3D Bioprinting of Spatially Heterogeneous Collagen Constructs for

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3D Bioprinting of Spatially Heterogeneous Collagen Constructs for Cartilage Tissue Engineering Stephanie Rhee, Jennifer L. Puetzer, Brooke Mason, Cynthia A. Reinhart-King, and Lawrence J. Bonassar ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00288 • Publication Date (Web): 18 Jul 2016 Downloaded from http://pubs.acs.org on July 22, 2016

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ACS Biomaterials Science & Engineering

3D Bioprinting of Spatially Heterogeneous Collagen Constructs for Cartilage Tissue Engineering 1

Stephanie Rhee, B.S., 1Jennifer L. Puetzer, PhD, 1Brooke N. Mason, PhD, 1

Cynthia A. Reinhart-King, PhD, 1,2Lawrence J. Bonassar, PhD

1

2

Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY *Address Correspondence to: Lawrence J. Bonassar, PhD. Associate Professor Department of Biomedical Engineering 149 Weill Hall Cornell University Ithaca, NY 14853 (607) 255-9381 [email protected]

Keywords: 3D printing, freeform fabrication, cartilage, meniscus, hydrogel, mechanics, gradient

Running Title: 3D Printing of Cellular Collagen Hydrogels

1 ACS Paragon Plus Environment


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Abstract 3D printing of biological tissues has been of increasing interest to the biomaterials community in part due to its potential to produce spatially heterogeneous constructs. Such technology is particularly promising for orthopedic applications, which require the generation of complex geometries to match patient anatomy and complex microstructures to produce spatial heterogeneity and anisotropy. Prior research has demonstrated the capacity to create precisely shaped 3D-printed constructs using biocompatible alginate hydrogels. However, alginate is extremely compliant and brittle, and high density collagen hydrogels could be a preferable option for load-bearing applications. This research focused on developing and evaluating a method of printing soft tissue implants with high-density collagen hydrogels using a commercially available 3D printer, modified for tissue-engineering purposes. The tissue constructs, seeded with primary meniscal fibrochondrocytes, were evaluated using measures of geometric fidelity, cell viability, mechanical properties, and fiber microstructure. The constructs were found to be mechanically stable and were able to support and maintain cell growth. Furthermore, heterogeneous 3Dprinted constructs were generated, consisting of discrete domains with distinct mechanical properties. Keywords: 3D printing, freeform fabrication, cartilage, meniscus, hydrogel, mechanics, gradient


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Introduction Permeable three-dimensional biodegradable scaffolds are commonly used in tissue engineering, as they provide the initial support that the engineered tissue requires while the cells produce their own extracellular matrix, and eventually degrade resulting in a construct that biologically resembles native tissue1,2. However, in a load-bearing tissue such as cartilage, the geometric fidelity of the implants is of great importance to ensure proper mechanical function of the joint3. Previous work in the field of tissue engineering has led to the development of a number of methods for the manufacture of geometrically complex tissues3,4,5,6. However, these techniques are often unable to produce spatially heterogeneous constructs and therefore cannot reproduce the complex types, densities, and spatial arrangements of cells found in native tissues. 3D printing, with its capabilities for spatial control of material deposition, offers a solution which may address these issues of tissue heterogeneity3,7. As a biocompatible material, collagen hydrogels are commonly used in biomedical applications, and have long been used as tissue engineering scaffold material to support cells for functional tissue regeneration. A number of studies have injected collagen in vivo to form constructs that eventually integrate and become remodeled by the surrounding tissue. The main limitation of collagen hydrogels is that the mechanical properties of the material are not strong enough to bear loads in vivo and retain shape fidelity in vitro. However, the collagen concentration used in these hydrogels is typically low (1-3 mg/ml), and limited research has been done using collagen hydrogels of much higher concentrations for the purpose of cartilage tissue engineering.



