3D Printing Gel

The exciting new fields of both 3-dimensional printing and gelation/thermogelation allow for new opportunities in biomedical research—including the capacity to generate custom cell scaffolds, and potentially fabricate tissues, by placing cells where they are desired in a medium that supports their growth. This website describes how several of PolySciTech’s products can fit into this novel area of research. PolySciTech is a division of Akina, Inc.

3D Printing Background

3D printing was originally developed in 1984 by Chuck Hull and is an additive process of making a three-dimensional solid object from a digital model. 3D printing is achieved by laying down successive layers of material to form shapes. There has been recently a huge growth in the sales and use of 3D printing and the market for these increased by 29% from 2011 to year 2012. To print, the machine reads the design from an .stl file and lays down successive layers of material to build a series of cross sections. These layers, as decided by the CAD model, are joined or automatically fuse to create the final shape. This technique allows for the ability to create almost any shape or geometric feature.

The printer resolution (layer thickness and X-Y resolution) are defined as either dpi (dots per inch) or micrometers. Typical layer thickness is around 100 micrometers (µm). Higher end machines however (Objet Connex series and 3D Systems' ProJet series) can print layers as thin as 16 µm. The X-Y resolution of 3D printers is around 50 to 100 µm.

Among other uses, 3D printing has found promise in the biomedical field as a means to generate tissue scaffolds out of biodegradable polymers as well as potentially tissues by printing cells and matrix into a defined area.

Cube 3D Printer
Example 3D printer.
Cube (Cubify 3D Systems) plugs into any computer, prints parts in biodegradable PLA, and is priced starting at $1299.
Video
Biodegradable poly(lactic acid) (PLA) is molded into a hyperboloid object
(designed by George W. Hart) using a RepRap "Prusa Mendel" 3D printer.
(Sorry, Internet Explorer and Safari do NOT natively support this video format; get an add-on or use Chrome, Firefox or Opera.)
http://upload.wikimedia.org/wikipedia/commons/5/5d/Hyperboloid_Print.ogv.

The rest of this site focuses on the application of 3D printing toward biodegradable polymers as well as the potential for printing gels and other biomedical uses.

3D Printing Biodegradable Polymers

Some biodegradable polymers have been previously utilized for 3D printing in traditional “melt” state. These include Poly(caprolactone), Poly(L-lactide) and Poly(Lactide-co-glycolide). A recent article relates use of 3D printing for generating scaffolds of PLLA and PLGA. In this article biodegradable porous scaffolds were generated as potential bone graft materials. In this study 3D printing was used for fabrication rather than traditional techniques such as salt leaching/phase separation so as to allow for studying the effects of architecture and design on bone formation. Both Poly(L)-Lactide (PLLA) and Poly(lactide-co-glycolide) PLGA) were printed using image based design and indirect solid freeform fabrication. These structures were then seeded with BMP7 transduced gingival fibroblasts and implanted subcutaneously in mice for 4-8 weeks. MCT scans and histology revealed that the PLGA scaffolds had broken down after 4 weeks but the PLLA scaffolds maintained their architecture which improved bone ingrowth revealing the importance of choosing the right material for the scaffold usage. See more at Saito, Eiji, Elly E. Liao, Wei-Wen Hu, Paul H. Krebsbach, and Scott J. Hollister. "Effects of designed PLLA and 50: 50 PLGA scaffold architectures on bone formation in vivo." Journal of Tissue Engineering and Regenerative Medicine 7, no. 2 (2013): 99-111. PubMed link

Recent research has optimized the parameters for 3D printing with polycaprolactone. In this research synthesized PCL with a molecular weight near 79 kDa (similar to PolyVivo product AP11) was printed successfully using a Bioscaffolder (Envisiontec GmbH, Gladbeck, Germany) by extruding the molten polymer through a 23 Ga heated nozzle at 110 °C and strands were applied onto a collector plate in a layer-by-layer method at a speed of 350 mm/min. The strand pattern was rotated at 90° angles in between layers which created square pores. The distance between strands was 0.9 mm with a spindle speed of 200 rpm. The resultant elastic modulus of PCL is ~59 MPa but the modulus (compressive) of the scaffold was around 10 MPa. Note the density of crystalline and the amorphous PCL is 1.200 and 1.021 g/cm3, respectively. Seyednejad, Hajar, Debby Gawlitta, Wouter JA Dhert, Cornelus F. van Nostrum, Tina Vermonden, and Wim E. Hennink. "Preparation and Characterization of a 3D-printed Scaffold Based on a Functionalized Polyester for Bone Tissue Engineering Application." Functionalized Polyesters 7, no. 5 (2012): 87. (PDF link)

printed scaffold
Example 3D-printed scaffold (bar is 1mm)
Image from: Hajar Seyednejad,, Debby Gawlitta, Wouter J.A. Dhert, Cornelus F. van Nostrum,
Tina Vermonden and Wim E.Hennink. "Preparation and Characterization of a 3D-printed Scaffold
Based on a Functionalized Polyester for Bone Tissue Engineering Application."
Functionalized Polyesters 7, no. 5 (2012): 87.

