Cell-Nanomaterial Interactions: The role of the surface

1. Karla Brammer1 1Materials Science and Engineering, UC San Diego Nanomaterials Lecture Jan. 6th 2011 Cell-Nanomaterial Interactions: The role of the surface

2. Cell nanostructures Cells are typically 1-100�m in size, but their interior and exterior contains many nanometer size objects.

3. Primary cell elements Nucleus (�Mayor of the City�) - This is often referred to as the 'brain' of the cell. The function of the nucleus is to control all other activities that are carried on within the cell. Cytoskeleton (�Steel Girders�) - makes up the internal framework, like the steel girders that are the framework for buildings in a city that gives each cell its distinctive shape and high level of organization. It is important for cell movement and cell division. Cell Membrane (�City Border�) - Like a city perimeter, cell membranes surround the cell and have the ability to regulate entrance and exit of substances, thereby maintaining internal balance. These membranes also protect the inner cell from outside forces.

4. The cytoskeleton: Internal nanostructured scaffolding Strengthens cell & maintains the shape Allows the cell to adapt, reorganize and change shape Plays important roles in both intracellular transport, cell signaling, gene expression and cellular division. Provides "tracks" with its protein filaments for transport of organelles, molecules, vesicles, etc

5. Cell membrane nanostructure Outer membrane of cell that controls cellular traffic. Contains proteins called integrins that span through the membrane and allow passage of materials. Proteins are surrounded by a phospholipid (hydrophilic head and two hydrophobic tails) bi-layer.

6. The importance of integrins: Surface receptors Typically, integrin protein receptors inform a cell of the molecules in its environment and the cell evokes a response. Integrins performs outside-in signalling But they also operate an inside-out mode They transduce information from the external environment, extracellular matrix (ECM), to the cell as well as reveal the status of the cell to the outside, allowing rapid and flexible responses to changes in the environment.

7. Influence of external nanostructures: ECM The normal cell environment is comprised of a complex network of extracellular matrix (ECM) with nano�micro scale dimensions with intricate features. In many instances the capacity of a cell to proliferate, differentiate, and to express specialized functions intimately depends on the presence and maintenance of an intact extracellular matrix. As I mentioned in the beginning, cells can sense the surface. The normal cell envrionment ECM features determine adhesion complexes, cytoskeletal organization, and intracellular signalling To adhere cells to their substratum and transmit signals from the enviroment to the cell interior. Schematic illustration of the nanotopographic details of the TiO2 nanotube configuration interacting with a cell. As I mentioned in the beginning, cells can sense the surface. The normal cell envrionment ECM features determine adhesion complexes, cytoskeletal organization, and intracellular signalling To adhere cells to their substratum and transmit signals from the enviroment to the cell interior. Schematic illustration of the nanotopographic details of the TiO2 nanotube configuration interacting with a cell.

8. Mechanisms for stimulating cell behavior

9. Motivation Fabricating nano-features upon biomaterial surfaces provides features that are on the same scale as the bio-environmental features that interact with cells. Looking at what happens on different nanotextures can help to uncover signaling pathways that promote the desired cellular response.

10. Nanomaterial � �Particles with lengths that range from 1 to 100 nanometers in two or three dimensions.� ASTM E 2456-06 : Terminology for nanotechnology. ASTM International 2006 Biomaterial � �A non-living material intended to interface with biological systems to evaluate, treat, augment or replace any tissue, organ or function of the body.� Chester 1986 Nanobiomaterial� �A biomaterial substrate composed of nanometer-scale components.� Example : the inorganic bone matrix is comprised of apatite crystals with final dimensions 400 x 30 x 75 �. Nanomedicine � �The monitoring, repair, construction and control of human biological systems at the molecular level, using engineered nanodevices and nanostructures.�

11. For non-leaching materials, the body �reads� materials through its surface. Surfaces are uniquely reactive. Surfaces are different from bulk. Surfaces readily contaminate Especially in fluid, more so in biofluids Surface structures are often mobile Much higher surface/volume ratio for micro/nano

12. Surface chemistry Surface energy/tension/wettability Surface roughness Crystallinity Surface charge Size of features (nano vs. micro) Geometry of features Redox potentials/ion release Other-mechanical properties such as elasticity, doping

13. Time sequence of response to a surface Protein adsorption onto surface (<1 sec) Development of protein monolayer or multilayers (1-10 sec) Adsprbed proteins denature (10-30 sec) Cellular attachment to proteins (30 sec �minutes) Cellular secretions to stimulate tissue response (soon after)

14. Cellular events at the surface ( A ) Initial contact of cell with solid substrate of adsorbed proteins. ( B ) Formation of bonds between cell surfacee receptors and cell adhesion ligands (protien funtional groups). ( C ) Cytoskeletal reorganization with progressive spreading of the cell on the substrate for increased attachment strength.

