TISSUE INTEGRATION

CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims priority to U.S. Provisional Application No. 62/425,486, filed on November 22, 2016.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This work was made with government support under grant/contract No. R01 AR067306 awarded by the National Institutes of Health. The government has certain rights in this technology.

BACKGROUND

[0003] Titanium and titanium alloys have been widely used in implantable biomedical devices for having high corrosion resistance, cyto-compatibility, and suitable mechanical properties. For example, titanium has been used for dental implants, hip replacements, knee replacements, and other suitable implantable devices. However, titanium is bioinert in nature. As such, direct bonding, or osseointegration, of titanium with bones in animals or humans can be a slow process. For instance, dental implants of titanium typically bond with jaw bones in about six to eight weeks. During this time, however, a recipient of such a dental implant can be susceptible to pain, infection, or other undesirable effects.

SUMMARY

[0004] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

[0005] Osseointegration of implantable biomedical devices can be a slow process during which a recipient (e.g., a human or animal) can be susceptible to pain, infection, and other undesirable effects. Several embodiments of the disclosed technology are directed to forming a charge holding material onto a metallic surface of a biomedical device containing, for example, titanium (Ti), titanium alloy, zirconium (Zr), tantalum (Ta), stainless steel, cobalt-chrome-molybdenum (CoCrMo) alloys, or other suitable types of metal or metal alloy. During and/or subsequent to formation, the charge holding material can be polarized to impart surface charges onto the biomedical device. In one example, the charge holding material can include titanium oxide (ΤΊ02) nanotubes that are at least partially crystalline and semi-conductive (e.g., having a band gap of about 3.05 eV) formed on a titanium surface. In certain implantations, such titanium oxide nanotubes can be formed via electrochemical anodization in which the titanium oxide nanotubes are polarized during formation. In other embodiments, the titanium oxide nanotubes can also be formed and/or polarized via low-temperature solution chemical or other suitable techniques. In another example, the titanium oxide can also be formed on the titanium surface as an oxide layer not in nanotube form. In further examples, the charge holding material can also include zirconium oxide (Zr02) on a zirconium surface, tantalum oxide (Ta20s) on a tantalum surface, a chromium oxide in different chromium to oxygen ratio (e.g., CrO, Cr203, Cr02, Cr03, or CrOs) on a stainless steel surface or cobalt-chrome-molybdenum surface, or other suitable combinations.

[0006] Subsequent to implantation, the resulting biomedical device can form an electrical field in the surrounding implantation area having bones, bodily fluids, or other parts of a recipient based on the stored surface charge in the charge holding material. The inventors have recognized that such electrical field formed from the surface charge of the biomedical device can significantly improve osseointegration of the biomedical device with surrounding bones by a surprising amount. For example, in experiments conducted, a seven to eight fold improvement of osseointegration of titanium bone implants was observed. The significant improvement of osseointegration of such biomedical devices can thus reduce healing periods from implantation of such biomedical devices from a period of six to eight weeks to a period of two to three weeks. Without being bound by theory, it is believed that such improvement can be attributed to an improved ability of the implanted biomedical device to attract building block molecules (e.g., proteins) and cells (e.g., osteoblasts or osteocytes) from the surrounding implantation area using the formed electrical field from the stored surface charge. For example, protein molecules are often positively charged in the surrounding implantation area. As such, by having a negatively charged surface, the biomedical device with the surface charge can attract more proteins from the surrounding implantation area than one without such surface charge. Similarly, a positively charged surface can attract cells and biomolecules that are negatively charged to enhance the healing process.

