applications of inkjet technology for tissue engineering and

APPLICATIONS OF INKJET TECHNOLOGY

FOR TISSUE ENGINEERING AND REGENERATIVE MEDICINE

Patrick W. Cooley, David Silva, David B. Wallace

APPLICATIONS OF INKJET TECHNOLOGY FOR TISSUE ENGINEERING AND REGENERATIVE MEDICINE

Microfab Technologies, Inc. 1 August 2014

Table of Contents

1. PURPOSE ........................................................................................................................................................... 1

2. BACKGROUND ................................................................................................................................................... 1

2.1 Demand Mode Inkjet Technology ................................................................................................................ 1

2.2 Fluid Property Effects on Inkjet Dispensing Performance ........................................................................... 2

2.3 Advantages of Inkjet Dispensing .................................................................................................................. 2

3. TISSUE ENGINEERING AND REGENERATIVE MEDICINE APPLICATIONS .......................................................... 3

3.1 Cell Dispensing .............................................................................................................................................. 3

Inkjet Printing of Silk Nest Arrays for Cell Hosting .................................................................................................... 3

Microenvironments Engineered by Inkjet Bioprinting Spatially Direct Adult Stem Cells Toward Muscle‐ and Bone‐

Like Subpopulations .................................................................................................................................................. 4

Towards the Inkjet Fabrication of Artificial Cells ....................................................................................................... 5

Adult Rat Retinal Ganglion Cells and Glia Can Be Printed by Piezoelectric Inkjet Printing ....................................... 6

3.2 Growth Factors.............................................................................................................................................. 7

Precise Control of Osteogenesis for Craniofacial Defect Repair: The Role of Direct Osteoprogenitor Contact in

BMP‐2‐Based Bioprinting .......................................................................................................................................... 7

Spatially Directed Guidance of Stem Cell Population Migration by Immobilized Patterns of Growth Factors ........ 8

3.3 Scaffold ‐ Construct Fabrication ................................................................................................................. 10

Scaffold‐Free Inkjet Printing of Three‐Dimensional Zigzag Cellular Tubes ............................................................. 10

Inkjet Printing and Cell Seeding Thermoreversible Photocurable Gel Structures .................................................. 11

4.0 CONCLUSION ..................................................................................................................................................... 12

5.0 REFERENCES ...................................................................................................................................................... 13



Figure 1 Drop‐on‐demand inkjet printing schematic.

Figure 2 Drop‐on‐demand inkjet dispensing 60µm diameter drops at 4kHz. Sequence span 130µs delay left to right.

1. PURPOSE

This position paper discusses the application of inkjet dispensing technology for tissue engineering and regenerative medicine research and development. The bioprinting of hydrogels, biopolymers, cells and growth factors (stimulants and inhibitors) using nanoliters and lower volumes is also discussed. The processes and potential applications described in this paper are based on the developments of inkjet dispensing technology by Microfab Technologies and its customers and collaborators.

2. BACKGROUND

Inkjet printing technology is familiar to most people in the form of desktop office printers. This technology allows for the rapid deposition of picoliter to nanoliter volumes of fluids onto submillimeter features in a highly repeatable manner at a reasonable cost. For over 30 years piezoelectric inkjet dispensing is well documented for a multitude of applications well beyond the scope of this paper. These applications include the printing of electronic materials ranging from embedded passive circuits, solder bumping to solar and fuel cell fabrication.1,2,3,4 Other areas include microoptics, OLED displays, vapor detector calibration and 3D printing for free form fabrication.5,6,7,8 The technology has also been proven invaluable for the creation of DNA, protein and antibody microarrays9,10,11,12, proteomic analysis13,14, drug coating15,16, biosensor fabrication17, tissue engineering and regenerative medicine.18,19,20,21

2.1 Demand Mode Inkjet Technology

There is a broad range of diverse technologies that fall under the inkjet printing category. The physics and the methods employed within this group may differ substantially, but the end effect is repeatable generation of small droplets of fluid. Most of these methods fall into two general categories, continuous mode and demand mode. Only the demand mode category will be discussed in this paper, since the equipment at Microfab Technologies is used mostly in the drop‐on‐demand configuration.

In a drop‐on‐demand inkjet printer, the fluid is maintained at ambient pressure and a transducer is used to create a drop only when needed. The transducer produces a volumetric change in the fluid which generates pressure waves. The pressure waves travel to the orifice and are converted to fluid velocity, which results in a drop being ejected from the orifice.22,23,24 The transducer in demand mode inkjet systems can be either a structure that incorporates piezoelectric materials or a thin film resistor.25 In the later, a current is passed through this resistor, causing the temperature to rise rapidly. The ink in contact with the heated resistor is vaporized forming a vapor bubble over the resistor. This vapor bubble creates a volume displacement in the fluid in a similar manner as the electromechanical action of a piezoelectric transducer.

Figure 1 displays a schematic of a drop‐on‐demand type inkjet system. Figure 2 displays an image of a drop‐on‐demand type inkjet device generating 60µm diameter drops of butyl carbitol (an organic solvent) from a dispenser with a 50µm orifice at 4,000 drops per second.

Demand mode inkjet printing systems produce droplets approximately equal in diameter to the orifice diameter of the droplet generator.26 Figure 1 indicates demand mode systems are conceptually far less complex than



continuous mode systems. However, they require the transducer to deliver three or more orders of magnitude greater energy to produce a droplet. The rate of droplet generation in commercial demand mode inkjet systems is in the 4‐12kHz range. Photographic quality printers use droplet diameters less than 10µm while in other commercial printers drop diameters up to 120µm have been demonstrated.

