve the rheological constrains of printing.
  • the bioink may comprise collagen II, which is the main protein constituent of articular cartilage.
  • the bioink may comprise gelatin (hydrolysed collagen) which retains abundant Arg-Gly-Asp (RGD) sequence motifs, cell attachment sites recognised by many integrins. RGD motifs have been shown to promote cell adhesion, proliferation and stem cell differentiation.
  • the addition of photocrosslinkable methacrylate groups to gelatin to make GelMa may result in a hydrogel which retains some advantages of its collagen precursor while being amenable to bioprinting.
  • the bioink may further comprise hyaluronic acid (HA), which increases the viscosity of the hydrogel and may improve printability of the bioink.
  • HA hyaluronic acid
  • HA is additionally a key component of articular cartilage. It functions as a core molecule for binding keratin sulfate and chondroitin sulfate in forming aggrecan and contains binding sites for chondrocytes via the CD44 receptor.
  • HA hydrogels have been extensively studied as promising materials for the bioscaffold component of engineered articular cartilage, and have been shown to support the differentiation of mesenchymal stem cells towards a chondrocytic lineage in vivo.
  • the addition of HAMa to GelMa hydrogels has been shown to increase the mechanical properties of tissue engineered cartilage constructs after eight weeks in culture.
  • the bioink may comprise a GelMa/HAMa hydrogel.
  • the bioink may be seeded with stem cells, to enable growth and formation of biofabricated structures in situ.
  • the stem cells may be the patient's own stems cells to allow recapitulation of the population of cells that would normally inhabit that location and which is able to ultimately impart the normal functional and physical characteristics of that part of the tissue.
  • adipose stem cells are an attractive cell source for autologous implantation owing to their ease of harvest and abundance. When cultured with appropriate factors, they have been shown to undergo chondrogenesis in hydrogel scaffolds, producing collagen type II and aggrecan. Infrapatellar fat pad derived stem cells in particular have also been shown to undergo chondrogenesis.
  • the bioink may comprise human primary adipose stem cells, eg derived from human infrapatellar fat pad.
  • the concentration of cells in the bioink may be in the order of 10 x 106 cells/ml, which is similar to the density of chondrocytes in human articular cartilage.
  • the concentration of stem cells in the bioink is at least about 2 x 106 cells/ml.
  • the inclusion of living cells in the bioink places additional requirements and constraints on the device 10. For example, the extrusion pressure may be controlled or restricted to ensure that the maximum shear stress applied to the bioink during extrusion through the nozzle 4 is insufficient to adversely affect the viability of the living cells.
  • the material properties of the bioink is preferably selected by considering both printability and cell viability, eg a bioink that exhibits shear-thinning behaviour may reduce or eliminate damaging shear stresses at the extrusion point. Further, the type of radiation used for curing the bioink is selected to ensure the living cells are not denatured in the process.
  • the bioink may further comprise one or more morphogens such as growth factors.
  • the bioink may comprise ⁇ 3, BMP6, hEGF, hFGF-2, or combinations thereof. Direct extrusion control of the growth factors allow for 2D and 3D gradients to be set up within the printed structure, which could influence depth- dependent differentiation and phenotype of the biofabricated cells or tissues.
  • the bioink may further comprise a photoinitiator to induce crosslinking upon radiation.
  • the photoinitiator VA-086 may provide non-cytotoxic free- radicals.
  • the concentration of the photoinitiator VA-086 is preferably selected to reduce the formation of nitrogen gas bubbles produced during cross-linking, while still ensuring a sufficient rate of cross-linking.
  • the present invention also relates to methods for performing handheld free-form biofabrication on and/or in a wound site during surgery using the device 10. Specifically, the chambers 2a, 2b are filled with the materials required for the particular surgical application.
  • the user or surgeon then selectively deposits the contents of the chambers 2a, 2b in a direct-write fashion on and/or into the wound or defect by independently controlling the extrusion of content from each chamber.
  • the user may simultaneously or subsequently activate radiation source 14 in order to cure the extrudate in situ.
  • the user controls extrusion and/or the radiation source 14 via foot pedals.
  • the device 10 allows the user to deposit the bioink in a freeform manner.
