1 Characterization of Cell-Based Adhesive Biointerfaces September 11 (Mon) and 14 (Thurs) Dr. David Shreiber Dept. of Biomedical Engineering Prof. Moghe 125:583 Biointerfacial Characterization

2 SIGNAL TRANSDUCTION GENERAL CONTROL MECHANISM Stimulus Sensor Signal Transducer Regulator Effector (Response) ECM Another Cell Force (Form junctions/adhesion complexes) Integrins Cadherins Ig Superfamily of cell adhesion molecules (CAMs) Selectins Proliferation Tissue Formation Traction Migration Apoptosis Differentiation Cytoskeletal Elements Kinases G-Proteins

3 CELL ADHESION Most cells demonstrate anchorage-dependent growth. Adhesion can also affect migration, differentiation, and apoptosis. –Includes adhesion to materials for tissue engineering

4 FORCES IN BIOLOGICAL INTERACTIONS Electrostatic double layer: even though cell and bacterial membranes are negatively charged, and you think they’d repel each other, ions can associate with the cell membranes to form a kind of cloud around it. If two cells share the same cloud, there is measurable adhesion between them.

5 FORCES IN BIOLOGICAL INTERACTIONS Electrostatic double layer: even though cell and bacterial membranes are negatively charged, and you think they’d repel each other, ions can associate with the cell membranes to form a kind of cloud around it. If two cells share the same cloud, there is measurable adhesion between them.

6 FORCES IN BIOLOGICAL INTERACTIONS Of course, cells are not in water, and this force is sensitive to the ionic strength of the extracellular environment in vivo and culture medium in vivo.

7 FORCES IN BIOLOGICAL INTERACTIONS Then are the Van der Waals attractive forces, which are due to interactions between oscillating dipoles of the surface molecules. These forces are very powerful, but only for a small distance.

8 FORCES IN BIOLOGICAL INTERACTIONS If we combine these forces, we can describe the net forces acting on, essentially, colloidal particles. At least this is what the first people were studying who described this theory. It’s called DLVO theory, named after people from two research groups: Deryagin and Landau (group one) and Verway and Overbeek (group two). SUMMARY 1.Most cells and biological surfaces are negatively charged 2.In a fluid environment the surface negative charge attracts a layer of mobile counterions (cations) which is known as the "Electrical Double Layer" 3.As bacteria approach a surface or another cell the electric double layers begin to overlap causing an electrostatic repulsive force 4.As bacteria approach a surface they also experience an attractive force known as van der Waals force 5.The combination of these two forces dictates how a bacterial cell adheres and is described by the DLVO theory 6.The DLVO theory predicts that in a solution of physiological ionic strength bacterial cells will be held about 10nm away from a surface and will be unable to approach closer. 7.The electrical double layer shrinks if the ionic strength of the surrounding fluid is increased which allows cells to get nearer to surfaces. This is the reason why adding salt to a bacteria suspension causes the cells to flocculate 8.The stability of bacteria in suspension is very sensitive to the valence of the counterion, therefore, calcium has a greater effect on bacterial adhesion than sodium.

9 THERMODYNAMIC ASPECT OF CELL ADHESION

10 TISSUE SORTING What is the work of adhesion for a single liquid? W o/o = E o/A + E o/A - E o/o = E o/A + E o/A – 0 = 2E o/A Suppose W o/w >= W o/o and W o/w >= W w/w Add these to get W o/w >= (W o/o + W w/w )/2 or >=E o/A + E W/A Recall from previous slide that: W o/w = E W/A + E o/A - E o/w So in this case, in order for E W/A + E o/A - E o/w >= E o/A + E W/A, E o/w must be <= 0….the liquids are totally miscible If we change the conditions in blue above, we can get separation or sorting. O W W W W W O O OO O W W W O O O O O O W W W W O O O O O W W W O O O O O O O O W W W W W W W SORTING: W o/o >W o/w >=W w/w RANDOM: W o/w >W o/o >=W w/w SEPARATION: W w/w >=W o/o >>W o/w

