Two Photon Polymerization Biocompatible Photopolymers 3d Biointerfaces Biology Essay

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To allow cells to be cultured in vitro in a way that resembles the natural conditions more closely than flat TCPS, 3D microstructured scaffolds are needed.Two-photon polymerization (2PP) is applied as a tool for formation of high-resolution 3D scaffold structures with completely defined microarchitecture within biocompatible photopolymers. 2PP is a novel photolithographic technique using femtosecond laser pulses with enables free 3D microstructuring of liquid photopolymers due to the axial and lateral spatial confinement of the photoreaction to the focal volume of a focused laser beam. A set of photopolymers were tested with regard to 2PP processability and six different classes of (meth-) acrylated photopolymers (oligolactones, polyglycerines, urethane dimethacrylate, polyethylene glycol diacrylate, dextran, and hyaluronan) were found to be efficient 2PP materials. Based on 3D computer models 3D microstructures from different photopolymers were produced and tested for biocompatibility. The initial cell adhesion to the polymeric scaffolds and the response of bovine chondrocytes after three-day culture on 2PP derived microstructures was evaluated morphologically by CLSM after staining. 2PP derived scaffolds were fabricated in different sizes, starting from the 100 µm-range to the cm-range showing the actual possibilities to produce large volume scaffolds even for implantation purposes.

1) Introduction 

Tissues and organs consist of a complex organization of cells, extracellular matrix (ECM), and signaling molecules. In vivo, tissues are composed of repeating units on the scale of 100-1000 µm. The three-dimensional architecture of these repeating tissue units underlies the coordination of multicellular processes, emergent mechanical properties, and integration with other organs via the microcirculation. A key element of tissue structure in vivo is therefore the local cellular environment. The "microenvironment" (~10 µm) presents biochemical, cellular, and physical stimuli that synchronize the cell fate such as proliferation, differentiation, migration, and apoptosis. The goal of tissue engineering is to restore or replace the lost functions of diseased or lost organs. In the classical tissue engineering approach cell were taken from a patient, expanded in vitro on 3D scaffolds and re-implanted to the defect site. Successful application of living cells in regenerative medicine requires an understanding of how tissue structure relates to organ function. There is growing evidence that presentation of extracellular cues in a three-dimensional (3D) context can fundamentally alter cellular responses [].Thus, microenvironment studies that previously were limited to adherent two-dimensional (2D) cultures may not be appropriate for many cell types. In the special case of tissue engineering of articular cartilage the differentiation behavior of chondrocytes in 3D Scaffold has to be controlled.

Recent findings in oncogenesis and stem cell differentiation clearly show acute disparities in cell function between 2D and 3D culture and suggest that examining hierarchical biology in just two dimensions is insufficient. For example, enhanced chondrogenesis of embryonic stem cells has been observed when cells are cultured in 3D embryoid bodies as compared to monolayer culture [1 Tanaka 2004]. In an early example, Bissel and coworkers has demonstrated, that human breast epithelial cells develop like tumor cells when cultured in two dimensions, but revert to normal growth behavior when cultured in 3D analogs of their native microenvironment [2 Petersen 1992].

Cartilage tissue that lines our moveable joints serves to support mechanical loads and facilitates movement and flexibility. Unfortunately, cartilage heals very poorly after injury or trauma, often leading to gradual degradation over time. Its degeneration, which effects presently in the US alone over 36 million people, leads to serious and painful diseases and impairment of mobility and. In spite of tremendous efforts made in biomedical sciences cartilage has remained a particularly challenging tissue to engineer [3 Raghunath 2007]. Different strategies to engineer or regenerate cartilage tissue have been developed in the past decades. Typically, tissue constructs are assembled in vitro by combining cells and so-called scaffolds made from various biomaterials. In contrast to traditional cell culture on two-dimensional substrates such as tissue culture polystyrene (TCPS) the culture of mammalian cells on 3D scaffolds in vitro provides a defined platform for investigating cell and tissue physiology and pathophysiology outside the organism.

ECM [4 Badylak 2007]

The importance of the role of the ECM for the construction 3D tissue in vitro is highlighted, for example, by experiments using layer-by-layer techniques to deposit alternating layers of few nanometer thick ECM-analogous oppositely charged polyelectrolyte films made from fibronectin and gelatin on top of a layer of adherent mammalian cells. While an ECM-analogous layer allows a second layer of cells to adhere to the first cell layer and so forth to built up multicelluar aggregates, without the ECM-resembling layers, no second cell layer on top of the second was observed [5 Matsusaki 2007].