  • the user is able to modify various parameters of the extrusion on the fly, such as the rate of extrusion, thickness of the extrudate, ratio of extrusion from each chamber, the shape of the printed structure, thickness and number of layers, amount of UV curing, etc.
  • the device 10 facilitates a method for controlling the geometry, compositional gradients, and material properties of a free-formed biofabricated structure in two-dimensions and/or three-dimensions by independently controlling extrusion from each chamber of the device.
  • the device 10 may be dismantled, eg by removing the nozzle 4 and/or chambers 2a, 2b for sterilisation or replacement.
  • the device 10 may be used for promoting chondrogenesis of a chondral defect by applying the bioink on and/or in the chondral defect.
  • the present invention also encompasses biological structures which are biofabricated using the device 10, the free-form printing method as described, and/or the bioink as described.
  • biological structures include the structures printed (eg during surgery), and the biofabricated structures grown/formed (eg in vivo) from the printed structures.
  • Bioink was formulated from a HAMa/GelMa hydrogel, photoinitiator VA-086 and adipose stem cells. The bioink was printed and cultured to determine cell viability.
  • HA 0.5g 1200-1900 kDa
  • sterile deionised water 120 ml
  • methacrylic anhydride 3.75 ml, 5 molar equivalent of the hydroxyl groups per HA disaccharide repeating unit.
  • the reaction was stirred at room temperature overnight with the pH maintained at 8.0 by adding 5N sodium hydroxide.
  • HAMa was purified by dialysis (molecular weight cut-off (MWCO) 12- 14 kDa) for 48 hours, then freeze dried for 3 days.
  • the degree of functionalisation was calculated by nuclear magnetic resonance spectroscopy (1 H-NMR in deuterium oxide (D20)).
  • the solution was dialysed in distilled water (MWCO 12-14 kDa) at 40 °C for 7 days. After purification, the GelMa solution (a clear colour-less viscous liquid) was subjected to freeze drying resulting in a bright white product with 65%-70% yield. The degree of functionalization (73%) was calculated by 1 H-NMR in D20. The freeze dried product was stored in 4 e C in a dark and inert environment prior to use.
  • IPFP Human infrapatellar fat pad derived adipose stem cells
  • Photocuring of the printed bioink was achieved with a 365 nm UV source 14 (Omnicure LX400+, Lumen Dynamix LDGI) fitted with a 12 mm lens (25 mm focal distance).
  • the light source was fitted to the side of the device 10 and focused on the bioink as it was extruded from the nozzle 4.
  • the bioink was irradiated for 60 seconds at a radiation intensity of 130 mW cm- 2 immediately after printing, equating to a final UV exposure of 7.8 J cm- 2 .
  • Printing was carried out within aseptically contained class II laminar flow hoods.
  • the bioink was covered with culture media comprising Dulbecco's Modified Eagle Medium (DMEM), 10% Fetal Bovine Serum (FCS), 15 mM HEPES, 2 mM Glutamax, 5 ng/ml rhEGF, 1 ng/ml rhFGF-2, 100 U/ml Penicillin and 100 ⁇ g/ml Streptomycin.
  • DMEM Dulbecco's Modified Eagle Medium
  • FCS Fetal Bovine Serum
  • HEPES 1 mM HEPES
  • 2 mM Glutamax 2 mM Glutamax
  • 5 ng/ml rhEGF 1 ng/ml rhFGF-2
  • 100 U/ml Penicillin 100 ⁇ g/ml Streptomycin.
  • the printed cells were grown under standard tissue culture conditions (5% C0 2 , 37 °C) for 1 , 3 and 7 days before analysis. At these time points, the media was removed from the wells and the printed cells were washed twice
  • a live/dead viability/cytotoxicity kit for mammalian cells from Life Technologies was used to determine the viability of printed cells.
  • Digital photographs for quantification of cell viability were obtained on an Olympus IX70 inverted micro-scope using a SPOT Diagnostic RT-Slider camera and SPOT Diagnostic software.
  • Live/dead cell counts were obtained using the cell counter macro available in Image J software.
  • Table 1 and the graph in Figure 7 show the percentage viability obtained from mean average over four fields of view selected at random over each of three wells per time point (number is parenthesis shows the standard deviation). Each field of view contained more than 100 cells.