2.2 Fluid Property Effects on Inkjet Dispensing Performance

The properties of a fluid to be dispensed must be considered to obtain reliable jetting for the intended process and application.27 Surface tension and viscosity have the greatest effect on drop formation. Generally, a drop‐on‐demand piezoelectric dispenser will dispense a fluid (specific gravity ≈ 1.0) with the following properties:

Viscosity: 0.5‐40cp (Newtonian) Surface Tension: 20‐70 dynes/cm

Fluids with properties outside these ranges may be dispensed, but with increased difficulty (higher drive voltage, precise backpressure control) and decreased performance (slower and / or unstable drop, satellites, offset drop trajectory due to orifice face wetting). The fluid properties listed above are required at the orifice regardless if the fluid is heated or cooled. For high density fluids, such as molten metals, the values above should be converted to kinematic values. Newtonian behavior is not strictly required, but the flow conditions of the fluid properties at the orifice should be within the above range. Thus, a shear thinning fluid could have a low shear rate viscosity greater than the 40cp. Viscoelastic behavior which adds a component of stress inhibiting droplet breakoff is common for fluids with dissolved polymers. This includes biopolymers such as DNA, proteins, enzymes, etc, as well as biosorbable polymers, extracellular matrix materials, and polymers used for coatings. Viscoelastic effects increase with polymer concentration and molecular weight (length), creating operation limitations for both. In some cases, rheological measurements may indicate acceptable fluid properties, but the short time scales associated with inkjet dispensing result in an unstable response. Particle suspensions containing colloids, cells, etc., are acceptable as long as the particle / agglomerate size and density do not cause the suspension to depart from the fluid properties in the range specified above. Particles that are >5% of the orifice diameter (cells) may cause drop generation instability, but this may be acceptable in low concentrations. Fluid properties will also influence the spreading (lower surface tension = more spreading) on a substrate and ultimately affect the size, shape and morphology of the printed features. 2.3 Advantages of Inkjet Dispensing

Inkjet printing delivers subnanoliter drops onto a substrate in a precise and repeatable spatial and temporal manner using an order of magnitude less volume than conventional dispensing (micropipette / syringe / valve displacement dispensing). The digital nature of inkjet dispensing is advantageous for addressing on‐the‐fly the variability of printed patterns associated with tissue engineering applications. The printing of patterns is based on immediate user input via scripts, images or 3D scans, thus alleviating costly and time consuming masking and / or tooling requirements. The approach is amendable for addressing multiple materials, thus increasing process versatility to provide a greater flexibility in terms of design and the creation of custom one off prototypes.

As a noncontact printing process the volumetric accuracy of inkjet dispensing is unaffected by fluid interaction with the substrate while the risk of cross‐contamination is minimized. The droplets can be deposited separately or overlapping at a desired pitch to create microspot arrays, lines and 2D structures. Droplet accretion and stacking is used to increase the spot volume per position to create 3D structures. Multiple inkjet dispensers can delivery any combination of different fluids to the same location on a substrate. The ability to free‐fly droplets a millimeter or more provides for fluid dispensing into wells or onto other substrate features (e.g., features that are created to control wetting and spreading). Increasing printing throughput to address larger substrate features can be achieved by combining high resolution inkjet dispensing with other lower resolution freeform fabrication methods, such as extrusion or valvejetting. The combination of these methods demonstrates the flexibility associated with inkjet printing in the biomedical arena.



3. TISSUE ENGINEERING AND REGENERATIVE MEDICINE APPLICATIONS

Inkjet dispensing has proven valuable to tissue engineering (TE) and regenerative medicine for the ability to reproducibly deposit and fabricate architecture of cells, matrices and biochemicals in two and three dimensions (2D, 3D) to mimic native function. Creating the 3D structural orientation and mechanical properties of native tissue is critical for emulating function, as 3D tissue models have been found to be superior to 2D versions to emulate the cellular physiological environment.28,29 The repair, replacement and study of diseased / damaged tissues and organs require the structural orientation30, mechanical properties31 and chemical patterning32 to be analogous with the compromised native tissue in order to obtain meaningful studies from the appropriate function. The bioprinting approach is a method to advance the in vitro and in vivo study of biology and function of cells, tissues and organs by manipulating cells, hydrogels, polymers, extracellular matrix (ECM) and adjunct biochemicals into cell laden constructs.33,34,35,36 Inkjet printing offers an extraordinary degree of control in both tissue design and fabrication strategy, as droplets containing cells, scaffold materials and growth regulators can be deposited within a few microns of accuracy. The concurrent deposition of cells and other scaffold materials results in a more uniform distribution compared to cell sparse regions associated with conventional bulk cell seeding.37 These 3D fabricated tissues can be used in the association with animal testing as “humanized” xenografts,38 or individually for toxicology studies39 and cosmetic development40. These tissue modeling approaches may reduce or eliminate the need to perform costly in vivo animal testing studies. Engineered hepatocyte, skin, renal and other tissue models can be used for predictive studies41, toxicity assessments42, pharmacological compound screening43 and organ transplant studies44, or for the elucidation of disease development and progression where results are interpreted in the context of appropriate 3D cell to cell interactions45. For therapeutic tissues, the ability to use autologous cells can minimize artificial organ transplant rejection and the need for immunosuppressant drugs.46,47 Other therapeutic 3D printed tissues include grafting onto an impaired organ to supplement function or therapy delivery via peritoneal or subcutaneous implantation or extracorporeal tissues.48

