Two Photon Polymerization Biocompatible Photopolymers 3d Biointerfaces Biology Essay

To let cells to be cultured in vitro in a manner that resembles the natural conditions more closely than level TCPS, 3D microstructured scaffolds are needed.Two-photon polymerisation ( 2PP ) is applied as a tool for formation of high-resolution 3D scaffold constructions with wholly defined microarchitecture within biocompatible photopolymers. 2PP is a fresh photolithographic technique utilizing femtosecond optical maser pulses with enables free 3D microstructuring of liquid photopolymers due to the axial and sidelong spacial parturiency of the photoreaction to the focal volume of a focussed optical maser beam. A set of photopolymers were tested with respect to 2PP processability and six different categories of ( meth- ) acrylated photopolymers ( oligolactones, polyglycerines, urethane dimethacrylate, polythene ethanediol diacrylate, dextran, and hyaluronan ) were found to be efficient 2PP stuffs. Based on 3D computing machine theoretical accounts 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 civilization on 2PP derived microstructures was evaluated morphologically by CLSM after staining. 2PP derived scaffolds were fabricated in different sizes, get downing from the 100 Aµm-range to the cm-range demoing the existent possibilities to bring forth big volume scaffolds even for nidation intents.

1 ) IntroductionA

Tissues and variety meats consist of a complex organisation of cells, extracellular matrix ( ECM ) , and signaling molecules. In vivo, tissues are composed of reiterating units on the graduated table of 100-1000 Aµm. The 3-dimensional architecture of these reiterating tissue units underlies the coordination of multicellular procedures, emergent mechanical belongingss, and integrating with other variety meats via the microcirculation. A cardinal component of tissue construction in vivo is hence the local cellular environment. The “ microenvironment ” ( i?z10 Aµm ) presents biochemical, cellular, and physical stimulations that synchronize the cell destiny such as proliferation, distinction, migration, and programmed cell death. The end of tissue technology is to reconstruct or replace the lost maps of diseased or lost variety meats. In the classical tissue technology attack cell were taken from a patient, expanded in vitro on 3D scaffolds and re-implanted to the defect site. Successful application of populating cells in regenerative medical specialty requires an apprehension of how tissue construction relates to organ map. There is turning grounds that presentation of extracellular cues in a 3-dimensional ( 3D ) context can basically change cellular responses [ ] .Thus, microenvironment surveies that antecedently were limited to adherent planar ( 2D ) civilizations may non be appropriate for many cell types. In the particular instance of tissue technology of articular gristle the distinction behaviour of chondrocytes in 3D Scaffold has to be controlled.

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Recent findings in oncogenesis and root cell distinction clearly show acute disparities in cell map between 2D and 3D civilization and suggest that analyzing hierarchal biological science in merely two dimensions is deficient. For illustration, enhanced chondrogenesis of embryologic root cells has been observed when cells are cultured in 3D embryoid organic structures as compared to monolayer civilization [ 1 Tanaka 2004 ] . In an early illustration, Bissel and coworkers has demonstrated, that human chest epithelial cells develop like tumour cells when cultured in two dimensions, but revert to normal growing behaviour when cultured in 3D parallels of their native microenvironment [ 2 Petersen 1992 ] .

Cartilage tissue that lines our movable articulations serves to back up mechanical tonss and facilitates motion and flexibleness. Unfortunately, gristle heals really ill after hurt or injury, frequently taking to gradual debasement over clip. Its devolution, which effects soon in the US entirely over 36 million people, leads to serious and painful diseases and damage of mobility and. In malice of enormous attempts made in biomedical scientific disciplines gristle has remained a peculiarly ambitious tissue to engineer [ 3 Raghunath 2007 ] . Different schemes to engineer or renew gristle tissue have been developed in the past decennaries. Typically, tissue concepts are assembled in vitro by uniting cells and alleged scaffolds made from assorted biomaterials. In contrast to traditional cell civilization on planar substrates such as tissue civilization polystyrene ( TCPS ) the civilization of mammalian cells on 3D scaffolds in vitro provides a defined platform for look intoing cell and tissue physiology and pathophysiology outside the being.

