Nano Materials for Potential Cardiovascular and Other Biomedical Applications

Sungho Jin Professor of Materials Science University of California, San Diego Outline Introduction Nanotechnology for medicine using forefront new materials --- Nano Particles --- Nano Needles --- Nanostructured Substrates Potential Applications for Cardiovascular Research and Therapeutics Summary

Types of nanomaterials being studied for medical applications 1. Nano Particles --- Iron oxide mangetic nanoparticles --- Au nanoparticles --- Other shapes, e.g., core-shell coated, rod shape, hollow geometry, composites with quantum dot, etc. 2. Nano Needles --- Carbon nanotubes --- Sharp probes for ion channel study --- Nano-pipettes for single cell manipulations 3. Nanostructured Substrates --- Porous substrates --- Protruding arrays of soft or hard pillars Most of these nanomaterials are being fabricated and their medical applications being investigated at UCSD.

Strong Recent Trend --- Convergence of NanoTech and BioTech

Magnetic nanoparticles

(e.g., superparamagnetic Feoxide) --- actively investigated for targeted cancer treatment (magnetic hyperthermia), stem cell sorting & manipulations, guided drug delivery, gene therapy, DNA analysis, and medical diagnosis (magnetic MRI). Potential use of nanotubes/nanowires for nanoscale probing of cell behavior, biosensing, therapeutics, etc. Quantum dots for bio imaging, genome sequencing, etc. Au nanoparticles for imaging, photothermal cancer treatment (by laser). These are all very rapidly advancing fields. Many opportunities exist to design/develop diagnostic or therapeutic techniques, and instrumentations using these nanotechnologies.

Multifunctional Nanoparticles for Therapeutic and Imaging Applications (from NCI)

Nanoparticles -- can be designed to target specific cells or biomolecules with the ability to reach those targets by crossing tissue and membrane barriers. Multifunctional nanoparticles may combine a targeting agent specific for a protein biomarker, with conjugated agents for imaging, therapy, and reporting efficacy Magnetic core can provide mobility, imaging (MRI), and local temperature rise if needed.

Magnetic Nanoparticles
Fe-oxide nanoparticles have been used as magnetic recording media (audio or video tape, disk, etc.) Fe3O4(magnetite) or -Fe2O3 (maghemite) 6-12 nm diameter particles --- Need to be superparamagnetic to avoid agglomeration. Biocompatible. Magnetic nanoparticles --- actively investigated for targeted cancer treatment (magnetic hyperthermia), stem cell sorting & manipulations, guided drug delivery, gene therapy, DNA analysis, and medical diagnosis (magnetic MRI). Biomagnetics --- Potentially a huge field for science and applications of magnetism and magnetic materials!

 Different types of magnetic materials ---Magnetic Properties can be altered by chemistry, geometry and processing.

M Mr Hard Magnetic Hc H SuperParamagnetic

(Zero Hc, zero Mr, low )

Soft Magnetic

Magnetic nanoparticle synthesis and size control

--- Typically by reduction of Fe-salts, using micelle or other surfactant configurations. --- Experimental apparatus and conditions.
Gas inlet Reaction Temperature Cooling water 100 oC Oleic acid Oleylamine Metal precursor R.T. 1,2-Hexadecandiol Nanoparticle seed Phenyl ether Thermocontroller

Control of Magnetic Nanoparticle Size

TEM micrographs of Fe3O4 nanoparticles

20 nm

Fe3O4 Fe3O4 st As-nucleated After 1 particle enlargement seeds

Fe3O4 After 2nd particle enlargement

2-3 nm

5-6 nm

12-15 nm

TEM Micrographs for Chemically Synthesized Magnetic Nanoparticles at UCSD

For On-Going R&D For Potential Bio Applications Cancer treatment, gene delivery, neural regeneration, drug delivery, cell sorting, MRI. (a) Superparamagnetic Fe3O4 (c) Fe O Magnetic Nanoparticle Array
3 4

10 nm

(b) Silica-Coated Fe3O4

(c) Elongated Magnetic Nanoparticle

Composite Nanoparticles --- Quantum Dot+Magnetic Particle

Quantum Dot (Cd/Se with ZnS shell) Magnetic Nanoparticle

(a)

(b)

CdSe/ZnS qdots conjugation onto the magnetic nanoparticles. (a) Schematic illustration, (b) TEM micrograph showing the satellite conjugation of qdots on iron oxice particle. The scale bar is 5 nm.[From Wang et al, Nano Lett, 4, 409-413 (2004). ] --- Possibly configured and applied for combined medical applications of bio-imaging + magnetic MRI + magnetic hyperthermia treatment of cancer.

