1

Fabricating Tissue Replacements using aHybrid Inkjet-Electrospinner 3D Printer

Amal Duriseti and Uri Nator - University of California Los AnglesBob Loblaw and Anurag Dikshit - University of WaterlooJack Goff - Carnegie Melon

Abstract

In this paper we introduce a technique for producing mechanically superior fibrous scaffolding and controllingcell differentiation when fabricating tissue. This approach uses a inkjet head working in tandem with anelectrospinning head to deposit a layers of induced pluripotent stem cells interspersed with layers of growthfactor laced PCL. These implants have excellent mechanical properties, and outperform alternate gel based tissuemanufacturing strategies in both stiffness and tensile strength, making them suitable for applications like cartilagereplacement. Cells in these implants have high survival rates both in vitro and after incubation because the initialcell suspension can be relatively sparse, allowing each cell access to ample resources. The growth factor basedmethod of controlling cell differentiation has a highly localized effect in the implant, allowing for a high level ofprecision when controlling expressed tissue type. This study demonstrated the viability of this method in creatinga complex circulatory system - additonally, these findings indicate that not only can cell differentiation be used tocreate complex circulatory structures, but also that this artificial vascular system can improve cell proliferation andsurvival rates for other cells in the implant.

I. INTRODUCTION

In addition to the more obvious applications of3D printing in mechanical and industrial engineer-ing, researchers in the medical field have exploredthe possibility of using 3D printing to fabricatereplacement organs. While the intricacy and inher-ently variable nature of living tissue lends itselfwell to the strengths of 3D printing, the crucialyet subtle differences in makeup between tissuesof close proximity and the fragility of any solutionof cells used for deposition preclude transplantsof all but the most simplistic of organs 1. Thetwo biggest obstacles to any 3D printed organ arethe fragility of the deposit in both solution formand after addition. Because cells require proxim-ity to others, but paradoxically also require ampleaccess to resources (oxygen, food, etc...) in theirenvironment, a solution of bioactive material withsufficient cellular density can’t be sustained forlong prior to deposition. Additionally any cluster oftissue without intelligent differentiation (i.e. a welldeveloped circulatory system) will become necroticafter deposition4.

In this paper we explore a method of creatingmechanically superior tissue implants with a com-plex circulatory system. Traditionally most methodsof tissue fabrication have attempted to create abiodegradable matrix similar to that of the body’sown extracellular matrix (ECM) using ink-jet 3Dprinting techniques2. Cells are embedded in thismatrix in sufficient numbers to replace their housingas the body breaks it down, ensuring the implantturns into healthy tissue. This method has the ad-vantage of generating bio-active tissue artificially,at the cost of a fragile implant. During the timeactive cells replace the matrix, a patient must becareful to limit stress placed on the implant areato avoid damaging the structure of the matrix.Additionally, it is unclear as to whether the tissuematrix produced by the implant seed cells will beas strong, if the initial 3D printed matrix is non-fibrous. This technique also has the disadvantageof limited control over what hormones and growthfactors seeded cells see within the matrix. Thismakes controlling cell differentiation within tissuechallenging2. To address these shortcomings, we

2

attempt to create an initial biodegradable implantlaced with cell differentiation hormones and stemcells with a fibrous structure similar to the ECM.This artificial structure is achieved using a novelcombination of electrospinning and ink-jet 3D print-ing technologies.

II. MATERIALS AND METHODS

A. Printer Design and Additive Manufacturing Pro-cess

Our hybrid 3D-printing testbed was constructedby incorporating an electrospinning mechanism ontoan ink jet printing platform. Below is a diagramshowing the setup:

2

Figure 1: Experimental Apparatus

The inkjet printing platform used consisted of anXYZ step motor driven CNC base with a solenoidinkjet deposition system. The suspension containingthe non-differentiated stem cells was supplied tothis print head using pressurized air. The printheadpositing and deposition were controlled with customsoftware build using Microsoft’s .NET Framework.

The electrospinning head was mounted in tandemwith the XYZ plotter housing the inkjet depositionapparatus. The electrospinning mechanism used aDC voltage supply to draw fibres from an arrayof reservoirs containing ECM fibers laced withdifferent combinations and concentrations of growthfactors and differentiation hormones.