Ecm Ecm

Ecm [6 Tibbitt 2009]

Cancer ecm [7 Ingber 2008]

Chondrocytes embedded randomly within various hydrogels favored proliferation at lower seeding density and matrix biosynthesis at higher cell density.

Establishing the influence of microscale organization on cell fate has practical as well scientific implications. For tissue engineering applications, one might expect more rapid matrix synthesis by chondrocytes in high density seeded scaffolds [8 Albrecht 2006].

In the success of tissue engineering, three dimensional (3D) scaffolds play important roles as extracellular matrices onto which cells can attach, grow, and form new tissues [9 Nam 2004].

Traditional scaffold fabrication techniques have involved the production of porous polymer monolithic constructs as substrates for cell attachment. [10 Leong 200311 Andersson 2004] [12 Andersson 200413 Khademhosseini 2006]

Factors governing scaffold design are complex and include considerations of

matrix architecture,

pore size and


stability versus porosity,

surface properties and

degradation rate.

The scaffold must provide sufficient mechanical strength and stiffness to guarantee structural integrity during the development step and subsequent cell culture. Furthermore, scaffold architecture should enhance initial cell attachment; it must also enhance the mass transfer of metabolites and provide sufficient space for proliferating cells.

To date, two methods of incorporating cells into scaffolds are being explored: (i) seeding of cells onto the surface of the scaffold following fabrication and (ii) the incorporation of cells into the scaffold fabrication process.


Three stages: simple woodpile scaffolds for the evaluation of the 2PP processibility and biocompatibility.

Second stage, a 2.5 D "letter case" structure, an array of different size micro cavities to evaluate cell morphology on substrate with different geometry.

Third stage, 3 D microarchitectures. First: a cubic lattice. Two different unit cells were compared with respect to the stability of the produces scaffold and, more important, to the processing time (Fig. 3).

Although the cubic lattice is highly suitable for producing large volume scaffolds in short processing time and is easily be adapted to the needs of the cells by scaling the computer model of the scaffold unit cell, it is not optimal from the secundam-naturam paradigm: straight edges, acute angles, sharp turns and inhomogeneous curvature distribution along the scaffold surface are contra-naturam and should be prevented in biomorphic cell supports. The biomorphic geometry that mimics the secundam-naturam substrate would be one that is continuous through all space, partitioned into two not-necessarily-equal sub-spaces by a non-intersecting, two-sided surface. Minimal surface geometry is not only ideal to describe such a space but is also the preferentially assumed geometry in natural and pathological or manipulated cells. Therefore a 3D scaffold unit cell based on the "Schwarz P" triply periodic minimal surfaces (TPMS) was models using AutoCAD [14 Rajagopalan 2006]. [15 Srinivasan 2005]. 2PP-derived structures fabricated on the basis of the "Schwarz P" minimal surface showed excellent stability in combination with high porosity (>75 %) (Fig.5). The surface-to-volume ratio of TPMS scaffolds is optimized and enables sufficient cell attachment and growth. Fluid transport properties within porous TPMS scaffolds is extremal as well, facilitating easy diffusion of nutrients to and waste products from the cells [16 Torquato 2004].

Scaffold Materials


biocompatible polymers acrylates methacrylates

Degradation PEG PLA scaffold [17 Burdick 2003]

[18 Wichterle 1960]

[19 Nguyen 2002]

[20 Stampfl 2005]

[21 Schuster 2006]

[22 Correa 2009]

However, complex architectures with tunable microscale features have become possible not before mouldless CAD-based manufacturing (synonyms are rapid prototyping (RP) and solid freeform fabrication (SFF)) technologies have emerged for fabricating complex shaped scaffolds [23 Tsang 2007]. Excellent reviews on the use of RP techniques, including conventional photolithographic patterning, electrochemical deposition, 3D printing and soft lithographic approaches, has been published throughout the last decade [24 Hutmacher 2004] [25 Borenstein 2007]. [26 Moroni 2008].

To create internally complex materials, these methods are repeated in a layer-by-layer fashion until a scaffold of the desired dimensionality is achieved.

Unlike conventional machining, which involves constant removal of materials, most RP techniques builds parts by selectively adding materials, layer by layer, as specified by a computer program. Each layer represents the shape of the cross-section of the model at a specific level.