ECM [ 4 Badylak 2007 ]

The importance of the function of the ECM for the building 3D tissue in vitro is highlighted, for illustration, by experiments utilizing layer-by-layer techniques to lodge jumping beds of few nanometer thick ECM-analogous oppositely charged polyelectrolyte movies made from fibronectin and gelatin on top of a bed of adherent mammalian cells. While an ECM-analogous bed allows a 2nd bed of cells to adhere to the first cell bed and so forth to construct up multicelluar sums, without the ECM-resembling beds, no 2nd cell bed on top of the second was observed [ 5 Matsusaki 2007 ] .

Ecm Ecm

Ecm [ 6 Tibbitt 2009 ]

Cancer ecm [ 7 Ingber 2008 ]

Chondrocytes embedded indiscriminately within assorted hydrogels favored proliferation at lower seeding denseness and matrix biogenesis at higher cell denseness.

Establishing the influence of microscale organisation on cell destiny has practical as good scientific deductions. For tissue technology applications, one might anticipate more rapid matrix synthesis by chondrocytes in high denseness seeded scaffolds [ 8 Albrecht 2006 ] .

In the success of tissue technology, three dimensional ( 3D ) scaffolds play of import functions as extracellular matrices onto which cells can attach, turn, and organize new tissues [ 9 Nam 2004 ] .

Traditional scaffold fiction techniques have involved the production of porous polymer massive concepts as substrates for cell fond regard. [ 10 Leong 200311 Andersson 2004 ] [ 12 Andersson 200413 Khademhosseini 2006 ]

Factors regulating scaffold design are complex and include considerations of

matrix architecture,

pore size and

geometry,

stableness versus porousness,

surface belongingss and

debasement rate.

The scaffold must supply sufficient mechanical strength and stiffness to vouch structural unity during the development measure and subsequent cell civilization. Furthermore, scaffold architecture should heighten initial cell fond regard ; it must besides heighten the mass transportation of metabolites and supply sufficient infinite for proliferating cells.

To day of the month, two methods of integrating cells into scaffolds are being explored: ( I ) seeding of cells onto the surface of the scaffold following fiction and ( two ) the incorporation of cells into the scaffold fiction procedure.

Considerations:

Three phases: simple woodpile scaffolds for the rating of the 2PP processibility and biocompatibility.

Second phase, a 2.5 D “ missive instance ” construction, an array of different size micro pits to measure cell morphology on substrate with different geometry.

Third phase, 3 D microarchitectures. First: a three-dimensional lattice. Two different unit cells were compared with regard to the stableness of the green goodss scaffold and, more of import, to the processing clip ( Fig. 3 ) .

Although the three-dimensional lattice is extremely suited for bring forthing big volume scaffolds in short processing clip and is easy be adapted to the demands of the cells by scaling the computing machine theoretical account of the scaffold unit cell, it is non optimum from the secundam-naturam paradigm: consecutive borders, acute angles, crisp bends and nonuniform 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 uninterrupted through all infinite, partitioned into two not-necessarily-equal sub-spaces by a non-intersecting, reversible surface. Minimal surface geometry is non merely ideal to depict such a infinite but is besides the preferentially false geometry in natural and pathological or manipulated cells. Therefore a 3D scaffold unit cell based on the “ Schwarz P ” triply periodic minimum surfaces ( TPMS ) was theoretical accounts utilizing AutoCAD [ 14 Rajagopalan 2006 ] . [ 15 Srinivasan 2005 ] . 2PP-derived constructions fabricated on the footing of the “ Schwarz P ” minimum surface showed first-class stableness in combination with high porousness ( & gt ; 75 % ) ( Fig.5 ) . The surface-to-volume ratio of TPMS scaffolds is optimized and enables sufficient cell fond regard and growing. Fluid conveyance belongingss within porous TPMS scaffolds is extremal every bit good, easing easy diffusion of foods to and waste merchandises from the cells [ 16 Torquato 2004 ] .

Scaffold Materials

Degradable

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 characteristics have become possible non before mouldless CAD-based fabrication ( equivalent word are rapid prototyping ( RP ) and solid freeform fiction ( SFF ) ) engineerings have emerged for manufacturing complex shaped scaffolds [ 23 Tsang 2007 ] . Excellent reviews on the usage of RP techniques, including conventional photolithographic patterning, electrochemical deposition, 3D printing and soft lithographic attacks, has been published throughout the last decennary [ 24 Hutmacher 2004 ] [ 25 Borenstein 2007 ] . [ 26 Moroni 2008 ] .