3.1 Cell Dispensing

Inkjet Printing of Silk Nest Arrays for Cell Hosting

Silk fibroin is considered an alternative polymer to encapsulate and protect cells for biosensors49, tissue engineering50,51,52 and therapeutic drug applications53. Suntivich, et al investigated a drop‐on‐demand inkjet printing approach for the fabrication of microscopic arrays of biocompatible silk “nests” to host live cells on glass and flexible polymer substrates.54 Silk array patterning was accomplished using a multiple layer‐by‐layer (LbL) deposition of ionomeric silk modified with poly‐(L‐lysine) and poly‐(L‐glutamic acid) side chains. The resulting ≈100µm diameter LbL structures were stabilized via ionic pairing with the formation of silk II secondary structures to create “nest” morphologies having well‐defined rims. The “locked‐in” silk nests with depleted central regions and elevated rims remained anchored to the substrate during incubation in cell growth media. The encapsulation, immobilization and growth of E. coli cells within the array of silk nests were also verified.

A JetLab II inkjet printer (MicroFab Technologies, Plano, TX, USA) fitted with a 50 μm orifice diameter nozzle was used to print 6 x 6 microspot arrays of silk‐polylysine (1.0 mg/mL, 0.05M NaH2PO4, pH 5.5) onto polystyrene coated glass cover slips (Figure 3). A solution of 1.0 mg/mL silk‐polyglutamic acid (0.05M NaH2PO4, pH 5.5) was over printed on top of silk‐polylysine dots creating the initial silk bilayers stabilized via ionic interactions. The printing Figure 3 Cell encapsulation by inkjet printed silk array process.



process was repeated to produce multiple silk bilayers (1−10) without using an intermediate wash step. Dispersions of E. coli cells in synthetic minimal media (SSM) were printed onto the center of the silk nest regions followed by additional silk bilayers for cell encapsulation.

The surface morphology of the printed silk bilayers was characterized using a Bruker Dimension 3000 scanning probe microscope (Bruker Biosciences Corp., Billerica, MA). The surface morphology of 1, 3 and 5 bilayer nests is shown in Figure 4 as having a thickness of 120nm, 340nm and 570nm, respectively. The characteristic coffee ring nest shape was thought to be a result of solution impact, outward microflow distribution, and the difference in evaporation rate between the center and the periphery of the deposited material during formation of the so called coffee‐ring structures.

Cell encapsulation within the printed silk arrays was validated by overprinting dispersions of E. coli cell into the center of the dried silk nest regions followed by the addition of silk bilayers to complete the encapsulation process. Optical microscopy revealed the E. coli cells were encapsulated consistently within the silk nests (Figure 5 left images). The cells were confined within the rim of the silk dots and did not spread to other areas on the substrate. AFM analysis also confirmed the cell encapsulation at a high density within the silk nests (Figure 5 middle, right images). The preservation of the cylindrical shape of E. coli cells was also apparent on the

nest shape regions of silk within the arrays. The silk nest approach has the potential for immobilizing different cell types within multiplexed arrays for biosensing and biodetection of various chemical and biological agents for tissue engineering, biosensing and pharmaceutical applications.

Microenvironments Engineered by Inkjet Bioprinting Spatially Direct Adult Stem Cells Toward Muscle‐ and Bone‐Like Subpopulations

Spatially defined patterns of immobilized growth factors were created using inkjet bioprinting to develop biologically relevant approaches to study stem cell behaviors. Phillippi, et al used this approach to manipulate the cell fate toward the osteogenic lineage while in register to printed patterns of bone morphogenetic protein (BMP) 2 contained within a population of primary muscle derived stem cells (MDSCs) isolated from adult mice.55 The approach was conducive to patterning the MDSCs to exploit the inherent myogenic potential of the MDSC into subpopulations of osteogenic or myogenic cells simultaneously on the same chip.56 When cells were cultured under myogenic conditions on BMP‐2 patterns, cells on pattern differentiated toward the osteogenic lineage, whereas cells off pattern differentiated toward the myogenic lineage. Time‐lapse microscopy was used to visualize the formation of multinucleated myotubes while immunocytochemistry was used to demonstrate expression of myosin heavy chain (fast) in cells off the BMP‐2 pattern. The spatially controlled multilineage differentiation of stem cells using inkjet printed patterns of immobilized growth factors was demonstrated. The

Figure 4 AFM cross section for A) 1, B) 3 and C) 5 printed bilayers (2000nm Z scale).

Figure 5 Optical and AFM microscopy of silk nestencapsulated E. coli cells. Left: optical; middle, right:AFM.



