Biomaterial Surfaces and Biointerfaces Surface Science II

Transcription

1 Biomaterial Surfaces and Biointerfaces Surface Science II April 29, 2013 Prof. Prabhas Moghe Biomedical Engineering; Chemical & Biochemical Engineering Rutgers University 1

2 Areas and Technologies where Surface Properties of Biomaterials Are Relevant: A Sampling-- can you think of some more? Development of Biosensors Point of Care Devices Biomaterials for Growing Living Cells (Cell Culture) Biomaterials (e.g. Catheters, Bandages) that Resist Microbial Growth and Infection Multifunctional Nanoparticles that Bind to Cancer Cells and Cause Ablation/Killing Hydrogels and Dressings for Wound Healing Scaffolds for Tissue Regeneration Vascular Stents that Prevent Blood Vessels from Occluding (Stenosis) Titanium implants for bone fracture fixation and hip prostheses 2

3 SURFACE PROPERTIES OF MATERIALS cell tissue fluid and proteins BIOMATERIAL Surface properties determine biomaterial-tissue interface. Properties: Biocompatibility Topography/Roughness Wettability Surface Mobility Crystallinity Chemical Composition 3

4 Methods to Characterize Biomaterial Surfaces Method Principle Depth analyzed Resolution Cost Contact Liquid wetting of material 3 20 A o 1 mm Moderate angles estimates surface energy ESCA / XPS X rays cause Electron emission A um V. Expensive AES Electron beam causes Auger e. emission A 100 A V. Expensive SIMS Bombarded ions cause surface secondary ion emission 10 A 1 um 100 A V. Expensive FTIR- IR radiation causes excitation 1-5 um 10 um Expensive ATR STM Quantum tunneling current betn 5 A 1 A Expensive probe & conducting material SEM Electron beam causes secondary 5 A 40 A Expensive electron emission CLSM Reflected /fluorescent images spatially determined 10 um 2000 um 0.5 um Expensive Other Methods: Quartz crystal microbalance (Measure protein or biomolecule adsorption on materials); Ellipsometry (Measure thickness of biointerface); Surface Plasmon Resonance (Kinetics of binding); Fluorescence Resonance Energy Transfer (Biomolecule-Biomolecule Binding) 4

5 Surface Contact Energetics -Molecules exterior to materials are most accessible to adjacent phases as well as incoming cells (Surface Reaction) -There is always a positive energy necessary to create a unit area of surface. Systems reach equilibrium by minimizing this surface area/energy. In solids, this happens by changing the nature of interface to one with lower (lowest) energy. -Energy minimization occurs when groups/chains in a biomaterial (e.g., polymer) rearrange to yield lowest interfacial energy. e.g. hydrogel migrates to/from surface of a graft copolymer exposed to water/dry air. 5

6 Determination of Surface Energetics Excess free energy per unit surface area is surface tension Young s Theory of the Spreading of Liquid Droplet: At equilibrium, surface energy sv = sl + lv cos lv sl sv 6

7 Techniques to measure contact angles Static Drop air Capillary air-bubble DuNouy Ring or Wilhelmy plate 7

8 Dynamic Contact Angle Measurements Dynamic contact analysis is done by increasing or decreasing the drop volume until the three-phase boundary moves over the surface. ADVANCING CONTACT ANGLE RECEDING CONTACT ANGLE a r 8

9 Thermodynamics of Spreading/Adhesion on Materials Interfacial free energy of adhesion = Cell-solid interfacial free energy - Cell-liquid interfacial free energy - Solid-liquid interfacial free energy. F adh = cs - cl - sl If F adh < 0, adhesion and spreading are energetically favorable F adh and Substratum free energy (wettability) F adh Very hydrophobic substrates s [erg. cm -2 ] 9

