Posters

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PC01

SELF-ASSEMBLY OF ALANINE TETRAPEPTIDE - POLY(OXYETHYLENE) DIBLOCK POLYMERS IN WATER

N. HIGASHI*, Y. KATA, M. NIWA

Department of Molecular Science & Technology, Faculty of Engineering, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan

E-mail: nhigashi (mail.doshisha.ac.jp)

We describe herein the aggregation properties of novel amphiphilic diblock polymers, which consist of the alanine tetrapeptide segment and the hydrophilic poly(oxyethylene) (POE) segment. L-L-L-L sequence (4L) and L-D-L-D alternative sequence (2LD) were employed as the alanine tetrapeptide segment. Three types of diblock polymers of 4L-10 having thePOE segment with n = 10, 2LD-10and 2LD-23 havingPOE segment with n = 10 and 23, respectively were prepared (FIG. 1). The aggregation behavior was revealed by means of spectroscopies, dynamic light scattering (DLS) and AFM.Alanine tetrapeptides with L-L-L-L sequence and L-D-L-D alternative sequence were prepared by a conventional stepwise condensation. Finally, Ala-POE was synthesized by condensation reaction of the activated carboxylic group of alanine tetrapeptide with the primary amino group of POE.4L-10 and 2LD-10 were found to form huge assemblies, whose diameters were 166 nm and 145 nm, respectively, but 2LD-23 did not give any assembly. CD and FTIR spectroscopies showed that 4L-10 formed random coil structure and 2LD-10formed loop structure with b-sheet-like or b-turn structure in water. In addition, FTIR data for the cast film of 2LD-10 showed the intermolecular hydrogen bonding among alanine tetrapeptide segments.These results suggest that not only hydrophobic\par interaction but also intermolecular hydrogen bonding play a vital role in stabilizing the assembled structure of 2LD-10.


PC02

AGGREGATION PROPERTY OF POLY(L-GLUTAMIC ACID) TRIBLOCK- POLYMERS HAVING POLY(OXYETHYLENE) SEGMENT AS A LOOP

N. HIGASHI*, K. SUGAMOTO, M. NIWA

Department of Molecular Science & Technology, Faculty of Engineering, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan

E-mail: nhigashi (mail.doshisha.ac.jp)

We describe herein the preparation of novel triblock polymers, consisting of two poly(L-glutamine acid) (PLGA) segments and a flexible POE segment that connects with PLGA segments, and their aggregation properties in aqueous solution.Poly(g-benzyl-L-glutamate)(PBLG)-POE-PBLG triblock polymerswere firstprepared by polymerization of BLG-NCA initiated with amino groups at both termini of POE. By removal of benzyl groups, PLGA segments connected by POE (PLGA-POE-PLGA)s were obtained. The aggregation behavior of these triblock polymers was examined in their aqueous solutions by means of conductmetric and potentiometric titration, CD spectroscopy and dynamic light scattering (DLS). As a result, the pKa value of P(LGA)n-POE-P(LGA)n (n=26), a typical polymer, was found to be 6.5 that is higher than that of the POE-free P(LGA)n (n=26), and the helix content of P(LGA)n-POE-P(LGA)n (n=26) was considerably enhanced, compared with that for the POE-free PLGA (n=26). These results indicate a strong interaction between PLGA segments, resulting from the formation of an assembly in water (FIG. 1). DLS measurements were carried out and the observed particle diameter was 128.3±37.0 nm at pH 4.0. PLGA-POE-PLGA had a tendency to aggregate readily with increasing chain length (n). This result suggests that the helical PLGA segments play an important role in forming aggregation.


PC03

BIOMIMETIC QUINONE-CROSSLINKED POLYSACCHARIDE HYBRID FIBER

H. YAMAMOTO, Y. KUBOE, H. TONEGAWA, A. NISHIDA, K. OHKAWA

Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Japan. e-mail: hyihpr2 (giptc.shinshu-u.ac.jp)

The paper reports the synthesis of the N-(Lys-Gly-Tyr-Gly) (GYGK)-chitosan using the water-soluble active ester method, the preparation of the GYGK-chitosan-gellan hybrid fibers, and the reinforcement of the hybrid fibers by enzymatic cross-linking between the N-grafted peptides chains of chitosan. The cationic polysaccharide chitosan was treated with the Boc-Lys(Z)-Gly-Tyr(Bzl)-Gly (4-hydroxyphenyl)- dimethylsulfonium methylsulfate active ester in DMF-0.15M AcOH to incorporate the peptides into the side chain amino groups. After removals of the protecting groups, the degrees of N-GYGK-substitution were estimated to be 2.0 - 10 molar % by changing the molar ratios of the amino groups and the active ester. The resulting cationic GYGK-chitosan was spun into the hybrid fibers with the anionic polysaccharide gellan in water. The tensile strengths of the GYGK10-chitosan hybrid fibers (243 MPa) were superior to those of the original chitosan-gellan fibers (126 MPa). The mechanical strengths of the hybrid fibers further increased to 450 MPa upon enzymatic oxidation using tyrosinase. The covalent cross-bridges were formed through the oxidative couplings of the quinones or the Michael-type addition by the amino groups of Lys and glucosamine. Based on these results, we concluded that the covalent cross-linking due to the enzyme oxidation between the grafted peptides significantly contributed to reinforcement of the polysaccharide hybrid fibers. By summarizing the total findings, we can illustrate the reinforcement mechanism of the hybrid fiber. To characterize the biomimetic hybrid fibers, the breaking stress value of the cross-linked GYGK10-chitosan-gellan fiber (450 MPa) was compared with two of the typical natural protein fibers. The value of GYGK10-chitosan-gellan fiber was in between silk (700 MPa) and wool (250-350 MPa). As compared with the natural marine quinone cross-linked fiber, byssus of blue mussel Mytilusedulis, (143 MPa), the value for the GYGK10-chitosan-gellan hybrid fiber is superior to that of byssus.The present results afford a new methodology for the hybrid material reinforcement achieved by the polymer modification inspired by a biological process.


PC04

POLYMER BIOMATERIALS. ONE-POT SYNTHESIS OF AMPHIPHILIC RUBBERY NETWORKS BASED ON POLY(2-HYDROXYETHYL METHACRYLATE-co-ISOBUTYLENE)

M. JANATA, L. TOMAN, J. SPĚVÁČEK, P. VLČEK, A. SIKORA, J. MICHÁLEK, B. MASAŘ, P. LÁTALOVÁ, D. CHMELÍKOVÁ

Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic

Amphiphilic networks consisting of hydrophilic poly(2-hydroxyethyl methacrylate) (PHEMA) and hydrophobic polyisobutylene (PIB) have recently received considerable attention, particularly in biomedical applications. It has been found that these networks exhibit excellent biocompatibility and biostability in vivo in rats,1 reduced protein adsorption in human plasma protein adsorption tests and reduced cell adhesion in human monocyte adhesion tests, thus suggesting hemocompatibility.2 PHEMA-PIB networks have been synthesized by radical copolymerization of HEMA with methacrylate-telechelic PIBs, e.g., MA-PIB-MA.3 Generally,4 methacrylate end-functionalized PIBs were prepared by combination of a controlled cationic polymerization of isobutylene and subsequent transformation of PIB, e.g., Cl-PIB-Cl → CH2=CH-PIB-CH=CH2 → HO-PIB-OH→ MA-PIB-MA. Recently, a novel method of one-pot synthesis of multifunctionalized PIB bearing pendant methacrylate groups along the chain (PIB(MA)n, where n = 3-10) was developed.5 Cationic copolymerization of IB and 3-isopropenyl-α,α-dimethylbenzyl isocyanate gives rise to the multifunctionalized PIB bearing pendant isocyanate groups along the chain (PIB(NCO)n).6 The isocyanate groups were subsequently transformed to methacrylate groups by the dibutyltin dilaurate-catalyzed reaction with HEMA giving PIB(MA)n.5

In the present work, we prepared PIB(MA)n (Mn ~ 8 000 with an average functionality Fn ~ 4 per chain) in a one-pot synthesis. The PIB(MA)n is then used as a polymeric crosslinking agent in radical copolymerization of HEMA giving rise to a rubbery PHEMA-PIB network (~ 60 wt % HEMA). The amphiphilic nature of the network has been demonstrated by swelling in both n-heptane and water. Furthermore, the dry network could be simply press-moulded to form rings, plates, pellets, etc., unlike the wellknown PHEMA materials, which cannot be press-moulded. We have characterized PIB(MA)n by SEC and 1H NMR and the PHEMA-PIB network by solid state 13C NMR and DSC analysis.

Acknowledgement: Financial funding of this research provided by the Grant Agency of the Czech Republic (Grant 203/04/1050) is much appreciated.

References

  1. D. Chen, J. P. Kennedy, M. M. Kory, D. L. Ely, J. Biomed. Mater. Res. 23, 1327 (1989).
  2. B. Keszler, J. P. Kennedy, N. P. Ziats, M. R. Brunstedt, J. K. Yunk, J. M. Anderson, Polym. Bull. 29, 681 (1992).
  3. B. Iván, J. P. Kennedy, P. W. Mackey, Polym. Prepr. 31(2), 217(1990).
  4. J. P. Kennedy, M. Hiza, J. Polym. Sci., Polym. Chem. Ed. 21, 1033 (1983).
  5. L. Toman, P. Vlček, J. Vacík, J. Michálek, Czech Patent 290817, September 12 (2002).
  6. L. Toman, P. Vlček, Czech Patent 291062, March 17 (2003).

PC05

SURFACE MODIFICATION OF PEEK USING PLASMA IMMERSION ION IMPLANTATION

M. BILEK, K. NEWTON-MCGEE, D.R. MCKENZIE

Applied and Plasma Physics Group, School of Physics, A28, University of Sydney, NSW 2006, Australia, m.bilek (physics.usyd.edu.au)

Polymers are ideal materials for many bio-applications, such as implantable medical devices and diagnostic arrays. Polymers, such as PEEK (Poly Ether Ether Ketone), are durable, strong and have densities close to that of tissue, making them suitable for use in implanatable devices. They are also cheap enough to be suitable for use in disposable diagnostic chips, such as protein arrays. For applications in these areas to reach their full potential however, methods of modifying the surface of polymers are required to optimise wettability and specific and non-specific protein binding characteristics.

In this paper we describe the use of a technique known as plasma immersion ion implantation (PIII) to chemically modify the surface of polymers. Surface modifications performed on PEEK have been characterised with contact angle measurements, attenuated total internal reflection infra-red (ATR-IR) spectroscopy and XPS. The effects of the modifications on the contact angle and surface chemistry are described. Although a wide range of modifications could be achieved, their time stability was found to depend on the density of cross-linking induced in the sub-surface regions by the ion treatment. These findings lead us to propose a method for ensuring that surface modifications on polymers are stable over long time scales.


PC06

EVALUATION OF MECHANICAL PROPERTIES OF NOVEL BIOMEDICAL POLY(ALIPHATIC/AROMATICESTER) COPOLYMERS

M. EL FRAYa, P. PROWANSb

aTechnical University of Szczecin, Polymer Institute, P-70322 Szczecin, Poland, e-mail: mirfray (ps.pl )

bPomeranian Medical Academy, Clinic of General and Hand Surgery, Szczecin, Poland, e-mail: pprowans (wp.pl)

Poly(aliphatic/aromatic-ester)s (PED) copolymers containing semicrystalline poly(butylene terephthalate) (PBT) and monomers from renewable resources, namely, dimer fatty acid (DFA) are new and interesting materials. Their preparation is made by an environment friendly method of transesterification and polycondensation from the melt. The exceptional properties of dimer fatty acids, causing an excellent resistance to oxidative and thermal degradation, makes it possible to prepare PEDs without phenolic, thermal stabilizers, a known irritant. This is a particularly important feature, because the presence of extractable substances in biomedical polymers lead to various changes over the polymer structure and properties and to toxic reactions in contact with the human body.

PED are stable during processing and sterilization. Due to the wide window of compositional/structural variations among PED it was possible to prepare polymers changing their physical appearance from soft elastomers to semi-rigid polymers.

Mechanicalproperties (static and dynamic) and invitro degradation results will be presented.The resulting nanostructured morphology contribute to different mechanical properties, locating these new PED materials between commercially available thermoplastic poly(urethane-ether) (used as internal and external blood contacting medical devices as catheters or elements of artificial heart) and poly(ester-ether) elastomers (used as medical devices, osteoinductive materials and scaffolds).


PC07

LONG-LIFETIME POLYMER-LANTHANIDE LUMINESCENT COMPLEXES

D. VÝPRACHTICKÝ, S. KUKLA, P. PAVLAČKOVÁ, V. CIMROVÁ

Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, CZ-162 06 Praha 6, Czech Republic

(e-mail: vyprach (imc.cas.cz), kukla (imc.cas.cz), pavlackova (imc.cas.cz), cimrova (imc.cas.cz) )

The use of lanthanide complexes as luminescent labels in fluoroimmunoassay has recently become competitive with radioimmunoassay, which is still the most widely used. The principle of this assay takes advantage of sensitized Eu3+ or Tb3+ luminescence. Since the background fluorescence of biological materials is usually short-lived, the Eu3+ and Tb3+ complexes present the advantage that their emitting states (4f) have long luminescence lifetimes. Indeed, a delay is set between the excitation pulse and the measurement of lanthanide luminescence. Time-gated fluoroimmunoassays are steady-state intensity measurements over a period of time following pulsed excitation. Commercial kits presently available use e.g. ETDA-Eu3+ chelates and b-diketone. To improve lanthanide luminescence efficiency, the encapsulating ligands (i.e. ligands which form a three-dimensional cage around the metal ion), were developed. The synthesisof such ligandsisbased, for example, on 2,2´-bipyridine, 2,2´-biquinoline cryptands, or on their N-oxide derivatives.Theidea is to use a polymer supramolecular structures (coils, micelles, tactic polymer complexes) instead of a low-molecular-weight encapsulating ligands [1-3]. We replaced the low-molecular-weight pyridine cryptands with poly(2- and 4-vinylpyridine)s, poly(2- and 4-vinylpyridine N-oxide)s, poly(4-vinylpyridine-block-styrene) and/or polycarboxylates. Energy donors 9-vinylcarbazole and carbostyril 124 were used in the ligand-to-metal energy transfer processes.

Thisresearch wassupportedbytheGrant Agency of the Czech Republic (Grant No. 203/04/1372) and by the Grant Agency of the Academy of Sciences of the Czech Republic (Grant No. IAA4050409).

[1] Y. Okamoto, T. K. Kwei, D. Výprachtický: Macromolecules 1998, 31, 9201.

[2] D. Výprachtický, V. Cimrová, S. Kukla, L. Machová: Collect. Czech. Chem. Commun. 2004, 69, 309.

[3] D. Výprachtický, V. Cimrová, Y. Okamoto, R. Kotva: Macromol. Symp. 2004, 212, 239.


PC08

Morphology and Gelation of Thermosensitive Chitosan hydrogels

K.E. Cromptona, R. PRANKERDb, K.A. GROSSa, J.S. FORSYTHEa

aSchool of Physics and Materials Engineering, CRC for Polymers, Monash University, Wellington Rd, PO Box 69M, VIC 3800, Ausralia, john.forsythe (spme.monash.edu.au)

bDepartment of Pharmaceutics, Victorian College of Pharmacy, Monash University, 381 Royal Parade Parkville, VIC 3052, Australia

Chitosan is soluble in dilute acidic solutions, but phase-separates at pH > 6 to form a hydrogel. However, on addition of glycerophosphate salt (GP) to a chitosan solution, the pH can be raised to neutral without causing phase-separation. The system becomes thermally sensitive, forming a gel above a certain temperature. To date, the only study of chitosan/GP morphology was conducted after freeze-drying, by SEM.1 This study aims to gain a better understanding of the bulk morphology of the chitosan/GP in its native, hydrated state by LSCM (Fig. 1) and DSmC (Fig. 2), and to examine some of the factors influencing morphology such as composition, and degree of deacetylation.

1. Ruel-Gariépy E, Chenite A, Chaput C, Guirguis S and Leroux J-C. Characterization of thermosensitive chitosan gels for the sustained delivery of drugs. International Journal of Pharmaceutics, 2000. 203: 89-98.


PC09

A CELLULOSE SUBSTRATE ORIENTATES MELANOMA TUMOUR EXPLANTS TOWARDS DIFFERENTIATION AND APOPTOSIS

B. DAVIDa, J.L. DUVALa, M-T RACHEDa, G. LEGEAYb, I. PEZRONc, R. WAROCQUIERa, M.D. NAGELa

aUniversité de Technologie de Compiègne, UMR CNRS 6600, Domaine Biomatériaux-Biocompatibilité, Centre de Recherches de Royallieu, B.P. 20529 - 60205 Compiègne, France, marie-danielle.nagel (utc.fr)

bCTTM, Département Matériaux, 20, rue Thalès de Milet, Technopole Université, 72000 Le Mans, France, glegeay (cttm-lemans.com)

cUniversité de Technologie de Compiègne, UMR CNRS 6067, Division Thermodynamique et Physico-chimie de Procédés Industriels, Centre de Recherches de Royallieu, B.P. 20529 - 60205 Compiègne, France, isabelle.pezron (utc.fr)

It is now widely known that surfaces influence whether cells grow, differentiate or die. Our previous studies demonstrated that contact with cellulose substrata might cause cells to decrease their growth, to differentiate and to undergo apoptosis. In the present study, we assess the capability of metastatic tumor mouse melanoma explants cultured on an original cellulose coating (CEL), to reduce their proliferation, differentiate, synthesize specific proteins and undergo apoptosis. Tissue culture polystyrene (tPS) was used as control. We defined wettability and surface free energy of CEL by measuring contact angles. The results showed that CEL displayed a stronger hydrophilic character than tPS whereas the dispersive contribution was slightly lowered by the cellulose coating. Overall, the surface free energy was greater for CEL than for tPS. Using anti-FN polyclonal and monoclonal antibodies, we also studied (ELISA) fibronectin adsorption and conformation (RGD, N-terminal, C-terminal, and collagen domain accessibility) on CEL compared to tPS. The amount of adsorbed FN was 2.5 times smaller on CEL than on tPS; gelatin, N-terminal and C-terminal domain accessibility was also 2.5 to 3 times smaller on CEL compared to tPS, but RGD accessibility appeared to be sixfold smaller on CEL than on tPS. This means that FN is poorly adsorbed on CEL, and also partly (60%) denatured. A significant decrease of explant proliferation and migration was clearly demonstrated on CEL compared to tPS. On the contrary, cell loading in melanin (used as a differentiation criterion) and apoptosis were increased. CEL appears then to be an interesting tool to orientate normal and cancer cell genetic programs towards differentiation and apoptosis, and to study the mechanisms involved is such processes.