An excellent review article discussing scaffold design including 3D techniques is available in full-text here:

Hutmacher, Dietmar W. "Scaffold design and fabrication technologies for engineering tissues—state of the art and future perspectives." Journal of Biomaterials Science, Polymer Edition 12, no. 1 (2001): 107-124. PDF link

As well as another one here

Sachlos, E., and J. T. Czernuszka. "Making tissue engineering scaffolds work. Review: the application of solid freeform fabrication technology to the production of tissue engineering scaffolds." Eur Cell Mater 5, no. 29 (2003): 39-40. PDF link

Thermogels / Gel Formers

There are two types of temperature responsive hydrogels. One is a “normal” type of gelation response in which the hydrogel-water solution melts to a liquid upon heating. This is due to a reduction of chain-to-chain entanglements and an improvement in overall polymer solubility. Gelatin behaves this way and is a solid at cool temperatures but a liquid at higher temperatures G. Acharya, C.S. Shin, M. McDermott, H. Mishra, H. Park, I.C. Kwon, K. Park. "The hydrogel template method for fabrication of homogeneous nano/microparticles." Journal of Controlled Release 141(3) (2010) 314-319 There is also a “reverse” gel response in which the hydrogel system transitions toward a solid phase upon heating. Several polymers, especially those with hydrophobic domains as their main form of holding the gel together, display thermoreverse properties.

Polymer LCST  (°C) (approx.)
Poly(N-vinylcaprolactam), PNVC 30
Poly(N-isopropylacrylamide), PNIPAM 32
Poly(silamine) 37
Poly(vinyl methyl ether), PVME 40
Poly(propylene glycol), PPG 50
Poly(vinyl methyl oxazolidone), PVMO 65
Poly(methacrylic acid), PMAA 75
Methylcellulose, MC 80
Poly(ethylene glycol), PEG 120
Poly(vinyl alcohol) PVA 125
Poly(siloxyethylene glycol) ~10-60
Poly(vinyl pyrrolidinone) PVP 160
Polyphosphazene derivatives ~33-100
PLGA-PEG-PLGA (1100-1000-1100 Da) triblock (AK12 / AK24) 17-25
PLGA-PEG-PLGA (1500-1500-1500 Da) (AK19) 40-45
mPEG-PCL (750-2500 Da) 20
Table 1
Shows several of these reverse thermogels and their LCST transition temperature.

Reported thermogels and LCST. Some data from: B. Jeong, S.W. Kim, Y.H. Bae, ""Thermosensitive sol-gel reversible hydrogels."
Advanced Drug Delivery Reviews 54(1) (2002) 37-

How does it work?

One classic misconception is that upon heating the polymers become hydrophobic. This is not necessarily the case as the polymers have hydrophobic and hydrophilic domains at all times but the change in temperature affects the relative impact of the organized/disorganized state of water molecules R. Pelton, Poly(N-isopropylacrylamide)
"(PNIPAM) is never hydrophobic." Journal of Colloid and Interface Science 348(2) (2010) 673-674
For a good theoretical background as to the driving principles of temperature response, a simple thermally responsive polymer, PEG, can be used as a general model to apply to other systems. PEG by itself displays a cloud point, a higher level temperature at which the polymer precipitates out of water, which varies by molecular weight but is reported to exist around 100-120 °C. PEG has a unique interaction with water in that when in dissolved state (or in any state other than freshly dehydrated) each PEG chain monomer is tightly bound to 2-3 water molecules and the water molecules participating in this binding are highly organized leading to a decreased entropy relative to the entropy of free water. The binding energy is an enthalpic term (ΔHf – heat of fusion). As temperature increases the total energy of the system as described as Gibbs free energy of the system (G=H-TS, G-Gibbs free energy, H-enthalpy, T-temperature, S-entropy) favors the entropic term rather than the enthalpic term and the water molecules prefer to be in unorganized form in free solution rather than bound to the PEG chain. J.M. Harris, "Poly (ethylene glycol) chemistry: biotechnical and biomedical applications" Springer, 1992 This process is shown schematically

schematic
Schematic of change in hydration status with heating.