RP techniques can be easily automated and integrated with imaging techniques to produce scaffolds that are customised in size and shape allowing tissue-engineered grafts to be tailored for specific applications or even for individual patients.

In fused deposition modeling (FDM) molten plastics or ceramics (e.g. poly(ε-caprolactone, PCL)) are extruded through a noozle and deposited layer by layer to form the 3-D scaffolds with feature sizes of approximately 250-700 µm [27 Zein 2002]. Here the size of the pores created is predetermined by the size of the polymer filament extruded through the noozle and the materials that can be used for this method are limited by the melting points and processing conditions involved.

In selective laser sintering (SLS) a laser beam raises the local temperature of the powder bed in order to fuse the particles, forming patterned structures within each layer. The resolution is currently approximately 300 µm [10 Leong 2003].

Photopolymerization involves the use of light energy to initiate a chain reaction, resulting in the formation of a solid from the original liquid photopolymer solution. In stereolithography (SLA) spatial patterning of the polymer is achieved by directing the position of the light using a laser beam or by exposing certain areas of an entire layer through a photomask. The stage is then lowered and the process repeated to form additional layers. Resolution is shown to be smaller 100 µm [28 Yang 2002].

From the material choice point of view there is a close connection between the SLA and 2PP applications in biomaterials. Many resins have been used for both processes because of the 365 nm ()

2PP Two-photon excitation provides a means of activating chemical or physical processes with high spatial resolution in three dimensions. Historically, the first applications were the 3D fluorescence imaging (two-photon laser scanning microscope (TPLSM) and optical data storage. The latter holds great potential of a new generation of rewritable polymeric data carriers with high information density. Today, more and more, lithographic microfabrication based on two-photon excitation becomes the dominant field of invention because of the versatility of the process to built 3D structures of almost any desired complexity and the great spectrum of materials being processed.

These applications take advantage of the fact that the two-photon absorption probability depends quadratic on intensity, so under tight-focusing conditions, the absorption is confined at the focus to a volume of order λ3 (where λ is the laser wavelength) [29 Goeppert-Mayer 1931]. Any subsequent process, such as fluorescence or a photoinduced chemical reaction, is also localized in this small volume.

The emergence of 2PP as new technology in 1997 [30 Maruo 1997] has brought the light curable resin into the realm of nanofabrication [31 Sun 2004]. The two-photon process has at least two advantagescompared to single-photon absorption used in conventional rapid prototyping. First, common polymers have negligible linear absorption in the red/near- infrared (NIR) region, so the laser penetrates deeply into materials and directly induces polymerization from inside without contaminating outside of the focal volume; secondly, the quadratic dependence of polymerization rate on the light intensity enables 3D spatial resolution, and the accuracy is better than that achieved in single photon process. Actually a near 100-nm lateral spatial resolution has been reported [32 Kawata 2001].

[33 Farsari 2005]

[34 Lee 2006]

[35 Wu 2006]

[36 Li 2007]

[37 LaFratta 2007]

[38 Maruo 2008]

[39 Anscombe 2010]

[40 Fourkas 2010]

Material classes

Negative type photoresists:

Hydrogel materials made of acryloylacetone, acrylamide, and N,N'-bisacrylamide have also been fabricated by 2PP [41 Watanabe 2002]. These materials undergo a tautomerization after exposure to UV light, resulting in controllable shrinkage of the structures. Watanabe et al. produced a cantilever by using this material with an irreversible photoactuated process.

Organically modified ceramics (ORMOCERs or ceramers often used as photocurable dental composites) have also been used in 2PP [42 Serbin 2003]. These silicate-based materials combine the best features of sol-gel processing and organic polymers. They have an inorganic (-Si-O-Si-) backbone functionalized with organic groups such as acyrlates or epoxides. The organic side chains can cross-link the resin into a durable, biocompatible solid. s.

Another material that has been used in MAP is polydimethylsiloxane (PDMS). This material is ubiquitous in soft lithography, where it is used for making 2D patterns with nanometer resolution. Ober and co-workers reported photopolymerization of PDMS by two distinct methods. The first was a photohydrosilylation reaction using a photoactive platinum catalyst. This method suffers from undesired thermal polymerization, which adversely affects the resolution that can be attained. The second method employed ITX to initiate cross-linking of the dimethylvinyl-terminated siloxane components.

