Patent ID: 12257775

DETAILED DESCRIPTION

3D bioprinting of materials can use a chemical cross-linking agent that hardens the printed material after being extruded from the print head. A print head attachment is described further below that allows the cross-linker to be supplied as a mist to the extruded printing material and any excess cross-linker extracted from around the print head. Extracting excess cross-linker helps prevent or reduce print errors that can result from a buildup of excess cross linker around the printing area. The print head attachment can be attached to existing print heads without requiring significant changes to the 3D printer.

FIG.1depicts components of a 3D bioprinter incorporating a 3D print head attachment system. The 3D bioprinter100has a print head assembly that includes a print head102and a print head attachment104. The print head assembly may be secured to an XY positioning assembly106by a print head mount108. A printing material can be controllably extruded108from the print head102onto a print stage110to additively form a printed object112. The print stage110may be connected to a Z positioning assembly114to raise/lower the print stage110as the object112is printed. It will be appreciated that the XY positioning assembly106and the Z positioning assembly114provide relative movement between the print stage110and the print head102. Although depicted as moving the print head in the XY direction and the print stage in the Z direction, the positioning assemblies may be connected in various arrangements to provide the relative movement between the print head and print stage.

The print head may be provided by, for example a syringe, a Luer-Lock dispensing needles, etc. The print head may be controlled by various means including pneumatically by a compressed air source116, mechanically by a motor or other types of actuators. The print head may be filled with a print material that is extruded, or otherwise deposited, on the print stage to form the object. The printing material may be selected from a wide range of materials that are cured, or hardened, by contact with another chemical, referred to as a cross-linker. The particular cross-linker used may vary depending upon the printing material used. For example, the printing material may be sodium alginate, chitosan, collagen, agarose, or other compatible biomaterial and/or hydrogels. The cross-linker may include, for example, calcium chloride (calcium ion), genipin, aldehydes or other chemicals capable of cross-linking the printing material.

As depicted inFIG.1, the print head attachment104may be fit over, or attached to, the print head102so that the print material exiting the print head at an exit nozzle of the print head passes through the print head attachment. Although depicted as exiting the print head102within the print head attachment, it is possible for the exit nozzle of the print head may extend past a bottom of the print head attachment. The temperature of the print material being extruded may be controlled, for example temperatures may range between 4°-20° C. The temperature of the print material may change the material's viscosity for improved control over the printability as well as control of the diffusion of crosslinking ions.

The print head attachment102comprises a mist delivery channel118that supplies a mist of the chemical cross-linker around the extruded print material108. The mist delivery channel118may be connected to a mist supply port120that allows the mist delivery channel to be connected to a mist supply source122, for example by a tube or tubing. The mist supply source120may comprises a tank holding the cross-linker solution. An ultrasonic atomizer124may be used to generate a mist of cross-linker droplets126. An air pump128may be used to supply a flow of the mist of cross-linker to the mist delivery channel118. The droplets generated may vary in size, however for calcium chloride used to cross-link sodium alginate a diameter of approximately 10-100 microns in size may be used. Similarly, various flow rates of the cross-linker mist may be provided by the air pump128at a rate of about 1 L/min (air and droplets mixed). The mist of cross linker is supplied to the mist delivery channel118that supplies the cross-linker to around the extruded print material. The mist delivery channel is located in proximity to the exit nozzle of the print head when present so that the mist delivery channel supplies a flow of atomized cross-linker around the printing material extruded through the print head. The mist delivery channel118may have an exit opening to promote laminar flow of the cross-linker mist about the extruded print material.

In order to prevent excess cross-linker from contacting the printed object or possibly pooling on the print stage, the print head attachment further includes an extraction channel130that extracts excess cross-linker from the print area. The extraction channel130may have an extraction connection port132that can be connected to a vacuum pump134to provide sufficient suction to extract the excess cross-linker which may be collected in a waste tank136, or possibly resupplied to the cross-linker solution tank122. Although the extraction flow rate may vary, a flow rate of about 3-5 L/min may be sufficient to extract enough excess cross-linker to prevent or reduce pooling of the cross-linker. The extraction channel enables even removal of the excess cross-linker without disrupting the interaction between the cross-linker and the extruded print material.

FIG.2Adepicts an illustrative 3D print head attachment. The print head attachment104was described above as having mist delivery channel with a single opening in proximity to the extruded material. It is desirable to promote flow of the cross-linker around the entirety of the extruded print material and as such a plurality of mist openings may be provided in proximity to the extruded material. The print head attachment200comprises a body202, which may be formed from various materials including both plastics and metals. The body202comprises a receiver opening204for receiving the print head. The receiver opening204may secure the attachment to the print head, for example by a friction fit, or an additional securing mechanism (not depicted) may be used. The particular shape of the receiver may depend upon the shape and size of print head used, however it generally comprises an opening into which the print head can be received that continues through the body to allow the print material to be extruded from the print head onto to the print stage.