Magnetic Nanoparticle Quantum Dot Dual Structure

H. Gu et al, JACS 126 ,5664 (2004). Facile One-Pot Synthesis of Bifunctional Heterodimers of Nanoparticles: A Conjugate of Quantum Dot and Magnetic Nanoparticles

Sulfur Coated FePt

TOPO+Cd(acac)2 (FePt coated with Amorphous CdS)

Crystalline CdS

FePt (magnetic) CdS (quantum dot)

Biotech applications --- Magnetic nanoparticles can be utilized as a localized, intra-cellular source of Programmable heat (by applied AC magnetic field) --- Active R&D projects in progress worldwide.

Example; Magnetic Hyperthermia --- selective

(localized) heating of tumor cells using magnetic nanoparticles which are targeted and attached onto the cancer regions. Typically uses ~100 KHz ac magnetic field.

Temperature rise induced by remote magnetic field (100 KHz used) --- In a liquid containing various percentage of magnetic nanoparticles. --- ~10 nm diameter Fe3O4 particles.

80 Avg. Temp. [oC] 70 60 50 40 30 20 10 0 0

6 % Volume Fraction 3% 1.5 % 0.75 % 45oC

(a)
target

PC-12 neural cancer cells with endocytosed magnetic particles.

(b)

0.15 %

200 400 600 800 1000 1200 1400 Time of AC Magnetic Heating [sec]

Nano-Bio Tech Biomedical Devices for Cancer Treatment Highly interdisciplinary (materials, devices, physics, chemistry, biology,
biomedical engineering, electronics, mechanical engineering, manufacturing ). Example --- Magnetic Hyperthermia Cancer Treatment using Targeted Magn. Nanoparticles (Jordan et al., Dept. of Radia. Oncology, Virchow Univ., Germany). [Progress in Clinical Trial]

Cancer Patient with injected or targeted magnetic nanoparticles

AC Excitation Magnetic Field Solenoid

Control Systems

Potential modifications/control of embryonic stem cells using nanotechnology

Cells (including stem cells) are known to be affected by --- Temperature --- Mechanical strain --- Chemicals, ions, drugs --- Biological agents (proteins, growth factors, etc) --- Electrical voltage impulses Use of nanomaterials and nanotechnology to artificially introduce these changes to single cells or multiple cells. To control differentiation and multiplication of hESC.

Carbon Nanotubes, Sharp Probes, etc. Fabricated at UCSD for Bio Applications

(a)

Sharp CNT Nanocone Tip Array for AFM Probe

Extremely sharp AFM Probe (~1 nm tip) --- For ion channel conductivity study

(b)

2 m

1 m

10 nm nm 10

(b)

Nanocone Tip Array

(c)

Substrate Biomolecules, DNAs, etc. Magnetic or gold nanoparticles

Substrate

Magnetic Particles for Targeted Drug Delivery (e.g., gene therapy, cancer chemotherapy applications)
Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo, F. Scherer et al., Nature/Gene Therapy 9(2), 102-109 (2002).

Gene therapy ---Correcting defective genes responsible for disease by inserting a normal gene to replace a non-functional gene, or repair. Associate gene vectors with superparamagnetic nanoparticles and targeted gene delivery by application of a magnetic field. (Coating of magnetic particles with colloidal chemistry and attach DNAs). Enhanced efficacy by several hundred-fold, reduction of the duration of gene delivery time to minutes. High transduction efficiency observed in vitro was reproduced in vivo with magnetic field-guided local transfection (in the gastrointestinal tract and in blood vessels). Magnetofection provides a novel tool for high-throughput gene screening in vitro and can help to overcome fundamental limitations to gene therapy in vivo. Cardiovascular disorders Many cardiovascular diseases are excellent targets for somatic gene therapy as drug therapy may have reached a plateau in its effectiveness. Use of gene transfer approaches for long-term control of the pathophysiological conditions being explored by various groups. Gene vectors (such as adeno-associated viruses (AAV)) are currently available that can efficiently transduce cells of the myocardium & vasculature. Magnetic vectors may also be explored.