10 CELLS INTERACT WITH PROTEIN-ADSORBED BIOMATERIALS Receptors Cell Proteins Polymer 10

11 Discussion of Research Paper 1 Tziampazis, Kohn, and Moghe, Biomaterials 21:511, 2000 Our study focused on the role of poly(ethylene glycol) (PEG) in actively regulating the biological responsiveness of protein adsorbed biomaterials. To this end, we designed PEGvariant biomaterials from a family of tyrosine/peg-derived polycarbonates to present surfaces ranging from low to intermediate levels of PEG concentration, below the PEG level requisite for complete abolition of protein adsorption. We analyzed the e!ect of PEG concentration on the amount, conformation and bioactivity of an adsorbed model protein, fibronectin, and on the attachment, adhesion strength and motility of L929 fibroblasts. Our results demonstrate that low levels of PEG can regulate not only the extent but also the conformation and speci"c bioactivity of adsorbed fibronectin. As the PEG concentration was increased from 0 to 6 mol%, the amount of adsorbed fibronectin decreased linearly yet the fibronectin conformation was altered such that the overall bioactivity of adsorbed fibronectin was uncompromised. We report that the degree of cell attachment varied with PEG concentration in a manner similar to the dependence of "bronectin bioactivity on PEG. In contrast, the nature of cell adhesion strength dependence on PEG paralleled the pattern observed for fibronectin surface concentration. Our studies also indicated that the rate of cell migration was inversely correlated with PEG concentration over a narrow range of PEG concentration. Overall, these results highlight the striking ability of PEG-variant biomaterials to systematically regulate the behavior of adsorbed cell adhesion proteins and, consequently, se!ect cell functions. 11

12 Relationship between substrate PEG content and surface hydrophobicity Tziampazis, Kohn, and Moghe, Biomaterials 21:511, O O O O CH 2 -CH 2 -C-NH-CH-CH 2 O-C O-PEG-O-C C O O R = CH 3 CH 2 R 1-f f Chemical structure of poly(dte-co-peg carbonate)s. In this work, the alkyl group, R, on all polymers was ethyl (E), and the Mw of PEG blocks was The molar fraction of PEG units in the copolymer was varied from 10 mole% down to 0 mole% PEG. 60 Effect of PEG concentration on the hydrophobicity of PEG-variant biomaterials. Lower receding contact angles indicate lower hydrophobicity (increased hydrophilicity). Receding Contact Angle (degrees) Unconditioned Protein conditioning %PEG 1k Content

13 PEG Interfaces Alter Protein Adsorption & Activity Figure 3. PEG-mediated regulation of the amount of fibronectin adsorption on PEG-variant biomaterials. As described in Materials and Methods, adsorbed hpfn was quantified using radiolabeled FN as a tracer. Adsorbed FN (mg/cm 2 ) PEG Content (%) Data Linear Fit R 2 = FN Cell Binding Activity FN Bioactivity (ELISA Absorbance) Figure 4. Bioactivity of FN adsorbed on PEG-variant biomaterials. A. Cumulative sur face bioactivity was ev aluated using fibronectin E LISA Specific Bioactivity Specific of Index Protein B. Specific FN bioactivity index was computed by normalizing FN bioactivity to adsorbed amount and expressing the values relative to the specific bioactivity of FN adsorbed on poly(dte carbonate) * * PEG Content (%) PEG Content (%) 13

14 Organization & Conformation of FN Surface Surface Roughness (nm) Index PBS FN * * * PEG content (%) * Figure 5. Topographic imaging of polymer surfaces before and after fibronectin adsorption. Fluidmode atomic force microscopy was employed to analyze the surfaces in a physiologically relevant state. Images depict the topography of a square area, 1 µm wide, as would be seen from the front with a 70 degree pitch. For all images the scale of the Z-axis (height) is 8nm.