15. Why so much interest in nanostructured surfaces? Cells in our body are predisposed to interact with nanostructured surfaces developing subtle biomimetism. Protein adsorption characteristics are dependent on the surface features of implanted biomaterials. Integrins are transmembrane cell receptors interacting with the protein layer adsorbed to the scaffold. These interactions are governed by molecular events at nanometer scale. Proliferation, migration, differentiation and extracellular matrix production by cells during tissue repair are dependent on protein adsorption on the surface of implanted biomaterials. Nanobiomaterials have an even greater increase in number of atoms at their surface and possess a higher surface area to volume ratio than conventional microscale biomaterials. Thus scientific developments of nanobiomaterials are multiple, from wound healing, bone implants to bone (or cartilage) tissue engineering scaffolds, to stem cell differentiation.

16. The ability of the substratum to influence cell orientation, migration and cytoskeletal organization was first noted by Harrison in 1911. Grew cells on a spider web. The cells followed the fibers of the web in a phenomenon called physical guidance. Observed variations in the behaviour of cells due to the solid support they grow on. Later, in 1964, it was first proposed that cells react to the topography of their environment. Surface shape and physical features themselves. Since then, numerous studies have shown that many cell types react strongly to topography, especially on the nanoscale. The role of a substrate is more than merely providing mechanical support� Act as intelligent surfaces. Providing chemical and topographical signals. Guides and controls cell behavior.

17. Topography shapes cell behavior Influencing cell behavior using substrate topography, particularly in the sub-micron regime, is an attractive strategy for regenerative medicine applications and advanced tissue engineering. Many researchers are interested in investigating and understanding which way a cell reacts to different kinds of solid supports. Emerging literature presents many interesting findings on the effects of nanotopography: Influences extracellular matrix organization Enhances cell adhesion Alters cell morphology Affects proliferation Initiates intracellular signaling Provides contact/physical guidance Mediates stem cell differentiation Incorporating topographical consideration into the design of scaffold environments is becoming increasingly important in light of these studies. With advances in nanofabrication technologies the promise of and the unknown information about topographical effects in manipulating the cell-substrate interaction is now being uncovered� Influencing cell behavior proliferation to differentiation using substrate or implant topography is an attractive strategy for regenerative medicine applications. Substrate topography at the submicron range is of particular interest because the size range is comparable to extracellular matrix structures. Emerging literature presents many interesting findings on how nanotopography enhances cell adhesion, alters cell morphology, affects proliferation, initiates intracellular signaling, provides contact guidance and mediates stem cell differentiation. Incorporating topographical consideration into the design of a biomimetic microenvironment for cell culture will become increasingly important in light of these studies and practical with advances in nanofabrication technologies. This Highlight underscores the promise of and the unknown information about topographical effects in manipulating cell-substrate interaction and advancing tissue engineering. Interactions of cells with nanotopography is a rapidly expanding field Nanostructures serve as biointeractive stages promoting cell attachment, proliferation, and organization, in addition to acting as delivery vehicles for bioactive molecules Cells respond in the different culture conditions, express genes unique to their scaffold environment Influencing cell behavior proliferation to differentiation using substrate or implant topography is an attractive strategy for regenerative medicine applications. Substrate topography at the submicron range is of particular interest because the size range is comparable to extracellular matrix structures. Emerging literature presents many interesting findings on how nanotopography enhances cell adhesion, alters cell morphology, affects proliferation, initiates intracellular signaling, provides contact guidance and mediates stem cell differentiation. Incorporating topographical consideration into the design of a biomimetic microenvironment for cell culture will become increasingly important in light of these studies and practical with advances in nanofabrication technologies. This Highlight underscores the promise of and the unknown information about topographical effects in manipulating cell-substrate interaction and advancing tissue engineering. Interactions of cells with nanotopography is a rapidly expanding field Nanostructures serve as biointeractive stages promoting cell attachment, proliferation, and organization, in addition to acting as delivery vehicles for bioactive molecules Cells respond in the different culture conditions, express genes unique to their scaffold environment

