In vitro biocompatibility of deposited chitosan films as a platform for living cells in BioMEMS systems

Transcription

1 1 In vitro biocompatibility of deposited chitosan films as a platform for living cells in BioMEMS systems ITC-irst, Italy C.Pederzolli, L.Lunelli, M.Vinante, S.Forti, R.Canteri, L.Vanzetti, L.Pasquardini, G.Speranza, M.Anderle Division of Physics and Chemistry of Surfaces and Interfaces University of Maryland J.J.Park 1,2, G.F. Payne 3,4, G.W. Rubloff 1,2 1) The Institute for Systems Research, 2) Department of Materials and Nuclear Engineering 3) Center for Biosystems Research, 4) Department of Chemical and Biochemical Engineering Park J.J. et al. AS+NS+PS+SE+TF+TuA3 Nano-Bio and Self-Assembly II

2 2 utline Motivation Physico-chemical material properties Cell adhesive properties and surface morphology of electrodeposited or air-dried chitosan films Surface chemical composition of deposited films Conclusions Future work

3 3 Motivation Naturally occurring chitosan is an amine-rich polysaccharide derived by deacetylation of chitin Chitosan represent an increasingly important biopolymer for its unique physico-chemical properties Electrodeposition of chitosan and conjugation of nucleic acids and proteins established in joint work aimed at biomems applications Cell attachment and growth would be highly attractive as part of the portfolio of capabilities we could exploit in biomems

4 Electrodeposited chitosan films as a platform for living cells in BioMEMS systems Chitosan is a linear polysaccharide of β-(1 4)-linked D-glucosamine and N-acetyl-D-gluocosamine H H H H soluble + + H NH 3 H NH H NH 2 H NH +x H + unsoluble x CH 3 y x CH 3 y Deposition of chitosan on gold cathode in response to an applied voltage DC Power Supply + - ph dependent electrostatic behavior ph dependent solubility Spatial selectively ptical Image Chitosan Solution Fluorescence Image electrode Magnification 20 patterning 4

5 5 Physico-chemical chemical characteristics of the utilized chitosan samples Chitosan available from a number of suppliers in various grades of purity, molecular weight and degree of deacetylation (DD: proportion of D-glucosamine units with respect to the total number of units) CHIT A CHIT B CHIT C (Sigma) (Aldrich) (Novamatrix) Company spec. >80% 75-85% 86% DD XPS 64% 62% 79% FTIR 49% 63% 43% Chitosan titration 52% 49% 75% Source Molecular weight (g/mol) Ground shells 15,000 to 1 million Ground coarse shrimp shells Not specified 190, ,000 50, ,000 DD is a structural parameter which influences physicochemical and biological properties DD values of chitosan appeared to be highly associated with the analytical methods employed

6 Cell adhesion and proliferation tests 6 Human osteoblasts (MG-63), mouse fibroblasts (NIH-3T3) and mouse monocytes (RAW 264.3) were seeded at the density of 4x10 4 and 1.5x10 4 cells/cm 2 respectively in cell culture medium supplemented with 10% of FBS Incubation at 37 C in air containing 5% C 2 for 24 hours (adhesion) and up to 10 days (proliferation) Cell attachment, spreading and proliferation were analyzed by optical microscopic observations Surface morphological analysis by AFM on neutralized chitosan samples Controls on cell culture polystyrene steoblasts MG-63 Fibroblasts NIH-3T3 Monocytes RAW μm 100 μm 50 μm

7 7 Human osteoblast (MG-63) adhesion on electrodeposited chitosan film. CHIT B, fixed voltage 2V for 2 min. Reflected light bright field AFM analysis (2x2 μm 2 scans) Roughness= 9.8nm NaH 1M Y [2 μm] Z [100 nm] 100 μm 100 μm X [2 μm] Roughness= 8.0nm NH 4 H 0.47 M Y [2 μm] Z [100 nm] X [2 μm]

8 8 Human osteoblasts (MG-63) adhesion on electrodeposited chitosan film. CHIT B, fixed current 0.3mA for 2 min. Reflected light bright field AFM analysis (2x2 μm 2 scans) Roughness= 3.6 nm NaH 1M Y [2 μm] Z [100 nm] 100 μm X [2 μm] 100 μm Roughness= 5.9 nm NH 4 H 0.47 M 200 μm Z [100 nm]

9 Human osteoblast (MG-63) adhesion on electrodeposited chitosan film. CHIT B, fixed current 0.3mA for 5 min. Reflected light bright field AFM analysis (2x2 μm 2 scans) Roughness= 32 nm NaH 1M Y [2 μm] Z [230 nm] 100 μm 100 μm X [2 μm] Roughness= 30 nm NH 4 H 0.47 M 200 μm Y [2 μm] Z [350 nm] X [2 μm] 9