15 100 Effect on Cell Adhesion Strength 80 Cell Attachment (%) * * * PEG Content (%) Average Cell Movement (µm/h) * * PEG Content (%) Figure 7. Extent of L929 cell adhesion on PE G-variant biomaterials. A. Fraction of cells attached to the biomaterial after one day of culture. Asterisks denote values that differ significantly (p<0.05) from the cell attachment data in the absence of PEG. B. PEG mediated regulation of cell adhesion strength. The critical level o f shear required to detach 50% of the attached cell popula tion was used as a single parameter measure of cell adhesion strength. 14

16 DTE (Hydrophobic) PEG (Hydrophilic) Discussion Moderately Hydrophobic Strongly Hydrophobic poly(dte-co-peg carbonate) Moderately Hydrophilic Strongly Hydrophilic Fibronectin Schematic Cell Binding Domain Low or no PEG Intermediate PEG High PEG 15

17 PEG Microdistribution Regulates Cellular Survival Soft Matter, 2010, 6,

18 Fig. 1 Chemical composition of PEG-containing polymers and variations in hydration and protein adsorption due to increasing PEG1k mol%. (a) The chemical structure of polymers used in this study. Polymers were composed of two different co-macromers: DTE and PEG (Mw ¼ 1000). The chemical formula was abbreviated as p(dte-co-y%peg1k c). PEG1k mole percentages used in this study were 5, 8, 10, and 20%. (b) Water contact angle measurement for 0 and 2 hours of hydration. (c) FBS adsorption measured by QCM-D on given substrates. The Voigt model was applied to obtain the adsorbed mass (mg cm2). m

19 Fig. 2 Increasing PEG content of polycarbonate polymers correlates with an increase in oxidation. (a) Measurement of residual H2O2 (mm) on polymer surfaces. (b) Protein carbonyl formation (nm) generated on polymer surfaces. *p < 0.05 vs. the condition without the addition of ebselen on the same polymer type. (c) Intracellular peroxide production (O.D.) from cells cultured on polymer substrates. *p < 0.05 vs. the condition with no addition on the same polymer type. p < 0.05 vs. the other conditions on the same polymer type. Ebselen was added to scavenge H2O2. DPI was added to inhibit the activity of NADPH oxidase.

20 Fig. 4 Diverging effects of PEG content on attachment and spreading of SaOS-2 cells. (a) Cell populations and (b) cell spreading on polymer substrates as measured at 24 h postseeding by light microscopy and image processes. *p < 0.05 vs. p(dtec); p < 0.05 between two test materials indicated by a line.

21 Fig. 5 Formation of PEG microdomains and water uptake characteristics of PEG containing polymers. (a) SANS; (b) SAXS; and (c) water uptake, Tg, Young s modulus, and elongation to failure of polymer samples.

22 Fig. 6 PEG-induced modulations of cell spreading via integrin a5. (a) Intracellular distribution of GFP integrin a5 fusion proteins. (b) Protein levels of integrin a5 quantified by the fluorescence intensity of antibodies raised against integrin a5 on polymer samples both without and with the presence of ebselen. *p < 0.05 vs. p(dtec) under the appropriate condition (i.e., with or without ebselen). p < 0.05 between samples indicated by a line. (c) Expression of integrin a5 mrna in cells cultured on polymer samples under different conditions as detected by in situ hybridization. *p < 0.05 vs. p(dtec) with no addition. p < 0.05 vs. the other conditions, on the same polymer type. (d) Relative changes in gene expression of integrin a5 at 24 hours after seeding on polycarbonate PEG substrates normalized by gene expression at 4 hours postseeding. A relative expression of 1 indicates no change in expression between 4 and 24 hours. *p < 0.05 for 10 mol% and 20 mol% PEG1k compared to p(dtec) and 5 mol% PEG1k.