On-going cardiovascular-related R&D using nanotechnology (at the Jin lab)

(John Gallagher, Karla Brammer, Brian Oh)

1. Remote pacemaker. 2. Tissue engineering for growth of cardio-myocyte on cyclically moving substrate. 3. Endothelialization of coronary stent surface to minimize Late Stent Thrombosis

1. Research Toward Remote Pacemakers

Current pace maker systems use wire connection between heart and the implanted, battery-operated control circuit system. Lead wires --- involve complicated surgery going through blood vessels, with a risk of foreign body rejection on lead wires. Substantial risks associated with leaving failed and unused leads in place --- Increased likelihood of infection or blood clot formation around the old and entangled pacing leads, and the leads can restrict the operation of the heart valves and hinder the implantation of new leads in the heart, etc. Pacemaker lead explantation(removal) --- complicated, time-consuming procedure with significant risk.

Foreign body response

Left, Pacing lead firmly attached to ventricular wall. Distal tip appears to have typical foreign body response, with collagenous sheath ranging from 0.25 to 1.1 mm in thickness around the electrode. Distal half of lead is buried deeply between trabeculae of right ventricle. Except for approximately 1 cm (located 3 cm from distal tip), pacing lead is encapsulated in collagenous tissue and attached to tricuspid valve and chordae tendineae. Right, Atrial portion of pacing lead is encapsulated except for proximal one-third.

RETO CANDINAS, M.D.; FIRAT DURU, M.D.; et al , Mayo Clin Proc. 1999;74:120-125 1999

Remote pacemaker without lead wires

It would be highly desirable to completely eliminate the pacemaker leads -- e.g., by Remote Pacing; --- Surgical installation of a pacemaker to patients become easier, with no issue of scar tissue formation around the lead, etc. --- No need to be concerned about the risk of pacemaker lead explantation or leaving the unused lead in place. --- Remote pacing can be explored -- by remotely stimulating human cardiomyocytes with a supply of voltage impulses to a simple, needle-shape, implanted electrode.

Functional Electrical Stimulations

Human and animal nerve systems -- Communicate and control body functions by voltage impulses which travel between brain, sensory nerves, muscles, organs, etc. Control of neural functions with electrical stimulations -Intriguing and can potentially serve as powerful remedy Evidence of clinical effect for relief/control of pain (headache), epilepsy, spasticity, movement disorders (para- or quadriplegic), bladder function, accelerated bone fracture healing, etc.

Galvanis experiment in 1790 -Muscle contraction from bi-metallic rod contacting frog leg nerves

Various types of Electrodes for FES

} Percutaneous stimulation using

lead wires

Transcutaneous stimulation with no lead wires Enables remote control/activation

Small wire electrode implanted --- Minimal chance of infection Highly locallized stimulation treatment minimizes side effects in unwanted regions

New Magnetic Actuator Alloy Wires --- Fast magnetization reversal & large induced voltage (V d/dt) New Sensor Alloys
B B

Wiegand wire

New alloys Fe20Cr4Ni Fe6Ni Fe-10Si-15B (amorphous) HC 10~20 2~10 0.1~10 4MS 15kG 20kG 14kG

Vicalloy 52Co38Fe10V HC~10 Oe, 4MS~16kG

Hard shell + soft core Complicated processing Expensive High output voltage

uniform structure easy to process & reproducible low cost (no cobalt) high output voltage & wide pulse lower drive field

Possible Stimulation or Repair of Damaged Neurons

Broken nerve

Remote electrode

Implanted voltage-generating wire electrode (e.g., helically magnetic Fe-6%Ni) is transcutaneously actuated by remote magnetic field --- To induce muscle movement or to stimulate for nerve regeneration.

(a) Use square-loop magnetic wire as a core

(b) Use helically anisotropic magnetic wire which generates end-to-end voltage

Voltage generated on remote AC magnetic field

Voltage generated on remote AC magnetic field

Fig. 3. Two approaches to supply remotely actuated voltage impulses to implanted sensor wires for heart pacing.

