Biodegradable Copolymers for Tissue Engineering: Preparation and - - PowerPoint PPT Presentation

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Biodegradable Copolymers for Tissue Engineering: Preparation and - - PowerPoint PPT Presentation

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TB TAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY Biodegradable Copolymers for Tissue Engineering: Preparation and Characterization and Cell Growth Bahattin M. Baysal, Alexandra Porjazoska, Oksan Karal- Yilmaz, Nilhan Kayaman-Apohan, Kemal

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

Biodegradable Copolymers for Tissue Engineering: Preparation and Characterization and Cell Growth

Bahattin M. Baysal, Alexandra Porjazoska, Oksan Karal- Yilmaz, Nilhan Kayaman-Apohan, Kemal Baysal, Maja Cvetkovska

  • Assoc. Prof. Dr. Kemal Baysal

TÜBİTAK RESEARCH INSTITUTE for GENETIC ENGINEERING and BIOTECHNOLOGY (RIGEB) http://www.rigeb.gov.tr Presented at the ICNT 2005, November 1, San Francisco

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

Cell-cell and Cell extracellular matrix interactions

İntegrins v3, v5

ECM

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

A cell interacting with the basal membrane formed by type IV collagen and laminin

  • Prof. Anne Ridley
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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

Signal Transduction from Integrin heterodimers

  • J. T. Price & E. W. Thompson Expert Opin. Ther. Targets (2002) 6(2):217-233
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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

RGD

 

MEMBRANE

Integrin heterodimers

ECM PROTEIN

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

Synthetic Polymers As Tissue Engineering Scaffolds

1)Poly(L-lactic acid-co-ethylene oxide-co-aspartic acid) terpolymer (PLLA/PEO/Pasp) 2)Poly(ethylene glycol)-poly(D,L-lactide-co-glycolide)- (polyethylene glycol) triblock copolymers modified with collagen

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

Synthesis of PLLA/PEO/PAsp Terpolymer

First step is synthesis of N-carboxy anhydride of -benzyl L- aspartate -benzyl L-aspartate + triphosgene  40°C, THF -benzyl L-aspartate of N-carboxy anhydride Asp(OBzl)-NCA

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

H2N CH C O Bz OH CH2 C O O

+ Cl3C

CCl3 O C O O THF 40 oC C O O C O CH NH Bz CH2 C O O

  • benzyl L-aspartate of N-carboxyanhydride

Asp(OBzl)-NCA 

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

O O O O CH 3 H 3C

+

Sn(II)O ct 110

  • C

_ _ _ _O CH CH 3 CH 3 C O _O _ CH C O _ _ _ _ _ y

L-Lactide poly(ethylene oxide) PLLA-PEO -PLLA m acrom er

H O _ _ _ O CH

3

CH

3

C O _ O _ C O _ _ _ _ _ _ y

3 3

CH

3

CH

H x CH 2 CH 2

_

_ O _ _ _ _ _ _ HO x CH 2 CH 2

_

_ O _ _ H _ _ _ _ _ _ _ _O CH CH 3 CH 3 C O _O _ CH C O _ _ _ _ _ y

PLLA-PEO -PLLA m acrom er

H O _ _ _ O CH

3

CH

3

C O _ O _ C O _ _ _ _ _ _ y

3 3

CH

3

CH

H x CH 2 CH 2

_

_ O _ _ _ _ _ _

+

C O O C O CH NH Bz CH 2 C O O

_ _ O CH CH 3 CH 3 C O _O _ CH C O _ _ _ _ _ y O _ _ _ O CH

3

CH

3

C O _ O _ C O _ _ _ _ _ _ y

3 3

CH

3

CH

x CH 2 CH 2

_

_ O _ _ _ _ _ _ _ _ NH CH C O Bz CH 2 C O O HN CH C O Bz CH 2 C O O _ _ _ _ _ _ _ _ _ _ H H

n n

PLLA/PEO /PAsp(O Bzl) terpolym er Asp(O Bzl)-NCA

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

Characterization

1) FTIR(Fourier Transform Infrared Spectroscopy) 2) 1H-NMR (Nuclear Magnetic Resonance) 3) ESEM (Environmental Scanning Electron microscope) 4) SEC ( Size Exclusion Chromatography)

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

1.34 6950 5200 PLLA/PEO/PAsp 1.35 8750 6500 PLLA/PEO/PAsp(OBzl) 1.58 7300 4600 PLLA/PEO/PLLA P.D. Mw (g/mol) Mn (g/mol) Polymer

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

ESEM micrographs of cross-section and side surface details of PLLA/PEO/PAsp terpolymer A star shaped fracture zone on the polymer microsection is seen, adjacent to the locus of failure. ESEM photos: Zulal Mısırlı, Boğaziçi Univ. Advanced Technologies R&D Center.