23 Applications of PEG Biointerfaces Plasma-micropatterning of albumin nanoparticles: Substrates for enhanced cell-interactive display of ligands María Pía Rossi, Jing Xu, Jean Schwarzbauer, Prabhas V. Moghea Department of Chemical and Biochemical Engineering, Graduate Program in Cellular and Developmental Biology, and Department of Biomedical Engineering, Rutgers University, 599 Piscataway, New Jersey Received 29 August 2010; accepted 11 October 2010; published 11 November 2010 The authors demonstrate a novel, efficient, and widely applicable approach to direct the patterning of ligand-functionalized organic nanoparticles derived from albumin on nonconductive, biodegradable polymeric substrates. In contrast to traditional deposition methods for inorganic nanoparticles, the approach involves oxygen plasma treatment of spatially restricted regions on a nonbiopermissive polymer. Albumin nanoparticles conjugated with a truncated fragment of fibronectin containing the Arg-Gly-Asp domain were successfully patterned and used as templates to elicit adhesion and spreading of human mesenchymal stem cells and fibroblasts. Attachment and spreading of both cell types into the plasma-exposed polymer areas was considerably more pronounced than with the ligand alone. The authors hypothesize that the underlying mechanism is oxygen plasma treatment-induced selective enhancement of ligand exposure from the deposited functionalized nanoparticles, which facilitates ligand receptor clustering at the cell membrane. The results highlight a promising nanoscale approach to modulate ligand presentation and spatially direct cell attachment and phenotypic behaviors American Vacuum Society. Biointerphases 5(4), 105, December 2010

24 FIG. 1. Schematic illustrating the microscale plasma-initiated patterning process. A A PDMS stamp is placed on the biocompatible, bioresorbable polymer surface and treated in oxygen plasma at a pressure of 660 mtorr for s and 50 W. Areas of the polymer exposed to the plasma undergo surface functionalization via the formation of end groups by interaction with the radicals, electrons, and ions in the oxygen plasma. B Biofunctional ANPs then preferentially adsorb to the exposed area of the material. C By exposing the polymer to the oxygen plasma for 60 s, rotating the stamp by 90, and exposing the polymer to the oxygen plasma for 60 s again, areas of differentially exposed polymer enabled the differential deposition of nanoparticles for cell attachment.

25 FIG. 2. Color online Biointerfacial characterization of spatially restricted patterning of bioactive albumin nanoparticles. A Fluorescence microscopy image of patterned nanoparticles on polydte-co-8% PEG1K carbonate. Briefly, after plasma-initiated patterning of the substrate with 660 mtorr oxygen at 50 W for 120 s, the nanoparticles were adsorbed onto the surface at 4 C overnight, then washed, fixed, and stained with mouse antihuman serum albumin as a primary antibody and FITC-conjugated Donkey antimouse secondary antibody for visualization under fluorescence. B Atomic force microscopy image of nanoparticle patterns on polydte-co-8% PEG1K carbonate. While nanoparticle monolayers are observed on one of the stripes the plasma-exposed area, minimal nanoparticle adsorption is observed on the other stripes unexposed or plasma-protected areas. C Scanning electron microscopy image of the nanoparticles patterned on the polymer substrates after plasma treatment. D High magnification scanning electron microscopy image of C. E Higher yet magnification of plasma-treated region shows organization of nanoparticles in D.

26 FIG. 5. Color online Human mesenchymal stem cells adhere and organize effectively on spatially patterned albumin nanoparticles. Fluorescent images green actin; blue DAPI show MSCs patterned with FNf-functionalized albumin nanoparticles A 4; B 20, compared to those patterned on FNf fragment control ı Cˇ 4ˇ ; ı Dˇ 20ˇ, and on positive control, wholelength fibronectin E 4; F 20. Patterning was sparse with ligand alone. Patterning with the FNf-ANPs is more effective than with FNf alone, yields more contiguous organization than whole-length fibronectin control, and cells clearly spread within the plasma-exposed stripes ı Cˇ and ı Dˇ.

27 Summary 1. Surface contact energetics of materials can be measured using contact angles. 2. The surface energetics of polymeric materials govern protein adsorption and cell adhesion and spreading. 3. Changing the PEG microdistribution within biomaterials can control the bioactive conformation of proteins, which in turn, can effect a qualitative change in the nature of cell adhesion, and a quantitative change in cell responses, including attachment/detachment kinetics leading to cell migration; cell adhesion-based survival or apoptosis. 4. Applications of PEG-based interfaces is in restricting protein adsorption and promoting specific cellular/tissue interactions with functionalized bioactive ligands.