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Although a number of hydrogels have been used as bioinks for cell-based tissue printing, two of the most common bioink materials are alginate8 and collagen9. While the materials are useful for printing, they suffer from limitations based on their mechanical properties. Alginate gels are somewhat stiff, but often brittle8, while collagen gels are extremely compliant, with moduli < 1.0 kPa. Recently, we have developed high density formulations of collagen gels with superior mechanical properties10 that can be used with tissue molding techniques11. However, the use of these higher concentration (10 - 20 mg/ml) collagen hydrogels for tissue printing applications has not been reported. With this in mind, the objectives of this study were to 1) evaluate a range of collagen concentrations used to formulate bioinks for tissue printing and 2) demonstrate the feasibility of creating multi-domain constructs with distinct compositions and mechanical properties.. The suitability of collagen formulations for tissue printing was evaluated by assessment of viability of printed cells, geometric accuracy of printed structures, as well as evaluation of mechanical properties and microstructure of collagen hydrogels.

Methods Fibrochondrocyte isolation and purification: While many types of cartilage have distinct structural and mechanical heterogeneity, this is particularly true of the meniscus, which varies in composition from inner and outer zones12,13 and in loaded and unloaded regions14,15. As such, we chose meniscal fibrochondrocytes as a source of cells for these structures. Fibrochondrocytes were isolated from 1-3 day old bovine joints (Gold Medal Packing, Oriskany, NY)4. The meniscus was harvested from the joint and cut into 1 mm3 pieces, which were then digested in a solution of 0.3% collagenase (Worthington Biochemical, Lakewood, NJ) in DMEM (Invitrogen,


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Carlsbad, NY) containing 100 units/ml penicillin and 100 µg/ml streptomycin (Invitrogen) for 18 hours, as previously described11. The digested cells were then centrifuged at 2500 rpm for 12 minutes. The supernatant was discarded and the cells were washed twice in a solution of phosphate buffered saline (Invitrogen) containing 100 units/ml penicillin and 100

µg/ml

streptomycin and centrifuging at 1500 rpm for 8 minutes. The cells were then counted and suspended in a solution of DMEM, containing 10% fetal bovine serum (VWR, Radnor, PA), 100 units/ml penicillin, 100 µg/ml streptomycin, 0.1 mM non-essential amino acids, 50 µg/mL ascorbate, and 0.4 mM L-proline.

Collagen hydrogel formulations: As described previously11, tendons were obtained from skeletally mature rat tails (Bioreclamation IVT, Westbury, NY) and solubilized in a solution of 0.1% acetic acid (Sigma, St. Louis, MO) at a concentration of 1g per 150mL solution16. The tendons were solubilized for 48 hours at 4˚C and then centrifuged at 9000rpm for 90 minutes to remove unsolubilized collagen, blood, muscle tissue, etc. The supernatant was then collected, frozen, and lyophilized for 96 hours. The resulting lyophilized collagen was reconstituted in 0.1% acetic acid, at a stock concentrations of 30 and 20 mg/mL. Collagen hydrogels were then synthesized by combining the stock collagen solution with a working solution containing sodium hydroxide, phosphate buffered saline and 10x phosphate buffered saline (all from Sigma). Primary fibrochondrocytes were immediately mixed into the collagen hydrogel at a density of 10x106 cells/mL and loaded into syringes for 3D printing.

3D bioprinting: 3D printing was conducted using the Fab@Home 3D printer (Seraph Robotics, Ithaca, NY, Figure 1). Several modifications were made to the printer, specific for use in the



