MECHANICALLY TUNABLE BIOINKS FOR BIOPRINTING

Abstract

A process for bioprinting wherein a matrix comprising a modified primary hydroxyl groups containing polysaccharide comprising repeating disaccharide units wherein in at least part of the disaccharide units the primary hydroxyl group is replaced by functional groups selected from carboxyl groups, halide groups or groups comprising sulfur or phosphorus atoms, like e.g. sulfate groups, sulfonate groups, phosphonate groups and phosphate groups, is used and bioink formulations.

Claims

1. A process for bioprinting wherein a matrix comprising a modified primary hydroxyl groups containing polysaccharide comprising repeating disaccharide units wherein in at least part of the disaccharide units the primary hydroxyl group is replaced by functional groups selected from carboxyl groups, halide groups or groups comprising sulfur or phosphorus atoms, like e.g. sulfate groups, sulfonate groups, phosphonate groups and phosphate groups is used as bioink.

2. The process in accordance with claim 1 wherein the polysaccharide has a helical secondary structure.

3. The process in accordance with claim 1 wherein in 20-99% of the disaccharide units the primary hydroxyl groups are replaced by functional groups.

4. The process in accordance with claim 1 wherein the bioprinting process is selected from inkjet-printing, syringe-printing or bioplotting and laser-printing.

5. The process in accordance with claim 1 wherein the functional group is a carboxyl group.

6. The process in accordance with claim 1 wherein the functional group is a halide group.

7. The process in accordance with claim 1 wherein the functional group is a phosphonate or phosphate group.

8. The process in accordance with claim 1 wherein the functional groups is a sulfate or sulfonate group.

9. The process in accordance with claim 1 wherein the matrix additionally comprises unmodified polysaccharides.

10. The process in accordance with claim 1 wherein either the modified polysaccharide or the non-modified polysaccharide or both are selected from the group consisting of a member of the carrageenan family, hyaluronic acid, heparin sulfate, dermatan sulfate, chondroitin sulfate, alginate, chitosan, pullulan and agarose.

11. The process in accordance with claim 10 wherein either the modified polysaccharide or the non-modified polysaccharide or both are agarose.

12. Bioink formulation suitable for bioprinting, comprising a modified primary hydroxyl groups containing polysaccharide comprising repeating disaccharide units wherein in at least part of the disaccharide units the primary hydroxyl group is replaced by functional groups selected from carboxyl groups, halide groups or groups comprising sulfur or phosphorus atoms, like e.g. sulfate groups, sulfonate groups, phosphonate groups and phosphate groups, for bioprinting processes and living cells.

13. Bioink formulation in accordance with claim 12 wherein the polysaccharide has a helical secondary structure.

14. Bioink formulation in accordance with claim 12 wherein in 20-99% of the disaccharide units the primary hydroxyl groups are replaced by functional groups

15. Bioink formulation in accordance with claim 12 wherein the living cells are selected from the group consisting of chondrocytes, osteoblasts, osteoclasts, skin epithelial cells, intestinal epithelial cells, corneal epithelial cells, astrocytes, neurons, oligodentrocytes, smooth muscle cells, endothelial cells, cardiomyocytes, pancreatic islet cells, kidney epithelial cells and nave cells obtained from umbilical cord.

16. Bioink formulation in accordance with claim 12 comprising as living cells stem cells selected from the group consisting of embryonic stem cells, somatic stem cells, reprogrammed pluripotent somatic cells, induced pluripotent cells and amniotic stem cells.

17. A process for three-dimensionally structuring biological materials wherein a bioink formulation as defined in claim 12 is processed by inkjet-printing, syringe-printing or bioplotting or laser-printing.

Description

EXAMPLES

[0068] Native agarose was obtained from Merck (Darmstadt, Germany), (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO, 99%), sodium bromide (NaBr, 99%), NaOCl solutions 15% v/v, NaBH.sub.4 (99.99%), NaCl (BioXtra >99.5%), Rhodamine 123, and phosphate buffer saline (PBS) were purchased from Sigma Aldrich (USA) and used as received. Ethanol technical grade was used as received. Deionized water was obtained from a laboratory ion exchanger.

[0069] Synthesis of Carboxylated Agarose:

[0070] 1 g of native agarose type 1 (Merck) was transferred into a three-necked round bottom flask, equipped with a mechanical stirrer and pH meter. The reactor was heated to 90 C. to dissolve the agarose and then cooled to 0 C. in an ice bath under mechanical stirring to prevent the solution from gelling. The reactor was then charged with TEMPO (0.160 mmol, 20.6 mg), NaBr (0.9 mmol, 0.1 g), and NaOCl (2.5 mL, 15% v/v solution) under vigorous stirring. The pH of the solution was adjusted to pH 10.8 throughout the duration of the reaction, and the degree of carboxylation was controlled by the addition of predetermined volumes of NaOH solution (0.5 M). At the end of the reaction NaBH.sub.4 (0.1 g) was added, and the solution was acidified to pH 8 and stirred for 1 h. The carboxylated agarose was precipitated by sequential addition of NaCl (0.2 mol, 12 g) and ethanol (500 mL), and the solid was collected by vacuum filtration and extracted using ethanol. Residual ethanol was removed by extensive dialysis against water and the carboxylated agarose was obtained as a white solid upon freeze-drying overnight.

