MOLECULAR LEVEL INTERACTIONS BETWEEN BLOOD COMPONENTS AND MODEL BIOMATERIALS STUDIED BY ATOMIC FORCE MICROSCOPY
Open Access
- Author:
- Agnihotri, Aashiish
- Graduate Program:
- Bioengineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 28, 2005
- Committee Members:
- Christopher Alan Siedlecki, Committee Chair/Co-Chair
Cheng Dong, Committee Member
Erwin A Vogler, Committee Member
William O Hancock, Committee Member
Bruce Ernest Logan, Committee Member - Keywords:
- Biomaterials
Fibrinogen
Atomic Force Microscopy (AFM)
Blood Material Interactions
Integrins - Abstract:
- A complete understanding of blood-biomaterial interactions is necessary for the development of blood-compatible biomaterials that can be used for making long-term implants. To this end, in this in vitro study, the interactions of purified blood components with model biomaterials have been studied with an objective of establishing relationships between the surface properties and the biological response. The principal technique that was used in this study is atomic force microscopy (AFM), which allows both visualization of biomolecules in physiologically relevant environments and the measurement of interaction forces from the microN to pN range. Tapping mode atomic force microscopy was used to study the time-dependent changes in the structure of fibrinogen under aqueous conditions following adsorption on two model surfaces: hydrophobic graphite and hydrophilic mica. Fibrinogen is a key plasma protein involved in initiation of thrombosis on synthetic surfaces, and its adsorption to the biomaterial surface and subsequent interactions with the blood platelets are of fundamental interest. Fibrinogen was observed in the characteristic trinodular form. Based on the differences in the relative heights of the D and the E domains, four initial orientation states were observed for fibrinogen adsorbed on both surfaces. On graphite, the initial asymmetric orientation states disappeared with spreading over time. Spreading kinetics of fibrinogen on the two surfaces was determined by measuring the heights of the D and E domains over a time-period of ~2 hours. The spreading of the D and E domains on graphite was analyzed using an ‘exponential-decay-of-height’ model and a two-step spreading model is proposed. With the objective of relating the observed post-adsorption structural changes in fibrinogen to the surface availability of active epitopes and extending AFM imaging studies to complex multicomponent protein films, the adhesion mapping mode of AFM was developed for biologically sensitive imaging. AFM probes were functionalized by covalently linking polyclonal antibodies against fibrinogen. Adhesion mapping mode of AFM was used to generate both topographic images and adhesion images. The efficacy of the functionalized probes was first established by performing adhesion mapping on patterned dual-component protein films formed by microcontact printing bovine serum albumin (BSA) on a mica surface and then backfilling with fibrinogen. Next, adhesion mapping was done on randomly distributed two-component protein monolayers generated by sequential adsorption of submonolayer amounts of fibrinogen followed by backfilling with bovine serum albumin. The interactions of platelet membrane integrin GPIIbIIIa in purified form as well as in intact unfixed platelets against fibrinogen adsorbed to model hydrophilic and hydrophobic surfaces were characterized by using the force mode of AFM. Force curves obtained with probes functionalized with integrins on adsorbed fibrinogen showed multiple rupture events over a large range of distance on both surfaces. On the hydrophobic surface, the rupture length range was 20-200 nm, whereas on the hydrophilic surface, the rupture length range was 20-400 nm. Rupture events in the force curves had contributions from non-specific protein-protein interactions, mechanical denaturation of fibrinogen domains, and specific ligand-receptor interactions of integrin and adsorbed fibrinogen. Periodicities in the debonding force distributions were used to estimate the debonding strength of a single integrin-fibrinogen pair at different loading rates. For loading rates of 10-60 nN/s, the debonding strength of a single integrin-fibrinogen pair was in the range of 50-80 pN on both surfaces. This result suggests that once the active epitope is exposed on a surface, binding of the platelet membrane integrin receptor follows the same kinetics, regardless of the surface properties. Polyurethane biomaterials are among the most blood-compatible materials available for biomedical devices. It has been suggested that the good blood compatibility of polyurethanes arises from nanoscale chemical heterogeneities at the surface as a consequence of the microphase-separated morphology. Tapping mode atomic force microscopy with phase imaging under aqueous conditions was used to characterize the interfacial properties of a series of poly(urethane urea) block copolymers with varying hard segment content. Topographic images showed the formation of nanometer-sized raised features on the surface, having lateral dimensions of 50-70 nm and heights of 10-15 nm. Phase images, reflecting the local distribution of the mechanical properties under aqueous conditions, were quite different from those obtained in ambient conditions, consistent with water-induced structural reorientation. Images under aqueous conditions suggest that there is little soft phase material at the polymer surface in the presence of water, while images acquired after dehydration of the samples show that the surface layer remains rich in hard domains, indicating that the films do not return to their original states over the time period studied.