B. Inkjet and Electrospinner SolutionThe solution fed to the ink jet head contained

a suspension of induced pluripotent stem cells.These stem cells were derived from adult fibroblaststhrough isolation and introduction of genes: Oct3/4,Sox2, Klf4, and c-Myc with a retroviral system.

These stem cells were suspended in a gel con-sisting of fibrinogen and collagen to add viscosity

and additional structural integrity to the implant.To ensure the stem cells had access to sufficientresources, but also had sufficient density to populatethe implant’s artificial matrix, the cell suspensionwas made to have a density of ≈ 4∗106 cells ml−1.

The solution feeding into the electrospinner headused PCL as its artificial polymer. Pluronic F-127was used as an additive to reduce the viscosity andsurface tension of the solution, allowing for greatercontrol over the printing process.

Multiple resevoirs fed to the electrospinning head- the main difference between the solutions theyhoused being the type and concentration of growthfactors suspended with the PCL polymer. Becausethese additives affected the viscosity and surfacetension of the solution, Pluronic F-127 was usedin different quanities for each solution to ensurehomogeneity across solutions.

C. Printing ProcessImplant fabrication involved deposition of a lay-

ers of artificial fibrous matrix alternating with de-position of the cell suspension containing the stem

3

cells. During deposition, the substrate was kept wetbecause electrospinning completely dries depositedfibers, turning them into a dessicant.

Cell differentiation was controlled during thedeposition of the electrospun layer. These implantswere designed with the goal of producing tissuewith a functional circulatory system, so the elec-trospinning head deposited PCL laced with vas-cular endothelial growth factor, which promotesdifferentiation into blood vessel cells, into a pre-generated 3D model of the desired structure of thecirculatory system. Elsewhere, the PCL matrix wasdeposited with fibroblast growth factor to encouragedifferentiation into connective tissue.

III. TESTING

A. Bio-viability and differentiation of Implant

The survival rate and degree of differentiationof implants was assessed a week after deposition.During this week, the implants were allowed to sitin a relatively nutrient rich and well oxygenatedsolution designed to mimic internal body condi-tions. To test for cell survival, the constructs wererinsed with PBS and examined under a fluores-cent microscope. Number of live cells stained withcalcein AM (fluorescing green) was compared tototal number of cells. Cell counts were conductedon implants with and without PCL deposited withvascular endothelial growth factor, testing whether adeveloped circulatory system can help maintain cellproliferation and survival.

To test for differentiation, flourescent moleculeswere bonded with ligands known to associate withproteins expressed only on endothelial cells. Aftera bath in these floursecet ligands, flourescence pat-terns for the implants were cross referenced with the3D model for endothelial growth factor laced PCLdeposition.

B. Mechanical Viability

Mechanical quality of hybrid scaffolds with andwithout cells was compared to that of implantsgenerated without electrospinning using alginate ora collagen/fibrin gel. The thickness of the sampleswas measured, and the maximum tensile strengthrelative to distance stretched was measured and usedto calculate Young’s modulus and ultimate tensilestrength UTS.

C. In-Vitro Testing

Because the solution bath designed to mimic bodyconditions couldn’t control for factors like immuneresponse and hormone proliferation external to theimplant, the behavior of the implants in vitro wasalso examined. Fibroblasts were harvested fromsubjects separately and reverted to pluripotent stemcells. They were then grown in sufficient quantitiesto make a cell suspension for the inkjet head of theprinting apparatus. After fabricating the implant, theprosthetics were inserted back into the subjects whodonated cells for the cell suspension.

The implants were removed from subjects at 2,4, and 8 weeks, and cell differentiation and survivalright were examined in the same way as for the onessuspended in a solution bath. The hybrid naturaland artificial ECM was examined for any signs ofimmunological response. Additionally, the subjects’implant areas were examined for any effects of thegrowth factors laced into the implant.