To make internally complex stuffs, these methods are repeated in a layer-by-layer manner until a scaffold of the coveted dimensionality is achieved.

Unlike conventional machining, which involves changeless remotion of stuffs, most RP techniques physiques parts by selectively adding stuffs, bed by bed, as specified by a computing machine plan. Each bed represents the form of the cross-section of the theoretical account at a specific degree.

RP techniques can be easy automated and integrated with imaging techniques to bring forth scaffolds that are customised in size and form leting tissue-engineered transplants to be tailored for specific applications or even for single patients.

In amalgamate deposition mold ( FDM ) molten plastics or ceramics ( e.g. poly ( Iµ-caprolactone, PCL ) ) are extruded through a noozle and deposited bed by bed to organize the 3-D scaffolds with characteristic sizes of about 250-700 Aµm [ 27 Zein 2002 ] . Here the size of the pores created is predetermined by the size of the polymer fibril extruded through the noozle and the stuffs that can be used for this method are limited by the thaw points and processing conditions involved.

In selective optical maser sintering ( SLS ) a optical maser beam raises the local temperature of the pulverization bed in order to blend the atoms, organizing patterned constructions within each bed. The declaration is presently about 300 Aµm [ 10 Leong 2003 ] .

Photopolymerization involves the usage of light energy to originate a concatenation reaction, ensuing in the formation of a solid from the original liquid photopolymer solution. In stereolithography ( SLA ) spacial patterning of the polymer is achieved by directing the place of the light utilizing a optical maser beam or by exposing certain countries of an full bed through a photomask. The phase is so lowered and the procedure repeated to organize extra beds. Resolution is shown to be smaller 100 Aµm [ 28 Yang 2002 ] .

From the stuff pick point of position there is a close connexion between the SLA and 2PP applications in biomaterials. Many rosins have been used for both procedures because of the 365 nanometers ( )

2PP Two-photon excitement provides a agency of triping chemical or physical procedures with high spacial declaration in three dimensions. Historically, the first applications were the 3D fluorescence imagination ( two-photon optical maser scanning microscope ( TPLSM ) and optical informations storage. The latter holds great potency of a new coevals of rewritable polymeric informations bearers with high information denseness. Today, more and more, lithographic microfabrication based on two-photon excitement becomes the dominant field of innovation because of the versatility of the procedure to construct 3D constructions of about any coveted complexness and the great spectrum of stuffs being processed.

These applications take advantage of the fact that the two-photon soaking up chance depends quadratic on strength, so under tight-focusing conditions, the soaking up is confined at the focal point to a volume of order I»3 ( where I» is the optical maser wavelength ) [ 29 Goeppert-Mayer 1931 ] . Any subsequent procedure, such as fluorescence or a photoinduced chemical reaction, is besides localized in this little volume.

The outgrowth of 2PP as new engineering in 1997 [ 30 Maruo 1997 ] has brought the visible radiation curable rosin into the kingdom of nanofabrication [ 31 Sun 2004 ] . The two-photon procedure has at least two advantagescompared to single-photon soaking up used in conventional rapid prototyping. First, common polymers have negligible additive soaking up in the red/near- infrared ( NIR ) part, so the optical maser penetrates profoundly into stuffs and straight induces polymerisation from inside without polluting exterior of the focal volume ; secondly, the quadratic dependance of polymerisation rate on the light strength enables 3D spacial declaration, and the truth is better than that achieved in individual photon procedure. Actually a close 100-nm sidelong spacial declaration has been reported [ 32 Kawata 2001 ] .

[ 33 Farsari 2005 ]

[ 34 Lee 2006 ]

[ 35 Wu 2006 ]

[ 36 Fifty-one 2007 ]

[ 37 LaFratta 2007 ]

[ 38 Maruo 2008 ]

[ 39 Anscombe 2010 ]

[ 40 Fourkas 2010 ]

Material categories

Negative type photoresists:

Hydrogel stuffs made of acryloylacetone, acrylamide, and N, N’-bisacrylamide have besides been fabricated by 2PP [ 41 Watanabe 2002 ] . These stuffs undergo a tautomerization after exposure to UV visible radiation, ensuing in governable shrinking of the constructions. Watanabe et Al. produced a cantilever by utilizing this stuff with an irreversible photoactuated procedure.