18. Inspiration New fabrication technologies and new nanotechnologies have provided biomaterial scientists with enormous possibilities when designing customized tissue culture supports and scaffolds with controlled nanoscale topography. The main challenge: To effectively design these scaffold for specific tissue engineering applications. Choose the appropriate combination of biomaterial composition, size, structure, etc. Be able to tailor towards applications as challenging and complex as� (a) wound healing (b) new bone growth and orthopedic implant technologies (c) stem cell differentiation (d) reconnect nerves/spinal cord injuries/brain damage

19. Control and design of surfaces for desired tissue interactions Nano lithographic surfaces/Nanomachined surfaces�precise topographies Rough surfaces�promote adhesion of cells through focal contacts. Porous surfaces�promote ingrowth of tissue for mechanical interlocking, also used for enhanced surface effects. Fiber-meshed surfaces�can generate gradients of pore networks, imitates ECM fibers.

20. Nanopattern fabrication The definition of nano-structures on the substrates relies on the clean room lithographic and Si processing methods. Lithography Usually, a computer-designed pattern is exposed by means of light, electrons, ions or imprinted. Carried out on a special light or electron-sensitive material or imprinted into a special deformable polymer, which is then used in subsequent pattern transfer processes as a mask, or, alternatively, used for cell culturing as it is.

21. Contact guidance Controlling cell adhesion, orientation and morphology through topographical patterning is a phenomenon that is applicable to a wide variety of medical applications such as implants and tissue engineering scaffolds. Wound healing Neuron alignment Effects on cells based on �ridge� features. Sharper angles orient cells better. Cells adhere better when aligned with surface texture. Possible explanations: Higher surface energy leads to preferential protein binding Alignment of adhesion plaques Abrupt edges lead to alignment or bridging Stochastic spreading of cells

22. Directed migration mechanism External guidance clues: Topography of the extracellular matrix. Protein adhesion on the features. Intracellular polarity machinery and adhesion receptors direct the adhesion and cytoskeletal remodelling that is necessary for lamellipodium formation (the moving front) to align.

23. Epithelial cells align Cell elongation and alignment on grooves and ridges of nanoscale dimensions was compared with the morphology and orientation of cells cultured on smooth substrates. It was found that ridges 70 nm wide induced human corneal epithelial cells to elongate and align along the topographic features. Valuable for the development of implantable prosthetics.

25. Morphological changes in cells Aside from grooves inducing the elongation of cells, geometric features (wells, pits, pillars) with dimensions in the nano range seem to effect the cell morphology, organization of the cytoskeleton and internal framework. In general, significant increases in cell spreading and intricate shape changes are measured in cells on nanotopographies compared to cells cultured on planar controls. This behavior seems to be related to the fact that the cell membrane in contact with the nanostructured surface will suffer tensile and relaxation mechanical forces that will rearrange its components, such as integrin complexes and thus signaling cell morphological changes.

26. Mechanotransduction Cells are exquisitely sensitive to forces of varying magnitudes, and they convert mechanical stimuli into a chemical response. Integrin�cytoskeleton linkage is sensitive to force. Close-up of a focal adhesion showing the balance of external and internal forces (Fext and Fcell, respectively) in driving stress at a mechanosensor. Depicted are actin stress fibers (red) anchored into focal adhesions (multicolored array of proteins) that bind to the ECM (blue) through integrins (brown). This balance of forces provides the stress necessary for mechanical sensing. This triggers a cascade of reactions that alter the balance of anabolic / catabolic events within the cell. This is a major regulator in cell homeostasis and development.

27. Mesenchymal stem cell differentiation Isolated from bone marrow. Self-renewing multi-potential cells with the capacity to differentiate into many distinct cell types. Osteoblasts (Bone) Chondrocytes (Cartilage) Adipocytes (Fat) MSCs play an important role in treatment for trauma, disease, or aging.

28. Differentiation by topography only �A key principle of bone tissue engineering is the development of scaffold materials that can stimulate stem cell differentiation in the absence of chemical treatment �� - Dalby

29. Polymer nanoscale order vs. disorder Defined e-beam lithography approach using polymethylmethacrylate (PMMA) . 120-nm-diameter, 100-nm-deep nanopits Demonstrated the use of nanoscale disorder to stimulate mesenchymal stem cells to produce bone mineral in vitro. Claimed that the focal adhesion complexes determined by the nanostructure effected the differentiation pathways.