PC10

THE Aggregation of mouse melaNoma cells INDUCED BY cellulosE substratE IS REVERSIBLE

M. Hindié, M.D. NAGEL

Domaine biomatériaux-biocompatibilité, UMR CNRS 6600, Université de Technologie de Compiègne, BP20529, Compiègne, France,mathilde.hindie (utc.fr)

The physical and chemical nature of a substratum's surface influence cell morphology and functional orientations of adhering cells. Cellulose substrata hinder serum adhesive protein adsorption and prevent cells spreading. Previous studies have shown that cells round in aggregates on cellulose substratum [Faucheux N. et al, Biomaterials 2004]. This work presents the ability of cells aggregated on cellulose to reverse aggregation when seeded on tissue culture polystyrene (tPS).

Materials and methods

Materials: E4M HPMC and 7LF CMC bi-layered coated polystyrene (CEL), and tPS. Cells : B16F10 melanoma cells grown in RPMI 1640 10% foetal bovine serum, at 37 °C with 10% CO2. 105 cells/cm2 were seeded either on CEL or on tPS and incubated for 48 hours. Aggregates were washed 3 times and incubated on tPS for an additional hour in serum-free medium. Staining: Cells were fixed by 3% paraformaldehyde (w/v), then permeabilised by Triton X100 1% (v/v). Mouse monoclonal anti fibronectin antibodies, rabbit polyclonal anti alpha V and anti alpha 5 integrins antibodies, and Cy3 coupled secondary antibodies were used. F-actin microfilaments were directly revealed by TRITC conjugated phalloidin.

Results

Aggregates secreted fibronectin. After 1 hour on tPS, cells escaped from aggregates, adhered and spread. Filamentous actin staining showed the beginning of cytoskeleton organisation in cell periphery. Alpha V and alpha 5 integrins, were basically visible on adherent cells, staining was concentrated in extremity of cells pseudopodes. Focal adhesion sites began to appear in cells runaway from aggregates.

Discussion & Conclusion

Our results demonstrate that cells aggregated for 48 hours secrete fibronectin and are able to reverse aggregation. Focal contacts appeared as soon as one hour after culture on tPS, which means adhesion proteins were present in aggregates. Substratum signal was mediated by integrins, which induce actin polymerisation and starting of cytoskeleton organisation.


PC11

NUCLEATION AND SELECTIVE GROWTH OF POLYMORPHS OF CALCIUM CARBONATE IN ORGANIC-INORGANIC HYBRID FILMS

J. RETUERT*1, Y. MARTINEZ1, J. L. ARIAS2

Center for Advanced Interdisciplinary Research in Materials (CIMAT), Santiago, Chile and 1Depto Quimica. Fac. of Physical and Mathematical Sciences, 2 Fac. of Veterinary, University of Chile. Av. Beauchef 850, P.O.Box 2777, Santiago, Chile

The biological systems use organic molecules (fundamentally proteins and polysaccharides) to control the nucleation and growth of minerals in an organic matrix. In this way such hybrid materials are formed as bones, teeth, and shells of mollusks by means of the biomineralization process. The biomacromolecules that participate in the production of these composites can act as insoluble structural directing agents. The study on the influence of structured organic surfaces and soluble organic macromolecules in the orientation and growth of inorganic salts is a fundamental aspect in biomimetic mineralization processes.

A fundamental factor for the heterogeneous nucleation of inorganic crystals in organic substrata is to increase the charge density in the surface of the matrix. In this work, our approach has been to study the crystallization of calcium carbonate (CaCO3) in organ-inorganic ternary hybrid films constituted by poly (monomethyl itaconate) (PMMI), silica (SiO2) and chitosan (CHI) or gelatin (GE). The ternary hybrid films possess a high mechanical stability and they don't disintegrate in water at difference of anyone binary combination of these precursors. By varying the composition of the films, it is possible to change the concentration of the functional groups present in the organic macromolecules and to study their influence and the morphology and the stabilization of a particular polymorph of the CaCO3 crystals that are formed in the surface. The glasses and particles with different morphology that you/they crystallize in the hybrid CHI/SiO2/PMMI films, only consist on calcite, while those that are formed in GE/SiO2/PMMI films are identified as vaterita and calcite. The stabilization of the less stable polymorph is controlled by increasing the concentration of gelatin in the GE/SiO2/PMMI films.

Acknowledgments: To Conicyt (Projects Fondap 1198000-2 and Fondecyt 1010525) and German Academic Exchange Service (DAAD) for a PhD scholarship to Y.M.


PC12

POSTSURGICAL ANTI-ADHESION FILMS BASED ON POLYGALACTURONATE WITH TWO DIFFERENT CROSSLINKERS

MING-WEI LEE, YNG-JIIN WANG*

Institute of Biomedical Engineering, National Yang Ming University, Taipei, ROC

Two anti-adhesion films were prepared separately from polygalacturonic acid (PGA) via (1) direct crosslinking with EDC, and (2) photo crosslinking of PGA-CIN which derived from the partial esterification of polygalcturonic acid with cinnamylbromide. With the EDC crosslinking, the carboxyl groups of PGA were activated and subjected to inter- and intra-chain nucleophilic attack of the hydroxyl groups. Thecross-linked PGA-EDC film had 86% gel content, and contained 47% water when immersed in normal saline. Thephoto crosslinked PGA-CIN film had 9% of its D-galacturonic residues modified with cinnamate group and displayed an absorption peak at 252 nm. Theextent of crosslinking reaction of PGA-CIN increased with light irradiation time and reached 32.5% in 12 min.

Both films (PGA-EDC and PGA-CIN) were evaluated for their anti-adhesion capabilities. Bothfilms did not show any evidence of cytotoxic effects since they did not induce any significant increase of cytoplasmic LDH release from the L929 cells in contact with them. It was found that about 30% of inoculated L929 cells attached to both films, less than the 40% for Seprafilm membranes.When implanted into rats, the PGA-EDC and PGA-CIN films exhibited most promising anti-adhesion potential as compared with commercial product SeprafilmTM. The results of animal implant studies are summarized in Table1.Furthermore, the implanted PGA-EDC and PGA-CIN films did not elicit any acute inflammatory reaction based on the results of histological examination and peritoneal fluid leukocytes analyses. Two newly developed anti-adhesion films based on polygalacturonatethus have great potential for future use in clinical applications.

Table 1

  3 days after surgery 7 days after surgery 14 days after surgery Combined data

Control

7/7* 6/7 5/7 18/21
SeprafilmTM 3/7 4/7 4/7 11/21
PGA-EDC 0/7 0/7 1/7 1/21
PGA-CIN 1/7 2/7 1/7 4/21

Note: *number of rats with adhesion/number of rats operated


PC13

EFFECT OF SURFACE MODIFICATION ON VIABILITY OF MURINE EMBRYONIC STEM CELLS

PATTANAWONG, S 1, HARRISON, J 2, MOLLARD, R 2, GROSS, KA 1, BEH, H 3, FORSYTHE, JS 1

1School of Physics and Materials Engineering, PO Box 69M, Monash University 3800, Australia

2Institute of Reproduction and Development, Melbourne, VIC, Australia

3 Manufacturing and Infrastructure Technology, CSIRO, Melbourne, VIC, Australia

This research investigates a biocompatibility of various biodegradable polymers, poly-L-lactide (PLLA), poly(glycolide) (PGA) and poly(lactide-co-glycolide) (PLGA) with murine embryonic stem (ES) cells and to ascertain the effects of surface modification. To this end, ES cell colonization and growth following a 48h culture period were assesessed. Contact angle measurements (water/ substrate), Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) were used to examine the surfaces of these materials. Cell viability indicated significant differences with respect to substrates used. Samples which were surface treated (with 0.1M KOH) showed higher cell viability compared to the virgin samples (Figure 1). The modified PLLA (PLLA/KOH) and modified PLGA (PLGA/KOH) presented the highest cell viability. SEM and AFM micrographs suggested that the surface erosion of substrates had occurred. The results from the contact angle measurements show that hydrophilicity of the substrates were changed, thus revealing a correlation between surface hydrophilicity and viability.

Figure 1 Mouse ES live cell no. (x100)/mm2 following 48 hrs growth on untreated and treated with 0.1M KOH substrates

The rate of embryonic stem cells colonization upon a given poly(α-hydroxy esters) is, therefore intimately linked to polymer compsition and hydrophilicity.


PC14

PHYSICAL CHARACTERIZATION OF LIPID MEMBRANES BETWEEN POLYELECTROLYTE FILMS

S.E. MOYA, E. DONATH, W.T.S. HUCK

Nanoscience Centre, Cambridge University, West Cambridge, Cambridge CB3 OFF, U.K.; sem61 (eng.cam.ac.uk)

Lipid membranes composed of mixtures of dipalmitoyl phosphatidyl choline (DPPC), dipalymitoyl phosphatidyl acid (DPPA) and cholesterol were assembled on polyelectrolyte (PE)-coated flat surfaces and colloidal particles. The assembly was studied by means of quartz crystal microbalance, confocal microscopy, flow cytometry, and freeze-fracture electron microscopy. Homogeneous lipid coverage was established within the limits of optical resolution. Flow cytometry showed that the lipid coverage was uniform. Freeze-fracture electron microscopy revealed that the lipid was adsorbed as a bilayer, which closely followed the surface profile of the polyelectrolyte support. Additional adsorption of polyelectrolyte layers on top of the lipid bilayer introduced inhomogeneities as evident from jumps in the fracture plane. QCM showed as well that extra assembled PE layers removes lipid from the surface. The stability of lipid membranes in presence of organic solvents could be study by means of QCM and Foerster energy transfer.


PC15

Biomimetic artificial muscle

V. Boudaa, L. Boudováb , D. Haluzíkováb

aCzech Technical University in Prague, Faculty of Electrical Engineering, Department of Mechanics and Material Science, Technická 2, 166 27 Prague 6, Czech Republic, e-mail: bouda (fel.cvut.cz)

bCharles University in Prague, The 1stFaculty of Medicine, Inst. of Sports Medicine, Salmovská 5, 120 00 Prague 2, Czech Republic, e-mail: lboud (lf1.cuni.cz)

To construct small but powerful drives (actuators) is one of the most important aims in the field of micro- or nano-electro-mechanical systems. The universal actuator in the animal kingdom is the muscle. Its efficiency is very high when compared with other piezo-ceramic, magneto-strictive actuators or electro active polymers. So, the use of its internal design is very attractive for engineers.

Mammalian muscles are built of micron-sized contractile units called sarcomeres, which contain two filament types: thin and thick. Swinging of the globular heads on the thick filaments drives the thin filaments toward the center of the sarcomere, thereby shortening the sarcomere. According to our new paradigm of the function of the sarcomere, interactions between the heads have the colloidal nature. They can be estimated as a sum of energy of electrostatic repulsion and of energy of van der Waals attraction. There are two states of the system of the heads: the relaxation and the contraction one. The relaxation state at low Ca2+ concentration has two important features. First, the thin filaments are free in movement typical for the relaxation state of the muscle. Second, the heads are regularly arranged and can control the process of evolution of the internal structure of the sarcomere.

At the transition to the contraction state, the concentration of Ca2+ ions increases. The induced relative increase of attractive van der Waals forces between the heads renders their coagulation. However, the coagulation is prevented because of the presence of thin filaments between the heads.The effect of the van der Waals forces is that the heads grip the thin filaments and shift them to the center of the sarcomere. The computed sum of the forces of the heads in a specific muscle approaches the force measured on the muscle in experiments.

The very simple mechanism of the sarcomere contraction can be used in the design of a new kind of biomimetic artificial muscle with very high performance. However, it seems that only nanotechnology and self-organizing processing of electro-conductive polymers can enable us to realize such a design.


PC16

OLIGOLACTIDES AS THE CROSSLINKERS FOR TYPE I COLLAGEN FIBERS

C.S. LIN, C.W. CHANG, Y.J. WANG

Institute of Biomedical Engineering, National Yang-Ming University,Taiwan

Natural biopolymers such as collagen and hyaluronic acid are ideal biomedical materials for tissue engineering, but usually lack of mechanical strength and are difficult to form a rigid 3-D matrix. Therefore, these biopolymers need to be cross linked in order to fabricate into a useful scaffold for tissue regeneration. The aim of this study is to synthesize hydrophobic, short-chain poly(lactide) and poly(glycolide), and utilize them as the crosslinkers for collagen fibers. We have prepared oligolactides with various chain length bearing -COOH or -OH functional groups on both end-terminals, and molecular weight well controlled in the range of 950 to 12,000. The -OH and -COOH terminal groups of the resulted oligolactides were activated with tresyl chloride and 1,1'carbonyldiimidazole, respectively, and then reacted with bovine collagen fibers. The results of nuclear magnetic resonance spectroscopy confirmed the structure of the synthesized oligopolylactide crosslinkers. The crosslinked collagen, on hydrothermal denaturation, behaves as a rubberlike material with thickness of about 40 to 60 μm. From the stress-strain relationship of the swollen denatured-collagen film, average molecular weights between points of crosslinking were determined. In the preliminary results, the fracture stress/strain of the collagen film were 7.52MPa/36.2%, whereas that of GA-treated film were 13.46MPa/51.6%, and oligolactide-treated film were 9.77MPa/52.5%. The extension ratio, at 20%, of membrane were selected to determine the crosslnking molecular weight. Based on the Flory-Rehner equation, the crosslnking molecular weight of GA crosslinked membrane was calculated to be 7000~7200, and oligolactides crosslinked membrane 7100~7200. The maximum number of crosslinkers introduced by 1% GA treatment was 8, and oligolatide treatment was 7. Higher GA or oligolactide concentration exert no effect on the crosslinking density. These results of GA-treated membrane are comparable with the previous results1. Fibrillar collagentreated with oligolactidedemonstrated significant decrease of solubility in aqueous solution at neutral pH and increases of fixationindexas compared to the noncrosslinked control.In addition, the crosslinked collagen fibers have a slight increased in density of collagen fibers and forms order structure as observed under transmission electron microscope

These data suggest that collagen fibers can be crosslinked by the end-group activated oligopolylactide to yield scaffold combining the properties of collagen and oligolactides. It therefore has great potential for medical and pharmaceutical applications.

1.V.Charulatha, A. Rajaram.J. Biomed. Mater. Res 36(4), 478-486, 1997


PC17

NMR STUDY OF PROTEIN-BASED POLYMERS WITH THERMOTROPIC PROPERTIES IN AQUEOUS SOLUTIONS

D. KURKOVÁa, J. KŘÍŽa, P. SCHMIDTa, J. DYBALa,J. C. RODRÍGUEZ-CABELLOb, M. ALONSOc

aInstituteof Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, CZ-16206 Praha 6, Czech Republic,

bDep. of Cond. Matter/E.T.S. I.I and cDep. of Anal. Chem., E. U. P., University of Valladolid, Valladolid, Spain

Polypentapeptidewith thermotropicproperties[(PG1V1G2V2)2-(PG1EG2V2)-(PG1V1G2V2)2]9 in D2O and H2O solutions was studied at various temperatures and at two different concentrations (5% and 0.5% wt), with respect to its structural changes during the temperature transition.

We found that thermotropic properties of investigated polymer are concentration dependent and therefore intermolecular interactions play a role in them. JNHCH coupling constants and 13C chemical shift values confirmed the random coil structure of the polymer below and above transition temperature. From JNHCH values used for calculation of Val1-αCH-NH and Val2-αCH-NH dihedral angles a conformation change (the αCH-NH bonds rotation of Val1 and Val2 residues of the polymer chain) was found during the heating. 13C chemical shifts did not confirm a characteristic specific change of the structure. Nearly constant value of hydrodynamic radii of the polymer at lower concentration did not confirm any change in the polymer coiling; the polymer apparently remains in the roughly extended form. Lower values of HOD T1 relaxation times and lower slope of its increase compared to T1 relaxation times of HOD without polymer suggest the interaction of the polymer with D2O (HOD).

The most pronounced intensity changes, corresponding to the increased rotation hindrances of the polymer, were observed for protons directly attached to the polymer backbone, whereas the more distant protons reach the immobilized state less easily.

Acknowledgement: The authors wish to thank to the Grant Agency of the Academy of Sciences of Czech Republic (Projects No.: AVOZ4050913, KSK4050111, IAA4050208).


PC18

Polypeptide translocation through a pore. a monte carlo study

A. SIKORSKI, P. ROMISZOWSKI

Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warszawa, Poland

In this work we studied a simple model of a copolymer (polypeptide) chain in the confinement. The model chain was restricted to a flexible [310] lattice. It was represented as a sequence of united atoms located at the positions of alpha carbons. The force field introduced into the model consisted of the long-range contact potential between amino acid residues with the excluded volume and a local helical potential. The chain was built of hydrophilic and hydrophobic segments. The properties of such chains were determined by means of the Monte Carlo simulations using a Metropolis-like algorithm. During the simulations we observed and tracked the motion of the chain during its threading through a hole in an impenetrable wall. The influence of the chain length, the sequence of residues and the temperature of the system on the structure of the chain during the translocation were investigated. The dynamic properties of the system such as the mean first passage time were also studied and discussed.


PC19

COMPUTER SIMULATIONS OF POLYPEPTIDE ASSOCIATES

S. KMIECIK, A. SIKORSKI, P. ROMISZOWSKI

Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warszawa, Poland

We built a simplified model of a protein-protein docking. In this model all heavy atoms of a polypeptide chain are represented. The positions of these atoms were restricted to vertices on a face-centered cubic lattice. No long-range atom-atom potential was introduced. We used only a contact potential in a form of an excluded volume. A series of Monte Carlo simulations were carried out for some examples of receptor-ligand systems taken from the Protein Data Bank. The process of docking was simulated step-by-step by random translations and reorientations of the ligand (smaller proteins) in the vicinity of the receptor surface. During the simulation the number of native ligand-receptor contacts was monitored and it was used as a criterion of the docking quality. The results of the simulations were compared with the previous ones obtained for a united atom model of the system.


PC20

ALGINATE MICROPARTICLES FOR DRUG RELEASE AND ENZYME IMMOBILIZATION: A DIMENSIONAL STUDY.

L. LAZZERI , M.G. CASCONE , L.P. SERINO, S. DANTI , P. GIUSTI

Department of Chemical Engineering, Industrial Chemistry and Materials Science, University of Pisa, Via Diotisalvi 2, 56126 Pisa, Italy.

Polymeric particles are currently employed in a wide range of applications, including drug delivery and enzyme immobilization. Both synthetic and natural polymers are used for the production of biodegradable micro and nanoparticles.

In the present work biodegradable polymeric particles were produced using alginate, a naturally occurring biopolymer extracted from brown algae. This biological macromolecule, that can be ionically cross-linked by the addition of divalent cations in aqueous solution, has several properties that enable it to be successfully used as a matrix for the entrapment and/or delivery of biological agents, such as drugs and enzymes.

In particular proteins can be loaded and released by alginate matrices without loss of their biological activity because of the relatively mild gelation process of alginate.

Alginate particles were produced by a water-in-oil emulsification process and calcium chloride (CaCl2) was used as cross-linking agent. The effect of changing different operative parameters, such as temperature, stirring rate, alginate concentration, surfactants concentration and CaCl2 concentration, on size and size distribution of the produced particles was studied. The morphological characteristics of the particles were investigated mainly by scanning electron microscopy (Fig.1). Model proteins, bovine serum albumin (BSA) and urease were encapsulated into the particles and in vitro test were performed to evaluate their release.