  • a small but significant decrease in cell viability between cultures encapsulated in the cured gel without bioprinting (“Control gel”) and control cultures (“Control”) was observed. This small decrease in viability, of about 3%, may be due to exposure to UV light, the presence of free radicals induced by initiation of the curing process or the effects of encapsulation in the gel itself.
  • Material properties of a bioink were characterised by testing the rheology of the bioink in response to shear stress, temperature and UV exposure, as well as the UV absorbance of the bioink.
  • Figure 8a illustrates the shear-thinning behaviour of the bioink, with numerals 22 and 24 referring to recorded viscosity and shear stress respectively.
  • the viscosity at low shear rates (less than 1 s "1 ) is relatively high (higher than 300 Pa.s), allowing for cells to remain suspended when held in the chamber 2a, 2b prior to extrusion.
  • the application of a shear force dramatically decreases the bioink viscosity.
  • the viscosity drops to less than 10 Pa.s.
  • This shear-thinning behaviour reduces the shear stresses of extrusion, ensuring the cells are not exposed to damaging shear stresses.
  • this shear-thinning behaviour can be described by the power law (Ostwald-de Waele) relationship:
  • K is the flow consistency index
  • n is the flow behaviour index.
  • FIG 8b shows the storage modulus (G') (reference numeral 26) and loss modulus (G") (reference numeral 28) for the bioink across the temperature range 2 e C to 45 e C.
  • the bioink is a weakly crosslinked hydrogel, with G' higher than G" over the temperature range examined. At and below room temperature range (22.5 e C), G' is much higher than G" indicating gel-like properties and formation of a bioink suitable for extrusion printing. However, the 22 e C temperature is positioned in the middle of the bioink's gelation region, meaning the viscosity of the bioink is strongly dependent on temperature within this range. The low viscosity, liquid-like behaviour at 37 °C allows for mixing of cells.
  • UV absorbance was measured on an Ultrospec 3300 pro UV/vis spectrophotometer (GE Healthcare). Absorbance at 365 nm was measured relative to a blank solution (i.e. DPBS) for each species over a range of bioink concentrations. Absorbance was plotted against concentration and the absorptivity (in units of cm -1 ) was taken as the slope.
  • Figure 9a shows that the absorbance of the various concentrations of bioink in DPBS follows the Beer-Lambert law, ie absorbance increases linearly with concentration.
  • the UV absorptivity of the bioink at the concentrations printed in Example 1 ie 100 mg/ml GelMa, 20 mg/ml HAMa
  • Figure 9b shows that the absorbance of the photoinitiator VA-086 also follows the Beer-Lambert law.
  • the UV absorptivity at the concentration of VA-086 printed for the cell viability example ie 0.5 wt%) is 0.410 cm -1 .
  • ⁇ 0 is the initial intensity
  • / is the intensity at a given depth through the solution
  • A is the absorptivity in units cm -1
  • d is the penetration depth through the solution.
  • the rate of photopolymerisation is related to the concentration of photoinitiator free-radicals, which in turn is proportional to the intensity of UV light.
  • the intensity of UV light decreases with increasing depth of the gel, an effect that may affect the spatial uniformity of the photocuring reaction and thus the spatiomechanical properties of the cured hydrogel. This effect was assessed by calculating the percentage of UV light transmitted (%7) as a function of penetration depth:
  • Figure 9c shows % T across a range of penetration depths. At a depth of 500 ⁇ within the bioink (the width of a typical printed strand), 70% of the UV light is being transmitted. Thus a 30% difference in the initial rate of photocrosslinking would be expected between the anterior and posterior sides of the irradiated strand. A depth-dependent difference in the degree of crosslinking (and thus the mechanical properties of the scaffold) is thus expected. In the present case, the difference in mechanical properties between the top and bottom of the scaffold is minimised because the UV exposure (and hence the degree of crosslinking) is high.
  • the inset plot in figure 9d shows the data for the first 1.25 J cm -2 exposure, corresponding to the first 10 seconds of curing by the device. It can be observed that the rate of increase in G' of the bioink is very low within the first 5 seconds of UV exposure, before rapidly increasing. This can be attributed to the kinetics of the photo-induced free-radical polymerisation, which proceeds in three main stages.