30. Cell sense the surface Cells are responding to the nanotopography even in a such a small range of dimensions or disorder� Protein adhesion (fibronectin and albumin) are affected by the surface features. Changes in adhesion formation: Impose morphological changes on cells. Impacts on cytoskeletal tension. Affects indirect mechanotransductive pathways. Surface nanotopography directly induces pronounced changes to cell shape, and consequently gene expression, which can potentially mediate differentiation of stem cells into various cell types. This is clear evidence that surface nanotopography directly induces pronounced changes to cell shape, and consequently gene expression, which can potentially mediate differentiation of stem cells into various cell types. This is clear evidence that surface nanotopography directly induces pronounced changes to cell shape, and consequently gene expression, which can potentially mediate differentiation of stem cells into various cell types.

31. Ceramic nanopores/nanotubes Electrochemical anodization Anodic Aluminum Oxide (AAO) TiO2 Nanotubes Controllable features Film thickness Pore size/diameter Wall thickness Interspaces

32. Nanoporous alumina (AAO) SEM of two-step anodization process for the fabrication of nanoporous alumina surfaces. Observed difference in cell morphology. Cells on flat surfaces are spherical whereas on nanoporous surfaces seem to be spreading Different adhesion mechanism on nano-features. Demonstrated: An increase in the alkaline phosphatase activity, bone forming ability on nanoporous surface. Enhanced matrix production of MSCs when they are cultured on nanoporous alumina substrates. I want to first discuss some relevant studies in the literature that use the nano surface structure as a mechanism of differentiation. I want to first discuss some relevant studies in the literature that use the nano surface structure as a mechanism of differentiation.

33. Mechanical interlocking � TiO2 nanotubes Osteoblasts (bone cells) Higher density of cells. 3-4X accelerated growth. Up-regulated mineralization. Intricate interaction. Our initial observation of using tio2 nanotubes in osteoblast or bone cell culture was just the beginnning of showing the beneficial effects of of using nanostructures in tissue engineering. These experiments for first conducted by our dr brian oh Comparative back-scattered SEM images of growing osteoblast cells on the surfaces after 48hrs. of culture. Comparative graph showing number of adhered cells as a function of culture time. ALP activity of MC3T3-E1 osteoblast cells cultured for 12, 24, and 48 h of incubation Micrograph showing the adhesion of the osteoblast cells (after 2h) on vertically nanoporous TiO2 nanotubes. The circles show cells are anchoring into the nanotubes. Our initial observation of using tio2 nanotubes in osteoblast or bone cell culture was just the beginnning of showing the beneficial effects of of using nanostructures in tissue engineering. These experiments for first conducted by our dr brian oh Comparative back-scattered SEM images of growing osteoblast cells on the surfaces after 48hrs. of culture. Comparative graph showing number of adhered cells as a function of culture time. ALP activity of MC3T3-E1 osteoblast cells cultured for 12, 24, and 48 h of incubation Micrograph showing the adhesion of the osteoblast cells (after 2h) on vertically nanoporous TiO2 nanotubes. The circles show cells are anchoring into the nanotubes.

34. TiO2 nanotubes for stem cell therapies In light of those studies we too explored the possibility of differentiaiton based on our nanotube surface. We observed that the stem cell fate was dictated by the nanotube dimensions. First there is a distict difference in the way that the proteins adsorbed to the surface first two hours of incubation Secondly, that it affected the way the cells could adhere to the surface. Thus affecting the morphology of the cells and internal cytoskeletal stress Which induced differentiation into osteoblasts or bone cells The PCR relative transciption levels showd the highest on 100nm surfaces. In light of those studies we too explored the possibility of differentiaiton based on our nanotube surface. We observed that the stem cell fate was dictated by the nanotube dimensions. First there is a distict difference in the way that the proteins adsorbed to the surface first two hours of incubation Secondly, that it affected the way the cells could adhere to the surface. Thus affecting the morphology of the cells and internal cytoskeletal stress Which induced differentiation into osteoblasts or bone cells The PCR relative transciption levels showd the highest on 100nm surfaces.

35. Mimicking bone properties Advantages of using nanoporous AlO2, TiO2, and other ceramic scaffolds. Bone (trabecular) is porous in nature. Nanoscale ECM properties have similar ceramic material properties Granular minerals and collagen fibers TiO2 crystal structure has same crystal structure as hydroxyapatite. Similar chemistry To help orthopedic implant �osseo�-integration into existing bone.