It was observed that the produced alginate particles are able to work as a release system for BSA. On the contrary the enzyme is not released but it is retained inside the particles in its active form.

Fig. 1


PC21

A SAFE AND EFFICIENT METHOD TO PRODUCE LOW-MOLECULAR-WEIGHT HYALURONIC ACID

F. GUILLAUMIEa, B. L. FUHLENDORFFa, K. SCHWACH-ABDELLAOUIa

aNovozymesBiopolymer A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark

Hyaluronicacid (HA) is a natural and linear polymer composed of b-1,3-N-acetyl glucosamine and a-1,4-glucuronic acid repeating disaccharide units with a molecular weight (MW) up to 6 MDa. It is a major constituent of the extracellular matrices and the synovial fluid. In the last decades, various fields of application including cosmetics, ophthalmology, rheumatology, tissue engineering and drug delivery have been explored, owing to the many important biological functions of HA and its unique physico-chemical properties. However, for some specific applications, the relatively high MW of HA is a limiting factor and the availability of low MW fractions would be highly desired. For food applications, low MW HA has been shown to penetrate the gastrointestinal barrier, thereby increasing the HA bioavailability. Moreover, low MW HA fractions are able to reestablish the HA content in the skin and can be thus be used in cosmetics as anti-aging and anti-wrinkle agents. Finally, they exhibit anti-inflammatory effect and have potential applications in the treatment of inflammatory diseases.

A range of methods have been developed for the depolymerization of HA involving for example heating, ultrasonication, UV/gamma irradiation, and chemical/enzymatic treatment. Ultrasonication is the preferred technology, as it leads to samples with a low polydispersity and offers additional advantages such as absence of purification after sample treatment, ease of handling, environmental friendly process, and high potential for upscaling. Accordingly, we present results on the ultrasonic degradation of HA originating from Bacillus subtilis fermentation into well-defined low MW fragments down to 50 kDa.


PC22

A STUDY OF THE INFLUENCE OF POLYSACCHARIDES ON COLLAGEN SELF-ASSEMBLY

TSAI, SHIAO-WEN1, LIU, RE-LIN 1, HSU, FU-YIN 2

1Institute of Biochemical and Biomedical Engineering, Chang-Gung University, TAIWAN

2Materials Research Laboratories, Industrial Technology Research Institute, TAIWAN

Type I Collagen is themost commonly used biomaterial in matrix construction for tissue engineering, and is known to affect the growth and differentiation of a variety of cells.Collagen, the most abundant structure protein in animal connective tissue, is found in the fibrils with diameter of about 30 to 300 nm.Individual molecules of collagen, which consists of triple helix of about 300nm in length and 1.5nm in diameter, will undergo self-assembly to form fibrils with a characteristic banding pattern of 67nm period when observed via electron microscopy. Numerous studies of collagen fibril assembly in vitro have been focused on the mechanism of initiation of the aggregation, the control of the diameter and length of the fibril, and the roles of short nonhelical telopeptides at the carboxyl and amino terminals. However, extracellular matrices not only contain collagen but also have proteoglycan, and other macromolecules. On the other hand, most of the scaffolds for tissue engineering are made of collagen and other macromolecules in order to improve the mechanical/biochemical properties of matrix. Therefore, in this work, we utilized alginate which with the negative charge under physical condition, or chitosan that with positive charge at pH 7.0,mixed with collagen solution in various amount, to compare the diameter and D-period of fibril with pure collagen after reconstitution at pH 7.2, PBS, 37℃.The collagen exists in different morphologies when in the presence of alginate or chitosan. The collagen fibrils will show in spindle shape and more widely when they are reconstituted in presence of 0.2% alginate, but these phenomena cannot observe in lower concentration of alginate. Alternately, the reconstituted collagen in the presence of chitosan, we observed there is no change in shapes. Although the morphologies and diameter are different in various reconstituted condition, but the D-periods are all keep the same no mater what kind of polysaccharides are added.These results indicate that the diameter, D-period and morphologies of collagen fibrils are affected not only by the incubated temperature and ionic strength but also be affected by the presence of charged macromolecules.The reconstitution rate as well as the sizes and morphology of collagen could be varied by adjusting the concentration of polysaccharides. Because cells behave differently to changes geometries of the extracellular matrix, the understanding of the effect of differentially organized collagen structure on cellular behaviors is an area that remains to be explored.

Acknowledgement: This work was supported by a grant from national Science Council, R.O.C. (NSC-92-2120-M-010-001)


PC23

OXIDATION OF UHMWPE IN A BIOLOGICAL ENVIRONMENT

S.FIORITO 1,2, C. GOZE 2, C. REIBEL 2, R.M. STREICHER 3

1Dept. of Experimental Medicine, University La Sapienza-CNR, Viale Marx 43, 00100 Roma, Italy

2GDPC, University Montpellier II CC26 , Place E. Bataillon, 34095 Montpellier Cedex 05, France

3Stryker SA, Clinical and Scientific Affairs, Florastrasse 13, CH-8800 Thalwil, Switzerland

Background: Gamma irradiation used for sterilization of UHMWPE is known to induce breaking of the polymers chains and to cause the production of long lived free radicals. The primary free radicals produced during irradiation are a combination of alkyl and allyl types. Immediately after irradiation, at room temperature, the alkyl radicals decay rapidly owing to oxidation or crosslinking or formation of allyl radicals. The decay rates of alkyl and allyl radicals depends upon the presence or absence of oxygen.If PE is stored in air or any oxidative environment, conversion of allyl and alkyl radicals into peroxy radicals takes place (Jahan MS, et al, J Biomed Mater Res 1991, 25: 1005). In biologic environments free radicals can react with oxygen from the tissue fluids or, if an inflammatory condition exists, with other reactive, highly oxidant species produced by inflammatory cells (Fiorito S. et al., J Biomed Mater Res 2001, 57:35). Aim of the study: To evaluate the oxidation rate of UHMWPE samples in a biological environment as compared to that occuring during storage in air. Materials and Methods: Free radicals were detected in UHMWPE samples by ESR (Electron Spin Resonance), immediately after removing the under vacuum packaging and after 5, 10, 15 days of immersion in inflammed synovial cell cultures, as well as in PE samples stored in an ambient environment at room temperature. Results: immediately after removing the packaging, typical ESR spectra due to the presence of allyl and alkyl radicals were observed in all PE samples. Conversion of allyl-alkyl radicals into peroxy radicals took place very fast, beginning from the 5th day in cell cultures. On the contrary, conversion of allyl-alkyl radicals into peroxy radicals occurred much more slowly in PE stored in air, beginning from the 30th day of storage in ambient environment.


PC24

3D NANOSTRUCTURED CONDUCTIVE BIOMATERIALS

J. SERRA MORENO, S. PANERO, B. SCROSATI

Chemistry Department, University of Rome "La Sapienza",00185 Rome, Italy

Template synthesis1 is a very useful technique to perform nanotubes inside the pores of a nanoporous membrane. The nanostructured substrates show high electronic conductivity due to the ordered structure.

We report herein the results on the electrochemical synthesis of redox polymers, based on polypyrrole (Ppy) and biologically active compounds (such as Heparin and Hyaluronan derivatives HA) inside the pores of an inert membrane (Fig.1). These new materials2-3, which combine biological activity with electrical properties, represent an excellent substrate for the development of biocompatible 3D scaffolds for specific cell functions.

Polymer nanotubules have been prepared by electrodepositing the Ppy within the pores of microporous alumina-based membrane.

By controlling the nature of the doping anions and the polymerization time, we can control the thickness of the Ppy along the pore walls of the template membrane The kinetics and mechanism of charge transport in the nanostructured polymers can be studied using the usual electrochemical techniques.

Due to the favorable characteristics shown by the Ppy-Hep and Ppy-HA substrates, we may hypothesize that these nanostuctured conductive polymers may emerge in different areas4. The possibility to modulate the shape and the structures (pore size, distribution and thickness) candidates the nanoporous materials as successfully tools in molecular medicine.

Fig.1 Scanning electron micrograph of Ppy nanotubes.

References:

1. C.R.Martin, Science, 266, 1961 (1994)

2. Z. Samec, A.Trojánek, J.Langmaier and E.Samcová, Electrochem. Comm. 5, 867 (2003)

3. J.Collier, J.P.Camp, T.W.Hudson, C.E.Schimdt, Biomed.Mater.Res., 5, 574 (2000)

4. M.J.Tierney and C.R.Martin, J.Electrochem.Soc., 137, 6, 2005 (1990)


PC25

CHARACTERISATION AND CELL RESPONSE OF CHITOSAN AND ALGINATE DERIVATIVES BLEND MEMBRANES BY DIFFERENT CROSSLINKING METHODS

M.D. NOTARA a, C.A. SCOTCHFORD a, D.M. GRANT a, G.F. ROBERTS b

a: Bioengineering group, School of Mechanical, Materials, Manufacturing Engineering and Management, University of Nottingham, Nottingham, UK

b: ChiTech Consultants Ltd, Southwell Notts, UK

Chitosanis a cationic polysaccharide, the second most abundant natural polysaccharide after cellulose. It has been thus far broadly used as a biomaterial in various applications because of its biocompatibility in these applications and the fact that it is relatively simple to process and modify. Alginate is an anionic polysaccharide of similar structure to chitosan proved as well to be biocompatible in a range of uses including food industry and drug delivery. When mixed in acidic conditions they form a polyelectrolyte complex. Nevertheless, when chitosan is solubilised in the form of an anionic polyelectrolyte derivative it is possible to achieve a homogeneous blend with sodium alginate in an aqueous environment.

In this work, two types of membranes are being studied. The first is a membrane of chitosan water soluble anionic condensation product with sodium formaldehyde bisulfate (CNaFBS) blended with sodium alginate and crosslinked with Glutaraldehyde and Calcium Chloride (blend1). The second is a blend of (CNaFBS) with Ammonium Alginate without any other crosslinking step (blend2). In both cases two blend ratio were prepared, 50% CNaFBS and 70% CNaFBS.

FTIR analysis suggested that formation of hydrogen bonds between -COO, -OH in NaAlg, and -OH in blend1 occurred. Other interactions are suggested by the FTIR spectra of the blend 2. X-ray diffractograms showed that the blend1 membranes exhibit a crystalline form due to the effect of calcium ion induced egg box structure while blend2 samples are rather amorphous. This is supported by the DSC data where blend1 has increased stability.The wettability of the material was also investigated using Dynamic Contact Angle analysis.

Initial in vitro assessment of cytocompatibility of these membranes was carried out using 3T3 fibroblasts. Neutral Red assay results showed good cell response in terms of cell viability. However the SEM images demonstrated poor cell attachment in both cases. The tests were repeated for samples treated with Fibronectin (FN) which improved the cell attachment on blend1 but not on the blend2 membranes. These results are confirmed by BCA total protein assay.


PC26

PREPARATION AND CHARACTERIZATION OF A STARCH-BASED BIODEGRADABLE MATERIAL

T. DIVERS*, I. PILLIN, J.F. FELLER, Y. GROHENS

L2PIC,Centre de Recherche, rue de Saint-Maudé, BP 92116 Lorient Cedex, France

This project is aimed at designing new biodegradable materials with high performances from well-controlled blends of wheat starch and poly(caprolactone) (PCL). Starch has been largely studied for non food applications and, actually, it could replace synthetic polymers coming from petroleum industry. However, it cannot be mixed with PCL in its native form as it would lead to poor mechanical properties (due to the large dimensions of starch granules and their broad distribution in size). Consequently, it is necessary to destructurize starch before mixing it with PCL, what can be achieved through chemical modification.

Among the available reactants to achieve this modification, formic acid (FA) was chosen because on the one hand it acts both as a reactant and a destructurizing agent and on the other hand its handle does not need the use of an organic solvent [1]. A previous study showed that reaction conditions (t, T, starch concentration) have to be well-controlled or else it induces starch depolymerization due to acid hydrolysis [2]. The second step of the biomaterial preparationconsists of mixing starch with PCL. Starch samples are first let to react with a PCL oligomer (Mw = 2000 g.mol-1, functionnalized with hydroxyl groups) to increase compatibility between starch and PCL but also to induce native starch destructurization. Then, commercial PCL (Mw = 80000 g.mol-1) and para-phenylene diisocyanate (PPDI) are added. PPDI can create crosslinks between starch and PCL oligomer or polymer, generating a network. PPDI brings cohesion to the material that is why it is important to determine its percentage to add to the reaction medium precisely. Indeed, too little PPDI induces too little cohesion but too much PPDI leads to a highly crosslinked material and can engender processing problems.

References:

[1]: J. Aburto, I. Alric, E. Borredon : Starch/Stärke 1999, 51, 132-135.

[2]: T. Divers, I. Pillin, J.F. Feller, G. Levesque, Y. Grohens : Starch/Stärke, in press


PC27

CONTROLLING GRAFTING-TO MODIFICATIONS OF POLYSTYRENE SURFACES AND THEIR INTERACTIONS WITH PROTEINS

D. LAZOSa, S. FRANZKAb, M. ULBRICHTa

aLehrstuhlfuer Technische Chemie II,
b
Institut fuer Physikalische Chemie, Fachbereich Chemie, Universitaet Duisburg-Essen, 45141 Essen, Germany

Controlling the interface properties of polymeric materials is of key importance for their biocompatibility which strongly depends on the interactions with proteins, cells and tissue. We are investigating the optimization of hydrophobic material's biocompatibility with a particular focus onto the detailed characterization of surface/protein interactions via creating a heterogeneous surface, which is based on a grafting-to modification of polyethyleneglycol (PEG) conjugates on hydrophobic surfaces.

Here we report about the modification of polystyrene (PSt) with photo-reactive a-4-azidobenzoyl-w-methoxy-PEGs (ABMPEG) and commercial PEG-polypropylene-glycol-PEG tri-block copolymers (PEG-PPG-PEG) as PEG conjugates. As PSt substrates we use thin spin-coated films and commercial microplates. Both were modified, and the influences of PEG conjugate concentration and adsorption time onto the degree of modification were studied in detail. Contact angle (CA) measurements showed the increased hydrophilicity and reduced hydrophobicity of the modified films as well as the irreversibility of the modification. Streaming potential measurements indicated a slight reduction of the z-potential as a function of the degree of modification. Surface plasmon resonance (SPR) measurements showed that Bovine Serum Albumin (BSA) adsorption is systematically decreasing with increasing PEG modification efficiency but it is not reduced to zero. Furthermore, we found that at the same CA, PSt surfaces modified with ABMPEG had less BSA adsorbed. We assume that ABMPEG has a very small hydrophobic head group and what is the reason for a well-balanced distribution of ABMPEG molecules on the PSt surface, in comparison to PEG-PPG-PEG which has a large hydrophobic anchoring group and a much more irregular distribution. This hypothesis had been supported by scanning force microscopy images. Currently, we investigate the adsorption of other proteins like Trypsin and Fibrinogen, which have a different size and shape compared to BSA. Furthermore, we are montoring the affinity interactions of adsorbed proteins with antibodies. All results will also be discussed in terms of the nanostructure of adsorption sites on PEG / PSt heterogeneous which can provide a conformational stabilization of PSt-adsorbed proteins by adjacent PEG molecules.


PC28

SYNTHESIS OF POROUS EMULSION TEMPLATED POLYACRYLAMIDES FOR ENZYME ENTRAPMENT USING HIGH INTERNAL PHASE CO2-IN-WATER EMULSIONS

J. LONG* G.A. HUTCHEON*, R. BUTLER+, A.I. COOPER+

*School of Pharmacy and Chemistry, Liverpool John Moores University, James Parsons Building, Byrom St. Liverpool, L3 3AF

+Department of Chemistry, Liverpool University, Crown St. Liverpool L69 3BX

The immobilization of enzymes is important as it offers a more stable and recoverable biocatalyst for industrial use than traditional unsupported systems. This study demonstrates that highly active biocatalytic preparations can be synthesised using an environmentally sound, dense gas templating process. a-Chymotrypsin was entrapped within the matrix of highly porous emulsion-templated materials that were synthesised via the polymerisation of concentrated CO2-in-water (C/W) emulsions. The method does not use any organic solvents, in either the synthesis or purification steps, thus no solvent residues are left in the materials. The use of the non-polar surfactant Tween 40 in conjunction with a co-surfactant such as PVA was found to produce a stable emulsion between the two phases enabling the production of highly porous, low-density polyacrylamide materials. Redox free-radical co-initiators facilitated the rapid low temperature polymerisation of the enzyme/surfactant/monomer minimising the risk of enzyme inactivation. The highly porous open-cell nature of the materials allowed for the facile lyophilisation of the remaining aqueous phase. The effect of changing the monomer to crosslinker ratio was studied for polymers with fixed enzyme content. Materials were produced with surface areas ranging from 1.51 to 3.88 m2/g. Enzyme activity was measured in organic media using a transesterification assay. The highest activities of around 0.45 mmol/mg of enzyme/min corresponded with the highest surface area materials. These activities were found to be over 13 times greater than those observed with the non-immobilized enzyme and several orders of magnitude higher than the non-emulsion templated controls. Experiments were also conducted to investigate the effect of CO2 density on the overall pore structure of the product. Polymerisations were initiated at pressures ranging between 60 and 150 Bar. SEM imaging indicates that the pore structure is affected by the change in reaction conditions. This may allow for the production of materials with a tuneable structure for the optimisation of immobilised enzyme activity.


PC29

STRUCTURE ANALYSIS OF TWO “MUTANT” PEPTIDE ANALOGUES OF A PART OF THE CENTRAL DOMAIN OF THE B-FAMILY SILKMOTH CHORION PROTEINS: INSIGHT INTO AMYLOID-LIKE FIBRIL FORMATION

V.A. ICONOMIDOU, S.J. HAMODRAKAS

Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, Athens 157 01, Greece

shamodr (cc.uoa.gr), veconom (cc.uoa.gr)

Chorionis the major component of silkmoth eggshell. An 18-residue peptide-analogue of a part of the central conservative domain of the B family of silkmoth chorion (eggshell) proteins (B peptide) self-assembles in vitro, in various conditions, and forms amyloid-like fibrils. To study in detail the molecular basis of chorion protein self-assembly mechanisms, two “mutant” peptides of the 18-residue peptide were carefully designed and synthesized. These “mutant” peptides were studied both in solution, under a variety of conditions, and in the solid state, utilizing X-ray fibre diffraction, T.E.M. (negative staining and shadowing), FT-Raman, ATR FT-IR and NMR spectroscopy. It is clear from these studies, that only one of the two “mutant” peptides, the B_m1 peptide, forms fibrils with amyloid characteristics. The fibrils have the same structure as the amyloid-like fibrils of the B peptide: they are organized into twisted and non-twisted ribbons (each ribbon consists of thin protofilaments (fibrils) that have the tendency to coalesce laterally among each other, and have a uniform diameter of approximately 30-40 Å), they bind Congo red and they exhibit a cross-β sheet type of structure. On the contrary, the B_m2 peptide self-assembles into rings with unknown yet properties. This study shows that, amyloid fibril formation is related to: a) the existence of specific types of aminoacid residues, which are strongly amyloidogenic and b) their specific positionsalong the protein sequence. Apparently, suitably designed substitutions of specific residues in a protein sequence, inhibit or favour amyloid-fibril formation.