Organically modified ceramics ( ORMOCERs or ceramers frequently used as photocurable dental complexs ) have besides been used in 2PP [ 42 Serbin 2003 ] . These silicate-based stuffs combine the best characteristics of sol-gel processing and organic polymers. They have an inorganic ( -Si-O-Si- ) anchor functionalized with organic groups such as acyrlates or epoxides. The organic side ironss can cross-link the rosin into a lasting, biocompatible solid. s.

Another stuff that has been used in MAP is polydimethylsiloxane ( PDMS ) . This stuff is omnipresent in soft lithography, where it is used for doing 2D forms with nanometer declaration. Ober and colleagues reported photopolymerization of PDMS by two distinguishable methods. The first was a photohydrosilylation reaction utilizing a photoactive Pt accelerator. This method suffers from unsought thermic polymerisation, which adversely affects the declaration that can be attained. The 2nd method employed ITX to originate cross-linking of the dimethylvinyl-terminated siloxane constituents.

Positive type photoresists:

The PAGs used for cationic polymerisation can besides be used in positive-tone photoresists, which promises to be a utile manner to manufacture 3D microfluidic devices. Marder, Perry, and colleagues demonstrated positive-tone 2PP with the instigator BSB-S2 in a random copolymer consisting of tetrahydropyranyl methacrylate, methylmethacrylate, and methacrylic acerb units. The tetrahydropyranyl ester groups were converted into carboxylic acids after the photoacid protolysis, doing them to be soluble in a basic developer. Channels 4 Aµm x 4 Aµm in cross subdivision have been made 10 Aµm below a surface by utilizing an mean power of 40 AµW from a Titanium: sapphire oscillator.

ORMOCERE [ 43 Haas 1999 ]

I? is the peak two-photon soaking up cross-section in 10-50 cm4 s per photon ( GM )

Energies

For one-photon soaking up, the soaking up chance is independent of the manner of excitement, i.e. uninterrupted moving ridge or pulsed, and there is no dependance on the pulsation breadth for the latter. By contrast, multiphoton soaking up graduated tables as pn/atn21, where P is the pulse energy in Joules, a is the topographic point size in cm2, and T is the optical maser pulsation breadth. There are two different operative powers, the mean power and peak power, where the latter is the operative parametric quantity in multiphoton soaking up.

2PP for scaffolds

Multiphoton excited polymerisation has attracted increasing attending as a powerful 3 D nano- and microfabrication tool Recently, a twosome of documents were depicting the usage of 2PP for the microfabrication of 3D scaffolds.

2PP was applied to model bioactivity into preformed photoactive stuffs. PEG-based stuffs have been studied extensively as tissue-engineering scaffold and are biocompatible and per se immune to protein surface assimilation. Poly ( ethylene ethanediol ) ( PEG ) -diacrylate ( PEGDA ) hydrogels are in themselves biologically “ clean slates ” into which desired bioactivity can be tailored via light-based patterning. [ 44 Hahn 2006 ] .

Using 2PP and extra Methacrylated RGD-ACRL, adhesion advancing peptide RGD or a low-molecular weight MW 600 PEGDA was allowed to spread into a preswelled MW 6000 PEGDA hydrogel. This prepolymer sample was irradiated with femtosecond pulsation in the nowadays of photoinitiator. The acrylate-derivatized medieties in the precursor solution were conjugated to the hydrogel in the irradiated parts, changing its cell-adhesion behaviour or its stiffness due to higher grade of crosslinking in the lighted parts. Site-specific RGD-functionalization of hydrogels was demonstrated to be utile for cell-guiding, while 3D site-specific control of the stiffness of hydrogels is of import to mime the mechanical belongingss of the extracellular matrix ECM ) because mechanical cues of the microenvironment are of import signals for direct the cell destiny ( proliferation, distinction, programmed cell death ) [ 45 Pek 2010 ] . [ 46 Nemir 2009 ] .