IV. RESULTS AND DISCUSSION

This research demonstrated the viability of anovel approach to both tissue scaffolding fabricationand cell differentiation control using 3D printing.PCL was used because of its ease of manufacture, itsbiodegradability , and its high mechanical quality.The apparatus used for deposition proved capable ofdepositing a consistently fibrous matrix layer, withminimal beading more typical of gel scaffoldingtechniques. The resulting matrix had a higher tensilestrength and greater thickness than implants of al-ternate manufacture, improving chances of implantintegration. Cells deposited in this matrix and incu-bated in a body-like bath demonstrated high survivalrates and were able to proliferate as well as replacethe artificial ECM with a natural one. Additionally,cell counts for the implants with PCL laced withvascular endothelial growth factor were higher forall test cases, indicating that some vascular behaviorwas achieved.

The technique of controlling cell differentiationby lacing the artificial ECM with targeted growthfactors also proved successful. The matching qualityof the florescent pattern to deposition XY coordi-nates was given by the formula, where δX and δYare the shortest distances in the X and Y directions

4

from a deposition cycle to an observed flourescencepoint.

Matching quality =δXavg + δYavg

largest sample dimension

The matching quality for samples was 0.032, whichcorresonds to a percent difference of less than 5

percent, indicating that the fluorescence patternsgenerated after ligand based isolation of differen-tiated cardiovascular and connective tissue matchedstatistically significantly with the 3D model usedto guide deposition of PCL. Below is a display ofthe computer model used to guide PCL depositioncompared to observed flourescence patterns:

2

Figure 2: 3D Model to Guide Endothelial Growth Factor Laced PCL

Figure 3: Observed Flourescence Patterns at 2 weeks(left), 4 weeks(middle), and 8 weeks(right)

In vitro testing proved sucessful from an implantsurvivability and differentiation standpoint. The hosthad no immunological response to the implant orthe PCL matrix. Additionally, the host’s naturalhormone balances didn’t affect cell differentiationwithin the implant. However, the effects of theimplant on surrounding tissue is cause for someconcern. High levels of both fibroblast and endothe-lial growth factor are linked to diseases most organsystems. Additionally, high levels of these hormonesmay be carcinogenic. That said, this study did nottrack effects over a long enough period of time toprovide useful data on the subject.

V. CONCLUSION AND FUTURE DIRECTION

This research demonstrated the viability of a hy-brid electrospinner-inkjet deposition system in pro-ducing implants with high mechanical quality andgood cell differentiation. Not only was it possible tocontrol differentiation to produce complex vascularstructures, but cell proliferation rates indicated thatthis differentiation improved implant bioavailability.Despite these promising results, this technologyis currently very limited. The method of growthfactor lacing demonstrated in this study limits dif-ferentiation processes to single hormone cultivation

5

cycles. As a result this method can’t differentiate ahomogeneous stem cell solution into all the tissuetypes needed for organs like a kidney or a liver. A3D printing technology with this capability couldstill use the growth factor laced PCL matrix, butit would have to use a more complicated inkjethead capable of depositing many different typesof already partially differentiated cells, and couldpossibly have to be organ specific. Future researchcould explore this idea.

The electrospinning process proved capabale ofselectively differentiating cells, but it also gave theimplant excellent tensile strength and stiffness. As aresult, implants generated using this method couldhave high stress applications like cartilage or con-nective tissue replacement. Although not supportedin this study, it is possible that greater controlover the structure of the PCL matrix could guidethe artificial ECM replacement to produce highlyordered tissue with mechanical quality equal to or

greater than natural tissue.Despite the many advantages of this tissue fab-

rication process, the health risks of inserting highconcentrations of growth factor into host were notaddressed in this study. Abnormally high levels ofany type of growth factor are almost universallybad and are generally linked to health compli-cations in other organ systems. Additionally, anygrowth factor, while necessary for development andmaintenance, is inherently carcinogenic because itencourages cell growth and reproduction. None ofthe subjects of this study exhibited any abnormalgrowth around the implant area or seemed no-ticeably unhealthy. However, because of the shorttimeframe in this study, this insufficient to ruleout health complications. Before this technologybecomes available to the public, further researchmust be conducted to determine this the safety ofthis method of cell differentiation.

2

6