LABORATORY OF TISSUE ENGINEERING

Doc. RNDr. Evžen Amler, CSc.
email: evzen.amler@lfmotol.cuni.cz
tel: +420241062387

Scientists:

RNDr. Lucie Koláčná, Ph.D.
MUDr. Milan Handl, Ph.D.
Mgr. Eva Prosecká

Ph.D. Students:

Mgr. Eva Filová
Mgr. Michala Rampichová
Mgr. Andriy Lytvynets

Technical Assistants:

Gracián Tejral
Hana Švecová
Jana Závodská

Two main research topics of the Department of Tissue Engineering are:
- protein engineering
- tissue engineering

In the field of protein engineering, our research is focused on molecular dynamics simulation, protein structure modeling and model verification, namely on molecular mechanism of Na,K-ATPase phosphorylation.
In the field of tissue engineering, research of the department is concentrated on the development of new three-dimensional scaffolds utilizing biodegradable materials and on the preparation of autologous chondrocyte-based artificial cartilages.
The Department of Tissue Engineering at the Institute of Experimental Medicine, Academy of Sciences of the Czech Republic closely collaborates with the Department of Biophysics, and Faculty of Textile Engineering, Technical University at Liberec.

PROTEIN ENGINEERING

Research focus:

• Molecular mechanism of Na,K-ATPase phosphorylation
• Structure and dynamics of the ATP-binding site on Na,K-ATPase
  Our research proved eight amino acid residues – Glu⁴⁴⁶, Phe⁴⁷⁵, Lys⁴⁸⁰, Gln⁴⁸², Lys⁵⁰¹, Gly⁵⁰², Phe⁵⁴⁸, Cys⁵⁴⁹ – forming the complete ATP-recognizing pocket of Na,K-ATPase. The shape of the ATP-binding pocket is probably stabilized by a hydrogen bond between Arg⁴²³ and Glu⁴⁷² which supports the connection of two opposite halves of the ATP-binding pocket.
• Molecular dynamics simulation and protein structure modeling
  modeling of the structure of the H₄-H₅ loop of Na,K-ATPase, ATP-binding site localization in the so-called N-domain (Arg³⁷⁸-Arg⁵⁸⁹), separated from the P-domain (Leub³⁵⁴-Asn³⁷⁷ and Ala⁵⁹⁰-Leu⁷⁷³) with the phosphorylation site (Asp³⁶⁹) 
• Verification of computational protein models using molecular biology techniques
  expression of the H₄-H₅ loop, mutations of selected amino acid residues and evaluation of their effect on ATP binding 
• Verification of computational protein models using advanced spectroscopic and fluorescence techniques, such as steady-state or time-resolved fluorescence spectroscopy or Raman spectroscopy
  binding studies of the ATP fluorescent analog 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (NTP-ATP)

TISSUE ENGINEERING

Research focus:

• Development of new three-dimensional scaffolds
  utilization of different biodegradable materials (composite scaffolds, textile-base knitted and non-woven scaffolds  and nanofiber-based scaffolds) 
• Preparation of autologous chondrocyte-based artificial cartilages and reparation of the osteochondral defects in experimental animals
• Biomechanical properties testing
  special contactless techniques enabling rapid evaluation of biomechanical properties of miniature pieces of tissue
• In vitro testing of developed scaffolds
  autologous chondrocyte isolation and cultivation and scaffold seeding, determination of parameters characterizing the chondrocytes and scaffolds
• In vivo testing of artificially developed tissues
  implantation of scaffolds into the condyles of femoral trochlea of experimental animals (rabbits and mini-pigs)
• Liposomes incorporated into nanofibres
  for controlled drug delivery and nutrient supply

Fig. 1.: Structure of the H 4-H5 loop of Na,K-ATPase with the ATP-binding site (N-domain) and the phosphorylation site (P-domain).

zoom picture

Fig. 2.: Detailed structure of the ATP-binding site with the hydrogen bond.

zoom picture

Fig. 3.: Confocal microscopy of our PGA/PVA non-woven scaffold seeded with autologous chondrocytes (14-day cultivation, propidium iodide staining, 200 x magnification).

zoom picture

Fig. 4.: Histology (haematoxylin-eosin staining) of osteochondral defect (rabbit knee) after 6 weeks of treatment with composite scaffold seeded with autologous chondrocytes.

zoom picture

List of current grants:

Name of the grant	Grant agency	Number of the grant	Principal investigator	Duration of the grant
Protein computer modeling and molecular dynamics simulation	GAČR	1ET40011010	Doc. Amler	2004-07
Molecular mechanism of Na,K-ATPase phosphorylation	GAUK	121/2005/B-BIO/2.LF	Doc. Amler	2005-07
Synthesis of new biomaterials and preparation of stem cell derived cells, and their aplications in for the treatment of diseases affecting human tissues derived from mesoderm: cartilage, bone, ligament and meniscus	MŠMT ČR	2B06130	Prof. Nečas	2006-11
Centre for cardiovascular research	MŠMT ČR	1M6798582302	Prof. Herget	2005-09
Nanofiber scaffolds with liposomes for tissue engineering	GA AVČR	IAA500390702	Doc. Amler	2007-11

1986 | 1987 | 1988 | 1989 | 1990 | 1991 | 1992 | 1994 | 1995 | 1996
1997 | 1998 | 1999 | 2000 | 2001 | 2002 | 2003 | 2004 | 2005 | 2006
2007 | 2008