[ 47 Cunningham 2006 ]

[ 48 Ovsianikov 2007 ]

[ 49 Schlie 2007 ]

[ 50 Tayalia 2008 ]

[ 51 Schade 2009 ]

[ 52 Wei? 2009 ]

[ 53 Claeyssens 2009 ]

[ 54 Ovsianikov ]

Scaffolds from crosslinked Protein

[ 55 Campagnola 2000 ]

Very pulling for edifice scaffolds crosslinked protein gels. Particularly the proteins of the ECM ( factor I, fibronectin, collagebn ) are interesting campaigners. Rose Bengale was used as a sensitising dye. [ 56 Basu 2004 ] . Even inside populating 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 aminic derivate of benzophenone dimmer even collagen, which was non crosslinkable utilizing photosensitizers, was used as edifice stuff for ecm-analogous mictrostrructures. [ 60 Pitts 2002 ] .

There are two by and large accepted manners in which proteins can be cross-linked by photoactivation: H atom abstraction and formation of vest O to photooxidize protein residues ( 31 ) . Given singlet O outputs of 75- 85 % for rose bengal, we can non govern out the engagement of vest O in the protein cross-linking because these measurings were non 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 Konig 2005 ]

[ 67 Xiong 2008 ]

[ 68 Sidorenko 2007 ]

[ 41 Watanabe 2002 ]

[ 69 Knoll 2006 ]

Metalic microstructures based on 2PP were described. The photopolymerizable stuff consists of a commercially available rosin ( Z7012C, JSR ) mixed with cinnamene in a 1:1 volume ratio. In the instance of two-photon soaking up, the chemical reaction progresses by extremist attacking14 and therefore is really effectual for cinnamene which has a vinyl group. A sensitising stannic chloride thin movie is produced on the polymeric microstructure and act as a binder with the metallic movie. Electroless plating utilizing silver nitrate consequences in metallic microstructures [ 70 Formanek 2006 ] . [ 71 Formanek 2006 ] .

BioMEM Integration

BioMEMS

miniaurized cell civilization systems

[ 72 Stubenrauch 2009 ]

[ 73 Frober 2009 ]

A

3-D fiction techniques are utile for analyzing structure/function relationships in theoretical account tissues.

Cell seeding

Lift

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

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

TPLSM

[ 75 Denk 1990 ]

[ 76 Martini 2006 ]

2 ) Experimental

2.1 Materials

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

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

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

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

All dissolvers and other chemicals ( 4- ( N, N-dimethylamino ) pyridine ( DMAP ) , 2-hydroxy-4`- ( 2-hydroxyethoxy ) -2- methylpropiophenone ( Irgacure 2959 ) , 2-isocyanatoethyl methacrylate ( IEM ) , stannic octoate, dibutyltin dilaurate ( DBTL ) , eosin Y ( EY ) , triethanol aminoalkane ( 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 2959A® ) was received from Ciba Speciality Chemicals, Inc. as a gift. The concentration of the photoinitiator in the precursors was 2 % ( w/w ) . No extra dissolver was used.

A

2.2 Two-Photon Polymerization

Near-infrared Ti: sapphire femtosecond optical maser pulsations were applied for the fiction of 3D constructions. The optical maser wavelength, pulse continuance and repeat rate was 750-790 nm, 100-120 degree Fahrenheit, and 80 MHz, severally.

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

a ) two beam splitter combinations dwelling each of a regular hexahedron polarizer and a I»/2-plate for spliting the optical maser power between different applications ( TPLSM, 2PP, nanotome ) and for fading the optical maser power in the 2PP experiment ; B ) a CCD camera for online-monitoring the 2PP procedure ; degree Celsius ) an akkusto-optical modulator ( AOM ) for rapid on/off control of the light.

The optical maser was focused into the light-sensitive samples by either a 100x high numerical aperture ( NA 1.4, oil submergence ) aim lens for bring forthing constructions with highest declaration ( & lt ; 500 nanometer ) or an nonsubjective lens ( Zeiss x 63, Plan-Neofluar ) with numerical aperture of 0.75 and a on the job distance of 2.2mm was used. ) for fast fiction of larger scaffold constructions ( sidelong declaration 2 Aµm, axial declaration 5-10 Aµm ) . The pick of the nonsubjective lens was governed by the demands of needed declaration of the one manus and the processing clip on the other manus. To bring forth 3-dimensional microstructures based on computing machine theoretical accounts extremist preciseness linear-motor-driven phases ( ABL 10100 Air Bearing Stage, Aerotech Inc. , Pittsburgh PA ) were used to travel the sample holder ( typical composing speeds from 100-3.000 Aµm/s ) .