method achieved has potential for application in regenerative medicine for elucidating the behavior of cells when subjected to immobilized biological patterns. For this study patterns were printed using a bespoke inkjet‐based bioprinting system fitted with a 20 µm diameter orifice drop‐on‐demand piezoelectric inkjet dispenser (MicroFab Technologies, Inc. Plano, TX, USA). The drop volumes of ≈14 pL resulted in spot sizes on the glass substrate of ≈75 µm in diameter. The bioink, consisting of 100µg/mL Cy3‐labeled BMP‐2 in phosphate buffered saline (PBS), pH 7.4 was printed onto fibrin‐coated glass slides. The printed patterns were 10 x 10 arrays having a 75 µm spot pitch with 1.75 mm between the squares. The squares were printed as 2 (28pL), 8 (112pL), 14 (196pL), and 20 (280pL) overprints of the bioink to

modulate the surface concentrations (Figure 6). The patterns were incubated at 37°C with 5% CO2 and 95% humidity in serum‐free Dulbecco’s modified Eagle’s medium (DMEM) with 1% penicillin streptomycin and were imaged every 24 hours up to 144 hours by epifluorescence microscopy using a Cy3 filter set. The spatial control of the differentiation of two lineages of MDSCs simultaneously in the same culture using an inkjet printing approach was demonstrated. MDSCs were cultured on 2x2 patterns containing four different surface concentrations of BMP‐2 under myogenic culture medium conditions. The MDSCs on all BMP‐2 patterns showed dose‐dependent alkaline phosphatase (ALP) response without myotube development (Figure 7). Outside the printed BMP‐2 patterns the cells began to

fuse and form multinucleated myotubes without exhibiting ALP activity indicating differentiation toward the myogenic lineage. These results demonstrate a proof‐of‐concept for spatial patterning of multiple lineages from a single population of stem cells simultaneously within the same culture well.

Towards the Inkjet Fabrication of Artificial Cells

A novel method for the fabrication of cell‐like microparticles comprised of a hydrogel core and a mesoporous silica shell was developed by Haufova, et al.57 The two step process involved 1) the inkjet fabrication of calcium alginate cores ranging from 40‐90µm containing immobilized nanoparticles and liposome based internal compartments; 2) a sol‐gel process for depositing a thick mesoporous silica layer on the microparticle surface using alkoxysilane precursors. The structure of the silica coating and the effect on diffusion rates of proteins with increasing molecular weight (lysozyme, myoglobin, ovalbumin and BSA) was elucidated. The silica shell was shown to act as a semi‐permeable barrier with the diffusion of small proteins while blocking larger proteins.

A drop‐on‐demand inkjet dispenser having an 80µm diameter orifice (MicroFab Technologies, Inc. Plano, TX, USA) was used to fabricate the alginate cores by dispensing sodium alginate (1.0% w/v) with 4 mg/mL suspended Fe3O4 nanoparticles (11nm) into a 1.0% w/v calcium chloride solution. The size range of the resulting microspheres was 40‐90µm. A silica shell was then formed on the rinsed and separated alginate microspheres that contained the Fe3O4 nanoparticles by suspension in neet n‐hexane followed by the addition of APTrMOS and then TMOS, each stirred for 1 minute after addition respectively. Diffusion rates and uptake kinetics of proteins with increasing molecular weight were measured for uncoated and silica coated alginate microspheres using lysozyme (14.7kDa), myoglobin (17kDa), ovalbumin (44kDa) and bovine serum albumin (66kDa).

Figure 6 2d patterns of cyanine 3‐labeled BMP‐2 printed onto fibrinfilms. (A): 2 (B): 8 (C): 14 (D): 20overprints.

Figure 7 Osteogenic differentiation in register to printed patterns. BMP‐2 overprints: 1) 2 ‐ 28pL 2) 8 – 112pL 3) 14‐ 196pL 4) 20 – 280pL.



This approach resulted in highly uniform microspheres varying 40‐90um in diameter with the potential to scale up or down by simply changing the orifice diameter (Figure 8). The diffusion rate of proteins across the silica shell were measured as a function of molecular weight, as the intended application is for artificial cells for in situ production and processing of biomolecules. The larger proteins (ovalbumin, BSA) were effectively blocked by the silica layer, while the smallest protein lysozyme passed rapidly. The intermediate sized protein, myoglobin was found to be semipermeable. The incorporation of iron oxide nanoparticles improved handling properties via magnetism, while the immobilization of liposomes in the alginate core indicated a potential role as artificial vacuoles. This proof of concept demonstrated the inkjet based fabrication of artificial cells having internal compartments to store reagents, immobilized enzymes and catalyst nanoparticle to facilitate reactions enclosed within an outer membrane which provided mechanical protection and transport regulation. Adult Rat Retinal Ganglion Cells and Glia Can Be Printed by Piezoelectric Inkjet Printing The study and development of alternative methods to promote functional recovery from neurodegenerative diseases and spinal cord and the brain injury is a significant focus of regenerative medicine. Thus far only embryonic neuronal cell types including hippocampal, cortical and motor neurons have been evaluated for their viability after thermal inkjet printing.58 At the time of this writing there were no other studies on the inkjet printing of viable cells derived from the eye or the CNS. Lorber, et al hypothesized that CNS embryonic and adult neuronal cells might be more susceptible to adverse effects of piezoelectric drop‐on‐demand dispensing, since CNS cells have a more limited ability to survive and regenerate in contrast to embryonic cells.59,60 To test this adult rat central nervous system (CNS), retinal ganglion cells (RGC) and glia were inkjet dispensed to assess the effects on viability and growth. High speed imaging of the printhead nozzle in the area where the cells experience the greatest shear stress and rate confirmed that there was no evidence of significant distortion or destruction of the cells during drop ejection and formation. Viability testing and culture of the printed and non‐printed RGC/glial cells indicated no significant difference in cell survival and RGC neurite outgrowth. Additionally, the use of a glial substrate significantly increased RGC neurite outgrowth of both control and printed cells. Retinal glial cultures were derived from retinal tissue of adult male Sprague Dawley rats (Charles River, Margate, UK). Purified glial cultures with astrocytes and Muller glia were prepared for dispensing via calcium/magnesium‐free phosphate buffered saline (PBS) wash followed by incubation for 5 min in a 1 x Trypsin‐EDTA followed by centrifugation with resuspension in DMEM (and 10% fetal calf serum). Dissociated retinal cultures were also obtained from Sprague Dawley rats using a papain dissociation kit (Worthington Biochemicals, New Jersey, USA) with resuspension in B27 supplemented Neurobasal‐A medium prior to printing. Piezoelectric dispensers having