Square loop magnetic alloy wire (e.g.,Fe-20%Cr-4%Ni, Fe-15%Cr-5%Mo)

(a)

M
Pulse field magnetized (e.g. 1 msec) 0

Elongated nanostructure for rapid switching and large induced voltage

H
Hc

Steep sidewall in M-H curve for rapid magnetic switching

500 nm

10

(a)
38.0
Fe-20Cr-4Ni D=0.05 cm, L=3.7 cm N=1000 turns coil f= 1000 Hz

(b)
Fe-20Cr-4Ni D=0.05 cm, L=3.7 cm N=1000 turns coil Hmax = 200 Oe

37.5 2.0

Volts

Volts

1.5 1.0

0.5
0 0 50 100 150 200 250 300

AC Applied Magnetic Field (Oe)

1 5 10 20 60 5000 Freq. of Appl. Magn. Field (Hz)

Fig. 4. Induced voltage impulse in the solenoid surrounding a square loop magnetic wire of Fe-20Cr-4Ni alloy as a function of (a) amplitude of applied AC magnetic field, (b) frequency of magnetic field applied.

End-to-End Voltage Impulse Generated by Helically Oriented Fe-6%Ni Needle

10 mil dia., 4 long wire, plastically twisted by 2 turns H=200 Oe, 60 Hz AC changing magnetic field (sinusoidal) Voltage directly from the wire ends (no solenoid used)
0.4

Voltage outputs (V)

0.3 0.2 0.1 0

Ha=200 Oe Ha=20 Oe

Voltage outputs (V)

0.3 0.2 0.1 0 -0.1 -0.2 -0.3 80 ms 800 s Time

0 180 360 540 720 Total Torsional Twist (degrees)

Electrical Stimulation of neonatal cardiac myocytes

Primary neonatal rat myocytes grown at low density. Myocytes are paced easily with the aid of a function generator and applying voltage impulses through two lead wires. Pacing demonstrated at 0.5 Hz frequency using 5-8 volts and 5 ms pulse width. Heart beat on-off control by simulated cardioplegic arrest (by incubating myocytes in a crystalloid cardioplegic solution) + electrical voltage pacing Remote pacing experiments with magnetic needles (or thin solenoids with magnetic needle cores) in progress.

Remote Pace Maker (with pulse rate sensor + voltage impulse generator)

(a)
Heart with implanted electrode needle Remote pace generator (e.g., magnetic pulse field train generator) Sensor outside the body for heart beat rate/ intensity sensing (piezoelectric, strain gauge, or pressure sensor) Chest and shoulder belts to hold pace generator in place, and to keep heart beat sensor in skin contact

(b)
Implanted pulse rate sensor either on the electrode or a separate piece (solenoid device, piezoelectric, strain gauge, pressure sensor, etc.) with RF signal emitting systems Remote pace generator RF signal sensor/ analyzer to detect irregular heart beats and initiate remote pacing (e.g., by activating magnetic field) Implanted electrode piece

RF signals

2. Culture of cardio myocytes on moving substrates

-- After myocardial infarction, some area of heart tissue becomes damaged. -- For repair/replacement of damaged heart tissues --- Heart tissue regeneration on beating heart would be desirable. However, cells are not easily cultured on moving substrates. -- Major hurdle would be, when you have created a construct, how to incorporate it into the heart, a dynamic environment which is not the case in a normal tissue culture environment. -- New, nanostructured substrates (e.g., TiO2 nanotubes) which enable strong adhesion of cells can be utilized. (based on our recent data with osteoblast and hepatocytes). -- Interaction of cells with nano topography is a rapidly expanding field. -- TiO2 nanotube substrates were constantly moved at ~ heartbeat frequency, and rat myocyte cell adhesion/growth was studied in the Jin lab.

Experimental set-up for myocytes adhesion on moving substrates

Disect and isolate neonatal ventricular myocytes

TiO2 nanotubes

Flat polished Ti

Rotating cantilever

Soft polymer tubing Site of impact

Motor Cell culture substrate

TiO2 nanotubes for accelerated cell growth

Vertically aligned TiO2 nanotube array on Ti (~80 nm dia, 300 nm tall)

Cross-sectional TEM of Aligned TiO2 Nanotubes (top view) Nanotube wall Spacing between nanotubes (~10 nm thick)

TiO2 nanotubes

Ti base
500 nm
100 nm

Effect of Nanotube Surface Layer on Osteoblast Cell --- Significantly Accelerated Growth by ~ 300 400% Number of Adhered Cells, 102/cm2 1000 800 600 400 200 0
On bare implant metal (such as Ti) On amorphous TiO2 nanotubes On crystalline TiO2 nanotubes

10 20 30 40 Incubation Time (hrs)

50

60

Comparative myocyte cell culture

Flat Ti

Glass culture plate

TiO2 nanotube

Number of cells attached with vs without mechanical pertubation

Mean number of Cells attached
70 60

# of cells

Nanotube

50 40 30 20 10 0

Nanotube

Nanotube TiO2 Flat Ti

Glass Flat Ti Flat Ti Glass

Glass

Control (No movement)