In order to take the non-illuminated and hence non-solidified stuff 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 ethyl alcohol was used, for OLDMA 1 and OLDMA 2 propanone, for ORMOCERE a 1:1 mixture of 4-methyl-2 pentanone and 2-propanol and for the water-borne precursors H2O was used.

For illustration, a 780 nm wavelength optical maser with a 100 degree Fahrenheit pulsation breadth at a repeat rate of 80 MHz possesses a spectral full breadth at half maximal ( FWHM ) of about 10 nm [ 31 Sun 2004 ] .

The wide optical maser spectrum associated with the ultrashort pulsation width brings about chromatic aberrance, which is overcome by use of apochromats.

Numeric Aperture

Laser scanning is the measure needed to change over pre-designed CAD patterns into existent constructions.

A Fig.1: Scheme of two-photon polymerisation set-up.

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

2.4 Cell civilization

A

2.4.1 Cell seeding

A A?

2.4.2 Cell civilization

A

2.4.3 Imaging

A

3. Consequences

A

3.1 2PP

The chief advantage of utilizing 2PP for 3D microfabrication of man-made scaffolds is the, in rule, free control of the 3-dimensional architecture of the mesoscopic biocompatible interface. The flexibleness is merely compromised, by now, by the file size of the computing machine theoretical account restricting the declaration of the minimum scaffold characteristic and the processing clip. Latter is strongly dependent on the optimized hatching and sliting parametric quantities but by far more by specifying proper unit cells as edifice blocks of the full scaffold construction. This is chiefly because of preliminary limitation of the filling scheme, the optical maser motion, severally, to equidistant x, y-slicingA and z-stepping. After the contours of one piece of defined thickness, which has to be adapted to the numerical aperture of the used aim lens ( e.g. for N.A. 0.75A the piece thickness is 6 Aµm, for N.A. 1.4 the piece thickness is 0.5 Aµm ) and, to a minor extent, to the scanning velocity because of decreased optical maser impact with lifting scanning velocity, are drawn, the inner of the construction is filled with a woodpile hatching form which appropriate distance is once more defined by the numerical aperture of the nonsubjective lens ( 2.5 Aµm for N.A. 0.75, 0.3 Aµm for N.A. 1.4 ) .

Clear, simple constructions are evidently favored, given rise for hunt for beginnings for theoretical account geometries: periodic crystal constructions, minimum surfaces and biomorphs.

The grading of the scaffold is a via media between possible declaration down to the nanoscale and the biological demands of the cell every bit good as the processing clip and the architectural stableness. As the woodpile construction of stapled beds of extraneous intersecting lines is the standard filling scheme in our 2PP control package we choose woodpiles with different distances ( from 10 to 150 Aµm ) as simple theoretical account constructions for proving the biocompatible of the stuffs by seeding with cells and measuring their response over 3-4 twenty-four hours civilization by CLSM after staining.

Support free topological cell french friess of missive instance construction ( 2 x 2 ten 0.15 mm3, Fig. XY ) were designed for happening ideal scaffold grading parametric quantities. As the diameter of bovine chondrocytes can change between 10 and 40 Aµm depending on the distributing extend an ECM correspondent scaffold pore size was estimated to lie in the scope 10 – 150 Aµm.

A Fig.2: Computer theoretical account of the missive instance topologicalA cell bit. Computer theoretical account of eight

“ Schwarz-P minimum surface ” unit cells.

A

A A Fig.3: Computer theoretical account of the three-dimensional lattice unit cells. Left: coop unit cell ; right: axes-of-coordinates-like unit cell. Size: 80x80x80 Aµm3, Thickness of the beams 10 Aµm. Scaffolds are formed by constructing x, Y and z-arrays of the unit cell.

The woodpiles and the topological bit are 2.5 dimensional constructions that could be produced, if with some attempt ( masks, alliance, repeats ) , by traditional microstereolithography every bit good.