Figure 8 Left image: optical micrograph of a silica coated alginate microsphere with encapsulated iron oxide nanoparticles; right image: Uptake kinetics of proteins by uncoated and silica coated alginate microcapsules. Dashed lines with open symbols = uncoated capsules, sold lines with closed symbols = silica coated capsules (230nm thickness); Myoglobin 10x = 2.3um think silica shell.



an orifice diameter of 50µm and 80µm (MicroFab, Texas, USA) were used to print purified retinal and dissociated glia cells. Viability testing was performed using a trypan blue test before (control) and after dispensing. The average maximum retinal cell and glia drop velocity was 1.3 m/s and 1.0 m/s, respectively. High shear rate and stress were nevertheless present in the fluid during the jetting process. The shear and extensional forces imposed on the jetted cells are likely to be highest during the early stage of ejection as they pass through the nozzle. The maximum fluid shear rates were estimated from the ratio of the drop

velocity to the nozzle diameter to be on the order of 105 s−1. It required 2–5 μs for the ejected drops to reach their maximum velocity, corresponding to an average acceleration of at least 106ms−2 (or 105 times that of gravity). However, observations of the cell motion when a drop was fully formed showed the acceleration in the later stages to be significantly lower. Magnified images (Figure 9) taken during the initial jetting phase (50 μm nozzle/retinal cells, 80 μm nozzle/glia cells), using an automated ultra‐fast freeze‐frame imaging showed no evidence of significant deformation once the cells are outside the printhead. Real‐time observation of the glass nozzle during jetting showed no sign of active cell disintegration despite the very high shear rate and acceleration to which the cells had been subjected (Figure 10). In addition no significant difference in the viability (glial 78% vs 69%, retinal 74% vs 69%) and number of surviving cultured printed and control cells was observed. A cell jetting study with realtime imaging dynamics demonstrated a drop frequency of 1.0 kHz with a drop velocity of 1.3m/s and 1.0m/s did not significantly affect the viability of printed adult RGC and glial cells which then retained their phenotype in culture. Although the cells were subjected to very high shear rates and acceleration during jetting, no significant distortion of the cell structures was observed either immediately before or after cell ejection. The observations suggest that either the cell membranes possess sufficient strength and elasticity to resist a brief period of high stress, or the geometry of the printhead nozzle used results in rather little shear or deformation of the cells during jetting. The printing of these cells in precise patterns may enable cell arrays to mimic the in vivo study of novel compound screening on cell–cell interactions. Demonstrating that printed glial cells maintain growth‐promoting properties opens new avenues for CNS grafts in regenerative medicine, such as printing light sensitive photoreceptors to create a retina to improve patient vision or alleviate blindness associated with disease or injury.

3.2 Growth Factors

Precise Control of Osteogenesis for Craniofacial Defect Repair: The Role of Direct Osteoprogenitor Contact in BMP‐2‐Based Bioprinting

The bone tissue reconstruction of large calvarial defects has been reported extensively using bone morphogenetic protein‐2 (BMP‐2).61,62,63 However, these treatments require enormous doses of BMP‐2, which is

Figure 9 Retinal and glial cells in drops exiting the dispenser orifice. Arrowindicates individual cells tracked for analysis. Scale bar = 100µm.

Figure 10 Top: Number of RGC surviving in culture;Bottom: Number of retinal glia cells surviving in culture.Control vs printed.



a potent morphogen. In addition the large dose applications of BMP‐2 do not facilitate the detail oriented repair required for intricate craniofacial structures. Inkjet bioprinting has been used previously for the precise customized low‐dose protein patterning to induce spatially regulated osteogenesis using significantly lower doses of BMP‐2 than conventional means.64 Further development of this approach would be to stack 2‐dimensional constructs to generate 3‐dimensional bone shapes. Therefore, this next step investigated the importance of direct contact between bioprinted BMP‐2 and the dura mater (a source of osteoprogenitors) in mediating calvarial healing. The BMP‐2 bioink was printed onto 5mm diameter acellular dermal matrix (ADM) discs (DermaMatrix; Synthes, West Chester, PA, USA) with a custom 2‐dimensional bioprinting system fitted with an inkjet dispenser having a 30µm diameter orifice from MicroFab Technologies (Plano, TX, USA). One semicircle of the dermal surface of each ADM disc was printed with BMP‐2 bioink. Each semicircle printed with BMP‐2 (Medtronic; Memphis, TN, USA) received 50 overprints of ≈ 14pL drops delivering a cumulative total of 155.4 ng of BMP‐2. Excess (unbound) BMP‐2 was rinsed from the discs by a sterile PBS bath for 24 hours. Notches were cut in the discs opposite the BMP‐2‐printed area