The body may include a mist connector206for securing tubing to in order to supply the mist of cross-linker. The mist connector206may be connected to a mist delivery channel208surrounding the receiver opening in the body202. A plurality of delivery openings210a,210bmay be connected to the delivery channel about the receiver opening in order to supply the mist of cross-linker around extruded print material. The print head attachment may further comprise an extraction connector212for connecting the attachment to a vacuum pump. The extraction connector212may be connected to an extraction channel214surrounding the receiver opening204. A plurality of extraction openings216a,216bmay be connected to the extraction channel about the receiver opening204in order to extract excess cross-linker from the print area.

FIG.2Bdepicts the 3D print head attachment ofFIG.2Ain use. Elements of the print head attachment described above with reference toFIG.2Aare not labelled inFIG.2Bfor clarity of the drawing. As depicted, print head, which may be a syringe filled with the printing material, is received within the receiver opening204in the attachment body202and is secured to the attachment. The print head controllably extrudes print material220to form a desired shape. Atomized cross-linker flows through the connection port206, into the delivery channel208and out of the delivery openings210a,210b. The flow of cross linker depicted by arrows222a,222bflows out of the openings and surrounds the extruded print material220causing cross links to be formed. Excess cross linker is extracted by a vacuum pump connected to the extraction connection port212. The excess cross linker is extracted through the plurality of extraction openings216a,216bin the extraction channel214.

FIG.3depicts a further illustrative 3D print head attachment. The print head attachment described above with regard toFIGS.2A and2Bmay provide acceptable printing results, however the mist delivery channel and openings as well as the extraction channel and openings may not provide desired printing results. The print head attachment described further below with reference toFIG.3andFIG.4has an improved shape of the mist delivery channel for promoting the laminar flow of the cross-linker mist 360° about the extruded print material. Similarly, the print head attachment has an improved shape of the mist extraction channel to reduce disruption of the laminar flow of the cross-linker mist about the extruded print material while still removing the excess cross-linker from the print area.

The print head attachment300may be formed as a single unitary piece of material having various channels and openings formed within it. The print head attachment may be formed using various manufacturing techniques including 3D printing and injection molding. The particular shape and size of the print head attachment may be varied according to the particular print head the attachment is designed for.

The print head attachment300has a receiver opening302into which a print head, such as a syringe can be received. The receiver opening302continues through the body as cylindrical body opening304for receiving a portion of the print head. The receiver opening and body opening may continue through a conical opening portion306for receiving a portion of the print head, such as a cone-tip needle attachment. The exit nozzle of the print head may extend through the conical opening and through an exit opening308. The exit nozzle of the print head may extend fully through the exit opening308, or it may remain within the exit opening308, or even within the conical opening306.

The print head attachment300comprises a mist delivery channel310. The delivery channel may be formed about a vertical axis of the body to provide a continuous 360° channel. The delivery channel310is connected to a delivery connection port312that allows a tube or hose to be connected to the print head attachment for providing a flow of cross-linker mist into the delivery channel310. The delivery channel310is in proximity to the exit nozzle of the print head when it is present, and supplies a flow of atomized cross-linker around the printing material extruded through the print head. The delivery channel310is depicted as having a substantially continuous opening314that substantially surrounds the exit opening308, however, it is possible to provide a plurality of discreet openings surrounding the exit opening308. The delivery channel310may have a cross sectional profile that descends downward at an angle of between 30-45° toward the delivery opening314to impart downward movement to the mist flow and promote laminar flow about the extruded print material. As depicted, the channel profile may have an enlarged upper chamber with the connection port312located towards a top of the enlarged upper chamber, which may help provide a better flow of cross-linker out of the delivery opening314.

The print head attachment300further comprises a mist extraction channel316that extracts excess cross-linker from the print area. Similar to the delivery channel, the extraction channel316may be formed about the vertical axis of the body to provide a continuous 360° channel. The extraction channel316is arranged in proximity to the exit nozzle of the print syringe when present to extract a flow of excess cross-linker from around the extruded printing material. The extraction channel316may have a substantially continuous extraction opening318that that substantially surrounds the exit opening308, however, it is possible to provide a plurality of discreet extraction openings surrounding the exit opening308. The extraction opening may be spaced apart circumferentially from the exit opening308. The bottom surface of the attachment between the extraction opening318and the exit opening308may have an arcuate profile320for helping with the extraction of excess cross linker from the print area. An extraction connection port322is connected to the extraction channel and allows a vacuum pump to be connected in order to extract the excess cross linker.