[0007] In certain embodiments, the formed charge holding material (e.g., titanium oxide nanotubes or titanium oxide in other forms, zirconium oxide, tantalum oxide, chromium oxide, or other suitable charge holding materials) can also be doped with a dopant for (1 ) modifying a band gap of the formed charge holding material; and/or (2) improving material compatibility of the formed charge holding material in an implantation area in the recipient. Example dopants can include zinc (Zn), magnesium (Mg), silicon (Si), sodium (Na), potassium (K), strontium (Sr), copper (Cu), iron (Fe), silver (Ag), or other suitable elements or oxides thereof typically present in the implantation area in the recipient. In other embodiments, the formed charge holding material can also be configured to have certain shape, size, or variants thereof such that the formed charge holding material can have certain structural orientation, morphology, band gap values, or other suitable mechanical and/or electrical characteristics.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figures 1A and 1 B are cross-sectional and top view of a portion of a biomedical device having surface charge for enhanced bone tissue integration in accordance with embodiments of the disclosed technology.

[0009] Figure 1 C is a cross-sectional diagram of the biomedical device when implanted into a recipient environment in accordance with embodiments of the disclosed technology.

[0010] Figure 2 is a schematic diagram of an example technique for polarizing titanium oxide nanotubes on the biomedical device 100 to impart electric charges to the titanium oxide nanotubes in accordance with embodiments of the disclosed technology.

[0011] Figure 3 shows scanning electron microscopy ("SEM") images of example titanium oxide nanotubes formed in accordance with embodiments of the disclosed technology at high and low magnification. [0012] Figures 4A-4C show SEM images of example titanium oxide nanotubes doped with Strontium (Sr), Magnesium (Mg), and Zinc (Zn), respectively, in accordance with embodiments of the disclosed technology.

[0013] Figures 5A and 5B show graphs of thermally stimulated depolarization current ("TSDC") versus temperature for nanotubes polarized at 300°C for one hour by applying an electric field of 2kV in accordance with embodiments of the disclosed technology.

[0014] Figure 5C shows a graph of stored charge versus time for example polarized titanium oxide nanotubes in accordance with embodiments of the disclosed technology.

[0015] Figures 6A-6E are photomicrograph showing histology images of implanted example biomedical devices with titanium nanotubes holding surface charge after four weeks (Figures 6A-6C) and ten weeks (Figures 6D and 6E) in which signs of osteoid or new bone formation was observed in accordance with embodiments of the disclosed technology.

[0016] Figures 7A-7D are SEM images of example stained porous titanium nanotube samples after four (Figures 7A and 7B) and ten (Figures 7C and 7D) weeks showing interfacial bonding between example biomedical device and bone tissue in accordance with embodiments of the disclosed technology.

[0017] Figures 8A and 8B are SEM images of example titanium oxide nanotube before and after electrothermal polarization, respectively, showing no signs of thermal or electrical degradation in accordance with embodiments of the disclosed technology.

[0018] Figures 9A-9F are SEM micrographs showing Control pure titanium (CpTi), titanium nanotube (TNT), and polarized titanium nanotube (TNT-P) samples after three days of culture in accordance with embodiments of the disclosed technology. Figures 9A-9C are low magnification images showing uniformity of osteoblast cells on the surface of the samples. Figures 9D-9F are high magnification images showing improved flattening of osteoblast on TNT-P samples.

[0019] Figure 10 is a graph showing optical density of Control pure titanium (CpTi), titanium nanotube (TNT), and polarized titanium nanotube (TNT-P) samples in Figures 9A-9F. [0020] Figures 1 1 A-1 1 C are computed tomography ("CT") scan images for Control pure titanium (CpTi), titanium nanotube (TNT), and polarized titanium nanotube (TNT-P) samples after five weeks in vivo. All three images show proper lodging of the samples into a femur bone of an animal.

[0021] Figures 12A-12C are photomicrographs showing histological analysis after five weeks in vivo for Control pure titanium (CpTi), titanium nanotube (TNT), and polarized titanium nanotube (TNT-P) samples, respectively. Figure 12A shows that the CpTi samples having presence of gaps between the samples and host tissue. Figure 12B shows that the TNT samples having some osteoid formation and fewer gaps as compared to CpTi. Figure 12C shows that the TNT-P samples having significant osteoid for formation and bone-implant interlocking.