to maintain orientation upon implantation. Eight male C57‐BL6 mice (Jackson Labs; Bar Harbor, ME, USA), 7 to 8 weeks of age were trephinated in the right parietal lobe to create a 5.0mm craniotomy. The craniotomy defect was filled with a BMP‐2 printed disc using the following treatments: BMP‐printed dermal surface toward dura, BMP‐printed dermal surface away from dura, unprinted dermal surface toward dura, and unprinted dermal surface away from dura (Figure 11). Radiographic evaluation and histology for analysis of osteogensis was performed 4 weeks after implantation (Figure 12). The percent ossification upon faxitron was compared between each of the 4 semicircular treatment conditions. Bone covering occurred on 66.5% (mean) of the surface area when the BMP‐2‐printed portion of the ADM was face‐down with the printed surface directly abutting dura mater. In comparison, the BMP‐2‐printed portion of the ADM generated bone covering an average of 21.3% of its surface area when it was face‐up with the printed surface away from dura. The unprinted portion of the ADM generated an average of 18.6% osseous coverage when face‐down and 18.4% when face‐up. Thus this study

demonstrated the BMP‐2‐printed ADM must be in direct contact with the source of osteoprogenitor cells (dura mater) to maximize the amount of osteogenesis. Although the results further indicate the potential of inkjet bioprinting BMP‐2 for craniofacial defect repair, the orientation of the treated construct must be taken into account when designing more complex bioprinted osseous structures.

Spatially Directed Guidance of Stem Cell Population Migration by Immobilized Patterns of Growth Factors

Understanding how chemotactic methods influence cell population migration is valuable for elucidating the potential effects on tissue development and repair in tissue engineering applications, such as maximizing stem

Figure 11 Schematic orientation of implanted ADM discs within the defects indicated bywhite bone on edges. The defect on the left has ADM with the dermal surface (****)facing away from the dura, and the defect on the right has ADM with the dermal surfacefacing towards the dura. The four treatment conditions are labeled A (BMP‐printed dermalsurface away from dura), B (unprinted dermal surface away from dura), C (BMP‐printeddermal surface towards dura), and D (unprinted dermal surface towards dura).

Figure 12 Faxitrons comparing ossification between discs with BMP‐printed surface up (away from dura) and down (toward) dura. The arrows indicate the border between BMP‐printed and unprinted semicircles. The printed semicircle is medial in both faxitrons.



cell ingrowth from a wound site source into tissue engineered constructs. Miller, et al investigated the effect of exogenous growth factor gradients immobilized to an extracellular matrix material on the guidance of stem cells for greater than 1 day with a region >1.0mm.65 They printed patterns of heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) onto fibrin substrates at low‐to‐high, high‐to‐low, and uniform concentrations. Time lapse video microscopy and vision‐based cell tracking analysis was used to record and interpret the proliferation and migration of mesenchymal stem cells on the HB‐EGF patterned substrates. It was anticipated that the low‐to‐high gradient would be the most effective to direct cell guidance based on recognized chemotaxis studies. 66,67,68 However, cell guidance was similar regardless of the printed pattern treatment suggesting a uniform distribution of immobilized growth factor may be as effective for controlling cell migration as complex concentration gradient. The indifference was attributed to: 1) cell diffusion and cell proliferation associated with a loss of contact inhibition; 2) spatially controlled proliferative growth factor cues; and 3) spatially directed growth factor migration cues. The growth regulator HB‐EGF was printed onto 18mmx18mm fibrin coated cover slips using a custom inkjet‐based bioprinting system with a 20 µm diameter orifice drop‐on‐demand piezoelectric inkjet dispenser (MicroFab Technologies, Inc. Plano, TX, USA). The printed growth regulator contained 100 mg/mL HB‐EGF (R&D Systems, Minneapolis, MN) in 10 mM sodium phosphate, pH 7.4. Spot arrays as shown in Figure 13 were printed (75µm x 80 µm pitch) onto the fibrin coated coverslips as a uniform concentration (13 overprints ≈ 300pL), a low‐to‐high concentration gradient (1 to 25 overprints ≈ 23pL to

575pL), and a high‐to‐low gradient (25 to 1 overprints≈ 575pL to 23pL). The control consisted of an unprinted fibrin coverslip. Unbound growth factor was removed by rinsing 3x in PBS followed by storage in serum‐free Base DMEM with 1% penicillin/streptomycin (PS) (Invitrogen, Carlsbad, CA, USA) in a cell culture incubator at 37 °C, 5% CO2. The overprinting method reduced the spread of HB‐EGF maintaining a spot size on the fibrin coating of 50µm to 60µm. Mouse mesenchymal stem cells (C3H10T1/2 clone 8) in serum free media were then seeded onto the HB‐EGF spot patterns (0.5ml of ≈25,000 cells/mL). The cells were allowed to attach to the seeded spot patterns coverslips for 1.5 hours in an incubator at 37 °C, 5% CO2. The coverslips were then rinsed with serum‐free Base DMEM and secured to the bottom of a 35mm Petri dish with 4 mL of media containing 0.2% CS, 1% PS, and 1 mg/mL aprotinin followed by imaging using time‐lapse microscopy. After 4 days in culture the images of the cell response on the control and printed patterns were recorded are shown in Figure 14. The cell density and front movement was minimal for the control. The cell fronts

on the uniform, high‐to‐low, and low‐to‐high HB‐EGF patterns displayed more significant movement. The relative positions of the cell migration fronts over 72 hour in response to the control and printed patterns are displayed in Figure 15. The control groups had relatively small migration front movement and lower cell density than the printed HB‐EGF treatments. The

Figure 13 Printed HB‐EGF patterning and dimensions. Photo inset displays actual uniform printed Cy3‐HB‐EGF fibrin coated coverslip.