  • the first stage the presence of oxygen (both dissolved in the bioink and diffusing from the atmosphere) impedes the photopolymerisation reaction as the oxygen consumes the photoinitiator free-radicals.
  • the rate of gelation increases to yield a rapid increase in complex viscosity during 2 to 10 J cm -2 exposure.
  • the third stage >10 J cm -2
  • the rate of reaction slows as the bioink gels and diffusion rates decrease.
  • Example 1 a device UV curing time of 60 seconds resulted in a photocrosslinked hydrogel of 15000 Pa storage modulus, which was stable in culture for at least 7 days. Accordingly, in some embodiments of the present invention, the UV exposure is performed only once, after the complete structure is printed. In other embodiments, the device 10 deposits bioink in a layer-by-layer approach, whereby each layer is partially cured using a short (eg about 5 seconds) UV exposure via the UV source 14 before the next layer is deposited.
  • a short eg about 5 seconds
  • Performance of device 10 was analysed by testing stability of extrusion under various extrusion pressures and the characteristics of extruded lines, including the definition of 2D patterns and material gradients.
  • PDMS polydimethylsiloxane
  • Figure 10a shows the extruded volumes of PDMS with respect to the applied pressure (for clarity, data from only one nozzle opening 6a is shown).
  • Numerals 30, 32, 34, 36 and 38 refer to data obtained from applied pressures of 5 kPa, 10 kPa, 20 kPa, 30 kPa and 40 kPa respectively.
  • the linear relationship between the extruded volume and extrusion time, for all applied pressures, illustrates the stability of extrusion via the device 10. The extrusion rate for each applied pressure was measured from the slope of these plots.
  • Figure 10b shows the extrusion rates with respect to the applied pressure via each of the nozzle openings 6a, 6b of the device 10 using the PDMS standard.
  • the extrusion rates increase linearly with applied pressure.
  • the extrusion rates for one of the nozzle openings 6a were slightly higher than the other nozzle opening 6b, likely due to fabrication defects.
  • the extrusion of the bioink formulation as shown in Figure 10c, is also highly stable at extrusion pressures in the range 125-200 kPa.
  • Numerals 40, 42, 44, 46 and 48 refer to data obtained from applied pressures of 125 kPa, 150 kPa, 175 kPa, 190 kPa and 200 kPa respectively.
  • bioink extrusion rate is non-linear with respect to applied pressure due to its shear-thinning rheological properties.
  • Figure 1 1 a shows optical micrographs of representative device-printed lines of the bioink at various extrusion pressures while mounted on a robotic stage to standardise draw rate to 30 mm s ⁇ 1 . The thickness of the lines increases with extrusion pressure. Continuous lines were formed in a range of applied pressures of 140 to 165 kPa. This is further illustrated in Figure 1 1 b, which characterises four regimes of device printing. In regime I ( ⁇ 100 kPa pressure) there is no extrusion. In regime II (100-140 kPa) discontinuous droplets are extruded.
  • FIG. 1 c shows two cross-hatched layers of the GelMa/HAMa bioink formulation printed by hand using the device 10. It is envisaged that during the handheld biofabrication process, the user will employ visual feedback to modify line dimensions in a free-form manner.
  • Colorimetric gradients were created by controlling the relative extrusion rates from each chamber 2a, 2b while the device was manipulated by a three-axis robotic system.
  • Each chamber contained a hydrogel (GelMa 10 wt%) coloured with either blue or red food dye.
  • the printed gradient was quantified by image analysis using Image J software: the red and blue RGB values were measured for lines printed at each ratio. The relative red or blue colour of each printed line was calculated as the ratio of the red and blue RGB values.
  • Figure 12a is a photograph of handheld printing of the red-dyed hydrogel onto a glass-slide using the device 10 with sufficient control to print the word "biopen”.
  • Figure 12d (scale bar 400 ⁇ ) is a mosaic of five optical microscope images showing the handwritten word "biopen” from Figure 12a.
  • Figure 12b shows a spiral printed in the same dyed hydrogel onto a PET petri-dish. The uniformity of the spiral (printed while the device was mounted on a robotic stage) illustrates the consistency of printing achievable with the device 10.
  • Figure 12c shows a 2D blue-red gradient created by controlling the relative extrusion rates of each chamber 2a, 2b during printing.