[0022] Figures 13A-13C are SEM images showing interfacial bonding between the implanted samples and surrounding bone-tissue area for Control pure titanium (CpTi), titanium nanotube (TNT), and polarized titanium nanotube (TNT-P) samples, respectively. TNT-P sample in Figure 13C shows improved interfacial bonding evident from absence of gaps in contrast to the other samples shown in Figures 13A and 13B.

[0023] Figure 14 is graph showing a histomorphometric analysis of osteoid formation give weeks post-surgery for Control pure titanium (CpTi), titanium nanotube (TNT), and polarized titanium nanotube (TNT-P) samples. Inset shows osteoid area percentage of CpTi and TNT samples in a reduced y-axis.

[0024] Figures 15A-15D are SEM images of example titanium oxide nanotubes without any dopants, doped with Strontium (Sr), doped with Magnesium (Mg), and doped with Zinc (Zn) in accordance with embodiments of the disclosed technology.

[0025] Figure 16 is a graph showing optical density values measured using (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) ("MTT") Assay after three and seven days of culture at a wavelength of 570 nm in accordance with embodiments of the disclosed technology.

[0026] Figures 17A and 17B are SEM images of osteoblast cell proliferation after three and seven days on un-doped, Mg doped, Sr doped, and Zn doped nanotube samples. DETAILED DESCRIPTION

[0027] Various embodiments of implantable biomedical devices having imparted surface charge for enhancing bone tissue integration and associated methods of manufacturing are described below. In the following description, specific details of components are included to provide a thorough understanding of certain embodiments of the disclosed technology. A person skilled in the relevant art will also understand that the disclosed technology may have additional embodiments or may be practiced without several of the details of the embodiments described below with reference to Figures 1 A-17B.

[0028] Titanium and titanium alloys have been used in dental, orthopedic, and other biomedical applications due to low toxicity, good corrosion resistance, suitable mechanical properties, and excellent biocompatibility of titanium. Nevertheless, the use of titanium as an orthopedic implant has a two-fold challenge. First, titanium is a biologically inert material (bioinert) that often results in scar tissue formation around a titanium implant. Post implantation, the scar tissue in a physiological environment can ultimately lead to loosening of the implant. Secondly, titanium has a significant mechanical property mismatch with natural bone and thus can negatively affect bone regeneration in response to compressive stress due to stress shielding.

[0029] Without being bound by theory, it is believed that progress of bone healing is related to osteoblasts i.e., bone forming cell activity on an implant surface. The inventors have recognized that nano-structural modifications of implants can promote osteoblast cell proliferation and thus improve bone-tissue integration. In particular, by imparting surface charge to formed nanostructures on implants can significantly improve osseointegration of such implants with surrounding bones by a surprising amount. The significant improvement of osseointegration of such implants thus reduce healing periods of implantation, as described in more detail below with reference to Figures 1A-17B.

[0030] Figures 1A and 1 B are cross-sectional and top view of a portion of an implantable biomedical device 100 having imparted surface charge for enhanced bone tissue integration in accordance with embodiments of the disclosed technology. As shown in Figure 1A and 1 B, the biomedical device 100 can include a substrate 102 having a surface 103, a charge holding material 104 on the surface 103, and an electrode 106 in contact with the titanium oxide nanotubes 104 and spaced apart from the substrate 102. In certain embodiments, the biomedical device 100 can be a dental implant, a hip replacement, a knee replacement, a vertebra spacer, a pin, a nail, a screw, a bracket, or other suitable types of biomedical device. Though particular components of the biomedical device 100 are shown in Figures 1A and 1 B, in other embodiments, the biomedical device 100 can also include surface protection layers or other suitable materials and/or configurations.

[0031] The substrate 102 can include a bulk metal or metal alloy with a utile shape. In certain embodiments, the substrate 102 can be constructed from titanium (Ti), stainless steel, tantalum (Ta), zirconium (Zr), a cobalt-chrome-molybdenum (CoCrMo) alloy, or other suitable biocompatible metals or metal alloys. Though the substrate 102 is shown in Figures 1A and 1 B as a homogenous body, in other embodiments, the substrate 102 can also include a laminated structure having, for example, a ceramic material (e.g., calcium phosphate), a metal, a polymer, or other suitable materials formed in a stacked configuration. In further embodiments, the substrate 102 can also have heterogeneous structure/composition in a width, depth, or length direction. In any of the foregoing embodiments, at least the surface 103 of the substrate 102 is metallic and thus electrically conductive.