Figure 14 The C3H10T1/2 cells were seeded at the bottom of the patterns andimaged over time. The images shown are after 4 days in culture for thecontrol, uniform pattern (13 overprints), high‐to‐low gradient (25‐1overprints) and low‐to‐high gradient (1‐25 overprints).



movement of the migration front for printed patterns was similar regardless of pattern type. The cell population expansion rates were calculated by dividing the measured cell counts over time by the initial cell count for each pattern. The cell count changes for the control patterns were 3 times the initial cell count. In contrast, the cell counts were 7.9, 8.2 and 7.3 times the initial values for the uniform, high‐to‐low and low‐to‐high gradient patterns, respectively. These results indicate that the migration of “non‐sheet” forming tissue‐based stem cells is governed by complex interactions between multiple forces and

not by single cell directional growth factor cues alone. The dynamic interactions between cell population diffusion, proliferation and directed migration (printed growth regulator) govern the net movement of a stem cell population away from their initial source. This new knowledge has potential impact for designing tissue engineered scaffolds which incorporate spatial patterning of growth factors.

3.3 Scaffold ‐ Construct Fabrication

Scaffold‐Free Inkjet Printing of Three‐Dimensional Zigzag Cellular Tubes

Xu, et al investigated drop‐on‐demand inkjet printing technology for the fabrication of three‐dimensional (3D) cellular tubes in a zigzag pattern to establish methodology to create vascularization in thick tissue constructs.69 A custom 3D inkjet bioprinting system was used to fabricate complex 3D constructs of alginate containing cells (Figure 16). The resulting tubes having an overhang structure containing fibroblast (3T3) cells with a viability greater 82% (or 93% with the control effect considered) after a 72 hours in culture.

NIH 3T3 mouse fibroblasts (ATCC, Rockville, MD, USA) were cultured in Dulbecco’s Modified Eagles Medium (DMEM; Sigma–Aldrich, St. Louis, MO, USA) supplemented with 10% Fetal Bovine Serum (FBS; HyClone, Logan, UT, USA) at 37°C and 5% CO2. Prior to inkjet printing the confluent flasks of 3T3 fibroblasts were washed twice with Dulbecco’s phosphate buffered saline (PBS; Cellgro, Manassas, VA, USA), and incubated with 0.25% Trypsin/EDTA (Sigma–Aldrich, St. Louis, MO, USA) for 5 min 37°C and 5% CO2 followed by centrifuging at 1,000 rpm for 5 min. The cells were resuspended in DMEM at a concentration of 6x106 cells/mL. The cell suspension was mixed 1:1 with the 2% (w/v) sodium alginate (Sigma– Aldrich) in DMEM at a volume ratio of 1:1, resulting in a final solution containing 1% (w/v) alginate and 3x106 cells/mL. The alginate and 3T3 cell solution was printed into a 2% (w/v) solution of calcium chloride dihydrate (Sigma–Aldrich, St. Louis, MO, USA) for gelation. A 0.4% trypan blue stain (Sigma–Aldrich, St. Louis, MO, USA) was used for live dead cell viability testing.

The alginate and 3T3 cells were printed at 50Hz using a 120 µm diameter orifice inkjet dispenser (MJ‐ABL‐01‐120‐6MX Microfab Technologies, Plano, TX, USA) attached to a Z motion stage on a Cartesian printing system as shown in Figure 16. The orifice was maintained at a 5 mm offset distance above the top of the construct surface

Figure 15 (A) C3H101/2 cell migration trajectories after 72 h for arepresentative experiment with different colors representing differentcell lineages. (B) The 90% cell migration front positions as calculatedover time. (C) The relative cell population expansion (the ratiobetween the current cell count and the initial cell count) over time.

Figure 16 Schematic of the 3D inkjet zigzag pattern constructprinting system.



and CaCl2 fluid level during fabrication. The Z‐stage moved down vertically at a constant speed of 0.2mm/s to create the tube in the gelation fluid along the Z direction, while X,Y printing a 3 mm circle at 200mm/min. This resulted in a single deposited layer of cells and alginate of approximately 70µm thickness. The printhead was moved 20µm after each layer of printing to create the approximately 65°offset for the overhang of the zigzag structure. A total of 210 layers were printed resulting in a 10mm high structure. The printed cellular tubes were cultured in complete DMEM prior to assessment of cell viability (Figure 17). Cell viability after 24 hours in culture was 87% compared to 91% for the control. After 3 days cell viability was 82% vs 86% for the control. Lahooti, et al reported cell death in alginate microspheres related to nutrient transport kinetics and waste product accumulation.70 However, the post‐printing cell viability achieved in this study is equal to and higher than those of similar alginate based drop‐on‐demand inkjet studies.71,72 This study demonstrated drop‐on‐demand inkjet dispensing can to print complex tubular structures to establish vascularization for a variety of tissue engineering applications.