  • the relative extrusion rates of the red and blue inks could be adjusted, beginning with 100% blue (left-side) and ending with 100% red (right-side) while keeping the total extrusion rate constant.
  • Figure 12d quantifies the quality of the printed gradient, as obtained via the image analysis of Figure 12c.
  • the x-axis shows the relative extrusion pressure between chamber 2a (containing the blue ink) and chamber 2b (containing the red ink).
  • the y- axis shows the relative red or blue colour of the printed lines (as obtained from RGB intensity). The relative colour is linearly proportional to the relative extrusion pressure, as shown by the trendline.
  • the device 10 used for this example had a co-axial nozzle configuration (as illustrated in Figure 6).
  • the inner opening 6b was supplied with a GelMa/HAMa hydrogel seeded with mesenchymal stem cells obtained from sheep infrapatellar fat pad, at a concentration of 2.5 ⁇ 10 6 cells/ml.
  • the outer opening 6a was supplied with a GelMa/HAMa hydrogel and 0.5 wt% photoinitiator VA-086. The user was therefore able to extrude a biofabricated structure having living cells in the core section and a supporting UV-curable shell. Multiple layers of this structure could be deposited in situ to build up a 3D printed bioscaffold.
  • BB group 1 x 1 x 0.1 cm blocks of bioscaffolds were printed one week before the surgical procedure and incubated in stromal media. During the surgical procedure, 8 mm diameter cylinders were cored out of the pre-constructed block and inserted in the defect.
  • MF group five holes were created in the defect area using a 1 .0 mm microfracture awl and confirmed by bleeding bone.
  • a thin layer of fibrin glue was sprayed on top of the bioscaffold to prevent its mobilization from the defect.
  • the sheep were allowed to weight bear after surgery, and were euthanized after 8 weeks. Stifle joints were opened and disarticulated and the femoral condyles explanted and photographed.
  • Osteochondral blocks with the repaired defect in the centre and host bone- cartilage margin of at least 10mm in the periphery were then collected using high precision bone saw.
  • the block was bisected in the coronal plane (as shown in Figure 13d) to allow for biomechanical and biochemical analysis. One half was used fresh for biomechanical compression testing (indentation test), while the other half was processed for histological analysis.
  • Specimen thickness and biomechanical data are summarised in Figures 15b to 15e.
  • the mean thickness measured for different intervention strategies and the host cartilage ranged between 0.7 mm and 1 .0 mm with quite large standard deviation (Figure 15b).
  • the instantaneous Young's modulus for the HH and BB groups were similar around 0.5 MPa and slightly lower than the host cartilage of 0.6 MPa.
  • the Young's moduli of the MF and C groups were generally lower than the HH, BB and host cartilage.
  • the HH Group showed a similar Safranin O staining compared to the other groups.
  • Collagen II immunohistochemistry staining showed that new tissue formation within the defect was mainly made of hyaline cartilage, with the exception of the MF group, in which fibrocartilage was evident in the defect area (Figure 16a).
  • Embodiments of the present invention provide apparatus and methods that are useful for in situ, free-form biofabrication, which address the practical constraints of the operating theatre. [0094] Embodiments of the present invention additionally or alternatively provide apparatus and methods for in situ fabrication of 3D tissue scaffolds as well as for delivery of primary human stem cells for tissue reconstruction.
  • Embodiments of the present invention additionally or alternatively provide an ergonomic, manually operated device that is useful for free-form, on-the-fly, surgical sculpting of substitute tissue to achieve a desired structure, with improved surgical dexterity that allows for deposition within crevices or beneath overhangs in native tissue.
  • Embodiments of the present invention additionally or alternatively provide a device for free-form biofabrication having a small form factor and is easy to sterilise and keep sterile, so that it may be easily brought into and out of the surgical field.
  • Embodiments of the present invention additionally or alternatively provide an affordable alternative to conventional bioprinting machinery, for example, where components of the device are 3D printed, and the device is operated using simple pneumatic equipment.
  • biofabrication means the fabrication of biological structure (such as living tissue) from raw materials, which may include organic and/or inorganic molecules, biocompatible matrices, living cells, etc.
  • bioink means a flowable material comprising a biocompatible matrix suitable for supporting cellular activity during biofabrication.