Positive type photoresists:

The PAGs used for cationic polymerization can also be used in positive-tone photoresists, which promises to be a useful way to fabricate 3D microfluidic devices. Marder, Perry, and co-workers demonstrated positive-tone 2PP with the initiator BSB-S2 in a random copolymer consisting of tetrahydropyranyl methacrylate, methylmethacrylate, and methacrylic acid units. The tetrahydropyranyl ester groups were converted into carboxylic acids after the photoacid protolysis, causing them to be soluble in a basic developer. Channels 4 µm x 4 µm in cross section have been made 10 µm below a surface by using an average power of 40 µW from a Ti:sapphire oscillator.

ORMOCERE [43 Haas 1999]

δ is the peak two-photon absorption cross-section in 10-50 cm4 s per photon (GM)


For one-photon absorption, the absorption probability is independent of the mode of excitation, i.e. continuous wave or pulsed, and there is no dependence on the pulse width for the latter. By contrast, multiphoton absorption scales as pn/atn21, where p is the pulse energy in Joules, a is the spot size in cm2, and t is the laser pulse width. There are two different operative powers, the average power and peak power, where the latter is the operative parameter in multiphoton absorption.

2PP for scaffolds

Multiphoton excited polymerization has attracted increasing attention as a powerful 3 D nano- and microfabrication tool Recently, a couple of papers were describing the use of 2PP for the microfabrication of 3D scaffolds.

2PP was applied to pattern bioactivity into preformed photoactive materials. PEG-based materials have been studied extensively as tissue-engineering scaffold and are biocompatible and intrinsically resistant to protein adsorption. Poly(ethylene glycol) (PEG)-diacrylate (PEGDA) hydrogels are in themselves biologically "blank slates" into which desired bioactivity can be tailored via light-based patterning. [44 Hahn 2006].

Using 2PP and additional Methacrylated RGD-ACRL, adhesion promoting peptide RGD or a low-molecular weight MW 600 PEGDA was allowed to diffuse into a preswelled MW 6000 PEGDA hydrogel. This prepolymer sample was irradiated with femtosecond pulse in the present of photoinitiator. The acrylate-derivatized moieties in the precursor solution were conjugated to the hydrogel in the irradiated regions, altering its cell-adhesion behavior or its stiffness due to higher degree of crosslinking in the illuminated regions. Site-specific RGD-functionalization of hydrogels was demonstrated to be useful for cell-guiding, while 3D site-specific control of the stiffness of hydrogels is important to mimic the mechanical properties of the extracellular matrix ECM) because mechanical cues of the microenvironment are important signals for direct the cell fate (proliferation, differentiation, apoptosis) [45 Pek 2010]. [46 Nemir 2009].

[47 Cunningham 2006]

[48 Ovsianikov 2007]

[49 Schlie 2007]

[50 Tayalia 2008]

[51 Schade 2009]

[52 Weiß 2009]

[53 Claeyssens 2009]

[54 Ovsianikov]

Scaffolds from crosslinked Protein

[55 Campagnola 2000]

Very attracting for building scaffolds crosslinked protein gels. Especially the proteins of the ECM (fibrinogen, fibronectin, collagebn) are interesting candidates. Rose Bengale was used as a sensitizing dye .[56 Basu 2004]. Even inside living cells protein crosslinking via photosensitizer reaction was demonstrated. [57 Basu 2005] .[58 Pins 2006]

Using enzymes like alcalic phosphatise (AP) catalytically active microstructures have been made by 2PP crosslinking via Rose Bengae crosslinking .[59 Allen 2005]

Using a specially designed photocrosslinker based on a amino derivate of benzophenone dimmer even collagen, which was not crosslinkable using photosensitizers, was used as building material for ecm-analogous mictrostrructures. [60 Pitts 2002].

There are two generally accepted modes in which proteins can be cross-linked by photoactivation: hydrogen atom abstraction and formation of singlet oxygen to photooxidize protein residues (31). Given singlet oxygen yields of 75- 85% for rose bengal, we cannot rule out the involvement of singlet oxygen in the protein cross-linking because these measurements were not made in an oxygen-free environment [60 Pitts 2002].