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printing of collagen hydrogels. An infrared heat lamp (Zoo Med Laboratories, San Luis Obispo, CA) was attached to the printer, directed towards the printing surface, and a ceramic baseplate was added to promote heat conduction. The IR heat lamp was regulated by electronic temperature controls (McMaster-Carr, Elmhurst, IL) to keep the baseplate temperature at 37˚C. The 3D printer was encased in aluminum foil to increase heat retention, while the deposition tool containing the printing nozzle and syringe was packed in ice and also encased in aluminum foil to prevent premature polymerization. 3D models for printing were generated by converting CT scans into stereolithography

files

(.STL)

using

CAD

Figure 1. Printing process of sheep meniscus (a) CT scan of meniscus (b) print path of meniscus (c) deposition of collagen hydrogel during printing (d) 3D printed meniscus. Geometry assessment of constructs (e) Constructs scanned using Cyberware 3D Scanner (f) The geometry of the test construct: half-cylinder.

software3 or created using SolidWorks software (Dassault Systemes SolidWorks Corp, Velizy, France). Based on a number of changeable parameters, a processing path was developed for the 3D printer to follow in order to create the construct.

3D bioscanning: Methods to assess the geometric accuracy of printed constructs were based on those described previously7,17. Printed half-cylinder samples were lightly coated in charcoal powder (Sigma) and scanned using a 3D Cyberware scanner and CyScan software (Cyberware, Monterey, CA). The constructs were placed on a rotating platform at a 45 degree angle to the


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scanner while the lateral scan was conducted (Figure 1E). The point cloud scan data was then processed using open-source MeshLab software, and Geomagic Qualify software (Geomagic, Morrisville, NC) was used to determine the percentage of the data that was within 2 mm of the respective points on the original model to obtain a measure of geometric fidelity.

Cell viability: Viability of cells within the constructs was evaluated using Live/Dead assay (Life Technologies, Grand Island, NY). Pieces of the constructs were harvested and placed in a 1mL tube with 1mL phosphate buffered saline, .25 µL ethidium homodimer solution, and .5 µL calcein AM solution. Images of the red ethidium-stained dead cells and green calcein-stained live cells were captured using fluorescence microscopy and image analysis was conducted using ImageJ software (National Institutes of Health, Bethesda, MD) to determine percent viability.

Confocal Reflectance Collagen Imaging The internal structure of collagen gels was visualized with confocal reflectance microscopy as described previously18. Briefly, a Zeiss LSM700 inverted laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) was used to imagesamples with both confocal reflectance to visual collagen and fluorescence imaging to capture autofluorescent cells by splitting a 488nm laser. Optical slices 1 micron in depth were collected with a long working distance LCI PlanApochromat 25×/0.8 IMM Korr objective (Carl Zeiss). To determine the resolution of printed interfaces between distinct collagen domains, FTIC- and rhodamine-label 5 µm microbeads (Sigma) were suspended in a concentration of 10 × 106 particles/ml, then printed into neighboring rectangular domains. The constructs were images in dual confocal reflectance and fluorescence modes as described above.



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Confined compression testing: Mechanical properties of printed constructs were assessed as described previously5. Tests were performed on an Enduratec EL2100 mechanical testing frame (Bose, Eden Prairie, MN) using a 1 kg load cell. Samples 1mm thick were cut out of the constructs using a 4 mm dermal biopsy punch and placed in a 4mm confined compression chamber. A porous platen was then placed on top of the sample and a bath of phosphate buffered saline added to the chamber. A custom-built holder was then lowered to touch the top of the platen and ten 50 micron displacement steps (5% strain) were imposed. Resulting stress relaxation data were fit to a poroelastic model to calculate equilibrium compressive modulus19.

Statistical Analysis: The effects of process variables (heating, collagen concentration, construct size, time in culture) on construct shape fidelity and cell viability were assessed by two way ANOVA with Tukey HSD posthoc test for pairwise comparisons.

The effect of collagen

concentration on constructs modulus was assesses by Spearman’s linear correlation analysis.