Furthermore, it appears that, nature, after millions of years of molecular evolution, has sensibly designed chorion proteins to form amyloid-like fibrils, in order to play an important functional role:� to protect the oocyte and the developing embryo from a wide range of environmental hazards.


PC30

DESIGN OF EROSION TIME-CONTROLLABLE HYDROGELS USING POLYROTAXANES FOR CARTILEGE TISSUE ENGINEERING

T. OOYAa,b, W. TACHABOONYAKIATc, M. KATOd, T. KURUSHIMAd, NOBUHIKO YUIa,b

aSchool of Materials Science, Japan Advanced Institute of Science and Technology, Ishikawa 923-1292, Japan. yui (jaist.ac.jp)

bThe21st Century COE Program, JAIST, Japan

cJapan Science and Technology Corporation, Innovation Plaza Ishikawa, Japan

dJapan Tissue Engineering Co., Ltd., Aichi, Japan

Wehave studied biodegradable polyrotaxanes, in which many a-cyclodextrins (a-CDs) are threaded onto a poly (ethylene glycol) (PEG) chain capped with bulky end-groups via ester linkages. These hydrolyzable polyrotaxanes were utilized as a cross-linker for preparing PEG-polyrotaxane hydrogels (PEG-PRX gels) for biomedical applications. Inclusion complexation between a-CD and the ester linkages achieved prolonged time to complete hydrogel erosion from day to month. In this study, cationic group-introduced porous PEG-PRX gels were designed and evaluated for a biodegradable scaffold of chondrocyte cultivation. Amino-modified porous PEG-PRX gels (Fig. 1) were prepared by immersing the PEG-PRX gels containing sodium bicarbonate in ethylenediamine-containing DMSO solution,followed by gas foaming in citric acid aqueous solution. Chondrocytes proliferationwas significantly increased by the amino-modification. Results of histochemical studies suggested that the amino-modification increased the production of cartilage like matrices with glycosaminoglycans. Furthermore, these porous PEG-PRX gels with cultivated chondrocytes were completely disappeared 28 days after subcutaneously implanting to rabbit without any serious inflammation reactions. These results suggest that the amino-modified porous PEG-PRX gels are promising as a scaffold of cartilage regeneration.

Fig. 1 Amino-modified porous PEG-PRX gels for chondrocyte cultivation.

PC31

MULTIVALENT MOLECULAR RECOGNITION BASED ON FAST SLIDING MOTION OF SACCHARIDE-POLYROTAXANE CONJUGATES

T. OOYAa,b, H. UTSUNOMIYAa,b, M. EGUCHIa,b, N. YUIa,b

aSchoolof Materials Science, Japan Advanced Institute of Science and Technology, Ishikawa 923-1292, Japan. yui (jaist.ac.jp)

bThe21st Century COE Program, JAIST, Japan

Saccharide, one of the ligands for targeting, in saccharide-polyrotaxane conjugates could easily diffuse in buffers via sliding and rotational motion of a-cyclodextrins (a-CDs) along a poly(ethylene glycol) (PEG) chain (Fig. 1). A maltose-polyrotaxane conjugate with appropriate threading number of a-CDs exhibits high mobility of maltose in aqueous conditions.1 A high mobility of such saccharide groups in the conjugates can contribute to preserving ordered structure of the bulk water clusters.2 The association constant (Ka) between the conjugate and Concanavalin A (Con A) was in the range of 6 powers. The features of high mobility of the ligands and preserving the water clusters led to gaining a large negative enthalpy. Therefore, the supramolecular motion of the polyrotaxane is thermodynamically favorable for multivalent interaction in biological systems.

Fig. 1 Structural image of sliding and rotational motion of a-CDs along a PEG chain in maltose-polyrotaxane conjugates. Maltose groups were conjugtaed with hydroxyl groups of a-CDs in the polyrotaxane. High mobility of the maltose-conjugated a-CDs was confirmed by spin-spin relaxation time (T2).1

References


PC32

BEHAVIOR OF PHASE SEPARATION ON HOLOGRAPHIC GRATINGS USING SILOXANE-CONTAINING PHOTOPOLYMER SYSTEMS

Y. H. CHOa, Y. KAWAKAMIa

aGraduateSchool of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan, E-mail: chohee (jaist.ac.jp)

Transmission volume holographic gratings were fabricated by irradiation of Nd-YAG laser (l= 532nm) using photopolymerizable materials, which are consisted of multifunctional acrylate as a radically polymerizable monomer (RPM) and epoxides of various structures as cationically polymerizable monomers (CPM). Ketocoumarin dye and diaryliodonium salt were used to generate not only radical species but also cations as a photosensitizer and a photoinitiator, respectively.

As CPM materials, various structures of siloxane-containing epoxides were designed to improve the performance of holographic gratings derived from the incompatibility of the siloxane component. Holographic gratings with well-fabricated and clearly phase-separated morphologies were obtained as shown in Figure 1. In these systems, polymerization rate and diffusion rate of monomers play an important role in the formation of holographic gratings. That is, holographic gratings in Figure 1(a) were more clearly formed with over 95% diffraction efficiency than those of Figure 1(b) due to its adequate polymerization rate and fast diffusion rate. Moreover, in Figure 1(b), its diffraction efficiency could be controlled by changing the laser intensity related to polymerization rate.

(a) (b)

Fig. 1. AFM topology of transmission holographic gratings using siloxane- containing materials as CPM materials; (a) 3-Glycidoxypropyltrimethoxysilaneand (b) 1,3-Bis[2-(7-oxabicyclo[4.1.0]hept-4-yl)ethyl]-1,1,3,3-tetramethyldisiloxane.


PC33

CONFORMATIONAL GEOMETRY CHANGES OF SUPRAMOLECULAR ASSEMBLING SYSTEMS VIA pH VARIATION

HAK SOO CHOI,a,b TOORU OOYA,a,b SANG CHEON LEE,b KANG MOO HUH,a NOBUHIKO YUIa,b

aSchool of Materials Science and bthe 21st Century COE Program, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan. yui (jaist.ac.jp)

In this study, pH-dependent conformational changes for the supramolecular aggregate based on b-cyclodextrin-conjugated poly(e-lysine) (b-CDPL) and 3-(trimethylsilyl)propionic acid (TPA) would be clarified. The thermodynamics and the pH-triggered cyclic supramolecular aggregate formation would be evaluated by isothermal titration calorimetry (ITC) and zeta potential measurements, respectively. In addition, the geometric changes of the complexes with pH variation would be measured by static and dynamic light scattering (SLS, DLS) and transmission electron microscopy (TEM). This system showed well-controllable delicate pH-responsive supramolecular assembly formation around biological pH ranges, therefore, it would be preferred for applications such as pH-sensitive drug delivery and molecular sensing system.

Figure 1.pH-reversible properties of the supramolecular assembly based on cationic b-CDPL and anionic TPA molecules; hydrophobic aggregationsvia protonated TPA molecules(a),supramolecular aggregation via dual complexations, specific host-guest interactions and intermolecularionic interactions (b), andsol state via repulsive interactionsbetween the negative charged carboxyl groups of TPA(c).


PC34

ENCAPSULATION OF CALCIUM SULPHATE HEMI-HYDRATE (PLASTER OF PARIS) IN CROSSLINKED POLYCAPROLACTONE.

A. LA GATTA1*, A. DE ROSA1, P. LAURIENZO2, M. MALINCONICO2, C. SCHIARALDI2

1Seconda Università degli Studi di Napoli, Dipartimento di Medicina Sperimentale, Unità di Biotecnologia e Biologia Molecolare, Via L. De Crecchio, 8 - Napoli(Italy)

2Istituto di Chimica e Tecnologia dei Polimeri, Via Campi Flegrei, 34 - Pozzuoli (Na) (Italy)

*presenting author, e-mail: annalisa.lagatta (unina2.it)

Calcium sulphate hemi-hydrate (CHS) is widely used in dental and orthopaedic applications for filling bone cavities(1). However, its rapid physical erosion and resorption limits its potential applications in the bone regeneration process.

The aim of this research work was to develop a novel system in which CHS can be encapsulated in situ in a biocompatible and biodegradable polymeric network and slowly released. The polymer used is polycaprolactone-diol, MW 530 (PCL 530)(2).

In this work PCL 530 was chemically modified (functionalized) by reaction with methacryloyl chloride in order to make PCL susceptible of photochemical crosslink. FT-IR analysis was performed to monitor the reaction. The final product (PCLf) was intimately mixed with CHS in the presence of photoinitiator system. The injectable mixture was photo-crosslinked using a plasma arc lamp(C PCLf). Thermal analysis and solvent extraction of the product (C PCLf-CHS) clearly demonstrate the occurrence of crosslink. Moreover a qualitative visual inspection of the behavior of a tablet of C PCLf-CHS immersed in water shows a prolonged integrity when compared with a similar tablet made of not cross-linkable PCL-CHS.

The in vitro citotoxicity assays, according to ISO normative by direct and indirect method, showed the absence of citotoxicity for PCLf and PCLf-CHS.

References:

Sidqui M., Collin P., Vitte C and Forest N. , Biomaterials 16 (1995) 1327-1332

(2) Kweon. H.Y., Yoo M.K. Park.K and Cho C., Biomaterials 24 (2003) 801-808


PC35

INTERACTION OF HEMATIC PROTEINS WITH NATURAL AND SYNTHETIC POLYMERS SURFACES: A COMPARATIVE STUDY

AA. MOTTA, BD. MANIGLIO, AA. PENATI, AC. MIGLIARESI, AE. SERVOLI

aDpt. Materials Engineering and Industrial Technologies, University of Trento, Via Mesiano 77, 3850 Trento, Italy

bINSTM, National Consortium of Materials Science and Technology, Via Benedetto Varchi 59, 50132 Firenze, Italy

e-mail: antonella.motta (ing.unitn.it)

The interaction between materials and a biological environment is mainly dependent on phenomena occurring at the interface, where different dynamic biochemical processes and tissue reactions occur. These interactions are particularly intricate in the case of blood contacting biomedical devices; blood proteins, in fact, trigger and guide blood coagulation, the inflammatory and the immune response. Protein adsorption on the surface is an early event after the implantation, hence the protein layer becomes the interface dominating the device overall biological behaviour. Cellular phenomena as adhesion, proliferation, differentiation and apoptosis will be mediated by the adsorbed protein species, by the boundary strength with the surface and by their conformation and distribution.

The present study puts in comparison the behaviour of natural origin polymer, silk fibroin derived by Bombyxmori (whose biocompatibility and bioactivity has been demonstrated [1,2]) and of thermoplastic polyurethanes (polyether-based and polycarbonate-based) widely used in cardio-vascular applications, in contact with human plasma proteins.

Different parameters have been investigated, such as surface morphology before and after the incubation in plasma (both with AFM and ESEM), and adsorbed proteins, by means of mono-dimensional electrophoresis and wester blott analysis. The study has been focused on two proteins, fibronectin and fibrinogen, involved on the cellular adhesion and on the process of coagulation, respectively. Finally some AFM studies on the surface distribution of the adsorbed fibronectin have been performed, in order to evaluate the presence of preferential adsorption domains that can trigger cells adhesion.

Referecences:

Santin M, Motta A, Freddi G, Cannas M. J. Biomed. Mat. Research, 46, 382-389, 1999.

Motta A, Migliaresi C, Lloyd AW, Denyer S., Santin M. J. of Bioactive and Compatible Polymers,17, 23-35, 2002.


PC36

STRUCTURAL FEATURES OF RGD-CONTAINING PEPTIDES AS STUDIED BY QUANTUM CHEMICAL CALCULATIONS

J. CZERNEK, V. PROKS, F. RYPÁČEK

Department of Bioanalogous & Special Polymers, Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovský Sq. 2, Prague 6, CZ-162 06, Czech Republic; email: czernek (imc.cas.cz )

The cell adhesion activity of fibronectin and a number of other proteins has been localized to an RGD amino acid sequence. Short RGD-containing peptides have been shown to mimic a number of the properties of cell adhesive proteins, with differing conformations of the RGD motif resulting in changes in binding activity and integrin specificity. Ab initio quantum chemical calculations performed at the RHF/6-31G** level are applied to the description of geometrical characteristics of a series of peptides containing the RGD triad. The results are compared to the NMR and X-ray structures of the tenth type III module of human fibronectin. Importantly, it is shown that the local structure expressed by the (φ,ψ)-RGD dihedral angles of the highly potent peptide cyclo[ -RGDfV- ] (f denotes D-Phenylanine) closely matches the experimental results obtained in the solution NMR study.

Acknowledgement

This work has been supported by the Grant Agency of the Academy of Sciences of the Czech Republic (KJB4050311). Time allocation in the Czech Academic Supercomputer Centre and in the Mississippi Center for Supercomputing Research is gratefully acknowledged.


PC37

THE PREPARATION OF VALINE-RICH POLYPEPTIDE REPEATS BY SOLID PHASE PEPTIDE SYNTHESIS

V. PROKS, J. CZERNEK, F. RYPÁČEK

Department of Bioanalogous & Special Polymers, Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, Praha 6, CZ-162 06, Czech Republic

email: proks (imc.cas.cz )

The VVVEVVV and VVVKVVV (where V, E and K denote valine, glutamic acid and lysine, respectively) heptads are studied as the building blocks of the self-assembling hydrogel biomaterials. Their amino acid sequences belong to the class of so-called "difficult sequences" in peptide synthesis. The strong propensity of these amino acids to β-sheet formation is manifested in aggregation of the growing peptide chains already at early stages of their synthesis thus precluding its completion. An auxilliary protection at the peptide backbone has to be applied to distort the chain conformation and thus prevent its aggregation. The experimental conditions, kinetics and efficiency of the reaction ( Scheme 1) were studied in detail and are hitherto presented. Successful synthesis of above heptades through applicatioon of this method was documented.

Scheme 1:Auxilliary backbone protection in the synthesis of VVVEVVV

Acknowledgement

The work has been supported by the Grant Agency of the Academy of Sciences of the Czech Republic (KJB4050311).


PC38

the structure of multilayer protein/polyelectrolyte coatings and its effect on blood compatibility

M. HOUSKAa, E. BRYNDAa, A. BROUČKOVÁb, J.E. DYRb

aInstitute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, CZ-162 06 Praha 6, Czech Republic

bInstitute of Hematology and Blood Transfusion, U nemocnice 1, CZ-110 00 Praha 2, Czech Republic

Biological response to materials intended for contact with blood can be effectively improved by coating their surfaces with protein/polyelectrolyte assemblies using a Layer-by-layer technique. The technique is based on a controlled, alternating electrostatic deposition of oppositely charged components, resulting in stabile defined molecular layers of protein(s) or protein(s) and polyelectrolyte(s)1-4 on surfaces. Multilayer assemblies composed of albumin and heparin shade off the original surface and bring in passivating (albumin) and anticoagulant (heparin) effect. At least three layers of albumin or albumin/heparin are required to shield blood effectively from the adverse influence of the underlying surface. The multilayer films retain an essential degree of layered structure and we investigated in vitro how the number and ordering of the layers, and type of heparin affect the biological response. The systems with outermost heparin layer exhibit the highest anticoagulant activity but, in comparison with albumin, adhere more platelets. However, the systems with one or two outermost albumin layers over-coating the heparin layer retain ca. 70 - 80 % of heparin activity; overcoating with three and more albumin layers starts to diminish heparin activity essentially. It confirms that partial interpenetration of thrombin into the assembly and effective formation of TAT complex is possible even with albumin on top. On the other hand, the outermost albumin layer improves significantly interaction with platelets. The availability of heparin was also confirmed by experiments following incubation of modified surfaces in blood plasma, which after an initial minor decrease show a constant heparin activity.

1. E. Brynda, M. Houska, in: Y. Lvov and H. Möhwald (Eds.), Protein Architecture: Interfacing Molecular Assemblies and Immobilization Technology, Marcel Dekker, New York, 2000, pp. 251-285.

2. M. Houska, E. Brynda, J. Colloid Interface Sci. 188 (1997) 243.

3. E. Brynda, M. Houska, M. Jiroušková, J.E. Dyr, J. Biomed. Mater. Res. 51 (2000) 249

4. M. Houska, E. Brynda, K. Bohatá, J. Colloid Interface Sci. (2004, in press, available online at www.scincedirect.com ).


PC39

BIOMIMETIC ANCHORS FOR SURFACE-INITIATED ATOM TRANSFER RADICAL POLYMERIZATION (SI-ATRP)

X. FAN, P. MESSERSMITH

Biomedical Engineering Department, Northwestern University, 2145 Sheridan Road, Evanston, IL, 60208, USA. Email: philm (northwestern.edu)

Atom transfer radical polymerization (ATRP) is a versatile and robust approach to synthesizing homopolymers and copolymers with controllable molecular weight and architecture. Recently, surface-initiated ATRP (SI-ATRP) has been intensively utilized to graft well-defined polymer brushes from flat substrates, spherical nanoparticles, planar clay nanoplatelets, and cylindrical carbon nanotubes. In many emerging healthcare applications, there is a growing interest and need for surface modification strategies for controlling biological interactions between device surfaces and cells or tissues. With the ability to produce polymer coatings of controlled composition and structure, SI-ATRP is an attractive method for polymer modification of medical materials and devices. A key aspect of SI-ATRP strategies is the surface adsorption of an initiator molecule capable of chemically interacting with the surface. The nature of the interaction between initiator and surface is important because it will determine the density of initiating sites, and therefore polymer chains on the surface, as well as the robustness of the polymer anchor onto the surface.

In this work, we report our initial efforts to develop new biomimetic initiators for SI-ATRP. For inspiration we turn to mussel adhesive proteins (MAPs), which secreted by marine mussels to tenaciously attach to a variety of surfaces in seawater. It has been speculated that a catecholic amino acid, L-3,4-dihydroxyphenylalanine (DOPA), which is abundantly found in certain MAPs, plays a key role in the chemical interaction between MAPs and various metal, metal oxide, and polymer surfaces. Therefore, we designed and synthesized a bifunctional initiator with a catechol moiety as the surface anchoring end group, and a halogenated alkyl terminus that can initiate ATRP. The initiator was immobilized to TiO2 surfaces, followed by SI-ATRP of acrylate monomers to form surface bound polymer brushes. The resulting surface-bound polymer was demonstrated to have nonfouling properties, providing steric resistance to fouling of the modified surfaces by cells, proteins, and other particles. This strategy can be used to prepare a variety of polymer modified surfaces for both medical (diagnostics, devices, nanoparticle-based therapies) and nonmedical (paints and other particle dispersions, MEMS, quantum dots, nonfouling surfaces) technologies. The water-resistant properties of the anchoring component are anticipated to be useful for permanent attachment of polymer coatings in aqueous environments.