Other 2PP Applications

[61 Cumpston 1999]

[62 Kirkpatrick 1999]

[63 Serbin 2004]

[64 Wylie 2008]

[65 Kim 2005]

[66 König 2005]

[67 Xiong 2008]

[68 Sidorenko 2007]

[41 Watanabe 2002]

[69 Knoll 2006]

Metalic microstructures based on 2PP were described. The photopolymerizable material consists of a commercially available resin (Z7012C, JSR) mixed with styrene in a 1:1 volume ratio. In the case of two-photon absorption, the chemical reaction progresses by radical attacking14 and thus is very effective for styrene which has a vinyl group. A sensitizing stannous chloride thin film is produced on the polymeric microstructure and act as a binder with the metallic film. Electroless plating using silver nitrate results in metallic microstructures [70 Formanek 2006].[71 Formanek 2006].

BioMEM Integration


miniaurized cell culture systems

[72 Stubenrauch 2009]

[73 Fröber 2009]


3-D fabrication techniques are useful for studying structure/function relationships in model tissues.

Cell seeding


Printing of cells into 2PP derived scaffolds [54 Ovsianikov 2010].

Leong et al. described a photolithographically structured, mobile microcontainers that function like three dimensionally patterned, mobile microwells. The containers load themselves as they self-assemble from cruciform templates en masse at around 40 â-¦C, a temperature low enough to enable parallel loading of biological objects. These containers have porous walls and interact with their surroundings in all three dimensions.[74 Leong 2008]


[75 Denk 1990]

[76 Martini 2006]

2) Experimental

2.1 Materials

polyethylenglycol diacrylate (PEG-DA) was received from Sigma-Aldrich , UDMA,  H 

Abbrevations: Dex = dextran, Dex-GMA = glycidyl methacrylated dextran, HA = hyaluronic acid, HA-MA = methacrylated hyaluronic acid, PG2 = poly(glycerine), PG2-UM = urethane methacrylated polyglycerine, PEG (Mn = 200 g/mol, 400 g/mol, 1000 g/mol) = poly (ethylene glycols) PEG (Mn = 200 g/mol, 400 g/mol, 1000 g/mol)-UM = urethane methacrylated poly(ethylene glycols).

Dextran (Mn = 15.000-20.000 g/mol, Dex), glycidyl methacrylat (GMA), methacrylic anhydride (MA) and poly(ethylene glycols), PEG (Mn = 200 g/mol, 400 g/mol, 1000 g/mol) ) were obtained from Fluka, Buchs, Switzerland..

Hyaluronic acid sodium salt (Mw = 924.000, HA) was from Graeber GmbH, Ellerbek, Germany. Poly(glycerine) (Mn = 2000 g/mol, PG2) was purchased from Hyperpolymers GmbH, Freiburg, Germany.

All solvents and other chemicals (4-(N,N-dimethylamino)pyridine (DMAP), 2-hydroxy-4`-(2-hydroxyethoxy)-2- methylpropiophenone (Irgacure 2959), 2-isocyanatoethyl methacrylate (IEM), stannous octoate, dibutyltin dilaurate (DBTL), eosin Y (EY), triethanol amine (TEA), 1-vinylpyrrolidone (VP)) were obtained from Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany.

1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959®) was received from Ciba Speciality Chemicals, Inc. as a gift. The concentration of the photoinitiator in the precursors was 2% (w/w). No additional solvent was used.


2.2 Two-Photon Polymerization

Near-infrared Ti:sapphire femtosecond laser pulses were applied for the fabrication of 3D structures. The laser wavelength, pulse duration and repetition rate was 750-790 nm, 100-120 fs, and 80 MHz, respectively.

The experimental set-up consists, aside of the femtosecond laser, of following parts:

a) two beam splitter combinations consisting each of a cube polarizer and a λ/2-plate for dividing the laser power between different applications (TPLSM, 2PP, nanotome) and for attenuation the laser power in the 2PP experiment; b) a CCD camera for online-monitoring the 2PP process ; c) an akkusto-optical modulator (AOM) for rapid on/off control of the illumination.

The laser was focused into the photosensitive samples by either a 100x high numerical aperture (NA 1.4, oil immersion) objective lens for producing structures with highest resolution (<500 nm) or an objective lens (Zeiss x 63, Plan-Neofluar) with numerical aperture of 0.75 and a working distance of 2.2mm was used.) for fast fabrication of larger scaffold structures (lateral resolution 2 µm, axial resolution 5-10 µm). The choice of the objective lens was governed by the needs of required resolution of the one hand and the processing time on the other hand. To generate three-dimensional microstructures based on computer models ultra precision linear-motor-driven stages (ABL 10100 Air Bearing Stage, Aerotech Inc., Pittsburgh PA) were used to move the sample holder (typical writing velocities from 100-3.000 µm/s).