Results Process variables affecting geometric fidelity of 3D printed constructs Samples printed both in anatomic shapes and regular geometric shapes were inspected visually. Half-cylinders were printed and optically scanned to quantify the geometric accuracy of the prints. The scanning data showed that the collagen constructs fabricated with the heated printing setup had greater geometric fidelity than the constructs printed without (Figure 2A) at all concentrations below 20 mg/ml (p < 0.05). Additionally, heated printing setup was of particular benefit for hydrogels of lower collagen densities. For 17.5 mg/ml gels, heating increased shape fidelity from 66% to 79%, and or 10 mg/ml gels, heating increased fidelity from 10% to 39%.


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Figure 2. (a) Accuracy of print as a function of collagen density as indicated by the percentage of scanned positions that were within 2 mm of target height for heated and unheated printing. (b) Geometric fidelity of cylindrical constructs as a function of time in culture. (c) Geometric fidelity of cylindrical constructs of varying diameters immediately after printing. (d) Cell viability in printed constructs with time in culture. Date are represented by mean +/- standard deviation for n = 5 samples. The data from the 3D scans immediately after printing showed a general trend of increasing geometric fidelity with increasing collagen density. This increased shape fidelity was likely due to gels with greater collagen densities demonstrating greater viscosity, as the layers are better able to retain their printed shape and adhere together. However, the optimal performance was found between collagen concentrations of 15 mg/ml and 17.5 mg/ml (p 0.95). Further, neither cell count (Figure 2d) nor viability varied with time over 10 days in culture. The data from the confined compression tests indicated that the equilibrium modulus of the constructs increased linearly with increasing collagen concentration (R2 = 0.953; p = 0.001) (Figure 3). Increasing collagen concentration from 10 to 20 mg/ml increased the modulus by slightly more than a factor of 2. At the highest printable concentration, the modulus of printed gel was ~30 kPa.

Characterization of fiber microstructure by confocal reflectance microscopy The confocal reflectance images show increased cell dispersion throughout collagen with increasing culture time (Figure 4). At day 0, the cells were observed to be distribution primarily in pockets of the collagen. By day 10, greater infiltration of cell throughout the collagen and attachment on the outside of nascent collagen fibers was observed. Fewer cells were seen residing in the spaces between the fibers as compared to the number of cells situated on the

Figure 3. Compressive modulus of constructs immediately after printing of as a function collagen density. Construct modulus increased significantly with collagen concentration (p < 0.001). The shaded region reflects the average aggregate modulus +/- standard deviation reported for human medial meniscus.20 Data are represented as mean +/- standard deviation for n = 5 samples.

collagen.



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Figure 4. Confocal reflectance images of fibrochondrocyte-seeded constructs of varying collagen concentrations (a-c) 12.5 mg/mL, 15.0 mg/mL and 17.5 mg/mL collagen hydrogels seeded with fibochondrocytes were printed, respectively, and imaged immediately after printing. Cells were observed to be homogeneously distributed among collagen fibers. (d-f) After 10 days of culture, constructs made with 12.5 mg/mL, 15.0 mg/mL and 17.5 mg/mL collagen hydrogels were imaged. Fibrochondrocyte distribution appears to be less homogenous, with more cells adhering to the collagen fibers. Confocal reflectance showed a sharp, defined interface between the two adjacent domains, as evidenced by the clearly defined red and green fluorescing sections of the multi-domain construct (Figure 5). Closer inspection of the confocal images also showed very little mixing between the two hydrogels at the interface; as the red and green fluorescing microbeads are clearly separated, with the gap between the two regions being very small.


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Furthermore, the mechanical testing data showed that the separate regions of the multidomain constructs had different mechanical properties (Figure 6). Each domain had an equilibrium

Figure 5. Confocal images (a, b, c) and confocal reflectance image (d) of interface between domains of varying collagen concentrations with fluorescent microbeads immediately after printing (a,b). Sharply defined interface is observed with little penetration beyond the boundary (c) Minimal mixing of the two printing substrates at the interface (d) Collagen is depicted in white and is observed to be continuous though the separate bead populations remain distinct.