PC40

DESIGN OF NEW BIOMATERIALS WITH TUNABLE THERMO-MECHANICAL PROPERTIES

I. FOLTRANa, M.L. FOCARETEa, P. DOBRZYNSKIb, M. SCANDOLAa

aUniversity of Bologna, Department of Chemistry "G. Ciamician", via Selmi 2, 40126 Bologna, Italy

bCenterfor Polymer Chemistry, Polish Academy of Sciences, 41-800 Zabrze, Poland

Poly(lactic acid), PLA, and poly(3-hydroxybutyric acid), PHB, are bioerodible and biocompatible polyesters that have been extensively investigated for applications in the biomedical field both in the pure state and in binary blends. In addition to the natural (biosynthesized) isotactic PHB, both polyesters can be polymerized with different tacticity, yielding polymers that range from totally amorphous to highly crystalline, with vastly different physical properties. When PHB and PLA are blended, by careful selection of the blend components a wealth of different phase behaviors and solid-state properties can be obtained.

The aim of this work is to study the phase diagram and to analyze the different morphologies that can be obtained in blends of PLA with PHB, when tacticity and molecular weight of the components is changed. Knowledge of structure-property relations in the PLA/PHB system will allow to select the most suitable blend components and compositions to design biomaterials with tuned physical and mechanical properties and with the desired hydrolytic degradation behavior.


PC41

IN VIVO TISSUE ENGINEERING:

NOVEL CONCEPT FOR "IN SITU" REPAIR AND FORMATION OF BODY TISSUES

I.J. Zdrahala, R.J. Zdrahala, Ph.D.

R&I Consulting International, Eden Prairie, MN 55346, USA

Limitation or loss of function of an organ often represents life-threatening situation. Transplantations are medically successful but the "replacement" organ availability, its compatibility with the host and subsequent healing often pose serious questions. An In vitro Tissue Engineering, where harvested cells populate carefully prepared, often complex scaffold to generate an artificial organ, addresses problems mentioned above. Trauma associated with the implant introduction to the host often complicates the process.

A novel concept of In vivo Tissue Engineering, which mediates the healing and tissue regeneration process via providing in vivo formed porous, microcellular scaffold is proposed. This scaffold is then inhabited by propagating autologous cells or by introduction of encapsulated harvested cells (e.g. transformed "stem cells", etc.). Minimally invasive microsurgery, characteristic for its limited and controllable trauma, is utilized to deliver this system to a site to be repaired. The approach requires formation of the polymer, in situ and in a reasonable time, capable of forming polymeric foam with both open and closed cells. Consequently, the maximum protection to the encapsulated cells must be provided, if delivered to the site during the foaming of the scaffold.

The utilization of biodegradable polyurethanes is selected for this concept. Diversity of their Structure/Property relationship, recognized biocompatibility and controllable biodegradability justify their use as the materials of choice. Polysaccharides are used as the soft segments to affect controlled biodegradability. Finally, combination of polyurethanes with lactide and glycolide copolymers is used as cell encapsulant. A "Bio-RIM", a novel delivery system utilizing Reaction Injection Molding has been designed to mix and deliver, in combination with minimally invasive surgery, both scaffold components and encapsulated cells to form a basis for regenerated organ.

The use of this approach is envisioned for both soft and hard tissue repair and augmentation applications.


PC42

Functional polymer hydrogels for embryonic stem cells support

J. Kroupováa, b, P. Dvořáka, b, c, Daniel Horákc, d

aInstituteof Experimental Medicine, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-140 00 Prague 4, Czech Republic

bMendelUniversity of Agriculture and Forestry Brno, Zemědělská 1, CZ-613 00 Brno, Czech Republic

cCentrefor Cell Therapy and Tissue Repair, 2nd Medical Faculty, Charles University, V Úvalu 84, CZ-150 00 Prague 5, Czech Republic

dInstituteof Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovskéhonám. 2, CZ-162 06 Prague 6, Czech Republic

Embryonic stem (ES) cells are pluripotent cells that are able to give rise a wide variety of differentiated cells of the body. ES cells, including recently isolated human ES cells, have a great potential in treating many serious disorders by stem cell-based transplantation therapy. One potential way to repair damaged part of organ or tissue by ES cells is to grow them on three-dimensional polymer scaffolds. To address this issue, new polymer hydrogels were synthesized and tested. Cationic polymer slabs were synthesized by bulk radical copolymerization of 2-hydroxyethyl methacrylate (HEMA) and 2-(dimethylamino)ethyl methacrylate (DMAEMA) with ethylene dimethacrylate (EDMA), or 1-vinyl-2-pyrrolidone (VP) with N, N'-divinylethyleneurea (DVEU) or EDMA in the presence of sucrose as a porogen. Swelling studies on synthesized copolymers showed their high water content in the swollen state. Cytotoxicity studies with mouse ES cells line D3 demonstrated that synthesized materials were not toxic to ES cells. The presence or the absence of surface charges and the type of crosslinking agent did not affect undifferentiated character of ES D3 cells. The cationic copolymers showed, however, reduced proliferation of ES cells in comparison with unmodified PHEMA.


PC43

CHARACTERIZATION AND EVALUATION OF RABBIT CHONDROCYTES ON THE SYNTHETIC BIODEGRADABLE POLYMERS FOR ARTIFICIAL CARTILAGE

H.J. OHa, N.K. LEEa, S.N. KIMa, C.M. HONGa, S.H. HONGa

aBiolocis Evaluation Department, Korea Food and Drug Administration, 5, Nokbun-dong, Eunpyung-gu, Seoul, 122-704, Korea, ohojung (kfda.go.kr

Tissue engineering has arisen to address the extreme shortage of tissues and organs for transplantation and repair. One of the most successful techniques has been the seeding and culturing cells on three-dimensional biodegradable scaffolds in vitro followed by implantation in vivo. The biomaterials are attractive candidates for chondrocytes embedding and tissue-engineered transplantation in cartilage. We used PLA and PLGA as biodegradable polymers and rabbit chondrocytes were isolated and applied to the polymers. To evaluate the biocompatibility and biological safety of polymers, in vitro cytotoxicity and in vivo animal tests were investigated. Rabbit articular chondrocytes were isolated and characterized using MTT assay, alcian blue staining, immunohistochemical staning, and Western blot for type-II collagen. For biological and viral safety evaluation, we checked adventitous agents. Chondrocytes were seeded onto scaffolds to make artificial cartilage. After seeding the cells, we characterized chondrocytes using scanning electron microscopy(SEM).

PLA and PLGA showed excellent biocompatibility with L929 cell line and no toxic effects in animals. They were also biologically safe. Chondrocytes were characterized using cell proliferation assay and type-II collagen identification. Chondrocytes were easily dedifferentiated and lost their phenotype in monolayer culture after 2nd passage. Adventitious agents such as pyrogen, bacteria, fungi, mycoplasma, and adventitious viruses were not found. To evaluate the ability of polymers for delivering chondrocytes, after seeding the cells, HE staining, immunohistochemistry and SEM were investigated. In the artificial cartilage, SEM documented cell attachment to scaffolds.

In conclusion, biodegradable polymer, especially PLA holds promise as a suitable substrate for scaffolding material, and 0 and/or 1st passage rabbit chondrocytes are optimal for seeding on scaffolds. It is suggested that these artificial cartilages offer a promising approach to deliver chondrocytes to the cartilage defects.


PC44

OPTIMIZATION OF SCAFFOLDS BASED ON NATURAL POLYMERS

S. HANZELOVÁa, L. KUKOLÍKOVÁa, D. BAKOŠa, P. ALEXYa, W. ZHONGb

a Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava, Radlinskeho 9, SK- 812 37 Bratislava, Slovak Republic

bDepartment of Macromolecular Science, The Key Laboratory of the Molecular Engineering of polymers, Fudan university, Shanghai, P.R.China

Scaffolds based on natural polymers are currently receiving a great deal of interest for medical and pharmaceutical applications. Moreover, they have many different functions in the field of tissue engineering. They are applied as space filling agents, as delivery vehicles for bioactive molecules, and as three-dimensional structures that organize cells and present stimuli to direct the formation of a desired tissue.

The study has been focused on optimisation and properties testing of the membranes based on the complex of chitosan, functionalised hyaluronic acid and enzymatically treated collagen. The main parameters influencing the characteristics of membranes are for example molecular weight and degree of deacetylation of chitosan. Therefore, the experiments were performed with chitins of different origins, namely lobster and fungal chitin. The complex is crosslinked with starch dialdehyde derivatives. Usually, chemical crosslinking has been widely used to increase the mechanical and biological stability of biomaterials. Mechanical properties and physical properties based on this complex were studied to optimise their composition.


PC45

SPONGY MATRICES CONTAINING BIOPOLYMERS AS SCAFFOLDS FOR TISSUE REGENERATION

M.G. CASCONE a, L. LAZZERI a, S. DANTI a, L.P. SERINO a, M. ARISPICI b, P. GIUSTI a

a Department of Chemical Engineering, Industrial Chemistry and Materials Science, University of Pisa, Via Diotisalvi 2, 56126 Pisa, Italy.

b Department of Animal Pathology, Prophylaxis and Food Hygiene, University of Pisa, Via delle Piagge 2, 56124 Pisa, Italy

Matrices based on biodegradable polymers such as poly(lactic acid) (PLA) are widely employed as temporary scaffolds for tissue regeneration. The use of PLA offers several advantages in that it is degraded to products easily metabolised and excreted, it is biocompatible and easy to sterilize. However because of its poor hydrophilicity, PLA-based scaffolds show a low affinity for the cells. The addition of natural biodegradable hydrophilic macromolecules to PLA could represent an easy way to increase its hydrophilicity and thus to ameliorate its cell affinity. In the present work biological macromolecules were used to prepare matrices, based on PLA, having a porous three dimensional structure. The emulsion/freeze-drying method was used to produce gelatin/PLLA and agarose/PLLA spongy matrices. The structural characteristics of these sponges were investigated by scanning electron microscopy (SEM) and mercury intrusion porosimetry. In vitro tests based on the cell culture method were performed in order to test the affinity of the systems for cells and thus their potential use as scaffolds. Images obtained by SEM showed that all the produced materials were highly porous with interconnected pores (Fig. 1). In the case of gelatine-containing matrices it was observed a pore size distribution having a maximum around 20 mm, while in the case of agarose-PLA sponges a maximum was observed around 40 mm. The results of colorimetric tests (Alamar Blue and MTT) performed at different times after seeding showed the presence of viable cells inside both types of matrices. Cell adhesion and proliferation inside the matrices are at the moment under investigation by hystological analysis.

Fig. 1


PC46

THREE- DIMENSIONAL FIBRIN SCAFFOLDS

T. Riedela, E. Bryndab, P. Lesnýc, P. JENDELOVÁc, E. SYKOVÁc, J. E. Dyra, M. Houskab

aInstitueof Hematology of Blood Transfusion, U Nemocnice l, CZ-12800 Praha 2, Czech Republic

bInstituteof Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, CZ-162 06 Praha 6, Czech Republic

cCenterfor cell therapy and tissue repair, Charles University, 2nd Faculty of Medicine, Institute of Neurosciences, V Úvalu 84, CZ-15018, Praha 5, Czech Republic

Three-dimensional fibrin networks with various pore dimensions and fibrin fibres were prepared by changing fibrinogen concentration and thrombin activity in the initial solution. The fibrin gel formation was observed by turbidity measurement. The structure of freeze dried or ethanol step-by step dried samples was observed using scanning electron microscopy (SEM). Similar results obtained using the two drying procedures indicated, that the morphology shown by SEM was not modified very much by the preparation. At fibrinogen concentration 1 mg/ml, the network density decreased and the thickness of fibrin fibres increased (60 ®180 nm) with decreasing thrombin activity (0.2®0.004 U/ml). At the thrombin activity 0,01 U/ml, the network density increased with increasing fibrinogen concentration while thickness of fibrin fibres of about 170 nm did not changed. Roughly estimated interfibre distances varied from 0.1 mm in the densest networks up to 8 mm in the least dense networks.

Several types of cells (rat mesenchymal stromal cells, rat olfactory ensheathing glial cells and NE-4C neural stem cells, cloned from primary neuroectodermal cultures of p53- mouse embryos induced by retinoic acid) were cultivated on the surface of and also inside fibrin networks. All of these cell types are candidates for use in cell therapy of spinal cord injuries. The behaviour of these cells was evaluated for one week using fluorescent and confocal microscopy. Mesenchymal stromal cells seeded on the surface of fibrin gels grew in a thin layer on the surface, and only a few cells penetrated the gel. Olfactory ensheathing glial cells and neuronal precursors seeded inside the fibrin networks started to elongate and to form processes. However, with high concentrations of seeded cells, the gels were gradually degraded. The seedingof cells within a fibrin network appears to be a possible method for delivering them into central nervous system lesions.


PC47

Bioanalogous surfaces prepared by successive deposition of biological macromolecules

E. Bryndaa, M. Orlika, Z. Pientkaa, M. Houskaa, J. Pacherníkb, P. Dvořákb

aInstitute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, CZ-162 06 Praha 6, Czech Republic

bCenter for cell therapy and tissue repair, Charles University, 2nd Faculty of Medicine, Institute of Neurosciences, V Úvalu 84, CZ-150 18, Praha 5, Czech Republic

Intermolecular forces, mainly the electrostatic ones, were utilized for the layer-by-layer deposition of collagen IV (C), gelatine (G), laminin (LA), fibronectin (Fn), polylysin (PL), poly(ethyleneimin) (PI), serum albumin (A), dextran sulphate (D), and heparin (H) on polystyrene substrates (PS). The formation and stability of the molecular assemblies was observed in situ using surface plasmon resonanc. Morphology of the surfaces was studied by atomic force microscopy in aqueous solutions. Mouse embryonic stem cells line D3 were seeded on polystyrene surface coated with assemblies composed of various molecular layers.

Fig.1 Cell amount (related to the amount on clean PS) 48 h after seeding on 1 PS, 2 PS/C, 3 PS/C/Fn, 4 PS/C/D/C, 5 PS/C/LA, 6 PS/LA, 7 PS/Fn, 8 PS/G, 9 PS/G/Fn, 10 PS/G/D/G, 11 PS/G/LA, 12 PS/G/H/G/H, 13 PS/A/PL/Fn, 14 PS/A/PL/LA, 15 PS/A/PL, 16 PS/A/PI


PC48

PREPARATION AND CHARACTERIZATION OF POLY(CAPROLACTONE) POROUS SCAFFOLDS INTENDED FOR TISSUE ENGINEERING

K. HOEM, P. PLIKK, A.-C. ALBERTSSON

Royal Institute of Technology, Fibre and Polymer Technology

SE-100 44 Stockholm, Sweden

hoem (polymer.kth.se)

Resorbablepolyesters, e.g. poly(ε-caprolactone) (PCL), have acquired much attention over the last years for their potential and present use in biomedical applications. Not only is there a request for new polymers such as poly(1,5-dioxepane-2-one) [1], but there is also a continues need for the improvement of already existing materials. Our research group at the Department of Fibre and Polymer Technology has, via ring-opening polymerisation through a controlled coordination-insertion mechanism implemented a variety of alterations to conventional materials. These alterations include architectural changes, e.g. star-shaped polymers [2] and networks [3], surface topology variations, through for example annealing [4] and nanopatterning, along with copolymerisations [5] of the traditional monomers with a variety of common and novel monomers. These variations affect not only the morphology, but also the degradation and mechanical properties of the materials created.

Introducing a physical structure to the polymer material, such as the creation of a porous scaffold, gives yet another possibility to vary these properties. The main function of resorbable porous scaffolds in tissue engineering is to serve as an adhesive substrate for cell attachment and in growth, as well as physical support, while guiding and aiding the formation of new tissue.

The purpose of this study was to optimise the construction of porous scaffolds of poly(caprolactone) using different leaching techniques. Pore size and porosity were altered to find the optimum mechanical properties along with suitable conditions for cell proliferation.

Three different scaffold preparation methods were evaluated, of which two were salt leaching techniques with NaCl or NH4HCO3 along with PCL and the third was a polymer blend leaching technique with poly(ethylene glycol) and PCL.

The studies showed that the scaffolds prepared by either salt leaching technique yield higher porosities compared to the scaffolds prepared using a PEG-PCL mixture, because the polymer mixture allows lower weight ratio of porogen to polymer and because the polymer mixtures greater ability to pack tightly. The highest degree of porosity is seen for the scaffolds made using ammonium hydrogen carbonate as a porogen. The mechanical strength varies according to the porosity, where a higher degree of porosity gives weaker scaffolds.

References

[1] Mathisen, T.; Masus, K.; Albertsson, A.-C. Macromolecules 1989, 22, 3842

[2] Finne, A.; Albertsson, A.-C. Biomacromolecules 2002, 3, 684.

[3] Finne, A.; Albertsson, A.-C. J Polym Sci Part A: PolymChem2003, 41, 1296.

[4] Finne, A.; Andronova, N.; Albertsson, A.-C. J Biomacromolecules 2003, 4, 1451.

[5] (a) Stridsberg, K.; Albertsson, A.-C. A.-C. J Polym Sci Part A: Polym Chem 1999, 38, 1774. (b) Andronova, N.; Finne, A.; Albertsson, A.-C. J Polym Sci Part A: Polym Chem 2003, 41, 2412.

[6] Bornstein, J.; La Liberte, B.R.; Andrews, T. M.; Montermoso, J. C. J Polym Sci Part A: Polym Chem 1959, 24, 886-87.


PC49

SUSTAINED DRUG DELIVERY FROM POLYLACTIDE-COATED METAL CORONARY STENTS

M. LAPČÍKOVÁ,a M. ŠLOUF,a J. ZABLOCKI,b F. RYPÁČEKa

a Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, CZ-162 06 Praha 6, Czech Republic

b CV Therapeutics, Inc., Palo Alto, 94304 CA, USA

e-mail: lapcik (imc.cas.cz )

The cardiovascular diseases belong to most serious health risks. The coronary angioplasty with implantation of stents is one of the procedures used to solve the problem with contraction or closure of the coronary arteries. The complications related to the application of stents involve stimulation of intima smooth muscle cells, the excessive proliferation of which may cause thickening of the blood vessel wall and, eventually, in-stent restenosis. Controlled release of active agents, affecting the cell proliferation inside the artery, from polymeric matrices covering the metal stent is considered to be a promising way to overcome this problem. Polylactide-based materials (PLA) are widely used as prosthetic biomaterials as well as sustained-delivery matrixes. We investigated the feasibility of using very thin PLA coatings on stainless steel stents to provide a long-term sustained release of agents controlling cell-proliferation, such as CDK2 inhibitors.