True 3D microstructures Schwarz P surface

The function of optical maser parametric quantities

The optical maser wavelength has influence on the soaking up of the incident visible radiation since the soaking up of the photoinitiator Irgacure 369 extremums at 330 nanometers at shorter wavelength than the half of 730-790 nanometer. On the other manus the optical maser end product power lessenings with diminishing wavelength. Typically the two-photon soaking up at lower wavelength than 750 nanometer was to strong, the declaration diminutions and harm occurred. For most

2.2.2 2PP Processibility of Photopolymers

Important facet in the processibility of photopolymers for 2PP is the viscousness of the photopolymer, the solubility of a suited photoinitiator in the monomer/macromer solution and the denseness of polymerizable vinyl groups. In general, photopolymers or photoresists are good suited for 2PP when they are liquids and exhibit an unmoved high viscousness, 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 polymerisation 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 doing PEG -DMA a really hapless 2PP photopolymer ( about non processable ) while PEG -DA with 2 % Irgacure 369 is an first-class 2PP photopolymer and serves as standard because of its good handiness and biological inertness. Cell adhesion, nevertheless, is greatly repressed on non-modified PEG- DA due to its water-swollen hydrogel character.

UDMA

HV08 = OL-MA 1

HV12 = OL-MA 2

2.2.2.2 Organic soluble photopolymers

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

Although the extremum of soaking up of Irgacure 369A® is 330 nanometer and therefore a two-photon wavelength of 750-790 nanometer is non optimum for the excitement of this photoinitiator

photopolymer

UDMA

A Fig.5: Letter instance construction, item of three-dimensional lattice construction made and Schwarz-P surface array of UDMA.A 1000x REM image.

A

A REM Bilder von den Strukturen

A HV 08, HV 12

A Fig.6: Woodpile construction made from HV 08.

2.2.2.1 Water soluble biopolymers

The somewhat water-soluble photoinitiator Irgacure 2959 was tested for 2PP responsiveness in combination with water-soluble monomers ( AAm, HEMA ) and macromers ( HyaMA, AlgMA ) but was found to be non efficient in originating 2PP because of the fact, that Irgacure 2959 is non every bit soluble to bring forth a 2 % ( w/w ) solution as needed in 2PP of organic soluble photopolymers or that the soaking up extremum of soaking up has a to short wavelength ( 330 nanometer ) .

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

A

2.2.3 Cell response

A

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

A

Fig. 8: Woodpile construction made of HV 08 seeded with cells.

A

A

A A Fig.9: Woodpile construction made of HV 12 seeded with cells.

A

A

A

Fig.10: Detail of missive instance construction made of PEG-DA on glass supportA demoing an exclusion of bovine chondrocytes from the surface of the H2O swollen stuff. Low initial adhesion.

A

Similar non-adhesive behaviour was found for other hydrogel stuffs as PEG-DA and Polyacrylamide.A A

A

4. Discussion

4.1 2PP

Choosing different grades of methacrylation, photoinitiators and 2PP procedure parametric quantities like incident optical maser power, scan speed and repeat rate a broad scope of form controlled microstructures can be polymerized with different belongingss sing the mechanical stiffness, geometry, cell fond regard and cell growing.

4.2 Biocompatibility

A

Imagination: CLSM, TPLSM, REM, AµCT

A

A

Fig.11: 3D Reconstruction of a 2PP derived scaffold ( three-dimensional lattice ) utilizing TPLSM. 80 Aµm unit cell. UD -MA.A

A

Noch machen REM -Bild Large Pore Scaffold 5x5x2 millimeter

A

A

Fig.12: AµCT image of 2PP scaffold made from PEG-DA. Grid size 500 Aµm.

A

Contrast PEG -DA

Resolution 10 Aµm.

A

A Results on 3D consequence on the proliferation and distinction of chondrocytes as measured by qRT-PCR will be published in an approaching paper.

A

4.3 Upscaling

One of the most serious drawbacks of the 2PP engineering is the built-in series, voxel-by-voxel character of the fiction procedure ensuing in extended processing times.

Chiefly this has limited the production of larger scaffolds of highest declaration or specimen Numberss up to now. To get the better of this restrictions assorted up-scaling schemes has been established in recent old ages.

Wu et Al. saved processing clip in the microfabrication of their classical Venus micro statue by demoing that it was merely necessary, provided that the slice, hatching and laser motion parametric quantities provide necessary stableness, to enlighten the outer shell of the construction ( the contours ) by degree Fahrenheit pulsations while the interior volume was solidified utilizing a UV-lamp after the environing liquid rosin was washed off [ 35 Wu 2006 ] .