Note that a key component of this interaction is also the presence of hydrophobic groups. Although the hydrogen bonding status between water and the polymer at increased temperature is affected negatively, the hydrophobic interactions are affected very little. When these are present the strength of the hydrophobic interactions becomes greater than the hydrogen bonding thus causing the polymer chains to bind to each other.

These properties of reverse thermogelation apply to several Akina products including the following:

Cat# Polymer Description Concentration (tested) w/v% Onset Temperature (°C) (G'>0; or sharp transition) Max G' (Pa) Max G' Temp (°C) Max G"  (Pa) Max G" Temp (°C)
AO26 Poly(poloxamer 407)-Methylene-diphenyl-di-isocyanate linked 5% 12.5 435.7 45 43.85 45
AK12 Poly(lactic-co-glycolic acid)-b-Poly(ethylene glycol)-b-Poly(lactic-co-glycolic acid) (1,500:1,000:1,500 Da, 1:1 LA:GA) 20% 15 484.7 25 535.6 25
AK35 Polycaprolactone-b-Poly(ethylene glycol)-b-Polycaprolactone (MW ~ 1,000:1,000:1,000 Da) 20% 17.5 0.1623 27.5 0.2582 30
AK24 Poly(lactic-co-glycolic acid)-b-Poly(ethylene glycol)-b-Poly(lactic-co-glycolic acid) (1,500:1,000:1,500 Da, 3:1 La:Ga) 20% 20 25.53 25 40.81 25
AK36 methoxy-Poly(ethylene glycol)-b-Polycaprolactone 750-25000 20% 20 164000 42.5 7138 45
AO19 Poly(dimethylaminoethyl methacrylate-co-methoxy polyethylene glycol) 20% 25 431.1 45 339.4 45
AO18 Poly(vinylcaprolactone-co-methoxy polyethylene glycol methacrylate) (95:5) 10% 30 58110 45 9131 45
AO17 Poly(n-isopropylacrylamide-co-methoxy polyethylene glycol methacrylate(Mn 475Da)) copolymer (95:5) 1% 35 6.757 45 0.9252 45
AO25 Stearate-Modified Methyl Cellulose 5% 37.5 1022 45 118.3 45
AO23 Poly(n-isopropylacrylamide-co-acrylamide) copolymer (95:5) 2% 37.5 4.342 45 1.614 45
AO22 Poly(n-isopropylacrylamide-co-acrylamide) copolymer (90:10) 2% 40 5.754 45 1.371 45

Thermogels / Gel Formers for use in 3D printing

Recent research indicates great potential for use of 3D printing and scaffolds for tissue repair and other uses. Bioprinting, which is the printing of living cells in a specific pattern, is a state of the art method and has the potential to fabricate living organisms. Bioprinting systems are classified in three different categories based on the methods used.

  1. The laser-based system processes 2D cell patterning. Laser direct write (LDW) makes precise patterns of viable cells. These cells are suspended in a solution in donor slides and are transported to a collector utilizing the laser energy. The laser pulse creates a bubble and this creates shock waves. The shock waves push the cells toward the collector on petri dishes.
  2. Inkjet-based bioprinting uses living cells that are printed in the form of droplets through cartridges. This method enables printing either single cells or aggregate cells, depending on the process parameters.
  3. Extrusion-based printing is another technique to print living cells. It is “the extrusion of continuous filaments made of biomaterials.”

Each of these processes comes with its own advantages as well as limitations.

  1. Laser-based systems have high resolution and allow precise patterning of living cells.
  2. Inkjet-based systems are favored for cell encapsulation because of their versatility and affordability. The surfaces where the cells are printed and patterned do not have to be two-dimensional. Drawbacks to this method include cell damage and death as well as cell sedimentation and aggregation because of the small orifice diameter. The structural integrity of the printed structure is another concern.
  3. The extrusion-based method yields much better structural integrity and it is also the most convenient method to quickly make porous 3D structures. Limitations for this method also exist such as shear-stress-induced cell deformation and limited material selection which is due to the need for rapid cell encapsulation (Ozblat and Yu, 2012).

• Medical Implants

Patients have individual needs based upon their own anatomy and genetic makeup. Advanced manufacturing is very suitable for fabrication personalized implants and devices. Pattering technologies can be designed to copy the surroundings and the regulatory microenvironment of cells in vivo, and to modify the microenvironment in order to study the cellular response. Two-dimensional techniques have proved to be inadequate for some of the newer challenges of cell biology, biochemistry, and in pharmaceutical assays.