[0032] The electrode 106 can include any conducting material in electrical contact with the charge holding material 104. For example, in the illustrated embodiment, the electrode 106 includes a plate or foil constructed from copper, titanium, aluminum, silver, or other suitable conductive materials contacting the charge holding material 104. In other embodiments, the electrode 106 can also include individual conductive portions discontinued from one another and individually correspond to portions of the charge holding material. In further embodiments, the electrode 106 may be omitted, and a bodily fluid and/or other parts of a recipient (not shown in Figures 1A and 1 B) can act as an electrical terminal instead of the electrode 106.

[0033] The charge holding material 104 can include an electrical insulator (e.g., with a band gap not less than 5 eV) and/or semiconductor (e.g., with a band gap of 1 .0 eV to 5 eV) that can be polarized by an applied electric field to contain a surface charge on the surface 103 of the substrate 102. For example, when an electric field of certain strengths is applied to the charge holding material 104, electric charges tend not to flow through the charge holding material 104 as in an electrical conductor (e.g., a metal). Instead, electric charges only shift from corresponding equilibrium positions and thus resulting in dielectric polarization in the charge holding material 104. The dielectric polarization in the charge holding material 104 can thus store certain amounts of electric charges that create an electric field 108 at the surface103 of the substrate 102. Example amounts of the stored electric charges in the charge holding material 104 can be about 1 micro-Coulomb/cm ² to about 1000 milli-Coulomb/cm ² . Though particular polarity of the electric field 108 is illustrated in Figure 1A, in other embodiments, the charge holding material 104 can have the opposite polarization from that shown in Figure 1A.

[0034] In the illustrated embodiment, the charge holding material 104 includes a plurality of titanium oxide nanotubes 105 formed on the surface 103 of the substrate 102. As shown in Figures 1A and 1 B, the titanium oxide nanotubes 105 include a plurality hollow cylindrical shaped structures extending away from the surface 103 toward the electrode 106 and are spaced apart from one another in a generally evenly. The titanium oxide nanotubes can have a length of about 1 nm to about 10,000 nm and a diameter of about 20 nm to about 1000 nm. In other examples, the individual titanium oxide nanotubes 105 can also have other suitable shapes and/or arrangements. For instance, the titanium oxide nanotubes 105 can be arranged as clusters (not shown) on the surface 103. In another example, the titanium oxide nanotubes 105 can also be distributed randomly on the surface 103.

[0035] In further embodiments, the charge holding material 104 can include a plurality of nanowires, nanocoils, other types of nanotubes constructed from other suitable dielectric or semi-conductive materials. For example, in some embodiments, the charge holding material 104 can include silicon (Si), silicon oxide (S1O2), gallium nitride (GaN), tungsten sulfide (WS2), and/or other suitable types of materials. In yet further embodiments, the charge holding material 104 can include a layer of titanium oxide not in any nanostructure form. In other embodiments, the charge holding material 104 can also include zirconium oxide (Zr02) on a zirconium surface, tantalum oxide (Ta205) on a tantalum surface, a chromium oxide in different chromium to oxygen ratio (e.g., CrO, Cr203, Cr02, Cr03, or CrOs) on a stainless steel surface or cobalt- chrome-molybdenum surface, or other suitable combinations. In the description below, a substrate formed of titanium and a charge holding material 104 formed of titanium oxide nanotubes are used to illustrate embodiments of the disclosed technology. However, one skilled in the art would recognize that the disclosed technology can also be applied to other combinations of materials, structures, and/or configurations. [0036] In certain embodiments, the charge holding material 104 can also be doped with a dopant for (1 ) modifying a band gap of the charge holding material 104; and/or (2) improving material compatibility of the charge holding material 104 in an implantation area in the recipient. For instance, a dopant (e.g., silicon, boron, zinc, etc.) may be added to the charge holding material 104 to reduce a band gap of the charge holding material 104. In another instance, a mineral present in natural bones may be used as a dopant for improving material compatibility of the charge holding material 104. Example of such dopants can include zinc (Zn), magnesium (Mg), silicon (Si), sodium (Na), potassium (K), strontium (Sr), copper (Cu), iron (Fe), silver (Ag), or other suitable minerals or oxides thereof. Example dopant concentrations can be from about 0.01 % to about 50% by weight. In any of the foregoing embodiments, dopants can be present in an elemental form or as molecules in combination with oxygen, carbon, or other elements.