FIG.4depicts a cross section of the print head attachment ofFIG.3. A print head402is received within the opening302, cylindrical body opening304, conical opening306, and through the exit opening308. As depicted, the exit nozzle of the print head extends past the exit opening, however the exit nozzle may remain within the print head attachment. The print head is controlled to extrude the printing material404onto a printing platform406. As the printing material is extruded through the exit nozzle of the print head, the printing material is contacted with a flow of cross-linker mist, depicted by arrow408. The mist of cross-linker is supplied through the connection port312into the delivery channel310and out the delivery opening314located in proximity to the nozzle exit. The shape of the delivery channel310helps to promote a laminar flow of the cross-linker mist down and around the printing material as it is extruded. The excess cross-linker mist that remains in the print area is extracted by the extraction channel316as depicted by arrows410through the extraction opening318. The extraction profile320at the bottom of the print head attachment may help to extract the excess cross-linker without disturbing the flow of the cross-linker around the extruded printing material.

A print head attachment system as described above can be easily connected to existing print heads. The print heads may print an object by extruding a filament of a printing material. The extruded filament may have a wide range of sizes, such as for example between about 20 microns and about 2000 microns. The print material may include various compounds or materials including for example, living cells, micro particles, nano particles, etc. The print head attachment can improve the printing results when printing with print materials that are exposed to cross-linkers or other coating particles. The cross-linker may be delivered in the form of atomized mist droplets. The print head attachment may be used with sodium alginate as the print material, using calcium chloride (CaCl2)) as the crosslinking agent to form a solid thermoset elastomer, calcium alginate. The print head attachment may be used with other print materials and crosslinking agents. Ultrasonic atomization of the cross linker creates a fine mist of droplets externally from the attachment that can be delivered to the attachment using forced airflow. The droplets of crosslinking agent are provided into a cavity that is designed to promote 360° laminar flow of mist about the needle tip that is focused directly on the extruded biomaterial. Excess droplets of the cross linker may be removed with an external vacuum pump. The attachment may fit directly onto common bioprinting equipment such as syringes and Luer-Lock dispensing needles. Mist removal may be enabled via a cavity that features a narrow channel to promote even removal of the excess mist to reduce the disruption of interaction between droplets of the crosslinking agent and the print material being extruded. The extraction flow rate may be set at between 3-5 L/min. Droplets of the cross linking agent may be generated in the diameter range of 10-100 microns using an ultrasonic atomizing system and forced into the print head at ˜1 L/min (air and droplets, mixed) by an air pump. The temperature of the printing material may be controlled, for example within a temperature range of between 4-20° C., which may change viscosity of the printing material for improved printability and may enable improved diffusion of crosslinking ions. The attachment may fit directly onto common bioprinting equipment such as syringes and Luer-Lock dispensing needles.

FIG.5shows photographs of a 3D printed cylindrical constructs. One cylindrical construct502was printed with 5 wt % sodium alginate using 10 wt % CaCl2mist with a flow rate of 750 mL/min from a misting attachment in accordance with the current disclosure. A second cylindrical construct504was printed with 7 wt % sodium alginate using 10 wt % CaCl2mist from a misting attachment in accordance with the current disclosure. Both cylindrical constructs exhibited strong layer adhesion.

FIG.6shows photographs of 3D printing results with and without mist removal using a misting attachment in accordance with the current disclosure. All other parameters of the printing remained the same and used 5 wt % sodium alginate and 10 wt % CaCl2mist. As can be seen in the comparison between print results depicted in panels (a) and (e), panels (b) and (f) and panels (c) and (g), the use of the mist removal during printing results in a higher quality print. Although not wishing to be bound by theory, it is believed that the higher quality print results are, at least in part, a result of no or little liquid accumulation on the print stage when using the mist removal as can be seen in comparison of panels (d) and (h). The print head attachment and system has been described above with particular reference to supplying an atomized mist of a cross-linker around extruded print material. The same print head attachment and system may also be used with materials other than cross-linkers. For example, the mist drops may contain the cross-linker as described above, or may contain suspensions of other particles and liquids such as a coating to be applied to the extruded material. The particles may be the cross-linker as described above, or may be other particles such as a coating to be applied to the extruded material. The coating particles may provide various functionality such as a lubricant, an adhesive, a colorant, or other particles provide desired functionality. Further, the atomized mist supplied around the print material may include a combination of particles such as cross-linkers and coatings. The attachment may be used in various applications including fabricating tissue constructs using biocompatible polymers including sodium alginate in fabricating vascular and liver tissue, collagen in fabricating skin tissue and agarose and chitosan for various tissue engineering applications. Further, the above has described the print head attachment with respect to its use with extrusion based 3D printing. As described further below, a similar attachment may be used with other deposition processes in addition to, or as an alternative to, extrusion based 3D printing.