Well-defined release systems were prepared by coating of stainless-steel stents, (55 mm2), by a PLA film with a defined thickness and composition. PLA films were prepared in two steps: first an anchoring PLA layer was prepared by the in situ grafting polymerization of lactide on the activated metal surface; then a well defined film of PLA-drug composition was cast using spraying method. The drug was incorporated into polymer films from a polymer-drug solution. Poly(L-lactide) and poly(D,L-lactide) were used in one or multiple layers with different drug loadings. The release kinetics of the agents from coated stents in phosphate buffered saline of pH 7.2 was followed in stirred cells at 37 °C. The amount of released drugs was analyzed by HPLC.

The spray coating of stents gave good reproducibility of the films. Stable polylactide coatings on metal surfaces were made with a capacity of long-term release for the drug under study. The stabilizing role of the grafting layer was demonstrated by SEM. The PLA films with a thickness ranging from 0.5 to 1.5um were capable of controlling the release of CDK2 inhibitor for up to 60 days in a quasi-linear manner.

Acknowledgment:The research was supported by CV Therapeutics, Inc., Palo Alto, CA, USA.


PC50

DIFFERENT EFFECT OF CVT-313, A CDK2 INHIBITOR, ON VASCULAR SMOOTH MUSCLE CELLS FROM ADULT AND NEWBORN RATS

E. FILOVÁa, V. BRYJAb, L. NOSKOVÁa, L. BAČÁKOVÁa, M. LAPČÍKOVÁc, F. RYPÁČEKc

aInstitute of Physiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-142 20 Praha 4, Czech Republic

bInstitute of Experimental Medicine, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-142 20 Praha 4, Czech Republic

cInstitute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, CZ-162 06 Praha 6, Czech Republic

CVT-313 is a synthetic inhibitor of cyclin-dependent kinase 2 (CDK2) which blocks the transition from G1 to S phase of the cell cycle (1). Incorporation of CVT-313 into vascular prostheses or stents is expected to prevent the proliferation of VSMC and restenosis of the vascular lumen. After two-day cultivation in media with CVT-313, the population density of rat aortic VSMC was lower than in cells not exposed to the inhibitor. This growth inhibition was more pronounced in VSMC derived from newborn than adult rats (IC50 = 410 and 4000 ng/ml, respectively). Similar tendency was found in bromodeoxyuridine labelling index, a marker of DNA synthesis. The activity of CDK2, measured in cells exposed to CVT-313 by phosphorylation of histone H1 using [32P]ATP, decreased in newborn rat VSMC, whereas in adult rat VSMC did not change significantly (passage 5) or even increased (passage 25). As revealed by Western blotting, the contents of p21 and p27, i.e. natural inhibitors of CDK2, were higher in cells from newborn rats. After CVT-313 treatment, the content of p21 increased only in VSMC from newborn rats, whereas the content of p27 only in cells from adult rats. However, immunoprecipitation showed that after CVT-313 treatment, the amount of p27 associated with CDK2 increased only in newborn VSMC. These results indicate different sensitivity of VSMC from newborn and adult rats to CVT-313, which may be due to age-dependent differences in regulation of VSMC proliferation.

1. Brooks, E.E., Gray N.S., Joly A., Kerwar S., Lum R., Mackman R.L., Norman T.C., Rosete J., Rowe M., Schow S.R., Schultz P.G., Wang X., Wick M.M., Shiffman: J. Biol. Chem. 272 (46), 29207, 1997


PC51

ORIENTED ELECTROSPUN FIBRES FOR NEURAL TISSUE ENGINEERING

P. DALTONa, D. GRAFAHRENDb, D: KLEEa,b, M. MÖLLERa,b

aDepartmentof Textile and Macromolecular Chemistry, RWTH-Aachen, Veltmanplatz 8, Aachen 52062, Germany

bDeutschesWollforschungsinstitut, RWTH-Aachen, Veltmanplatz 8, Aachen 52062, Germany

Functionalized electrospun fibres are of particular interest in neural tissue engineering due to diameters of 200 nm to 2 microns, and centimetre lengths. Electrospun fibres also have a high surface area to mass ratio; potentially providing a significant surface-mediated signal for therapeutic cells while minimizing the quantity of degradation products. The aim of the research is to orient, functionalize and determine their efficacy as a scaffold for neural tissue engineering.

Electrospun fibres, formed from applying high voltages to polymer solutions, can be suspended in a highly oriented manner, when dual collection rings are used. Fibres formed after 60 seconds at 15 kV were determined to be single and bridge across the electrode, with lengths of up to 8 cm, and very low rate of fibre splitting; with an average diameter of 1.26±0.19 mm (Figure 1). At 25 kV, fibres were significantly split, although there was still significant orientation. A range of polymers have been collected in this manner, including poly(e-caprolactone) (PCL), P(D,L-Lactide)(PDLA), polyethylene oxide (PEO) and di-block copolymers (PEO-b-PDLA).

Fig. 1

It is proposed to introduce biomimetic functionalities using a self-organising molecular process, where tailor-made block copolymers are used as 'additives' to the solution that is to be electrospun. Preliminary in vitro investigations indicate that axons are guided by electrospun fibres with a diameter of 1.26±0.19 mm.


PC52

MICROPATTERNED SURFACES AS USEFUL TOOLS FOR CELL GUIDANCE

A. MAGNANI, D. PASQUI, F.M. PIRAS, A. CHIUMENTO, S. LAMPONI, R. BARBUCCI

Dept. of Chemical and Biosystem Sciences and Technologies, and Polo Universitario Colle di Val d'Elsa

Micropatterned surfaces containing geometrically defined bioadhesive and non-adhesive domains are useful tools to study cell attachment and growth for the creation of highly orientated cell pattern that mimic a functional tissue. An open question is whether the cell behaviour is influenced by the surface chemistry or topography or both. The aim of the work is to find a correlation between the chemistry and/or topography of the different domains and the cell response. Micropatterned surfaces with different chemical and topographical heterogeneity have been realised by photoimmobilisation of polysaccharides and polysaccharide-copper(II) ion complexes, or "grid-assisted" deposition of self-assembling molecules. The micropatterned surfaces have been characterised by Atomic Force Microscopy (AFM) and Time of Flight-Secondary Ion Mass Spectrometry (ToF-SIMS) which provided us with useful information on the chemistry and topography of the different domains.

The influence of chemical and topographical patterning on fibroblasts and endothelial cells behaviour have been evaluated in terms of cell adhesion, proliferation, migration and orientation as well as of cell functionality.The presence of integrin clusters along the cell borders and at the migrating leading edge together with actin stress fibres polarised along domain directions indicated that the cell adhesion is integrin-mediated and the cell migration and orientation guided by the micropatterns. The cell proliferation and functionality seem to be controlled mainly by the chemistry of the micropatterns. Different types and amounts of serum proteins have been detected on the different patterns by 2D-electroforesis. The different proteins that interact with the substrates play a key role in modulating the cell behaviour and may explain why some domains are cell adhesive, whereas others are cell repulsive.

This work demonstrates that micropatterned surface are suitable substrates for cell guidance, since they are able to spatially direct the cell growth. The control of surface protein interactions is a key element to drive all subsequent events leading to cell biomaterial response.


PC53

THYXOTROPHIC POLYSACCHARIDE BASED HYDROGELS FOR MEDICAL APPLICATIONS

G. LEONE, A. MAGNANI, S. LAMPONI, R. BARBUCCI,

Department of Chemical and Biosystem Science and Technologies and CRISMA,University of Siena, Via Aldo Moro 2, 53100 Siena, Italy

Some hydrogels show thixotropic properties, i.e. they become liquid upon being shaken (see ketchup) or agitated (see quicksand) and coagulate again when left in an undisturbed condition.

Polysaccharide hydrogels synthesised by cross-linking the hyaluronane, alginate or carboxymethylcellulose chains with an alkylic diamine, present this property. The similarity of both mechanical moduli (G' and G") before and after the mechanical up setting, in physiological solution, indicates the hydrogel resumes the original consistence and can be injected through the needle of a syringe. This phenomenon permits to utilise hyaluronan based hydrogels in the osteoarthritis treatment by injecting into the knee of rabbits for in vivo tests. The alterated cartilage resumed a structure very similar to the native one after a 50 days treatment.The hyaluronan hydrogel was also charged by ibuprophen-lysine, an anti-inflammatory drug. The release kinetics was studied in vitro and the effectiveness of the system in the osteoarthritis therapy was evaluated in-vivo. Furthermore, the hydrogels were modified to obtain a microporous structure The technique consisted in stratifying the cross-linked polysaccharide gel on a filter with pores of known diameter and forcing CO2 bubbles through the filter.

The pore diameter and density, and the wall thickness were analysed by SEM. The microporous hydrogels showed a lower water uptake and increased the mechanical properties. The influence of the loading technique on the release kinetics was also evaluated. In fact, loading the native hydrogel with the drug, then forming the micropores, the release occurred in 7 days. On the contrary, forming the pores in the hydrogel, then loading with the drug, ibuprophen-lysine was released in 24 hours. The microporous hydrogels, after the same mechanical up setting in a syringe, maintained the morphology even if the pore sizes were reduced.


PC54

CONTROL OF CHONDROCYTE ATTACHMENT TO A PHYSICALLY MODIFIED 3D POLYMER SCAFFOLD

E.A. PEARSON. C.J. ROBERTS, K.M. SHAKESHEFF

School of Pharmacy, The University of Nottingham, University Park, Nottingham, NG7 2RD, United Kingdom

Biodegradable poly(a-hydroxy acid) polymers have been widely investigated as candidates for tissue engineering biomaterials. Poly(DL-lactic acid) (PDLLA) is of particular interest due to its biocompatibility and suitability for porous scaffold formation. It is recognised however that the affinity for cell attachment to PLA is relatively low. We aim, through physical modification of PDLLA to modulate and improve specific cellular interactions. These scaffolds have also been engineered to simultaneously block non-specific cell interactions while promoting specific integrin interactions.

PDLLA porous 3D scaffolds were fabricated by a salt leaching process1. The modification of a PLA continuous phase with poly(ethylene glycol) (PEG) and an RGD peptide sequence was achieved using a method of physical entrapment2. Quantitative analysis of cellular adhesion, viability and morphology of ovine meniscal chondrocytes (OMCs) on the scaffolds was carried out using Hoescht DNA, Alamar blueTM assays and SEM respectively.

State-of-the-art surface analysis techniques were utilised to assess the spatial distribution of the surface modifying species within the porous scaffold.

   

Modification with a second polymeric component (PEG) was shown to be successful throughout the construct. A significant difference in both cellular viability and morphology was observed between the modified and unmodified scaffolds.

We have demonstrated a simple and rapid method of physically engineering a 3D polymer scaffold. This method has been employed to modify the polymer surface thus producing a differential cellular response in terms of viability, adhesion and morphology.

REFERENCES:

1Mikos, A. G., Thorsen, A. J., Czerwonka, L. A., Bao, Y., Langer, R., Winslow, D. N. and Vacanti, J. P. Preparation and characterization of poly(L-lactic acid) foams. Polymer, 35, 1068-1077 (1994).

2Quirk, R. A., Davies, M. C., Tendler, S. J. B., Chan, W. C., Shakesheff, K. M. Controlling biological interactions with Poly(lactic acid) by surface entrapment modification. Langmuir, 17, 2817-2820 (2001).


PC55

BIODEGRADABLE POLYMER SCAFFOLDS FOR NERVE TISSUE REGENERATION

H. DRNOVSKÁa, b, K. MULINKOVÁa, R. KOTVAa, M. ŠLOUFa, F. RYPÁČEKa, b

aInstitute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, CZ-162 06 Praha 6, Czech Republic

bCenter for Cell Therapy and Tissue Repair, 2nd Medical Faculty, Charles University, V úvalu 84, CZ-150 06 Praha 5, Czech Republic

Polymer biomaterials play a significant role as scaffolds supporting the implanted cells and functioning as a temporary substitute of extracellular matrix in tissue regeneration. Recent progress in stem cells research makes the development of specific functional biomaterials even more topical.Synthetic poly(α-amino acid)s (polyAA), due to their polypeptide backbone, have the potential to be degraded in biological environments by enzyme-catalyzed hydrolysis. The enzyme specificity can be controlled by selecting suitable copolypeptide composition.

Biodegradable hydrogels based on polyAA have been investigated as scaffolds for soft tissue implants, namely as scaffolds for neural tissue regeneration. This contribution focuses on the development of techniques for formation of a superporous structure in a highly swollen hydrogel based on methacrylate-poly(AA) hybrid matrix. Two types of porosity were sought: (a) large superpores facilitating cell seeding and penetration through the matrix (b) secondary pores, which can enabling the diffusion of nutrients and growth factors.

Crosslinkedhydrogels were prepared by radical copolymerization of methacrylated copolypeptides with 2-hydroxyethyl methacrylate (HEMA) using 2,2´-azobisisobutyronitrile (AIBN) as initiator. Methacrylated copolypeptide poly[N-(hydroxyethyl) glutamine-co-lysine-co-alanine] was synthetized by polymerization of respective N-carboxyanhydrides and subsequent side-chain modification. Using a porogen-leaching method with a solid organic porogen, the macroporosity was formed in gels. The porosity and pore architecture of aqueous-swollen gels was followed by a high-pressure (low-vacuum) scanning-electron microscopy.

Acknowledgements: Support of the Grant agency of the Czech Republic (grant No. 203/04/P124) and Research Centers Program of the Ministry of Education of the Czech Republic (grant No.LN00A065) is acknowledged.


PC56

POlYLACTIDE SURFACES WITH domain structures

D. KUBIES a,b, S. POPELKA a,b, V. PROKS a,b, E. FILOVÁc, L. BAČÁKOVÁ c, L. MACHOVÁa , J. HROMÁDKOVÁa, F. RYPÁČEK a

aInstitute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq.2, 162 06 Prague, Czech Republic

bCenter for Cell Therapy and Tissue Repair, 2nd Faculty of Medicine, Charles University, V Uvalu 84, 150 00 Prague, Czech Republic

cInstitute of Physiology, Academy of Sciences of the Czech Republic, Vídeňská1084, Prague, Czech Republic

Polymer scaffolds designed for application in cell therapy and tissue engineering should reflect requirements for tuned mechanical properties and have suitably designed three-dimensional architecture. Moreover, demands on the specific interaction between biomaterial and living cells call for exposition of specific structures on the polymer surfaces providing solid-state signals to the cells in order to modify adhesion, proliferation and differentiation of cells on polymer substrates.

In present work, a polyester-based scaffold was chosen as a support material for cell seeding. Surface properties of the polylactide (PLA) matrix were modified by amphiphilic block copolymers composed of hydrophobic polyester and hydrophilic poly(ethylene oxide) (PEO) blocks. The hydrophilic blocks allowed introduction of functional groups, either nonspecific or cell-specific, on the polymer surface while the polyester blocks served to stabilize the copolymer layer on the polymer support.

Amphiphilic and end-functionalized PLA-b-PEO copolymers were synthesized by controlled polymerization of lactide using the semitelechelic PEO as co-initiators. Solid-phase techniques based on building a protected peptide on a polymer support and using functional copolymers as final reactants were applied to the synthesis of well-defined copolymers capped with selected peptide sequences.

The spin casting from the copolymer solution was used to prepare polymer surfaces with exposed functional structures. The effect of the molecular parameters of copolymers on the surface properties, such as surface wettability, stability and morphology of the deposited layers was studied. Domain structure of copolymer films composed of PDLLA-b-PEO(MeO) was investigated by electron microscopy.

Acknowledgment: Support from the Grant Agency of the Academy of Sciences of CR (A4050202), Grant Agency of CR (grant No. 304/02/0579) and the Ministry of Education of CR (grant No. LN00A065) is acknowledged.


PC57

RGD-bearing POLYMERS FOR VASCULAR TISSUE ENGINEERING

L. Bačákováa, E. Filováa, D. Kubiesb, L. Machováb, V. Proksb, F. Rypáčekb

aInstitute of Physiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-142 20 Praha 4, Czech Republic

bInstitute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, CZ-162 06 Praha 6, Czech Republic

Adhesion and proliferation of rat aortic smooth muscle cells (RASMC) were studied in cultures on glass coverslips coated with thin polymeric films consisting of a bottom layer of poly(L-lactic acid), PLLA, and a top layer of poly(DL-lactic acid), PDLLA, mixed with a copolymer of PDLLA and poly (ethylene oxide), PEO. The content of PEO in the surface layer was 33%. A fraction of PEO chains (5% or 20%) was functionalized with integrin-binding Arg-Gly-Asp (RGD) motif in a form of oligopeptide Gly-Arg-Gly-Asp-Ser-Gly (GRGDSG). In serum-supplemented media, the number of VSMC initially attached to PLLA or PDLLA (6 and 24 hours after seeding), size of cell spreading area, formation of vinculin- and talin- containing focal adhesion sites, as well as subsequent proliferation (estimated by bromodeoxyuridine labeling index and growth curves), were similar as on standard cell culture supports (e.g., polystyrene or glass). Non-functionalized PEO chains rendered the surface non-adhesive for cells, whereas the presence of GRGDSG restored the cell adhesion and growth almost to the values found on PLLA or PDLLA (Fig. 1). Similar results were obtained in serum-free media, although the adhesion and growth of cells were generally lower. Nevertheless, these finding indicate that the GRGDSG sequence on the material is bound by adhesion receptors on RASMC, and this artificial "extracellular matrix analogue" could be used for vascular tissue repair and regeneration.

A B C

Fig. 1: RASMC on PDLLA (A), PDLLA-PEO (B) and PDLLA-PEO-GRGDSG (C). Medium DMEM with 10% serum, 24 hours after seeding.


PC58

POLYLACTIDE FIBROUS SCAFFOLDS FOR cartilage IMPLANT engineering

K. MULINKOVÁa, L. MACHOVÁa, P. LESNÝb, D. KUBIESa,F. RYPÁČEKa

a Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám.2, CZ-162 06 Praha 6, Czech Republic;

b2nd Faculty of Medicine, Charles University, V úvalu 84, 150 06 Praha 6, Czech Republic

E‑mail: mulinkova (imc.cas.cz )

Biodegradable polymers are widely studied as scaffolds fortissue engineering. In addition to biodegradability and biocompatibility, the scaffolds used in should have highly porous structure with interconnected pores and suitable surface chemistry to stimulate cell attachment, proliferation and differentiation.

In this work, we used poly(L-lactic acid) (PLLA) to fabricate highly porous three-dimensional (3D) interconnected fibrous network that could be used as a biodegradable scaffold for preparing an autologous cartilage graft of tympanic membrane for myringoplasty. PLLA fibres were prepared by solution spinning from a PLLA solution in dichloromethane. Fibres with various diameters ranging from 60 - 350 mm were prepared by varying the spinning conditions. Scaffolds with the interconnected fibre 3D structure were made by compression the PLLA fibres in a mould. The fibre bonding was achieved by applying solvent etching or gluing with a PDLLA solution. The morphology of fibres and internal geometry of scaffolds were examined with scanning electron microscopy (SEM). By using fibres with different diameters and variable moulding conditions, the method makes it possible to prepare a range of scaffolds, differing in mechanical properties, volume fraction of pores, and permeability for fluids. The suitability of scaffolds for cartilage engineering in flow-through reactor was tested by seeding and cultivation of isolated human chondrocytes.