In order to remove the non-illuminated and therefore non-solidified material after irradiation and to develop the micro structured specimen the sample holder glass slide was eluted with a developer depending on the solubility of the used photopolymers. For UDMA ethanol was used, for OLDMA 1 and OLDMA 2 acetone, for ORMOCERE a 1:1 mixture of 4-methyl-2 pentanone and 2-propanol and for the water-borne precursors water was used.

For example, a 780 nm wavelength laser with a 100 fs pulse width at a repetition rate of 80 MHz possesses a spectral full width at half maximum (FWHM) of approximately 10 nm [31 Sun 2004].

The broad laser spectrum associated with the ultrashort pulse width brings about chromatic aberration, which is overcome by usage of apochromats.

Numerical Aperture

Laser scanning is the step needed to convert pre-designed CAD patterns into real structures.

 Fíg.1: Scheme of two-photon polymerization set-up.

To promote sample adhesion, if necessary, the coverslips were bathed in a dilute solution of 3-(trimethyoxysilyl) propyl methacrylate.

2.4 Cell culture


2.4.1 Cell seeding


2.4.2 Cell culture


2.4.3 Imaging


3. Results


3.1 2PP

The main advantage of using 2PP for 3D microfabrication of synthetic scaffolds is the, in principle, free control of the three-dimensional architecture of the mesoscopic biocompatible interface. The flexibility is only compromised, by now, by the file size of the computer model limiting the resolution of the minimal scaffold feature and the processing time. Latter is strongly dependent on the optimized hatching and slicing parameters but by far more by defining proper unit cells as building blocks of the entire scaffold structure. This is mainly because of preliminary restriction of the filling strategy, the laser movement, respectively, to equidistant x,y-slicing  and z-stepping. After the contours of one slice of defined thickness, which has to be adapted to the numerical aperture of the used objective lens (e.g. for N.A. 0.75 the slice thickness is 6 µm, for N.A. 1.4 the slice thickness is 0.5 µm) and, to a minor extent, to the scanning speed because of reduced laser impact with rising scanning speed, are drawn, the inner of the structure is filled with a woodpile hatching pattern which appropriate distance is again defined by the numerical aperture of the objective lens (2.5 µm for N.A. 0.75, 0.3 µm for N.A. 1.4).

Clear, simple structures are obviously favored, given rise for search for sources for model geometries: periodic crystal structures, minimal surfaces and biomorphs.

The scaling of the scaffold is a compromise between possible resolution down to the nanoscale and the biological demands of the cell as well as the processing time and the architectural stability. As the woodpile structure of stapled layers of orthogonal intersecting lines is the standard filling strategy in our 2PP control software we choose woodpiles with different distances (from 10 to 150 µm) as simple model structures for testing the biocompatible of the materials by seeding with cells and evaluating their response over 3-4 day culture by CLSM after staining.

Support free topological cell chips of letter case structure (2 x 2 x 0.15 mm3, Fig. xy) were designed for finding ideal scaffold scaling parameters. As the diameter of bovine chondrocytes can vary between 10 and 40 µm depending on the spreading extend an ECM analogous scaffold pore size was estimated to lie in the range 10 - 150 µm.

 Fíg.2: Computer model of the letter case topological cell chip. Computer model of eight

"Schwarz-P minimal surface" unit cells.


   Fíg.3: Computer model of the cubic lattice unit cells. Left: cage unit cell; right: axes-of-coordinates-like unit cell. Size: 80x80x80 µm3, Thickness of the beams 10 µm. Scaffolds are formed by building x,y and z-arrays of the unit cell.

The woodpiles and the topological chip are 2.5 dimensional structures that could be produced, if with some effort (masks, alignment, repetitions), by traditional microstereolithography as well.

True 3D microstructures Schwarz P surface

The role of laser parameters

The laser wavelength has influence on the absorption of the incident light since the absorption of the photoinitiator Irgacure 369 peaks at 330 nm at shorter wavelength than the half of 730-790 nm. On the other hand the laser output power decreases with decreasing wavelength. Typically the two-photon absorption at lower wavelength than 750 nm was to strong, the resolution declines and damage occurred. For most

2.2.2 2PP Processibility of Photopolymers

Important aspect in the processibility of photopolymers for 2PP is the viscosity of the photopolymer, the solubility of a suitable photoinitiator in the monomer/macromer solution and the density of polymerizable vinyl groups. In general, photopolymers or photoresists are well suited for 2PP when they are liquids and exhibit an in-situ high viscosity, when they are solvent free and are crosslinkable, e.g. posses more than one, better three and more polymerizable vinyl groups.