Resins integrating conventional ultraviolet-absorbing instigators exhibit relatively low radiosensitivity as the instigators have little two-photon soaking up cross-sections ( I? ) although 3-dimensional informations storage and microfabrication have been illustrated utilizing two photon- initiated polymerisation of such photopolymer systems. Typical values are in the scope of 4 GM ( Irgacure 819 ) and 28 GM ( Irgacure 651 ) ) . [ 77 Schafer 2004 ] . Consequently, this attack requires still comparable high optical maser power, connected with limited scan velocity and its widespread usage remains impractical. Any serious upscaling of the 2PP procedure, hence, has to concentrate on the material side on the development of specially designed two-photon absorbers that exhibit big two-photon soaking up cross subdivision ( I? ) . Cumpston et Al. hold synthesized a two-photon absorbers with two-photon soaking up cross subdivision of 1250 GM and showed that a commercial triacrylic photopolymer ( SR9008 and SR454, Sartomer ) has an order-of-magnitude higher two-photon radiosensitivity, comparative to the conventional instigators examined.

Photoinitiators with high two-photon soaking up cross subdivisions has been focus of intense research in the last decennary and it was demonstrated that they were able to take down the polymerisation threshold laser power, that is, the necessary irradiation power to bring on polymerisation [ ] . The development of stuffs which exhibit enhanced two-photon soaking up that can originate photopolymerization has been revieved in a figure of documents. [ 78 Lin 2003 ] [ 79 Kuebler 2003 ] [ 80 Lu 2004 ] [ 81 Lu 2004 ] [ 82 Xing 2007 ]

Low cost PS microlaser [ 83 Wang 2002 ]

Particular Fluorine aminoalkane system [ 84 Belfield 2000 ]

Acylamide photopolymerization with xanthene dye photoinitiator [ 55 Campagnola 2000 ]

Large country 2PP microstructuring [ 85 Malinauskas ]

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

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

One attack for upscaling the 2PP procedure, that means for doing 2PP applicable to the macroscopic universe, is to construct up a hydrogel stuff via majority ( photo- ) polymerisation, later leting a functional monomer, e.g. acrylated adhesion advancing peptide, to spread into the hydrogel and modeling the bioactive molecule inside the gel. By this the chief polymerisation takes topographic point conventionally and merely the functionalization in certain countries of the hydrogel needs to manufacture utilizing 2PP [ 88 Lee 2008 ] .

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

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

Periodic constructions are fabricated by multibeam intervention lithography [ 91 Moon 2006 ] . Or by holographic techniques.

Or by utilizing parallel processing utilizing multibeam fiction.

Our scheme has focused on the proper pick of the nonsubjective lens and of the proper pick of the unit cell.

2PP constructions described in the literature do non transcend the millimeter

In the literature described up to 1 mm2.

Here series of 2 x2 x0.25 mm3 scaffolds.

For being applicable in macroscopic universe, e.g. tissue engineered implants, high denseness informations storage or waver-size 3D lithography the 2PP procedure has to be farther accelerated [ ] and expanded to mesoscalic specimen sizes ( 0.5-5 millimeter ) .

Assorted schemes have been developed for the assembly of mesoscalic specimen, 2PP derived and other microfabricated “ small things ” . Khaddemhosseini et Al. invented the possibilities to self-assemble mesoscalic 3d hydrogel concepts utilizing polyphase liquid-liquid systems. Microstructured hydrogels, prepared by UV crosslinking utilizing photomasks or microstereolithography were immersed in non-polar dissolvers. Thermodynamically goaded self-assembly followed by 2nd measure UV postcuring outputs in organized super-structures which could be used for developing functional multicellular concepts with different cell types arranged in a tissue specific manner [ 92 Du 2008 ]

Microrobotic attacks have been applied for the assembly of mesoscalic scaffolds for tissue technology by utilizing microgrippers and a lock-and-key architecture of the edifice blocks [ 93 Zhang 2005 ] .

From a stuffs point of position, 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 optical masers for commercial applications. Besides the high I? stuffs would enable polymerisation at the same time from multibeam split out of one end product

of a optical maser for batch production.

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

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