[0071] Rheological Characterization:

[0072] The viscosities of native agarose and carboxylated agarose solutions/hydrogels were characterized using a rotary rheometer (Kinexus, Malvern Instruments, Worcestershire, UK) with a 4 cone and plate geometry. Shear stress and viscosity were measured for shear rates from 0.01 to 10 000 s.sup.1 at 40 C. The viscosity was measured for different temperatures, from 60 to 5 C. with cooling at the rate of 1 C. min.sup.1, with a constant shear rate of 10 000 s.sup.1.

[0073] Mechanical Testing:

[0074] Native agarose (NA) and carboxylated agarose (CA) bulk hydrogels were cast in a 15 mm diameter cylindrical plastic vial and allowed to set overnight at 4 C. The bottom of the vial was cut open and the cylinder of hydrogel was pushed out of the vial. Hydrogel discs were cut to similar height and measured using a caliper before testing. Printed hydrogel samples were produced with an in-house developed 3D printer. A 15 mm diameter cylinder was printed with a height of 10 mm. The hydrogels were allowed to set overnight at 4 C. prior to testing. Compression testing of printed and bulk samples was carried out on a universal testing machine (Zwick, Germany) equipped with a 50 N load cell at a speed of 1 cm min.sup.1 until fracture of the hydrogels, and the data were exported and analyzed using Microsoft Excel (Microsoft, Redmond, Wash.).

[0075] Droplet Volume:

[0076] The volume of the droplet dispensed by the printer for different NA and CA formulations (NA 0.5, 1 and 2% w/v; 2% w/v CA28, CA60, and CA93) was determined by extruding 500 droplets into a tared 1.5 mL Eppendorf tube. The total weight of each dispensation (500 droplets) through a 300 nozzle for various gating times (450, 675, and 900 ms) was measured for three independent dispensations.

[0077] 3D Printer

[0078] The printer comprised four printer heads mounted to a three-axis robotic system (Isel, Eichenzell, Germany). Each head could be heated, pressurized, and controlled individually (Figure S6A, Supporting Information). The printer heads were composed of an electromagnetic microvalve (Fritz Gyger, Gwatt, Switzerland) connected to a pressurized bioink reservoir. The printing pressure could be varied from 0 to 300 kPa. For all experiments the air pressure was adjusted to 50 kPa. The printer head was designed to enable quick exchange of the attached microvalves between valves with small (150 m), medium (300 m), and big (600 m) nozzle diameters. The microvalve constitutes the basic dispensing unit of the system. By application of an electric current running through a magnetic coil, the valve ball is lifted magnetically against the mechanical force of a spring. The valve opens and allows a fraction of hydrogel-cell suspension to be squeezed out of the nozzle. The gating time could be varied from 450 s to 1 sec. By dropping the current, the magnetic force was reduced and the ball was pressed into the seat again closing the valve. The printing stage was cooled by circulating refrigerant cooled down to 10 C. using a cooling aggregate (TC45-F, Peter Huber Kltemaschinenbau, Offenburg, Germany). The print head carrying the carboxylated agarose formulation was heated up to 40 C. and the printing pressure set to 50 kPa. All 3D bioprinting experiments with Rhodamine stained hydrogels and with loaded cells were conducted using the 300 m nozzle. After printing the structures were held for 20 min at 4 C. to ensure the complete gelation of agarose.

[0079] Postprinting Viability Study on hMSCs:

[0080] Four different hydrogel precursor solutions were prepared by mixing 0.04 g mL.sup.1 NA or CA (28%, 60%, and 93%) with phosphate buffered saline (PBS). The agarose solutions were subsequently autoclaved at 121 C. for 15 min. Four million hMSCs from three independent donors (n=3) were pooled together and resuspended in 2 mL growth medium (Mesenpan; PAN Biotech, Aidenbach, Germany) supplemented with 2% v/v fetal calf serum (FCS) and 1% v/v solution of 10000 units of penicillin mixed with 10 mg streptomycin (Gibco, Life Technologies, Carlsbad, Calif.). The hMSCs were isolated from femoral heads of three independent donors. For determining cell viability, 500 L of each of the NA or CA solution was mixed at 37 C. and resuspended with equal volumes of the cell suspension resulting in a final cell concentration of 106 cells/mL.

[0081] The hMSC dispersed in either NA or CA formulation was loaded into the printer head and printed dropwise into a 96-well plate. For each type of hydrogel, three samples with a final volume of 100 pL were printed using the 300 um microvalve (Fritz Gyger, Gwatt, Switzerland) at an air pressure of 50 kPa and a valve gating time of 450 ms. As a control three samples with a volume of 100 L of each hydrogel type were pipetted into the well plate. Immediately after printing cell viability was assessed using a vital fluorescence staining assay. The staining solution contained 0.083 mg/mL 1-propidium iodide (P4170-10116, Sigma-Aldrich, St. Louis, Mo.) and 0.083 mg/mL-1 fluorescein diacetate (F7378-10G, Sigma-Aldrich, St. Louis, Mo.) in Ringer's solution. Each sample was imaged three times using an inverted microscope (DM16000B, Leica Microsystems. Wetzlar, Germany). Living and dead cells were counted using ImageJ.

[0082] Shear Stress During Printing:

[0083] The shear stress the cells were exposed to during the dispensing process was estimated using fluid dynamics model. In addition to the printing settings, nozzle size (300 m) and air pressure (50 kPa), the flow consistency index (K) and the flow behavior index (), which describe the rheological behavior of a hydrogel solution, were applied as input parameters for shear stress calculations. Values of K and were derived from the viscosity measurements of the hydrogel solutions.