Acknowledgment:Support from Grant Agency of the Czech Republic (304/02/0759) is acknowledged


PC59

DISTRIBUTION OF BIOMIMETIC GROUPS ON POLYMER SURFACES MODIFIED BY FUNCTIONAL AMPHIPHILIC BLOCK COPOLYMERS

E. CHÁNOVÁ, Š. POPELKA, L. MACHOVÁ, F. RYPÁČEK

Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, CZ-162 06 Praha 6, Czech Republic,

e-mail: chanova (imc.cas.cz )

Biodegradable polyesters, such as polylactide (PLA), are important biomaterials in the area of tissue engineering. Besides other properties, a specific distribution (pattern) of cell-adhesion structures, e.g. fibronectine-derived peptide sequences (RGDS), over the biomaterial surface is significant factor in controlling cell behaviour. Amphiphilic block copolymers of polylactide (PLA) and poly(ethylene oxide) (PEO) containing cell-adhesion peptide sequences at the end of PEO block are used in our laboratory for preparation of functional biomimetic surfaces of PLA-based biomaterials, that would facilitate cell migration, growth and differentiation.

As a model system for studies of surface topography of adhesion groups that may result from surface deposition of amphiphilic block copolymers and/or their self-organization at the biomaterial/water interface, we use analogous block copolymers with biotin as an adhesion peptide surrogate. Di-block copolymers composed of poly(DL-lactide), PDLLA, and poly(ethylene oxide), PEO, with biotin at the end of PEO block (PDLLA-b-PEO-biotin) with different PEO length were prepared by ring open polymerization of DL-lactide in toluene using α-biotinyl-ω-hydroxy-PEO as a macroinitiator and tin(II) 2-ethylhexanoate as a catalyst. The copolymers were characterized by 1H NMR spectroscopy and GPC analysis. The terminal-biotin group contents were calculated from 1H NMR spectra. PDLLA-b-PEO-biotin copolymers were spin-coated from mixtures with different ratio of non-/biotinylatedcopolymers on a support plate made by a spin-coated PLLA film on a silanized mica. Taking advantage of specific biotin-avidin interactions the surface distribution of biotin groups was observed by visualization of surface-bound avidin (MW 66 KDa) using atomic-force microscopy (AFM) in tapping mode.

Acknowledgment: Support by Grant Agency of the Academy of Sciences of the Czech Republic (grant No.: A4050202) is acknowledged.


PC60

ENGINEERING BONE USING POLY-DL-LACTIDE 96/4 SHEETS

A-L. VESALAa, M. KALLIOINENb, P. TÖRMÄLÄc, M. KELLOMÄKIc, T. WARISa, N. ASHAMMAKHIa,c

aDepartment of Surgery, Oulu University Hospital, P.O.Box 5000, FIN-90014, Oulu, Finland, e-mail: anna-liisa.vesala (ppshp.fi)

bDepartment of Pathology, Oulu University Hospital, Oulu, Finland

cInstitute of Biomaterials, Tampere University of Technology, P.O. Box 589, FIN-33101, Tampere, Finland, e-mail: nureddin.ashammakhi (tut.fi)

The aim was to evaluate the use of self-reinforced poly-DL-lactide 96/4 (SR-PLA96) sheets for cranial bone tissue engineering in experimental defects in rabbits. Square defects of 10 x 10 mm were created in the right parietal bone. SR-PLA96 implants (15x15 mm) were used to cover these defects in 12 New Zealand White rabbits. Similar defects were created in the left parietal bone, but no sheets were used (controls). The rabbits were killed after 6, 24, or 48 weeks. Histology and histomorphometry were used to evaluate healing of the defects. Defects covered with SR-PLA96 sheets showed more abundant bone formation than control (non-covered) defects. At 6 weeks, the defects were occupied mainly by fibrous tissue. At 24 weeks, healing with bone formation was more obvious in the covered defects. At 48 weeks, bone completely bridged defects covered with SR-PLA96 sheets, and incomplete bridging was seen in non-covered control defects. Hence, bone tissue engineering in experimental cranial bone defects in rabbits can be achieved using SR-PLA96 sheets to guide bone regeneration.

Keywords:Bioabsorbable; guided bone regeneration; polylactide; tissue engineering


PC61

Study of the Effect of Human Platelet Supernatant on Proliferation and Matrix Synthesis of Human Articular Chondrocytes in Monolayer (2D) and Three-Dimensional (3D) Alginate Cultures

C. GAISSMAIERa,b, J. FRITZc, T. KRACKHARDTa, I. FLESCHa, W.K. AICHERc, N. ASHAMMAKHId,e

aBG Trauma Centre, Eberhard-Karls University, Tuebingen, Germany

bNatural and Medical Science Institute, Reutlingen, Germany

cDepartment of Orthopaedics, Eberhard-Karls University, Tuebingen, Germany

dInstitute of Biomaterials, Tampere University of Technology, P.O. Box 589, FIN-33101 Tampere, Finland, e-mail: nureddin.ashammakhi (tut.fi)

eDepartment of Surgery, Oulu University Hospital, Oulu, Finland

Articular cartilage is rich in collagen type II fibres and proteoglycans and is characterized by low cell density. Chondrocytes have specific nutritional requirements and therefore cannot be expanded in vitro without the risk of generating fibroblastoid cells expressing type I collagen. Therefore, various growth conditions were tested for cartilage tissue engineering. Human articular chondrocytes were expanded in monolayer cultures and seeded in alginate beads in the presence and absence of human platelet supernatant (hPS). Transcript levels of genes encoding chondrogenic factors were determined by quantitative RT-PCR. The deposition of type I and II collagen as well as proteoglycan was detected by indirect immunohistochemistry. Addition of hPS activated chondrocyte proliferation in monolayer cultures but induced rapid dedifferentiation of chondrocytes towards a fibroblast-like phenotype. The expression levels of mRNAs encoding type II collagen, aggrecan and bone morphogenetic protein-2 were reduced in all samples tested. Seeding chondrocytes in alginate beads in the presence of hPS generated a cell population capable of type II collagen expression, even though hPS induced considerable type I collagen expression as well. We conclude that addition of hPS may accelerate chondrocyte expansion but chondrocytes maintained a chondrocytic phenotype only in the absence of hPS.

Keywords: Articular cartilage, chondrocytes, platelet supernatant, tissue engineering.


PC62

Induced Indentation using bioabsorbable poly-DL-lactide in experimental rabbit model persists for sufficient time

S. LÄNSMANa, A. KARTTUNENb, H. HIRVELÄa, T. PALOSAARIa, M. KELLOMÄKIc, V.ELLÄc, P. OHTONENd, P. TÖRMÄLÄ, Tc. WARISd, N. ASHAMMAKHIc,d

aDepartment of Ophthalmology, Oulu University Hospital, P.O. Box 5000, FIN-90401, Oulu, Finland, e-mail: satu.lansman (oulu.fi)

bDepartment of Radiology, Oulu University Hospital, Oulu, Finland

cInstitute of Biomaterials, Tampere University of Technology, P.O. Box 589, FIN-33101, Tampere, Finland, e-mail: nureddin.ashammakhi (tut.fi)

dDepartment of Surgery, Oulu University Hospital, Oulu, Finland

Purpose:To measure the amount and duration of indentation depth achieved with biodegradable poly-DL-lactide 96/4 (PLA96) and silicone sponge implants.

Methods: Thirty rabbits underwent a scleral buckling procedure. A PLA96 buckling implant was used in 15 and a silicone sponge buckling implant in 15 rabbits. A circumferential scleral buckling implant was sutured episclerally on the left eye of each rabbit, just temporal to the superior rectus muscle and 7 mm posterior to the limbus. CT scanning was performed at one week, and three and five months postoperatively.

Results:The PLA96 buckling implant (implant diameter 3-3.5 mm) used in this study created lower indentation than the silicone sponge implant (implant diameter 4 mm), possibly as a result of the surgical technique. The indentation created by the PLA96 implant decreased over time compared with the silicone implant. There were no complications related to either kind of implant.

Conclusion: Both silicone sponge implants and PLA96 implants caused indentation that decreased in a comparable manner over the follow-up period (5 months).

Keywords: Bioabsorbable, experimental, polylactide, retinal detachment, scleral buckling.

Brief summary statement: An experimental study: bioabsorbable implants made of poly-(L/D)-lactide 96/4 were sutured episclerally in 15 rabbits; in the control group silicone sponge implants were used. The indentation achieved decreased in both groups in a comparable manner over the follow-up period of 5 months. There were no complications related to either kind of material.


PC63

In vivo study of Poly-DL-lactide (PLDLA) 96/4 Scaffolds in Rats

S. LÄNSMANa, M. KELLOMÄKIb, P. TÖRMÄLÄb, T. WARISc, N. ASHAMMAKHIb,c

aDepartment of Ophthalmology, Oulu University Hospital, P.O. Box 5000, FIN-90401, Oulu, Finland, e-mail: satu.lansman (oulu.fi)

bInstitute of Biomaterials, Tampere University of Technology, P.O. Box 589, FIN-33101, Tampere, Finland, e-mail: nureddin.ashammakhi (tut.fi)

cDivision of Plastic Surgery, Department of Surgery, Oulu University Hospital, P.O.Box 5000, FIN-90401, Oulu, Finland

Background: Various materials for scleral buckling have been used over the years. Non-absorbable materialsare commonly used, such ashydrogel and silicone, either solid rubber or silicone sponge.Now also bioabsorbable synthetic materials are available for scleral buckling in cases when only temporary buckling effect is needed.

Aims:To study the poly-DL-lactide (PLDLA) 96/4 (96/4, molar ratio of DL‑lactide) scaffolds in vivo in the subcutaneous tissue of rats.

Material and methods: Cylindrical knitted mesh scaffolds were made of PLDLA 96/4 fibers, with each fiber made of 8 PLDLA filaments. All implants were sized 15 x 3.5 mm. Three types were evaluated: Dense (weight 30 g), ordinary (25 g) and loose (20 g). Four scaffolds (2 ordinary, one dense and one loose type) were implanted in the dorsal subcutis of each of 32 Sprague-Dawley male rats. The implants were retrieved after 3 days, 1, 2, 3, 6, 12, 24 and 52 weeks postoperatively and they were removed with 5 mm of surrounding tissues. One ordinary scaffold was examined for characterisation of mechanical properties. The rest were examined histologically for tissue reaction and ingrowth.

Results: No postoperative occurred. Tissue ingrowth reached the innermost part of the implants within three weeks. Fibrin was the first to fill in the scaffold followed by the cells and at last collagen fibers were found in the structure. The orientation of the collagen fibers inside the implant changed from non-oriented to highly oriented fibers making septae. The amount of macrophages increased over the follow-up period of 52 weeks, which ismost probably due to increasing implant material degradation. The material was not fragmented at 52 weeks.

Conclusions:Upon implantation in rats, fibrous tissue ingrowth proceeds from all sides of the scaffold filling it completely at 3 weeks. The collagen fibers get more organized by time. Single fibers of the material were not fragmented at 52 weeks.


PC64

MULTIFUNCTIONAL CIPROFLOXACIN-RELEASING BIOABSORBABLE MATERIALS ARE SUPERIOR TO TITANIUM IN PREVENTING STAPHYLOCOCCUS EPIDERMIS ATTACHMENT AND BIOFILM FORMATION IN VITRO

S-M. NIEMELÄa, I. IKÄHEIMOb, M. KOSKELAb, M. VEIRANTOc, E. SUOKASd, P. TÖRMÄLÄc, T. WARISa, N. ASHAMMAKHIa,c, H. SYRJÄLÄe

aDepartment of Surgery, Oulu University Hospital, P.O. Box 5000, FIN-90401 Oulu, Finland, e-mail: saniemel (mail.student.oulu.fi)

bClinical Microbiology Laboratory of Oulu University Hospital, Oulu, Finland

cInstitute of Biomaterials, Tampere University of Technology, P.O. Box 589, FIN-33101, Tampere, Finland, e-mail: nureddin.ashammakhi (tut.fi)

dLinvatec Biomaterials Ltd., Tampere, Finland

eDepartment of Infection Control, Oulu University Hospital, P.O. Box 21, FIN-90029, Oulu, Finland, e-mail: hannu.syrjala (ppshp.fi)

Background: Antibiotic coating systems have been successfully used to prevent bacterial attachment and biofilm formation. Our purpose was to evaluate whether bioabsorbable polylactide-co-glycolide (PLGA) 80/20 on its own, and PLGA together with ciprofloxacin (PLGA+AB) have any advantages over titanium in preventing Staphylococcus epidermidis attachment and biofilm formation in vitro.

Materials and methods: Cylindrical specimens of titanium, PLGA and PLGA+AB in triplicate were examined for S. epidermidis ATCC 35989 attachment and biofilm formation after incubation with a bacterial suspension of ca. 105 cfu/ml for 1, 3, 7, 14 and 21 days, using scanning electron microscopy. Growth inhibition properties of PLGA and PLGA + AB cylinders were tested on agar plates.

Results: On days 1, 3 and 21, no bacterial attachment was seen in 19.5%, 9.2% and 41.4% of the titanium specimens, in 18.4%, 28.7% and 34.5% of the PLGA specimens and in 57.5%, 62.1% and 57.5% of the PLGA + AB specimens, respectively. During the whole study period no biofilm was observed on 74%-93% of the titanium specimens, 58%-78% of the PLGA specimens and 93%-100% of the PLGA+AB specimens. PLGA + AB showed clear bacterial growth inhibition on agar plates while PLGA and titanium did not show any inhibition.

Conclusions: PLGA + AB bioabsorbable material was superior to titanium in preventing bacterial attachment and biofilm formation and may have clinical applicability, for example, in prevention of infection in trauma surgery or in the treatment of chronic osteomyelitis.

Keywords: antibiotic, bioabsorbable, biofilm, ciprofloxacin, polylactide-co-glycolide


PC65

MACROPOROUS HYDROGELS BASED ON COPOLYMERS OF 2-HYDROXYETHYL METHACRYLATE WITH POSITIVELY AND/OR NEGATIVELY CHARGED COMONOMERS FOR PROTEIN IMMOBILIZATION

M. PŘÁDNÝ1,2, A. ARTYUKHOV1,3, J. MICHÁLEK1,2, E. SYKOVÁ2, J. VACÍK1,2

1Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, 162 06 Prague 6, Czech Republic

2Center for Cell Therapy And Tissue Repair, Charles University, V Úvalu 84, 150 06 Prague 5, Czech Republic

3D.I. Mendeleev University of Chemical Technology of Russia, Miusskaya Sq. 9, 125047, Moscow, Russia

E-mail: pradny (imc.cas.cz )

Crosslinkedmacroporous hydrogels based on copolymers of 2-hydroxyethyl methacrylate (HEMA) with charged comonomers were prepared and characterized by the porosity, pore size and water content in pores and walls. Morphology of hydrogels was studied by scanning electron microscopy. Four series of hydrogels were used: Copolymers of HEMA with sodium methacrylate (MANa) (Series 1) and 2-(methacryloyloxyethyl)trimethylammonium chloride (MOETACl) (Series 2), terpolymer of HEMA with MANa and MOETACl (Series 3) and polyelectrolyte complex obtained by the reaction of the crosslinked Series 1 hydrogel with linear poly(MOETACl) (Series 4).

All the hydrogels were prepared by polymerization of monomers under pressure in the presence of fractionated sodium chloride particles. Three fractions of NaCl with grain sizes were used: below 0.03 mm, 0.03-0.05 mm and 0.05-0.09 mm. The materials are subsequently tested from the viewpoint of biocompatibility and suitability for preparation of implants into the central neural system. First studies showed that some investigated materials are very promising for the envisaged purpose.

The immobilization of proteins (albumin and avidin was carried out in a physiological solution in the presence of 0.67 M phosphate buffers (pH 5-8). A sample of hydrogel was put into a solution with a known concentration of protein and maintained there for 12 h. Then the concentration of protein in the solution was determined by standard Breford´s spectroscopic method. The capacity of hydrogel carrier for protein immobilization was calculated. A major part of protein (95%) was immobilized during first 3-4 h and this time period does not depend on the structure of carrier. However, the amount of immobilized protein strongly depends on the composition of hydrogel carrier in order Series 2>>Series 3~Series 4>Series 1 for albumin and Series 1>>Series 3>Series4>Series 2 for avidin due to the low isoelectric point of albumin (pI 4.9) and high pI of avidin (10.5). Albumin interaction is highest with positively charged groups (Series 2) and avidin interaction is highest with negatively charged groups (Series 1). The amount of immobilized protein increases with inceasing amount of ionogenic comonomer in hydrogel. Protein immobilization by the hydrogel carrier is strong, reversible desorption does not proceed. As follows from almost identical morphologies of hydrogel without protein and hydrogel with immobilized protein, aggregation of protein does not occur and individual protein molecules are not observed on electron micrographs at given resolution.

The study was supported by the Grant Agency of the Academy of Sciences of the Czech Republic S4050005 and Ministry of Education, Youth and Sports (project LN-00A-065).


PC66

KERATINOCYTES CULTURED ON HYDROGELS FOR EXPERIMENTAL AND THERAPEUTICAL PURPOSES

J. MICHÁLEKa,b, B. DVOŘÁNKOVÁb,c, K. SMETANAa,b,d, M. PŘÁDNÝa,b, E. KRUMBHOLCOVÁa,b, J. VACÍKa,b ,

aInstitute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, CZ-162 06 Praha 6, Czech Republic

bCentre of Cell Therapy and Tissue Repair, 2nd Medical Faculty, Charles University, V Úvalu 84, CZ-150 00 Praha 5, Czech Republic

cBurn Centre, 3rd Medical Faculty, Charles University, Šrobárova 50, CZ-100 34 Praha 10, Czech Republic

dInstitute of Anatomy, 1st Medical Faculty, Charles University, U Nemocnice 3, CZ-120 00 Praha 2, Czech Republic

Cultivation of keratinocytes for the treatment of severe skin loses has practised for more than twenty years. Confluent epidermal grafts are transferred attached to textile to the patient's wound bed. However this procedure has got several disadvantages: - it takes a long time to prepare these confluent grafts; - the enzymatic detachment of grafts negatively influenced their viability; - after application, the grafts can be destroyed by infection or dried up. Therefor, we developed a new methodology of cultivation of keratinocytes on the surface of hydrogels. In this case a subconfluent growth of keratinocytes can be transferred to the patient and no enzymatic treatment of cells is necessary. After the up side down application of keratinocytes, hydrogel protects transplanted cells and make an optimal environment. The adhesion of cultured cells to a hydrogel support must be strong enough to enable cell proliferation, but not to strong to enable the colonization of a wound bed by transplanted cells.

Parallel we have introduced in vitro testing of cell migration from the surface of cultivation support and their ability to colonize a new surface (simulating the wound bed). This methodology brings a quick information of possibility to use any polymer as a support for keratinocyte transfer and excludes the necessity of an animal model.

Keratinocytes cultured on poly(2-hydroxyethyl methacrylate) were successfully used in clinical practice for the treatment of more than thirty patients with burn injury or trophic defects.