}
Agitated

Effect of TiO2 naotubes on myocyte attatchment

Area of myocyte spreading on 3 types of culture surfaces (after 72 hrs)
Area of cell spreading (m)
800 600 400 200 0 Flat Ti Glass

TiO2 Nanotube

Surface type

3. Prevention of Late Stent Thrombosis (LST): A Cellular Approach (by Karla Brammer (GSR))
http://www.smh.com/img/hv_institute/stent_eluting.jpg

Issue --- There is a clinical problem of late stent thrombosis (blood clotting, blockage of blood vessel, heart attack, etc.) due to a lack of endothelialization of the inner stent wall, where the stent fails to be fully integrated into the vessel wall. Proposed Solution --- A possible novel solution to the clinical problem of late stent thrombosis is to pre-endothelialize the stent with the patients own vascular endothelial cells prior to stent implanting. (Perhaps preventing restenosis, thus eliminating the need for drug-eluding stents?). To test this hypothesis --- Culture endothelial cells on conventional biocompatible, metallic, implant material with different surfaces (flat vs nanostructured). Look for --- attachment, adhesion, and spreading characteristics of endothelial cells on the different surfaces Determine --- the endothelialization of cells on the surfaces for potential use in stent applications. Some in vivo animal tests may follow.

7/9/07

(a)

Stent longitudinal cross-section Thin cultured endothelial cells

(b)

Stent crosssection

(c)

(d)

Artery vessel

Lumen

Fig. 7

Background: Endothelial Cells

Endothelial cells form the inner lining of a blood vessel and provides an anticoagulant barrier between the vessel wall and blood The endothelial cell is a multifunctional cell with critical basal and inducible metabolic and synthetic functions Endothelial cells retain a capacity for cell division and movement. They proliferate and migrate readily and can be stimulated easily. Endothelial cell injury, activation or dysfunction is a hallmark of many pathologic states including atherosclerosis (fatty substance deposit), loss of semi-permeable membrane function, and thrombosis.

Pure Ti vs. TiO2 nanotubes A comparative study of culture substrates

Growing cell that Adheres on TiO2 nanotubes Cell nucleus TiO2 nanotube array Spacing between TiO2 Nanotubes

Ti substrate

Figure 3. Biocompatible Ti chips with vertically aligned TiO2 nanotubes. Previous work with an osteoblast cell line proves that the pronounced topological feature and increased surface areas plays a significant role in the adhesion/propagation of osteoblast cells. The TiO2 nanotubes allow for filopodia of growing cells to actually go into the nanotube pores, producing a type of interlocking cell structure. Large surface areas, small pores, gaps between nanotubes are thought to facilitate long term survival and function of primary hepatocytes Super Hepatocyteswith enhanced functionality -- 3-D configuration and albumin secretion function better than real liver! There are pathways for fluid present between the nanotubes. Use of TiO2 nanotube surface is now being explored for pre-endothelialization experiments.

Experimental Procedure
Plate 120,000 primary bovine aorta endothelial cells in 1ml media per each one well of a 12-well plate containing a 1.27cm x 1.27cm square Ti or TiO2 nanotube chip. Preformed a flouroscein diacetate (FDA) stain of viability to observe morphology/spreading of cells at time points 6, 12, 24, and 48 hours after plating
6 hours 12 hours 24 hours 48 hours 6 hours 12 hours 24 hours 48 hours

VS.

Pure Ti

TiO2 Nanotube

Flat Ti substrate

TiO2 nanotube substrate

FDA(flouroscein diacetate) viability of Bovine Aorta Endothelial Cells (BAECs) on Ti flat vs TiO2 nanotube substrates after 24 and 48 hours of culture were compared. --- An aggregated, disrupted, and possibly detached status of cells on the Ti surface was seen. --- A flat monolayer of cells on the TiO2 surface similar to natural endothelium were observed.

Figure 2. SEM micrographs of BAECs on flat Ti (a,c) vs TiO2 (b,d) surfaces after 2 hours of culture. Loose, detached filipodia are present on the Ti substrate (arrow) while filipodia are able to probe and survey (arrows) on the TiO2 surface as they are even seen protruding into the pronounced features of the nanotubes (white dashed circles).