Three-dimensional structures are very important for in vitro experiments. This has been proved by multiple studies. For example, “hepatocytes retain many of their liver-specific functions for weeks in culture in-between two layers of collagen gel, whereas they lose many of these functions within a few days when cultured as a monolayer on the same gel.” Advanced manufacturing techniques have been developed or modified to include cells in the fabrication process. These include biolaserprinting, stereolithography, and robotic dispensing (which is also known as 3D fiber plotting (3DF) or bioplotting). The rapid advancements in this field is proven by the establishment of the journal Biofabrication, and the establishment of the International Society of Biofabrication in 2010.

• Scaffolds

Additive tissue manufacturing is still in its infancy. Many biodegradable materials have been manufactured and employed to design and fabricate scaffolds and matrices. These include polymers (both natural and synthetic), ceramics, and composites. These materials, however, usually require process parameters that are not conducive to the direct inclusion on the cells. Therefore, hydrogels are gaining the most interest in the manufacturing of tissues. They are polymeric networks that absorb the water while they remain insoluble and preserve their characteristic three-dimensional structure. Hydrogels can do this due to the large number of physical or chemical links between the polymer chains. Hydrogel structures with viable cells have been manufactured with just simple and isotropic designs. Also, the sizes have been limited to only a few millimeters. The imposed requirement for mechanical properties are 'self-supporting' and 'handleable'.

One of the factors that determines the biocompatibility of hydrogels is hydrophilicity. This makes hydrogels attractive for use in medicine and pharmacy as drug and cell carriers. One disadvantage of hydrogels is that their mechanical strength isn’t as high as load-bearing tissues. This is one of the primary limiting factors in advancing tissue manufacturing to larger scales. Obvious ways to increase the strength and modulus of the gel are to increase the polymer concentration and cross-link density.

Nanocomposite gels are a class of hydrogels which exhibit mechanical properties that are superior to conventional hydrogels. There have also been advancements in the strength of hydrogels outside biomedical engineering which exhibit novel chemical structures that have improved mechanical properties, with increased toughness and strength while still containing high water volume fractions. Advanced manufacturing techniques also offer a higher level of control over the scaffold architecture and range of materials that can be processed. Scaffolds for tissue engineering are usually prepared from ceramics, polymers, or a composite of the two. (Melchels, et.al, 2011)

• Biobots

Cells, tissues and organs are not the only items on the horizon for bioengineers. Researchers have also made biobots, which are part gel, part muscle. These tiny structures are poised to someday travel within the body to sense toxins and deliver medication. These biobots are made from the heart muscle cells and work is being conducted to regulate the muscle contractions. (New York Times 8/18/2013)

PolySciTech Products

• Traditional Melt/Solvent Printing

Several products offered by PolySciTech already hold promise for the capacity to be processed by 3D printing for creating bioscaffolds. These include poly(caprolactone) (a polymer used in Baker et. al. 2012, Sultana et. al. 2013, and Christensen et. al. 2012.) Poly(lactide) (a polymer used in Serra et. al. 2013), and Poly(lactide-co-glycolide) (a polymer used in Hoque et al. 2011).

PolySciTech markets a wide variety of these polymers and you can see them broken down by MW/monomer ratio/endcap and polymer type here. PolySciTech provides these as bulk formulations (powder/chunks) not rod or spool formatted.

• Thermal-Gel Printing

Additionally 3D printing can be achieved using thermogelation polymers available from PolySciTech as follows:

Cat# Description Publication
AO023 Poly(N-isopropylacrylamide-co-acrylamide)
P(NIPAM-AM)
95:5 NIPAM:AM
Dropwise gelation-dehydration kinetics during drop-on-demand printing of hydrogel-based materials
Polymers for Gel-Printing

 

Co-development project with Hatch51 for commercially available 3DCellMaker bioprinting system

Akina, Inc. has launched a joint project with additive engineering firm Hatch51 for the creation of a 3D gel-printing system optimized for printing of Akina’s synthetic thermogelling 3DCellmaker. The printer will allow for low-temperature, gentle, printing of the thermogelling polymer solution so as to allow for printing of live-cell loaded solution to form 3D structures and tissues. Such a system has promise for a wide array of future applications. Those interested in the hardware portion of the system are encouraged to contact Hatch51 directly regarding this.

 

The End