[0037] Figure 1 C is a cross-sectional diagram of the biomedical device 100 in Figures 1 A and 1 B when implanted into a recipient environment 1 12 in accordance with embodiments of the disclosed technology. As shown in Figure 1 C, upon implantation, the biomedical device 100 comes into contact with various electrolytes 1 14 (e.g., a bodily fluid, blood, etc.) in an implantation area 1 18 of the recipient environment 1 12, which can be in vitro or in vivo. The implantation area 1 18 can also contain various building block molecules that can promote osseointegration of the biomedical device 100 with surrounding bones of the recipient environment 1 12. For example, as illustrated in Figure 1 C, the implantation area 1 18 can contain protein molecules 1 16 (shown as circles in Figure 1 C). In other examples, the building block molecules can also include cells such as chondrocyte precursor cells (e.g., paraxial mesoderm and sclerotome) or other suitable molecules.

[0038] Experiments have shown that the imparted electric charges 1 18 on the surface 103 of the biomedical device 100 can significantly improve osseointegration of the biomedical device with surrounding bones of the recipient environment 1 12 by a surprisingly amount. For example, as described in more detail later, in certain conducted experiments, a seven to eight fold improvement of osseointegration of titanium bone implants was observed. Without being bound by theory, it is believed that such improvement can be attributed to an improved ability of the implanted biomedical device 100 to attract building block molecules (e.g., proteins 1 16) from the surrounding implantation area 1 18 based on the electrical field 108 induced by the stored surface charge in the charge holding material 104. For example, as shown in Figure 1 C, the protein molecules 1 16 are often positively charged in the surrounding implantation area 1 18. As such, by having a negatively charged surface, the biomedical device 100 with the surface charge can attract more proteins 1 16 from the surrounding implantation area 1 18 than one without such surface charge. The significant improvement of osseointegration may thus reduce healing periods due to implantation of the biomedical device 100 from a period of six to eight weeks to a period of two to three weeks. As a result, pain, infection, and other undesirable effects of implanting the biomedical device 100 into the recipient environment 1 12 may be reduced.

[0039] Figure 2 is a schematic diagram of an example technique for polarizing titanium oxide nanotubes on the biomedical device 100 to impart electric charges to the titanium oxide nanotubes in accordance with embodiments of the disclosed technology. As shown in Figure 2, the biomedical device 100 is in direct contact with a pair of polarizing electrodes 120a and 120b coupled to a voltage source 122 (e.g., a battery). In certain embodiments, the biomedical device 100 is also submerged in an electrolyte (not shown), such as ethylene glycol based solution containing hydrogen fluoride (HF).

[0040] During polarization, the voltage source 122 applies a direct current (DC) voltage to the polarizing electrodes 120a and 120b while the biomedical device 1 00 is heated (as indicated by the arrows 124) at a select heat rate (e.g., 5°C/min) from room temperature to a target polarizing temperate (e.g., 300°C) for a polarizing period (e.g., one hour). Upon expiration of the polarizing period, the biomedical device 100 can be cooled down to room temperature at a select cooling rate (e.g., 10°C/min) while the voltage source 122 continues to apply the DC voltage. Though particular technique for polarizing the biomedical device 100 is shown in Figure 2 for illustration purposes, in other embodiments, the biomedical device 100 can be polarized via other suitable techniques. In further embodiments, one or more of the heat rate, target polarizing temperature, polarizing period, or cooling rate can be adjusted based on and to achieve a target polarization level (e.g., a charge density in the charge holding material 104 shown in Figure 1A).