(a)

(b)

500 nm

500 nm

Figure 3. SEM micrographs after 6 hours of incubation. (a) BAECs are deficient in ECM material on the flat Ti controls compared to (b) where a coarse ECM material is deposited on and filled in the nanotubes on the TiO2 surface.

Polystyrene

Flat Ti

TiO2 Nanotubes

50 m

50 m

Figure 4. Immunofluorescent images of cytoskeletal actin for BAECs after 24 hours of culture. The lower magnification images show that the nanotube surface increases cell to cell interactions. The higher magnification images reveal that the nanotube surfaces trigger more organized, prominent lamellipodia for increased cell locomotion.

Polystyrene

Flat Ti

TiO2 Nanotubes
50 m 50 m

Figure 5. Immunofluorescent images of cytoskeletal actin for BAECs after 48 hours of culture. The BAECs on the nanotube surface are more elongated and mobile. The nanotube surface generates greater cell communication, interaction, and movement where BAECs on flat controls are spread wider with more stationary morphology. Unstable and broken cell extensions are seen on the Ti surfaces.

Cell Spreading Area (m2)

1000 800 600 400 200 0

* **
Figure 6. Analysis of BAEC morphology on polystyrene tissue culture plates, flat Ti, and TiO2 nanotubes.

Polystyrene Ti

TiO2 Nanotube

Minor/Major Axis Ratio

0.6 0.5 0.4 0.3 0.2 0.1 0.0

The spreading area and minor/major axis ratio are calculated from at least 100 cells.

**

Polystyrene Ti

TiO2 Nanotube

Figure 7. Immunofluorescent staining of vinculin at focal adhesions. (a) Cells on flat Ti and (b) Cells on TiO2 nanotubes.

--- In the endothelium, nitric oxide (NOx) is continuously synthesized as an important vasodilator (vascular endothelium relaxation factor) which inhibits platelet aggregation. --- On the other hand, the release of endothelin-1 counteracts NOx and is a vasoconstrictor that promotes platelet aggregation.

NOx/Endothelin-1 Ratio

2.0 1.5 1.0 0.5 0.0 Polystyrene Ti

NOx/Endothelin-1 ratio of endothelial cells on different culture substrates. --Higher ratio on TiO2 nanotubes indicates a reduced platelet aggregation and risk of LST.

TiO2 Nanotube

Comparative time-dependent migrational assay for bovine aortic endothelial cells on different culture substrates
Cells behind the moving front

Polystyrene

100 m

Polystyrene, flat Ti controls, vs TiO2 nanotubes with 50, 70, 100 nm diameter. The red dotted line indicates the start line of the cell migration.

}
Flat Ti

}
24 hours migration

50 nm Nanotube 70 nm Nanotube 100 nm Nanotube

The cells on the 100nm diameter TiO2 nanotube surface have traveled the greatest distance after 24 hours and are much more elongated (indicating a migrational morphology with enhanced movement and a higher mobility rate compared to smaller sized nanotube surfaces and flat Ti controls).

Before migration

12 hours migration

 Primary bovine aorta endothelial cells were able to interact more efficiently with the TiO2 nanotube surface with enhanced cellular migration and functioning as compared to a flat Ti surface. The nanotubes also stimulated an increase in ECM deposition and more organized lamellipodia on the nanotube surface. The TiO2 nanotubes increased the functionality of the cells by substantially increasing the NOx/endothelin-1 ratio in the media. The nanotube structure with enhanced endothelialization, much increased extra cellular matrix formation, and substantially raised level of nitric oxide-endothelin ratio, may be useful as a vascular stent material with a reduced probability of late stent thrombosis.

Figure 8. NOx/Endothelin-1 ratio with respect to the polystyrene tissue culture dish.

Summary
TiO2 nanotubes adherent on Ti surface --- introduced to provide large-surface area, nanoscale base microstructure and cavity configuration, which produces mechanically locking structure for improved cell adhesion. The TiO2 nanotubes significantly enhanced the adhesion and growth of osteoblast cells (in vitro) by ~300 400% as compared to non-nanostructure surface. In vivo rabbit implant tests indicate a much enhanced osseointegration of new bones on TiO2 nanotube implant surface, with ~x6 improved adhesion strength than conventional sand-blasted Ti implanted surface. The TiO2 nanotubes enhance endothelial cell migration and nitric oxide (NOx) formation as an vasodilator, which can be useful for inhibiting platelet aggregation and LST.