Droplet-based deposition techniques can be used both as a 3D printing techniques as well as for other applications, including in pharmaceutical development, high-throughput chemical processes, etc. Droplet based deposition techniques may be used for 3D bioprinting and enables precise deposition of biocompatible polymers and living cells, which may be referred to as bioinks, to fabricate complex in-vitro tissue models.

Existing systems crosslink bioink droplets after printing to form rigid structures; however, crosslinking after printing may result in too rapid gelation of the droplets as a result of too much crosslinker or too slow gelation of the droplets. Too rapid or too slow gelation can lead to poor adhesion or shape fidelity, respectively. Furthermore, improper gelation can inhibit cell proliferation. The fabrication of complex tissue and organ constructs by, for example, depositing living tissue spheroids may be limited by the rate and extent of fusion the deposited spheroids, which in turn may depend upon the gelation rate. A misting attachment similar to that described above may be used to facilitate the crosslinking of printed bioink droplets before deposition onto the print stage, offering controllable proper gelation of the bioink.

FIG.7depicts a misting attachment for a droplet-based deposition process. The misting attachment700is similar to the print head attachment described above. The misting attachment700may be attached to, or otherwise coupled to, a droplet deposition head702that deposits drops of material704onto a surface (not shown). The misting attachment700comprises a mist delivery channel706that delivers a supply of mist of crosslinker, or other suspensions of particles, around the droplets as they pass through the attachment. The mist delivery channel706may be connected to a delivery port708that can be used to provide a flow of the mist, whether it is a crosslinker, which could be atomized, or other suspensions of particles. The attachment700further comprises a mist extraction channel710through which excess mist can be extracted. The extraction channel710may be connected to an extraction port712that can provide a vacuum to extract the excess mist. The mist may enter a channel through which material droplets are deposited, as depicted by arrows714and then be extracted away from the droplets704as they are deposited as depicted by arrows716.

FIG.8depict simulation results of mist concentration within a misting attachment in accordance with the misting attachment ofFIG.7. As depicted, the mist concentration is high in a path that the deposited droplets pass802prior to being extracted through the mist extraction channel. The concentration of the mist below the attachment is relatively low. The length between the mist delivery channel and the mist extraction channel can be set so that the material droplets are exposed to the mist for an appropriate length of time. The appropriate length of time may depend upon the material being deposited, the misting agent, as well as the desired properties, such as gelation amount, coating amount, etc. for a particular application. In addition to the length between the mist delivery channel and the mist extraction channel, the flow rate and concentration of the mist may also be varied to adjust exposure levels to a desired or appropriate amount.

The misting attachment may direct the flow of mist to provide even, 360-degree contact with the droplets as they are deposited. The geometry of the attachment may provide a uniform mist concentration on the centerline of the attachment, or through the path of the deposited droplets. The mist flowrate and concentration may be adjusted to modify the extent of exposure which may adjust the crosslinking rate. Adjusting the flowrate and concentration may be used to control the mechanical properties of the deposited materials. The temperature of the bioink may be adjusted (4-37° C.) to improve diffusion of the crosslinker mist if desired.

The crosslinker, or the suspended particles, may be collected within the attachment device to prevent accumulation of liquid on the print stage. The geometry of the outlet channel may prevent disruption to the printed droplets as they exit the attachment.

The misting attachment for droplet-based deposition techniques may be used in a variety of applications, including for example, fabricating tissue spheroids for in-vitro tissue and organ regeneration, printing or otherwise manufacturing biocompatible beads for drug delivery, coating bioprinted droplets with functional substances (i.e. conductive, hydrophilic, hydrophobic).

FIG.9depicts a porous scaffold printed using the misting attachment described above with reference toFIG.7. The droplet based printing process used a 3 wt % sodium alginate with a crosslinking misting agent of 10 wt % CaCl2. As depicted the scaffold exhibits high shape fidelity and has good co-droplet adhesion.

It will be appreciated by one of ordinary skill in the art that the system and components shown inFIGS.1-9may include components not shown in the drawings. For simplicity and clarity of the illustration, elements in the figures are not necessarily to scale, are only schematic and are non-limiting of the elements structures. It will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.