Acrylated compounds tend to be more reactive than methacrylic monomers or macromers. E.g. the threshold of polymerization between PEG- DA and PEG- DMA of comparable molecular weight differs between 1-10 mW for PEG- DA and 100- 300 mW for PEG- DMA making PEG -DMA a very poor 2PP photopolymer (almost not processable) while PEG -DA with 2% Irgacure 369 is an excellent 2PP photopolymer and serves as standard because of its good availability and biological inertness. Cell adhesion, however, is greatly repressed on non-modified PEG- DA due to its water-swollen hydrogel character.


HV08 = OL-MA 1

HV12 = OL-MA 2 Organic soluble photopolymers

If possible Irgacure 369 ® was used as a photoinitiator. Irgacure 369 is soluble in ethanol, acetone PEGDA, UDMA, OL MA 1/2, polyglycerines, PEG 400-IEM, and Ormocere,

Although the peak of absorption of Irgacure 369® is 330 nm and therefore a two-photon wavelength of 750-790 nm is not optimal for the excitation of this photoinitiator



 Fíg.5: Letter case structure, detail of cubic lattice structure made and Schwarz-P surface array of UDMA.  1000x REM image.


 REM Bilder von den Strukturen

 HV 08, HV 12

 Fíg.6: Woodpile structure made from HV 08. Water soluble biopolymers

The slightly water-soluble photoinitiator Irgacure 2959 was tested for 2PP reactivity in combination with water-soluble monomers (AAm, HEMA) and macromers (HyaMA, AlgMA) but was found to be not efficient in initiating 2PP because of the fact, that Irgacure 2959 is not as soluble to produce a 2 % (w/w) solution as needed in 2PP of organic soluble photopolymers or that the absorption peak of absorption has a to short wavelength (330 nm).

As photoinitiator for the water-borne precursors a combination of Rose Bengale with triethanolamine as coinitioator was used.


2.2.3 Cell response


Fíg.7: Cubic lattice scaffold made of UD -MA seeded with cells.


Fíg. 8: Woodpile structure made of HV 08 seeded with cells.



  Fíg.9: Woodpile structure made of HV 12 seeded with cells.




Fíg.10: Detail of letter case structure made of PEG-DA on glass support showing an exclusion of bovine chondrocytes from the surface of the water swollen material. Low initial adhesion.


Similar non-adhesive behavior was found for other hydrogel materials as PEG-DA and Polyacrylamide.  


4. Discussion

4.1 2PP

Choosing different degrees of methacrylation, photoinitiators and 2PP process parameters like incident laser power, scan velocity and repetition rate a wide range of shape controlled microstructures can be polymerized with different properties regarding the mechanical stiffness, geometry, cell attachment and cell growth.

4.2 Biocompatibility


Imaging: CLSM, TPLSM, REM, µCT



Fíg.11: 3D reconstruction of a 2PP derived scaffold (cubic lattice) using TPLSM. 80 µm unit cell. UD -MA. 


Noch machen REM -Bild Large Pore Scaffold 5x5x2 mm



Fíg.12: µCT image of 2PP scaffold made from PEG-DA. Grid size 500 µm.


Contrast PEG -DA

Resolution 10 µm.


 Results on 3D effect on the proliferation and differentiation of chondrocytes as measured by qRT-PCR will be published in an upcoming paper.


4.3 Upscaling

One of the most serious drawbacks of the 2PP technology is the inherent serial, voxel-by-voxel character of the fabrication process resulting in extended processing times.

Mainly this has limited the production of larger scaffolds of highest resolution or specimen numbers up to now. To overcome this limitations various up-scaling strategies has been established in recent years.

Wu et al. saved processing time in the microfabrication of their classical Venus micro statue by showing that it was only necessary, provided that the slicing, hatching and laser movement parameters provide necessary stability, to irradiate the outer shell of the structure (the contours) by fs pulses while the inner volume was solidified using a UV-lamp after the surrounding liquid resin was washed away [35 Wu 2006].