Experiments

[0041] Certain experiments were conducted to study effects of imparting surface charge on an implantable biomedical device (or "implant") on osseointegration. In particular, experiments were conducted to form titanium oxide nanotubes on a metal substrate of titanium with and without dopants. The titanium oxide nanotubes are also polarized to contain electric charges having a charge density. The experiments showed that surface properties of example titanium implants can be altered via forming T1O2 nanotubes ("TNT") thereon and followed by introduction of a surface charge to the T1O2 nanotubes to form a biomaterial with enhanced bioactivity. The experiments showed that the imparted surface charge provided enhanced osteogenesis and early stage osseointegration in-vivo. Application of the surface charge appeared to accelerate bone healing by increasing rates of osteoblast cell proliferation, as described in more detail below.

Fabrication of Ti02 nanotubes

[0042] Fabrication of TNT was performed using CpTi disks (1 mm thickness and 9.5mm diameter) in ethylene glycol based medium containing hydrogen fluoride (HF). The TNT samples were kept between a pair of platinum plates. To polarize the samples, a DC voltage was applied through wires using a picoammeter (Model 6487, Keithley Instruments, OH). The samples were heated with a controlled heating rate of 5°C/min from room temperature to a polarization temperature (Tp) of 300°C and maintained at the polarization temperature for one hour in a DC electric field (Ep) of 2 kV/cm. After period of one hour at polarization temperature, the samples were cooled down to room temperature while the applied electric field is maintained. The polarized samples were then heated at a rate of 5°C/min from room temperature to 450°C and TSDC was measured using the picoammeter. Charge storage was calculated using the equation below:

Qp - l/β I / (T}dT

J where J(T) is a depolarization current density at temperature T, and β is a heating rate during TSDC measurements. The samples were characterized using SEM before and after polarization.

Contact Angle Measurements

[0043] A sessile drop method was used to measure contact angles on the surface of the samples using a face contact angle set-up with a microscope and a camera. A

0.5—1 .ΟμΙ droplet of distilled water was suspended from a microliter syringe tip and the surface of the sample was moved up towards the tip of the syringe. Images were then collected with the camera. The contact angle between the drop and the sample was measured using the magnified image collected from the computer. Contact angle measurements were taken for TNT-P, TNT, and CpTi samples. in vitro and in vivo study

[0044] Bone cell-material interactions were studied using human fetal osteoblast (hFOB) cells for three days using circular disc samples of 12.5 mm in diameter and 3 mm in thickness. Cell seeding onto the disc shaped samples was performed in 24 well plates. A 1 : 1 mixture of Ham's F12 Medium and Dulbecco's Modified Eagle's Medium (DMEM/F12, Sigma, St. Louis, MO), with 2.5 mM L-glutamine (without phenol red) was used as a base medium. The medium was added with 10% fetal bovine serum (HyClone, Logan, UT) along with 0.3 mg/ml G418 (Sigma) and the cultures were kept at 34 °C under an atmospheric condition of 5% CO2. The cell medium was changed every two days throughout the experiment.

[0045] MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay was performed to evaluate cell proliferation. A 5 mg/ml MTT (Sigma) solution was prepared by dissolving MTT in PBS followed by filter-sterilization using a filter paper of 0.2 m pore. Dilution of MTT was performed in 1 :9 ratio of MTT and DMEM/F12 medium. Then 1 ml of diluted MTT solution was added to each sample and kept for incubation for a period of two hours for formation of formazan crystals. After incubation, 1 ml of solubilization solution of 10% Triton X-100, 0.1 N HCI, and isopropanol was added to dissolve the formazan crystals. 100 μΙ of the resulting supernatant were transferred into a 96 well plate in triplicate and read by a microplate reader at 570 nm.