11 CELL/TISSUE SORTING

12 FORCES IN CELL ADHESION

13 CELL ADHESION: STRENGTH From Sundar

14 ADHESIVE DYNAMICS From Sundar

15 ADHESIVE DYNAMICS

16 ADHESIVE DYNAMICS

17 MEASURING ADHESION

18 ATOMIC FORCE MICROSCOPY

19 SURFACE FORCE APPARATUS

20 OPTICAL TWEEZERS (LASER TRAP) A particle encountering the laser beam will be pushed towards the center of the beam, if the particle's index of refraction is higher than that of the surrounding medium. In a ray optics picture we realize how light is deflected in the particle, resulting in a gradient force that pushes the particle vertically to the propagation of the laserbeam, towards the largest intensity of light (the middle of the laserbeam). By focusing the light, the gradient force pushes the particle backwards as well. If this force overcomes the propagation force of the laserbeam, the particle is trapped.

21 OPTICAL TRAP

22 MICROPIPETTE ASPIRATION

23 controlled conditions MEASURING CELL ADHESION

24 HOW CAN YOU MEASURE BULK ADHESION STRENGTH IN VITRO? Parallel plate flow chamber Cone and plate viscometer Rotating cylinder

25 SCHEMATICS OF ASSEMBLIES

26 CELLS CAN EXERT FORCES ON A SUBSTRATE THROUGH ADHESIONS

27 TENSEGRITY AND CELL ADHESION

28 FORCES DURING MIGRATION

29 TISSUE EQUIVALENTS Self-assembled biopolymer networks with entrapped tissue cells of interest. Cells exert traction on network resulting in compaction of the network and/or locomotion of the cells. Fibroblast Cell traction-induced alignment of network

30 ICTA - Methods Lexan Mold Medium with defined soluble factors Coverslip Stainless Steel Well

31 FREELY COMPACTING VS. CONSTRAINED GELS Freely Compacting Unstressed Constrained at Ends Stressed Presence of stress -> network alignment -> cell alignment

32 DYNAMIC REGULATION OF ADHESION

33 IN WHAT TISSUES DO CELLS REGULARLY EXPERIENCE MECHANOTRANSDUCTION?

34 MECHANOTRANSDUCTION It’s well known that shear flow affects endothelial cell behavior. How can you study this mechanotransduction system in a controlled manner? This would be the intima later of a vessel, which is lined by a confluent layer of endothelial cells.

35 LAMINAR FLOW IN A PIPE Anyone know what this flow rate law is commonly called?

36 SHEAR FLOW ON ENDOTHELIAL CELLS Anyone know what this flow rate law is commonly called? This defines the shear stress experienced by endothelium as a function of vessel radius and flow rate.

37 LAMINAR VS. TURBULENT Re low = laminar Re high = turbulent Laminar Turbulent D is on the order of microns Small reagent volume, Shorter reaction times “Low Reynolds number flow” All mixing is through diffusion In ‘Microfluidics’ …

38 IN VIVO FLOW PREDICTIONS

39 MECHANOTRANSDUCTION

40 HOW CAN YOU MEASURE BULK ADHESION STRENGTH IN VITRO? Parallel plate flow chamber Cone and plate viscometer Rotating cylinder

41 FLOW AFFECTS CELL SHAPE

42 RESPONSE OF ENDOTHELIAL CELLS TO SHEAR Just about every aspect of endothelial cell behavior can be regulated in part by mechanical signals.

43 RESPONSE OF ENDOTHELIAL CELLS TO SHEAR Just about every aspect of endothelial cell behavior can be regulated in part by mechanical signals.

44 MECHANOTRANSDUCTION IN VITRO eME bubble medium stir bar

45 ECM  CELLS Cells adhere to matrix by specific receptors that, just by binding, can initiate specific signal cascades Forces exerted on ECM are transduced to the cells, and forces by cells can remodel the matrix The distribution and strength of adhesion sites (either on cells or matrix/biomaterials) can control cell growth, differentiation, migration, traction etc…

46 CELL ADHESION AND BIOMATERIAL DESIGN Obviously the type and number of cell adhesion sites can be critical in optimizing a tissue engineered product Biomaterials can, therefore, be more than just “biocompatible/bioinert” – they can be “bioactive”. Fortunately, or unfortunately, after most cells adhere to a substrate, they begin to secrete their own matrix (especially fibronectin), which pretty much wipes out any specificity the biomaterial may provide.