Resins incorporating conventional ultraviolet-absorbing initiators exhibit comparatively low photosensitivity as the initiators have small two-photon absorption cross-sections (δ) although three-dimensional data storage and microfabrication have been illustrated using two photon- initiated polymerization of such photopolymer systems. Typical values are in the range of 4 GM (Irgacure 819) and 28 GM (Irgacure 651)). [77 Schafer 2004]. Consequently, this approach requires still comparable high laser power, connected with limited scan speed and its widespread use remains impractical. Any serious upscaling of the 2PP process, therefore, has to focus on the material side on the development of specially designed two-photon absorbers that exhibit large two-photon absorption cross section (δ). Cumpston et al. have synthesized a two-photon absorbers with two-photon absorption cross section of 1250 GM and showed that a commercial triacrylic photopolymer (SR9008 and SR454, Sartomer) has an order-of-magnitude higher two-photon photosensitivity, relative to the conventional initiators examined.

Photoinitiators with high two-photon absorption cross sections has been focus of intense research in the last decade and it was demonstrated that they were able to lower the polymerization threshold laser power, that is, the necessary irradiation power to induce polymerization []. The development of materials which exhibit enhanced two-photon absorption that can initiate photopolymerization has been revieved in a number of papers. [78 Lin 2003] [79 Kuebler 2003] [80 Lu 2004] [81 Lu 2004] [82 Xing 2007]

Low cost ps microlaser [83 Wang 2002]

Special Fluorine amine system [84 Belfield 2000]

Acylamide photopolymerization with xanthene dye photoinitiator [55 Campagnola 2000]

Large area 2PP microstructuring [85 Malinauskas]

Individually controlled multi-laser processing for 2PP [86 Obata]

Laser beam shaping effects on 2PP [87 Winfield 2008]

One approach for upscaling the 2PP process, that means for making 2PP applicable to the macroscopic world, is to built up a hydrogel material via bulk (photo-) polymerization, subsequently allowing a functional monomer, e.g. acrylated adhesion promoting peptide, to diffuse into the hydrogel and patterning the bioactive molecule inside the gel. By this the main polymerization takes place conventionally and only the functionalization in certain areas of the hydrogel needs to fabricated using 2PP [88 Lee 2008].

Another altenative approach in the same line of development is the establishment of positive tone 2PP photoresists, which contain chemical groups, which can be cleaved upon irradiation rendering the material better soluble in a developer. A positive-tone resists using a special two-photon absorbing photo-acid generators (PAGs). The crosslinkable epoxide resin ()[84 Belfield 2000].[89 Yu 2003]. [90 Zhou 2002].

Laseroptical developments try to upscale the 2PP process by different techniques:

Periodic structures are fabricated by multibeam interference lithography [91 Moon 2006]. Or by holographic techniques .

Or by using parallel processing using multibeam fabrication.

Our strategy has focused on the proper choice of the objective lens and of the proper choice of the unit cell.

2PP structures described in the literature do not exceed the mm

In the literature described up to 1 mm2.

Here series of 2 x2 x0.25 mm3 scaffolds.

For being applicable in macroscopic world, e.g. tissue engineered implants, high density data storage or waver-size 3D lithography the 2PP process has to be further accelerated [] and expanded to mesoscalic specimen sizes (0.5-5 mm).

Various strategies have been developed for the assembly of mesoscalic specimen, 2PP derived and other microfabricated "little things". Khaddemhosseini et al. invented the possibilities to self-assemble mesoscalic 3d hydrogel constructs using multiphase liquid-liquid systems. Microstructured hydrogels, prepared by UV crosslinking using photomasks or microstereolithography were immersed in non-polar solvents. Thermodynamically driven self-assembly followed by second step UV postcuring yields in organized super-structures which could be used for developing functional multicellular constructs with different cell types arranged in a tissue specific way [92 Du 2008]

Microrobotic approaches have been applied for the assembly of mesoscalic scaffolds for tissue engineering by using microgrippers and a lock-and-key architecture of the building blocks [93 Zhang 2005].

From a materials point of view, synthesis of two-photon chromophores with even higher two-photon cross-sections is required, so that photopolymerization may be induced by less expensive picosecond, nanosecond and even CW lasers for commercial applications. Also the high δ materials would enable polymerization simultaneously from multibeam split out of one output

of a laser for batch production.

Fíg.12: Micrograph of 2PPderived scaffold made from PEG-DA. Grid size 500 µm; beam thickness about 60 µm; scaffold size: 1 x1 x0.2 cm3.