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modulus consistent with that observed in constructs printed entirely with the respective density hydrogel. These findings were consistent across a range of printed density gradients, producing distinct domains that varied in stiffness by 16% for the 12.5 mg/ml to 15 mg/ml gradient to 130% for the 12.5 mg/ml to 17.5 mg/ml gradient.

Figure 6. Equilibrium modulus of each domain in multi-domain constructs. Moduli of neighboring domains are indicated by color intensity key (right).

Discussion The goals of this work were to develop a 3D bioprinting method using high density collagen hydrogels and to characterize the geometric fidelity, cell viability, and mechanical properties of printed constructs. The data show that heating of the deposition surface greatly enhanced the geometric accuracy of the print, with optimal results obtained with 15 and 17.5 mg/ml gels. Cell viability was independent of collagen gel concentration and the compressive modulus of printed gels increased linearly with collagen concentration. Printed constructs maintained viability and geometric fidelity over 10 days in culture. Over this period of time, cell distribution within constructs changed, with cells being primarily distributed in pockets within


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the collagen throughout the constructs at the time of printing, but by 10 days are homogeneously mixed throughout the collagen and associated with emerging collagen fibers. This research found that the application of heat and the formulation of the high-density collagen hydrogels were critical in the bioprinting process. The use of heated elements in 3D printing has been used previously in the printing of plastics and other synthetic polymers21. However, the application of heat while printing with biopolymers seeded with live cells has not thoroughly been explored. Our development of a heated bioprinting chamber achieved tissue constructs that demonstrated significantly greater geometric fidelity as compared to constructs printed without. Without the addition of heat to polymerize printed parts, the samples collapsed under the weight of newly-printed layers. While environmental heating of the deposition space enhanced print fidelity, it may have also played a role in heating the deposition tool, contributing to the clogging of the tool and poor accuracy of prints of the 20 mg/ml gel. The properties of printed constructs were consistent with previous studies of collagen gels of similar concentration made by molding or casting. For example, 10 mg/ml molded collagen gels seeded with either meniscal fibrochondrocytes22 or auricular chondrocytes23 both had compressive moduli of ~10 kPa, consistent with the 13 ± 4 kPa seen in the current study. Further, studies of cast collagen gels showed a linear dependence of compressive modulus on collagen concentration in the range of 8 – 20 mg/ml10, similar to the current study. The behavior of cells seeded into collagen gels during the printing process was also similar to that reported previously. Although chondrocytes and fibrochondrocytes are known to contract collagen gels of lower concentrations (1 – 5 mg/ml)24,25, the shape of samples in the current study did not change over 10 days in culture. Although there were clear changes in matrix organization after 10 days, previous studies have demonstrated that in such relatively



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short cultures (i.e. less than 2 weeks) there is little change in matrix composition due to synthetic activity of fibrochondrocytes seeded into high density collagen gels11,22,26,. Notably, other studies of fibrochondrocyte behavior in higher density collagen gels indicated that contraction occurred primarily after 2 weeks in culture11,20. Consistent with the current studies, this previous work also showed that this time frame was also associated with a change in distribution of cells, which at later times in culture are decorated on the surface of emerging collagen fibers. Cells associated with fibers also appeared aligned along these fibers, suggesting some direct interaction with the collagen gel. Fibrochondrocytes are known to prominently express integrin subunits α1, α2 and β1,27 which collectively confer the ability to bind to collagen. Binding of other cell types to collagen gels is known to be mediated by fibronectin, present in gels presumably through serum in culture media.28 Meniscus fibrochondrocytes also express integrin subunit α5, which in combination with β1 would enable further interaction with the collagen gel via fibronectin. Collectively, these data show that the mechanical properties and cellular behavior of printed constructs is similar to that of constructs made by other shaping techniques such as molding or casting. Such similarities were also noted in comparisons of molded and printed constructs made from alginate17,29, which broadly suggests that the printing process in general does not compromise the properties of hydrogels. The geometric accuracy of printed collagen gels was similar to that previously noted for 3D printed alginate constructs. In studies of whole meniscus printing with alginate17, ~80% of scanned constructs were within 2 mm of target dimensions. Printing of porcine heart valves using an alginate/polyethylene glycol (PEG) bioink showed accuracies of 70-90% using a similar threshold7. Printing of regular geometric shapes with alginate, polyethylene glycol diacrylate (PEG-DA), and gelatin, showed similar levels of accuracy for thick (> 1.5 mm) constructs