36. ECM Collagen fibers It is the main component of connective tissue, and is the most abundant protein in mammals. Abundant in cornea, cartilage, bone, blood vessels, the gut, and intervertebral disc. �Tropocollagen� is approximately 300�nm long and 1.5�nm in diameter.

37. Hydrothermal TiO2 nanotubes: Enhanced mineralization Hydrothermal reaction in autoclave chamber of Ti foils in 10M NaOH solution. 8nm diamter, 150nm tall tangled nanotubes. Could be advantageous for imitating the natural ECM environment of collagen type I fibers having fibrous and tangled nanoscale geometry. This is an important aspect to bone scaffold designs in order to present host cells with an innate ECM interface where it triggers repopulation and resynthesizing a new matrix.

38. Nanofiber designs Electrospinning technique: Nanofibrous scaffolds are promising candidate scaffolds for cell-based tissue engineering. More closely mimics the niche-like unit (ECM fibrils) for facilitating proper cell behavior. Electrospinning generates loosely connected 3D porous mats with high porosity and high surface area which can mimic ECM. Can be randomly or aligning on the surface based on ground collection plate Made mostly of organic polymers. Can be bioresorbable/biodegradable Natural materials or synthetic The standard laboratory setup for eletrospinning consists of a spinneret, typically a syringe, connected to a high voltage (5-50kV) directed current power supply , a syringe pump and ground collecter. When a voltage is applied to a solution an eletric field is generated between a charged polymer liquid and grounded collecter. As the power is increased, the charged polymer solution is attracted to the fiber collector. Once the voltage reaches a critical value, the charge overcomes the surface tnesion of the polymer cone formed on the capillary tip of the syringe, known as a taylor cone. As the jet dries out in flight the mode of current flow changes from ohmic to convective as the charge migrates to the surface of the fiber. The jet is then elongated by a whipping process caused by electrostatic repulsion initiated at small bends in the fiber until it is finally deposited on the grounded collector. Finally, ultrafine fibers are produced accululating randomly or aligning on the surface of the fiber collector. The standard laboratory setup for eletrospinning consists of a spinneret, typically a syringe, connected to a high voltage (5-50kV) directed current power supply , a syringe pump and ground collecter. When a voltage is applied to a solution an eletric field is generated between a charged polymer liquid and grounded collecter. As the power is increased, the charged polymer solution is attracted to the fiber collector. Once the voltage reaches a critical value, the charge overcomes the surface tnesion of the polymer cone formed on the capillary tip of the syringe, known as a taylor cone. As the jet dries out in flight the mode of current flow changes from ohmic to convective as the charge migrates to the surface of the fiber. The jet is then elongated by a whipping process caused by electrostatic repulsion initiated at small bends in the fiber until it is finally deposited on the grounded collector. Finally, ultrafine fibers are produced accululating randomly or aligning on the surface of the fiber collector.

39. Nanofiber advantage Scaffold architecture affects cell binding and spreading. The cells binding to scaffolds with microscale architectures flatten and spread as if cultured on flat surfaces. The scaffolds with nanoscale architectures have bigger surface area for absorbing proteins and present more binding sites to cell membrane receptors. The adsorbed proteins further can change the conformations, exposing additional binding sites, expected to provide an edge over microscale architectures for tissue generation applications.

40. General remarks Toxicity must be avoided but inertness is not a high priority in nano/biomaterials. Bioactivity: positive interaction and effects on the human body. Metallics, alloys and ceramic biomaterials are useful for hard tissue (bone) but not suitable to replace soft tissues because of markedly different mechanical properties. Must match the material properties to the body material Consider modulus, shear, wear particles, fatigue, strength Applicable for 3-D geometries Conventional polymers are used for many of today�s pliable disposable or biodegradable medical devices. New functional biomaterials are anticipated�

41. Take Home Message The eukaryotic cell is unimaginably complex and remarkable. Controlling interactions at the level of natural building blocks, from proteins to cells on the nanoscale, facilitates the novel exploration, manipulation, and application of living systems and biological phenomena. The nanostructured surface of a material can improve its interaction with cells and provide a desired response. Nanostructured tissue scaffolds and biomaterials are being applied for improved tissue design, reconstruction, and reparative medicine.

42. Questions