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Abstract

L'invention concerne un dispositif et des procédés de biofabrication de forme libre, le dispositif comprenant une pluralité de chambres d'extrusion qui peuvent être tenues à la main et actionnables individuellement, une buse d'extrusion comprenant une pluralité d'ouvertures qui sont très proches les unes des autres et respectivement en liaison fluidique avec la pluralité de chambres d'extrusion, l'une des chambres d'extrusion contenant une bioencre qui comprend des cellules vivantes qui, lors de l'utilisation, peuvent être délivrées de manière contrôlée in situ avec un biomatériau structurel pour former une structure biofabriquée.
PCT/AU2016/050886 2016-09-22 2016-09-22 Appareil et procédé de biofabrication manuelle de forme libre WO2018053565A1 (fr)

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CN109514858A (zh) * 2018-11-02 2019-03-26 四川大学华西医院 一种多通道3d打印喷头及采用该喷头制造管道的方法
CN110870938A (zh) * 2018-09-04 2020-03-10 上海叁钛生物科技有限公司 皮肤原位打印用打印头装置
WO2021020668A1 (fr) * 2018-10-10 2021-02-04 주식회사 클리셀 Dispositif de prévention de photopolymérisation pour prévenir la photopolymérisation d'un biomatériau à l'intérieur d'une buse de pulvérisation et d'un distributeur, et bio-imprimante 3d comprenant celui-ci
CN112587281A (zh) * 2020-12-25 2021-04-02 天津强微特生物科技有限公司 一种手持式皮肤原位打印氧化固化装置
EP3932437A1 (fr) * 2020-07-03 2022-01-05 Fundació Institut de Bioenginyeria de Catalunya (IBEC) Système d'impression permettant d'obtenir des fibres biologiques
WO2023113354A1 (fr) * 2021-12-13 2023-06-22 전남대학교산학협력단 Kit de composition d'encre biologique pour tissu biologique tubulaire, son procédé de fabrication, procédé de construction de tissu biologique tubulaire l'utilisant, et tissu biologique tubulaire construit par ledit procédé
KR102590675B1 (ko) 2023-03-28 2023-10-19 주식회사 티센바이오팜 노즐 및 이를 포함하는 인공육 제조 시스템

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CN110870938A (zh) * 2018-09-04 2020-03-10 上海叁钛生物科技有限公司 皮肤原位打印用打印头装置
WO2021020668A1 (fr) * 2018-10-10 2021-02-04 주식회사 클리셀 Dispositif de prévention de photopolymérisation pour prévenir la photopolymérisation d'un biomatériau à l'intérieur d'une buse de pulvérisation et d'un distributeur, et bio-imprimante 3d comprenant celui-ci
US11370169B2 (en) 2018-10-10 2022-06-28 Clecell Co., Ltd. Apparatus for preventing photocuring of biomaterial in discharge nozzle and dispenser and bio 3D printer including the same
CN109514858A (zh) * 2018-11-02 2019-03-26 四川大学华西医院 一种多通道3d打印喷头及采用该喷头制造管道的方法
EP3932437A1 (fr) * 2020-07-03 2022-01-05 Fundació Institut de Bioenginyeria de Catalunya (IBEC) Système d'impression permettant d'obtenir des fibres biologiques
WO2022003203A1 (fr) 2020-07-03 2022-01-06 Fundació Institut De Bioenginyeria De Catalunya (Ibec) Système d'impression permettant d'obtenir des fibres biologiques individuelles de forme libre et à largeur contrôlée
CN112587281A (zh) * 2020-12-25 2021-04-02 天津强微特生物科技有限公司 一种手持式皮肤原位打印氧化固化装置
WO2023113354A1 (fr) * 2021-12-13 2023-06-22 전남대학교산학협력단 Kit de composition d'encre biologique pour tissu biologique tubulaire, son procédé de fabrication, procédé de construction de tissu biologique tubulaire l'utilisant, et tissu biologique tubulaire construit par ledit procédé
KR102590675B1 (ko) 2023-03-28 2023-10-19 주식회사 티센바이오팜 노즐 및 이를 포함하는 인공육 제조 시스템
KR20240145864A (ko) 2023-03-28 2024-10-07 주식회사 티센바이오팜 노즐 및 이를 포함하는 인공육 제조 시스템

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