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

ESEM micrographs of fractured surface details of PLLA/PEO/PAsp terpolymer Fractured surface has an oriented texture. This is probably due to the semicrystalline nature of the polymeric sample.

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

The results of characterization showed that; 1) PLLA/PEO/PAsp is crystalline 2) Its surface structure is brittle Due to its inherent brittle behaviour, the terpolymer has been blended with a high molecular weight copolymer (PLGA) in two different compositions: (1) (PLLA/PEO/PAsp)/PLGA 25/75 (w/w) blend (2) (PLLA/PEO/PAsp)/PLGA 50/50 (w/w) blend

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

Synthesis of Poly(L-lactic-co-glycolic acid) Copolymer

Ring Opening Polymerization

Catalyst:Sn(II)Oct.

Monomer/Catalyst, mole ratio, 1/1000

The feed molar ratio of the L-lactide/glycolide= 85/15

Rxn temperature: 115°C

Rxn time: 24 hours

Mw: 95000 g/mol

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

Porous Film Fabrication

  • Solvent casting particulate leaching technique
  • Polymers (PLGA 85/15, (PLLA/PEO/PAsp)/PLGA in two different

compositions (25/75; 50/50))

  • Polymers/dissolved in chloroform
  • Polymer/salt ratio:0.18
  • 1ml of polymer solution was placed into Teflon molds (d:22mm,

h:10mm) packed with NaCl particles(size:200-400µm).

  • Solvent allowed to evaporate
  • Entrapped salt particles were removed by immersing the films in

distilled water for 48 hour.

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

The hydrolytic degradation of the porous polymeric mixtures ((PLLA/PEO/PAsp)/PLGA; 25/75 and 50/50) were investigated in PBS at pH=7.4 37°C

Degradation

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

Figure 5

20 40 60 80 100 10 20 30 40 50 60 20 40 60 80 100 Degradation Time(days)

Mass and Molecular weight loss for the 25/75 (PLLA/PEO/PAsp)/PLGA mixture: (o) mass loss; (•) Mw loss

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

20 40 60 80 100 20 40 60 80 100 10 20 30 40 50 60 Degradation Time (days)

Mass and Molecular weight loss for the 50/50 (PLLA/PEO/PAsp)/PLGA mixture: (o) mass loss; (•) Mw loss

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY ESEM micrograph of (PLLA/PEO/PAsp)/PLGA 50/50 blend before degradation ESEM photos: Zulal Mısırlı, Boğaziçi Univ. Advanced Technologies R&D Center.

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY ESEM micrograph of (PLLA/PEO/PAsp)/PLGA 25/75 blend before degradation

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY ESEM micrograph of (PLLA/PEO/PAsp)/PLGA 50/50 blend after degradation

  • f 10 days.
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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

  • An experiment in which cell viability was measured and compared
  • n different materials.
  • L929 cells placed on TC Plastic, gelatin or fibronectin rapidly

proliferate, giving high absorbance values after 72 hours.

  • PLGA (85/15) copolymer or (PLLA/PEO/PAsp)/PLGA 25/75 blend

allowed the attachment and proliferation of a significant amount of cells.

  • The number of L929 cells on the blend was slightly higher than the

85/15 PLGA copolymer.

Neutral Red cell viability assay

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY ESEM micrograph showing the cross section of the 25/75 blend 72 hours following seeding with L929 cells, showing a group of L929 cells attached to a surface inside a pore in the blend structure as well as the outer surface.

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY Cell viability assay by neutral red uptake. 150,000 L929 cells were seeded per 22 mm diameter polymer surface or glass coverslip and grown for 72 hours in a CO2 incubator. TC Plastic; tissue culture plastic, FN;fibronectin, 85/15; Poly(L- lactic-co-glycolic acid) copolymer, Blend;(PLLA/PEO/PAsp)/PLGA 25/75 mixture

0,0 0,5 1,0 1,5 2,0 2,5

85/15 Blend TC Plastic Gelatin FN

540 nm Abs

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

ESEM results showed that PLLA/PEO/PAsp is crystalline and its surface structure is brittle.

  • The hydrolytical degradation of polymeric blends was performed up

to 48 days and the results indicated that the degradation occurs by chain scission.