10 10 Surface chemical analysis by Tof-SIMS on electrodeposited chitosan films. Principal component analysis (PCA) fixed current, 2minutes fixed current, 5minutes CHIT A CHIT B fixed voltage The surface chemical composition varies changing the deposition methods

11 CELL ADHESIVE CHITSAN PRPERTIES ELECTRDEPSITED CHITSAN FILMS Adhesion on chitosan MG-63 NIH-3T3 : no RAW E+04 9.E+05 cells/cm 2 8.0E E E E E E E+04 round semi-spreaded spreaded aggregates/cm 2 4.0E E E E+03 cells/cm 2 8.E+05 7.E+05 6.E+05 5.E+05 4.E+05 3.E+05 2.E+05 NaH 1M 1.0E+04 1.E E+00 control 2Vx2' 0.3mAx2' 0.3mAx5' 0.0E+00 0.E+00 control 2Vx2' 0.3mAx2' 0.3mAx5' 9.0E+04 cells/cm 2 8.0E E E E E E E E+04 round semi-spreaded spreaded aggregates/cm 2 4.0E E E E+03 NH 4 H 0.47 M Cell Cell adhesion is is strongly dependent on on the the cell cell type type 0.0E+00 control 2Vx2' 0.3mAx2' 0.3mAx5' 0.0E+00 11

12 Summarizing 12 The extent of osteoblasts cell adhesion is fairly low on electrodeposited films made of CHIT A and B with not significant differences between them The obtained results show that several factors have influence on cell adhesion besides the DD value The utilized neutralization reactions (NaH 1M, NH 4 H 0.47M) result in a different cell surface affinity on both fixed voltage and current films A clear change of the cell adhesive behavior it s observed moving from fixed voltage to fixed current deposited films The fixed voltage samples (neutralized with NH 4 H) present a slightly better cell adhesion that probably could be correlated with the surface chemical composition steoblast cell adhesion doesn't depend on the surface morphology as observed with the AFM analysis The utilised deposition methods give chitosan films with different superficial chemical composition To clarify the influence of chemical and morphological properties of deposited chitosan films on cell adhesion, further data were collected using air-dried chitosan films.

13 Human osteoblast (MG-63) adhesion on air-dried chitosan films. NH 4 H 0.47 M neutralization CHIT A CHIT B CHIT C 100 μm 100 μm 100 μm Roughness= 7.3 nm Roughness= 3.1 nm AFM analysis (2x2 μm 2 scans) Roughness= 19 nm 13

14 Surface chemical analysis by Tof-SIMS on air-dried chitosan films. Principal component analysis (PCA) CHIT A CHIT C CHIT C fragments 2% 94% CHIT B CHIT A and B fragments The surface chemistry of CHIT C air-dried films is characterized for the presence of CH fragments meanwhile the CHIT A and B show also nitrogen containing fragments as expected 14

15 15 CELL ADHESIVE CHITSAN PRPERTIES AIR-DRIED CHITSAN FILMS Adhesion on chitosan MG-63 NIH-3T3 : no RAW E+04 9.E E E+04 round spreaded 4.0E+03 8.E+05 7.E+05 cells/cm 2 6.0E E E E E+04 aggregates/cm 2 3.0E E E+03 cells/cm 2 6.E+05 5.E+05 4.E+05 3.E+05 2.E E+04 1.E E+00 control CHIT A CHIT B CHIT C 0.0E+00 0.E+00 control CHIT A CHIT B CHIT C PRLIFERATIN CHIT A CHIT B CHIT C Human osteoblasts (MG-63) Mouse fibroblasts (NIH-3T3) detached colonies detached colonies no detached colonies CNTRL yes detached colonies no yes Mouse monocytes (RAW 264.3) yes Not determined Not determined yes

16 16 CNCLUSINS Chitosan starting material is not well defined: the DD values vary considerably with the analytical methods employed Cell adhesion observed to limited extent on deposited films (air-dried better than electrodeposited) Several factors affect the chitosan cell adhesive properties: the tested cell type sources (purity) amine site density (DD value) surface morphology (fibers vs. globulars) surface chemical composition These results suggest that chitosan with different surface properties can be deposited, modulating the in vitro level of cell attachment and spreading

17 FUTURE WRK Covalent immobilisation of biomolecules (i.e. adhesive peptides) to improve the chitosan cell adhesion properties Definition of the correlation between the deposition method and the film nanostructures Analysis of the adsorbed protein layer mediating the cell adhesion Cell growth on patterned surfaces Realisation of a cell-adhesive substrate A two step chemical reaction procedure was developed in order to attach a RGD adhesion factors to an activated surface. C.Pederzolli et al., Development and Characterization of RGD peptide coatings for cell adhesion in preparation 17