This study was supported by Ministry of Education, Youth and Sport of the Czech Republic, projects LN00A065 and 111100005 and by Grant S4050005, Czech Academy of Science.


PC67

ADHESION AND PROLIFERATION OF 3T3 CELLS ON PLASMA‑MODIFIED POLYMERS

P. ADÁMKOVÁa, K. KOLÁŘOVÁa, V. ŠVORČÍKa, B. DVOŘÁNKOVÁb,c E. KRUMBHOLCOVÁ b,d, J. MICHÁLEKb,d, T. FENCLOVÁd, M. PŘÁDNÝb,d

aDepartment of Solid State Engineering, Institute of Chemical Technology, Technická 5, CZ-166 28 Praha 6, Czech Republic

bCentre of Cell Therapy and Tissue Repair, 2nd Medical Faculty, Charles University, V Úvalu 84, CZ-150 00 Praha 5, Czech Republic

cBurnCentre, 3rd Medical Faculty, Charles University, Šrobárova 50, CZ-100 34 Praha 10, Czech Republic

dInstitute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, CZ-162 06 Praha 6, Czech Republic

The progress in transplantation medicine has led to ever growing demand for organs and tissue suitable for transplantation. In such a situation the importance of artificial prostheses increases. Besides of ceramics, glassy and metallic materials also polymer are used in tissue engineering. Their use, e.g. as a carrier in cultivation of skin cover cells, has been studied.

In this study polymeric films (polyethylene, polyethyleneterephtalate, polystyrene and polyhydroxyethylmethacrylate) were modified in Ar-plasma discharge. Physico-chemical properties of pristine and plasma discharge modified polymers were studied by ATR FTIR spectroscopy (chemical structure), goniometry (wettability) and SEM microscopy (surface morphology). The adhesion and proliferation of mouse fibroblasts line 3T3 in cultures on modified polymer substrate was investigated in vitro.

By interaction of polymers with plasma discharge occurs the ablation of material, the changes in surface morphology are evident, new chemical structures are produced (especially carbonyl and ester groups) and consequently growths a surface wettability of modified polymers. The plasma modification increases cell adhesion and proliferation especially by unporal polymers.

This work was supported by Ministry of Education, Youth and Sport of the CR, project No. 22310002 and by Grant Agency of the CR under the project No. 106-03-0514.


PC68

CELLS ADHESION ON THIN CARBON LAYERS

O. KUBOVÁa, B. DVOŘÁNKOVÁb,c, V. MACHOVIČd, V. ŠVORČÍKa

aDepartment of Solid State Engineering, Institute of Chemical Technology, Technická 5, CZ-166 28 Praha 6, Czech Republic

bCentre of Cell Therapy and Tissue Repair, 2nd Medical Faculty, Charles University, V Úvalu 84, CZ-150 00 Praha 5, Czech Republic

cBurn Centre, 3rd Medical Faculty, Charles University, Šrobárova 50, CZ-100 34 Praha 10, Czech Republic

dCentral Laboratories, Laboratory of Molecular Spectroscopy, Institute of Chemical Technology, Technická 5, CZ-166 28 Praha 6, Czech Republic

The use of artificial polymers in medicine as substitutes of bones, joints, eye lens, heart valves, arteries and skin, damaged by pathological or degenerative processes, is ever increasing. At the same time, further investigation of suitable materials is under way with aim to develop new ones with better biofunctionality and biocompatibility. Their use, e.g. as a carrier in cultivation of skin cover cells, has been studied.

In this work was studied the carbon deposition on polyethyleneterephtalate (PET). The carbon layers were prepared by RF magnetron sputtering (0-90 min). For characterization of these layers on polymer surface were used AFM microscopy(surface morphology), Raman spectroscopy(chemical structure) and goniometry(wettability). The adhesion of mouse fibroblasts line 3T3 in cultures on polymer substrate with thin carbon layers was investigated in vitro.

X-ray diffraction didn't showed crystal structure in the carbon layers. From Raman spectra of carbon layers on the polymer surface is evident very intensive peak at 1380 and 1558 cm-1 which is typical for amorphous carbon. AFM microscopy of carbon surface shows that the surface is very homogenous. Surface roughness increases with increasing time of the carbon deposition (the differences are between 0,6 and 2,1 nm).Carbon deposition has a positive effect on 3T3 cells adhesion; the greatest amount of adhered cells is on the PET, which has been sputtered 30 min.

This work was supported by Ministry of Education, Youth and Sport of the CR, project No. 22310002, by Grant Agency of the CR under the project No. 106-03-0514 and by Grant Agency of the AS CR under the project A 5011301.


PC69

synthesis OF biodegradabLE Aromatic-aliphatic copolyesters and poly(ester-amide)s

D. CHROMCOVÁ, J. VITÁSEK, L. BASLEROVÁ, J. TOLAR, J. Brožek, I. PROKOPOVÁ, J. Roda

Department of Polymers, Institute of Chemical Technology, Technická 5, 166 28 Praha 6, Czech Republic, e-mail: Jiri.Brozek (vscht.cz)

Nowadays considerable efforts are focused on the preparation of new biodegradable polymers. These materials could potentially find application as both commodity and specialty materials. Our research activity is focused on modification of two types of materials based on poly(e-caprolactam) and poly(ethylene-terephthalate), which are practically non-biodegradable due to high content of crystalline phase and rigidity of polymer chain, respectively. On the basis of known correlations between polymer structure and their ability to biodegradation it is possible to design new types of polymers based on industrially produced monomers, which can be biodegradable.

Thus by incorporation of ester units into polyamide chains, poly(ester-amide)s has been prepared with miscellaneous physico-chemical properties. Series of poly(ester-amide)s of different composition have been prepared by the anionic copolymerization of e-caprolactone with e-caprolactam (CL) initiated with Grignard compounds. e-Caprolactone was also used for synthesis of aromatic-aliphatic copolyesters from postconsumer PET bottles. A set of copolyesters containing 20-60 mol% aliphatic ester units was prepared by the reaction of e-caprolactone with glycolyzed PET followed by polycondensation. Due to increased flexibility of aromatic-aliphatic copolyester chains compared to the aromatic ones, the copolymers show enhanced sensitivity to biodegradation.

Thus prepared materials were characterized by the content of incorporated ester units in resulting copolymer and by means of thermal methods (TGA, DSC and DMA). Mechanical properties were also determined. Foils for biodegradation tests were prepared by melt pressing.

The biodegradability of prepared polymers was tested for hydrolysis in buffer solution (pH = 7) at 60°C, treatment with selected species of ligninolytic fungi and composting test.

The research was supported by grant No. 203/03/0508of the Grant Agency of the Czech Republic.


PC70

Novel Polymeric Micellar pH-Sensitive Drug Delivery System for anticancer antibiotic Doxorubicin

M. Hrubý*, Č. Koňák, K. Ulbrich

Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, CZ-162 06 Praha 6, Czech Republic

*corresponding author, email: mhruby (centrum.cz)

A novel polymeric micellar pH-sensitive system for the drug delivery of doxorubicin (DOX) is described. Polymeric micells were prepared by self-assembly of amphiphilic diblock copolymers in aqueous solutions. The copolymers consist of biocompatible hydrophilic poly(ethylene oxide) (PEO) block and hydrophobic block containing covalently bound anthracycline antibiotic DOX.

The starting block copolymers poly(ethylene oxide)-block-poly(allyl glycidyl ether) (PAGE-PEO) with very narrow molecular weight distribution (Mw/Mn ca 1.05) were prepared by anionic ring opening polymerization using sodium salt of poly(ethylene oxide) monomethyl ether as macroinitiator and allyl glycidyl ether as functional monomer. The copolymers were covalently modified via reactive double bonds by the addition of methyl sulfanylacetate. The resulting ester was subsequently reacted with hydrazine hydrate forming hydrazide. The hydrazide was furthter reacted with DOX forming pH-sensitive hydrazone bond between the drug and carrier.

The drug release kinetics was studied in aqueous buffers at pH 5.0 (close to pH in endosomes) and 7.4 (pH of blood plasma). The drug was released much faster at pH 5.0 than at 7.4. The micellar behavior in aqueous solutions at pH 5 and 7.4 was studied by static (SLS) and dynamic light scattering (DLS).

The authors acknowledge financial support of the Grant Agency of the Academy of Sciences of the Czech Republic (grants # A4050403, A1050201 and B4050408).


PC71

CONJUGATES OF POLYELECTROLYTE COMPLEXES WITH SURFACTANTS AND PHOSPHOLIPIDS AS NEW CARRIERS FOR DRUG DELIVERY

A. ZINTCHENKO, Č. KOŇÁK

Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, CZ-162 06 Praha 6, Czech Republic

Smart nanoparticles for drug delivery is a very attractive area in development of drug carriers. Polyelectrolyte complex nanoparticles are shown to be promising tool to deliver drugs to target cells. The major requirements for such systems are high circulation stability in the blood steam and the interactions with different species in the organism (proteins, surfactants, cell walls, etc.). In this study we try, on the one hand, to study the interactions of polyelectrolyte complexes with surfactants and phospholipid membranes. On the other hand, the products of such interactions might be quite effective vehicles to carry the drugs to distant targets.

The interactions of the complexes between poly(L-lysine) (PLL) and sodium poly(methacrylate) (Na-PMA) with negatively charged surfactant sodium dodecylsulfate (SDS) were studied by light scattering. The addition of SDS to complex solution leads to increase in Rh of the particles and does not lead to aggregation of the particles. The increase in Rgof the particles is much smaller in comparison to the changes in Rh. Such situation corresponds to swelling of the particles and formation of the polymer shell around it. The colloid stability of the final particles increases greatly in comparison to initial and PLL/SDS complexes formed by the same concentrations of the components. It decreases slowly with time. The colloid stability is independent of mixing ratio of initial complex and increases with the molecular weight of Na-PMA. According to the findings we were able to propose the mechanism of such interactions. The small molecules of SDS penetrate to the complex particle and exchange the poly(methacrylate) anions in the complex. Highly entangled Na-PMA species are not able to release fast from the particle. They lead the particle to swelling and form the stabilizing shell around it. The shell increases the colloid stability of such ternary conjugates. During the time the chains of Na-PMA slowly migrate to the boarder of the particle and release into the bulk. These lead to decrease of the colloid stability. Since many drugs show amphiphilic behavior, the formation of ternary conjugates with high stability could be achieved, which could be promising carriers in drug delivery.

The interactions of positively charged DNA/PLL complexes with phospholipid vesicles were studied by light scattering and fluorescent spectroscopy. Dipalmitoylglycerophosphatidic acid (DPPA) sodium salt and mixed vesicles of DPPA and dipalmitoylglycerophosphatidylcholine (DPPC)/DPPA (80/20 mol.) were applied. The main goal of this study is to mimic the interactions of DNA/PLL complexes with cell membranes in the organism and to obtain the coated complex particles with improved characteristics for gene delivery. The study is concentrated on changes in structural parameters of the particles in the solution and exchange of DNA as a result of interaction with lipid membrane.


PC72

THERMOSENSITIVE ORGANIC-INORGANIC HYBRID SYSTEMS

M. LUTECKIa, L. MATĚJKAa

aInstitute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, CZ-162 06 Praha 6, Czech Republic

Hydrogels based on N-Isopropylacrylamide (NIPA) have been the subject of investigation for many years. The main interest in these materials is the result of their sensitivity to different environmental stimuli, like pH and temperature changes. It is well known that at increasing temperature PNIPA gels exhibit discontinuous phase transition similar to coil-globule transition of peptides. The critical temperature for this process is known to be approximately 32oC. Below this temperature, immersed in water gel is in swollen state, keeping water inside thanks to strong hydrophilic interactions between water molecules and nitrogen atoms of PNIPA network. Above critical temperature phase separation takes place, hydrophobic interactions become predominant and water is expelled from the interior of gel.� This feature makes PNIPA-based gels very interesting subject as promising drug delivery systems.

The poster presents the approach to increase the efficiency of swelling and deswelling kinetics of these systems by introducing inorganic domains into polymer matrix. Series of gels modified with tetramethoxysilane (TMOS) and 3-methacryloxypropyltrimetoxysilane (MPTMOS) were synthesized. Foregoing Si-based precursors forming silica or silsesquiotanes were selected in order to investigate the effect of different attachment of inorganic domains to organic phase. Presence of double bond in the structure of MPTMOS ensures the covalent attachment to the organic phase, while hydroxyl groups are responsible for creating the network in sol-gel process. In turn, TMOS, without a covalent bonding to the polymer, form inorganic domains dispersed in polymer matrix. In this case N,N�-methylenebis(acrylamide) (BAA) must be used as a crosslinker of organic phase.

Subsequently, similar systems were synthesized under heterogeneous conditions, by carrying out the reaction above the lower critical solution temperature (LCST). This ensured the microphase separation of PNIPA, resulting in highly porous structure and further improvement in deswelling and reswelling rate.


PC73

MODELING OF VOLUME PHASE TRANSITIONS IN HYBRID BIOPOLYMER GELS

K. DUŠEK, M. DUŠKOVÁ-SMRČKOVÁ*

Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic

162 06 Prague, Czech Republic

Hybrid biopolymer gels are composed of disordered synthetic hydrophilic chains and built-in biopolymer motifs. A certain class of disordered hydrogels can undergo abrupt swelling transitions triggered by external stimuli (temperature, ionization degree, etc.). The feasibility of the transitions is determined by interplay of ionic and weak interactions, or by a complex interaction function without assistance of ionized groups, or by combination of both.

Chemically built-in biopolymer motifs can undergo order-disorder transitions, such as unfolding-folding or dissociation-association (coiled coils), upon change of external conditions. The accompanying change in quality and quantity of interactions affects the state of chains of the hydrophilic disordered polymer: the conformational change of the motif is thus amplified by the gel structure and results in swelling transition. Conversely, continuous swelling changes of the hydrophilic gel can trigger the conformational change of the motifs which by feedback mechanism causes a jump-wise swelling change of the gel.

We have modeled swelling behavior of a hydrogel with built-in biopolymer motifs undergoing folding-unfolding transition induced by increasing tensile force. The motifs are chemically attached to the gel chains at both ends as crosslinks. The behavior of the motifs roughly corresponds to that of immunoglobulin motifs in the cardiovascular protein titin. The unfolding force is generated by osmotic pressure produced by the synthetic gel. Thus, the synthetic gel plays initially an active role in unfolding-refolding transition of the motifs, followed by the gel amplification function. The model of this hybrid gel is based on a model of polyelectrolyte network with finite extensibility of chains and on the concept of surface interactions. In the folded state, the interaction is limited to the (small) surface of the folded domain and, upon unfolding, all chain segments in the domain take part in the interactions. When the continuously changing swelling pressure reaches a value corresponding to the critical unfolding force, the motif unfolds and new interacting surface is exposed. Since, usually, the newly exposed surface is enough hydrophilic to assist further swelling, the unfolding proceeds at once by none-or-all mechanism. The detailed analysis of the model has revealed that this motif-induced transition can trigger a classical collapse transition which is accompanied by large volume changes (expansion and shrinkage). Ideally, this nanoscopically self-assembled gel can function as a heat pump operating between two sufficiently distant temperatures. The high specifity of interaction of the motifs can be utilized in gel-based sensor devices and in drug delivery.


PC74

Functional Block Copolymers through Living Polymerization of Amino acid N-carboxyanhydrides

L. Machová, M. Dvořák, R. Kotva, V. ŠKARDA, D. KUBIES, F. Rypáček

Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovský Sq. 2, CZ-162 06 Prague 6, Czech Republic

E‑mail: machova (imc.cas.cz)

Block copolymers containing functional polypeptide blocks draw an increasing interest, particularly, in the area of biomedical applications. In this work we present approaches to the synthesis of poly(amino acid)/poly(lactide), PAA-b-PLA, and poly(amino acid)/poly(ethylene oxide), PAA-b-PEO, block copolymers adopting living polymerization of amino acid N-carboxyanhydrides (NCA).

Controlled living polymerization of NCA was carried out using N-acyl-NCA derivatives as co-initiators with tertiary amines as base catalysts.[1] The co-initiator provides for growing centers on which the polymer chains start to grow. The growing polymer chains propagate solely by the activated-monomer mechanisms via N-acylated NCA end group, while the other polymer end remains protected by the acyl group of the co-initiator. The kinetic data and molecular characteristics of the polymers are consistent with a living polymerization process.

PAA-b-PLA block copolymers carrying RGD-peptide function at the end of PAA block were prepared by applying subsequently two living ring-opening polymerizations, of α-amino acid-NCA and lactide, respectively. The RGD peptide sequence was incorporated at the end of hydrophilic PAA block by combining the initiation and capping steps in the synthesis of PAA. [2]

For the synthesis PAA-b-PEO-b-PAA copolymers, bis-amino-PEO is used a terminating agent in polymerization of NCA. The resulting block copolymers were characterized by 1H NMR and SEC.

Functional amphiphilic block copolymers with polypeptide segments were applied in preparation of cell-adhesive brush surfaces on polyester-based biomaterials, and their self-assembly behavior is studied.

Acknowledgment: Support by grants from Grant Agency of CR (304/02/0759) and Ministry of Health of CR (IGA-NDZ7448-3) is acknowledged.

References

[1] Rypáček, F., Dvořák, M., Kubies, D., Machová, L. US.Pat.: 6,590,061 (2003)

[2] Rypáček, F., Machová, L., Kotva, R., Škarda, V., Polym. Mater. Sci. Eng. 84, 817, 2001


PC75

STRUCTURAL FEATURES OF A HYPERTHERMOSTABLE ENZYME IN SOLUTION AND ADSORBED ON "INVISIBLE" PARTICLES

S. KOUTSOPOULOS, J. VAN DER OOST, W. NORDE

Laboratory of Physical Chemistry and Colloid Science and Department of Microbiology, Wageningen University, The Netherlands

We present the most straightforward method to study structural features of adsorbed macromolecules in situ using common spectroscopic techniques. Conformational characteristics and the adsorption behavior of a hyperthemostable enzyme from the hyperthermophilic microorganism Pyrococcus furiosus were studied by circular dichroism, steady-state and time-resolved fluorescence spectroscopy, and calorimetry in solution and in the adsorbed state. The adsorption isotherms were determined on two types of surfaces: hydrophobic Teflon and hydrophilic silica nanoparticles were specially designed so that they do not interact with light and therefore do not interfere with spectroscopic measurements. The enzyme was irreversibly adsorbed and immobilized in the adsorbed state even at high temperatures. Adsorption offered further stabilization to the heat-stable enzyme and in the case of adsorption on Teflon its denaturation temperature was measured at 133°C, i.e., the highest experimentally determined for a protein. Studying the conformation and biological function of enzymes upon interaction with surfaces is important not only for applications in biocatalysis and biotechnology but also in understanding important biochemical processes. With this study we also suggest that nature may employ adsorption as a complementary mode to maintain structural integrity of essential biomolecules upon changes in temperature.


PC76


PC77


PC78


PC79