  • For the 25/75 (PLLA/PEO/PAsp)/(PLGA) sample, the mass loss and

molecular weight loss is faster than the 50/50 (PLLA/PEO/PAsp)/(PLGA) sample because of the high (PLGA) content in the mixture.

  • The cell seeding experiments showed that cell growth supporting

characteristics of the 25/75 (PLLA/PEO/PAsp)/(PLGA) blend was slightly higher than the 85/15 PLGA copolymer which was used in producing the blend.

Conclusion

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

2) Synthesis of poly(ethylene glycol)-poly(D,L- lactide-co-glycolide)-poly(ethylene glycol) triblock copolymer PEG-PDLLG-PEG, and its modification with type I collagen.

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

1. Transformation of the PEG carboxylic groups into more reactive acyl halide groups, 2. Coupling reaction with OH terminated PDLLG, 3. Activation of PEG-PDLLG-PEG with N-hydroxysuccinimide to produce a polymer more reactive to proteins and peptides, 4. Attachment of type I collagen to the PEG-PDLLG-PEG copolymer.

The synthesis consists of several steps:

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

Preparation of PEG-coupled PDLLG includes two steps:

1) Bis carboxy methyl PEG (PEG-COOH) was reacted with thionyl chloride producing highly reactive bis-chlorocarboxy methyl PEG (PEG-COCl) 2) Reacting bis-chlorocarboxymethyl PEG with OH terminated PDLLG produced carboxyl terminated PEG-PDLLG-PEG triblock copolymer.

Activation of PEG-PDLLG-PEG triblock copolymer with N-hydroxysuccinimide (NHS):

PEG-PDLLG-PEG and NHS were reacted in the presence of dicyclohexylcarbodiimide (DCC) and PEG-PDLLG-NHS was obtained.

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

Attachment of collagen onto PEG-PDLLG- PEG triblock copolymer: For modification of PEG-PDLLG-NHS copolymer, type I collagen from calf skin (Mw : 140 000 g/mol) was used. The final product, PEG-PDLLG-Col, was used as a substrate for cell growth experiments.

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

Polymeric Samples

Mw gmol-1 Mn gmol-1 PEG-COOH 1 160 1 110 PDLLG 23 460 14 560 PEG-PDLLG-PEG 25 900 21 700 PEG-PDLLG-NHS 37 650 25 240

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

L929 cells on PDLLG

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

L929 cells on PEG-PDLLG-Col

SEM photos: Feriha Şirvancı, Marmara Univ.

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

L929 cells on PEG-PDLLG-NHS

SEM photos: Feriha Şirvancı, Marmara Univ.

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY Cell viability assay by neutral red uptake. 150,000 L929 cells were seeded per 22 mm diameter polymer films and grown for 72 hours in a CO2 incubator.

0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1

PDLLGA PEG-PDLLGA-NHS PEG-PDLLGA-Col

Biopolymers 540 nm Abs

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

A triblock copolymer of PEG-PDLLG-PEG was synthesized and characterized. A suspension of this PEG-PDLLG-Col polymer was poured onto PDLLG films formed in Teflon molds and a new polymeric film was prepared. Similarly, films of PDLLG and PEG-PDLLG-NHS were made. The biocompatibility of PEG-PDLLG-Col was compared with the latter materials by assessing the attachment and growth of L929 mouse fibroblasts on these films in vitro. Both SEM analysis and neutral red uptake assay following a 72- hour incubation indicated that collagen modification significantly increased the number of cells on PDLLG.

Conclusion

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

This work was supported by EUREKA Project BIOPOLYMER no:!2202, Turkish-Macedonian Science and Technology program for 2001 – 2003 and 2003 – 2006.

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

Molecular Cell Biology Lab Aslı Kumbasar PhD. Zelal Adıgüzel MSc. Müge Serhatlı MSc. Ömer Kaçar BSc. Polymer group

  • Prof. Dr. Bahattin M. Baysal
  • Assoc. Prof. Dr. Nilhan K-Apohan

Okşan Karal-Yılmaz PhD. F.Y.R. Macedonia Polymer Group

  • Prof. Dr. Maja Cvetkovska

Aleksandra Porjazoska MSc. Electron microscopy

  • Assoc. Prof. Dr. Feriha Ercan
  • Dr. Serap Şirvancı

Zulal Mısırlı (ESEM)

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY

THANK YOU FOR LISTENING

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TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY TÜBİTAK, RIGEB, MOLECULAR CELL BIOLOGY LABORATORY