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printed with all three materials30. In the current study 15 and 17.5 mg/ml collagen gels yielded 74-78% accuracy with this threshold, which is similar to these previous studies. A secondary goal of this study was to demonstrate the ability to deposit adjacent domains with distinct composition and mechanical properties. The results from our attempts at multidomain 3D printing demonstrate the ability to print constructs with multiple cell-seeded materials in mechanically discrete domains. One previous study19 developed 3D printing methods for mechanically heterogeneous constructs and used tunable PEG-DA hydrogels to create regions of variable stiffness. These constructs were first printed and then surface-seeded, resulting in limited cell distribution throughout. However, their results indicated that the printing methods were not cytotoxic to cell growth, as we also found when printing with cell-seeded materials. Additionally, the study found that the addition of collagen into their PEG-DA hydrogels did not promote cell integration in their constructs. This is contrary to our results that demonstrate a promotion of cell growth and spreading with increasing collagen concentration. Another more recent study31 also examined the potential of 3D printing for tissue engineering, using multiple cell-seeded materials to build constructs. This study indicated that the 3D bioprinting method is non-destructive to the cells being printed, which we also observed while printing with hydrogels seeded with fibrochondrocytes. Upon visual inspection of the microscopy images shown, their data also demonstrate minimal mixing of the different printing materials used. This observation is also consistent with our confocal reflectance images, in which our multi-domain constructs displayed sharply defined interfaces between boundaries and negligible mixing of different materials at interfaces. Notably the current study demonstrates the ability to control the mechanical properties of adjacently printed domains. For orthopedic and cartilage engineering, control of domain mechanical properties would be necessary in order to be



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able to accurately 3D print complex structures. The mechanical tests that we performed on our constructs confirmed that the mechanical stiffness found for each domain were consistent with those of single-domain constructs made of the respective materials. Although ability to produce controlled domains of seeded collagen hydrogels is potentially quite powerful, there are several limitations to be considered and improved. The print accuracy of this system is on the order of millimeters. But if these methods are to be used to create tissue implants with precise anatomical features or finely tuned gradients, the print accuracy should be improved further to be on the cellular scale. Additionally, the techniques used to measure mechanical properties of distinct domains are limited to the spatial resolution of the samples cut for mechanical testing (~5 mm). As such, measurements of stiffness gradients were quite coarse.

Recent studies32 have used microscopy-based techniques to map local

mechanical properties of soft materials to a resolution of ~20 µm and application of these techniques to the constructs fabricated in the current study would be of great interest.

Conclusions This study demonstrates the development of printable formulations of collagen hydrogels and the advantages of heated deposition to improve geometric accuracy. Collagen concentration was shown to affect shape fidelity and mechanical properties of printed constructs, with minimal effect on cell viability. Printed gels were sufficiently robust to enable printing of heterogeneous constructs with distinct compositional and mechanical domains.

Acknowledgments 18 ACS Paragon Plus Environment

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The authors would like to thank Dr. Bryan Brown and Dr. Jeff Lipton for their technical assistance in this work. This work was funded by NIH NIH/NCI U54CA143876.



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Table of Contents Graphic. 3D bioprinting of collagen hydrogels enables the fabrication of anatomically shaped constructs and structures with compositionally and mechanically distinct microdomains.


3D Bioprinting of Spatially Heterogeneous Collagen Constructs for

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