Inkjet Printing and Cell Seeding Thermoreversible Photocurable Gel Structures

The creation of well‐defined scaffold structures for cells to adhere and grow is essential in order to collect meaningful data during metabolic and proliferative activity studies. Fabrication techniques such as inkjet dispensing, are being developed to build scaffolds and other structures with complex internal architecture and controlled morphology and chemistry. To develop adequate mechanical properties for these support structures it is desirable that the printed scaffold fluid undergoes a phase change (gelling) after printing. The gelling phase transition must be isometric to preserve volume and aspect of the printed drops, yet be minimally exothermic to reduce the damage to previously deposited or encapsulated cells. Numerous inkjet printing studies have used physical gelling precursors including sodium alginate, which solidifies in the presence of Ca2+ ions, and thermal gelling polymers, such as poly(N isopropylacrylamide) (pNIPAm).73,74 Unfortunately, these physical gels often lack the strength and stability to be a useful tissue scaffold. Instability may occur due to limited cross‐links resulting in a gel with low elastic moduli and potential dissolution in the presence of sequestrants and conditions within the implanted environment. In response, DiBase et al developed a bioink containing acrylate functionalized Pluronic F127 for improved physical / chemical gelation and triethanolamine and eosin Y for photoactivated chemical cross‐linking.75 The physical gelation ensures a rapid phase change immediately after printing followed by a secondary chemical crosslinking to increase the stability and reduce material segregation. Complete curing of the gel is then achieved using a free radical biocompatible photopolymerization reaction. This system uses optical spectrum radiation for crosslinking to minimize potential damage to cells associated ionizing radiation. Pluronic (poly(ethylene glycol‐b‐propylene glycol‐b‐ethylene glycol)) Mw 12,600 g mol_1 containing a PEG content of 70% w/w was purchased from Sigma Aldrich (Gillingham, Dorset, UK) and used as received. Triethanolamine and poly‐ (ethylene glycol) diacrylate (PEGDA) with Mw = 575 g mol_1 were purchased from Sigma Aldrich (Buchs, Switzerland), eosin Y from Fluka (Buchs, Switzerland) and used as received. The Pluronic F127 diacrylate was synthesized as described elsewhere76 and mixed into the gel bioink with PEGDA, triethanolamine, eosin Y, (18, 2.0, 2.0, 0.02 % w/w aqueous, respectively). This resulted in a gel bioink having a viscosity of 30 mPa s at 5°C transitioning to over 150 mPa s at RT (Figure 18). The inkjet printing experiments were performed using a bespoke MPP‐1000 printing platform (School of Materials, University of Manchester) fitted with a MJ‐ATP‐ 01 inkjet dispenser having a 80 µm diameter orifice (Microfab Technologies, Plano, TX, USA) and a JetDrive III

Figure 17 Close‐up of printed cellular tube with magnified view showing cells embedded in the surface.

Figure 18 Viscosity of Pluronic F127 solutions as afunction of temperature and concentration.



controller (Microfab Technologies, Plano, TX, USA) to provide the electronic drive pulse. The printhead with polymer bioink solution and the substrate were maintained at 5°C and 20°C, respectively. The resulting ≈250pL drops were printed onto silanized glass slides on a 100µm pitch to create a series of square wells (500µm x 500µm) with 100µm wide walls and ≈150µm height (4 printed layers). The printed samples were crosslinked using a L.E. Demetron (SDS Kerr, Sybron Dental Specialties, Danbury, CT, USA) light source with a wavelength of 460nm. Human fibroblast cells (HT1080 fibrosarcoma) were obtained from the UK Centre for Tissue Engineering, University of Manchester, UK and were cultured at 37°C, 5% CO2 in Dulbeccos modified Eagles medium (DMEM, Gibco, Invitrogen, Paisley, UK) containing 4.5 gL‐1 glucose, GlutamaxTM, 10% foetal bovine serum (FBS, Gibco) and 1% penicillin and streptomycin and 50 mg L‐1 ascorbic acid V. Trypsinized cells suspensions were prepared containing 1 x 106 cells mL‐1. The cells suspensions were printed into the fabricated well structures at 5 and 20 drops per position resulting in 1.25 and 5 cells per well, respectively. Intracellular fluorescent dye CFSE (Carboxy Fluorescein Succinimidyl Ester) was used for visualization of the printed cells within the wells. The top two images in Figure 19 (top a and b) display the multi‐chamber grid structure after 5 drops of a cell suspension (1 x 106 cells mL‐1 with ≈0.25 cells per drop) were printed into each. The middle and bottom images of Figure 19 (bottom a, b, c, d) show cells printed into wells at 5 and 20 drops per well (1.25

and 5 cells per well) after 24 hours in growth media. A bioink that gels by tandem rapid physical gelation followed by a photoactivated chemical cross‐linking was developed. This ink is suitable for dispensing using a drop‐on‐demand inkjet printing system with a printhead held at 5°C. Multilayer structures on a substrate held at room temperature were created having dimensions consistent with models developed for the interaction of overlapping drops. Post photocrosslinked gel structures were seeded with fibroblast cells and incubated with cell growth for more than 48 hours. This approach presents a new inkjet scaffold fabrication method with potential applications in tissue engineering and cell based bioengineering. 4.0 CONCLUSION This position paper primarily reviews several inkjet dispensing applications to deposit living cells, growth factors, hydrogels and other biomaterials for basic research to address challenges in tissue engineering and regenerative medicine using equipment provide by Microfab Technologies. The greatest value of this paper is providing inspiration through example to tissue engineering and bioprinting developers for their research and development efforts. Although the focus of this paper is on applications using equipment provided by Microfab Technologies, a search engine, such as Google Scholar, would source many other inkjet dispensing applications adaptable to the Microfab’s inkjet dispensing technology. This paper will be revised on an ongoing basis to include information as latest developments arise.

Figure 19 Top a and b: 5 drops of cell suspension printed intophotocured multi‐chamber bioink grid (500x500um). Middleand Bottom: a, b ‐ cell seeding 5 drops, c, d ‐ 20 drops after 24hours in culture. Left side: optical. Right side: fluroescence.



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applications of inkjet technology for tissue engineering and