[0046] All samples were fixed using a fixative of 2% paraformaldehyde/2% glutaraldehyde in 0.1 M cacodylate buffer for overnight at 4°C. Post-fixation, 2% osmium tetroxide (OsO- was added for two hours at room temperature. Dehydration to the fixed samples was performed in a series of ethanol (30%, 50%, 70%, 95%) and finally with 100% ethanol for three times, followed by a drying procedure using hexamethyldisilane (HMDS). Post drying, the samples were observed under SEM after gold coating.

[0047] A total of six male Sprague-Dawley rats weighing between 280 and 300g were used. All rats underwent bilateral surgery. Prior to surgery, the rats were housed in individual cages with alternating twelve-hour cycles of light and dark in temperature and humidity controlled rooms for acclimatization. Following acclimatization, the rats were anesthetized and monitored by pedal reflex and respiration rate to maintain proper surgical anesthesia. Using a drill bit, a defect in the distal femur was created similar to the diameter of the implant samples. The defect cavity was washed using saline solution to rinse out any remaining bone fragments. Following implantation, the incision was closed. Betadine solution was applied at the incision site post-surgery to prevent infection. Pain reduction in the form of buprenorphine (0.03 mg kg-1 ) were given to the rats prior to surgery. Pain reduction via meloxicam injection was given post-surgery.

[0048] At necropsy, two sets of samples were harvested: one for push-out tests and CT scan analysis, and another for histological analysis. A series of radiographic exposures of the bone samples (acquired using the X-ray energy source on the IVIS® Spectrum CT) were analyzed by computed tomography ("CT") to generate a 3D volume. Scans were performed using a 40 pm voxel size and 150 pm resolution. 3D images of the defects were reconstructed from the scans. Push-out tests were performed to determine the interfacial shear modulus between the tissue and the implant using a universal material testing machine (Instron, PA, USA) in compression using a 300lb load cell. The shear modulus was calculated from the stress-strain plots of the push out test experiments.

[0049] For histological analysis, bone-implant samples were fixed in 10% formalin solution. Fixed samples were then dehydrated in series of ethanol (70%, 95% and 100%), 1 : 1 ethanol-acetone mixture, and finally 100% acetone. Following dehydration, samples were embedded in Spurs resin, cut into thin sections (n=3 for each sample) using diamond blade, mounted on glass slides and stained using modified Masson Goldner's trichrome staining method. Stained implant-tissue sections were then observed under a light microscope. Stained samples were then characterized under SEM (FEI Quanta 200, FEI Inc., OR, USA), which was maintained at low operating voltage of 5 kV.

Experimental Results

[0050] Figure 3 shows a SEM image of example titanium oxide nanotubes anodized using 1 vol.% HF, 0.5 wt.% NH4F, 10vol.% Dl water in ethylene glycol electrolyte at 40V for one hour. As shown in Figure 3, the resulting nanotubes were approximately 1 pm in length and 100±15 nm in diameter. Figure 2 shows the SEM images of ΤΊ02 nanotubes with the dopants. Figures 4A-4C show SEM images of example titanium oxide nanotubes doped with Strontium (Sr), Magnesium (Mg), and Zinc (Zn), respectively.

[0051] Figures 5A and 5B show the TSDC spectra of titanium oxide nanotubes polarized at 300°C for one hour. As shown in Figures 5A and 5B, a current density is very low until 200°C, then slowly starts increasing before reaching a peak value and starts decreasing again. The maximum current density observed was around 400- 440°C. The polarized charge was calculated to be about 1 -60 mC/cm ² with a maximum current density in the range of about 3-125 μΑ/cm ² . Figure 5C shows a graph of stored charge versus time for example samples having polarized titanium oxide nanotubes. Figure 5C shows a shelf life of stored charge on the TNT-P samples over time via TSDC measurement at different time. As shown in Figure 5C, no significant change in stored charge is observed over a period of two weeks.

[0052] Figures 6A-6E are photomicrograph showing histology images of implanted example biomedical devices with titanium nanotubes holding surface charge after four weeks (Figures 6A-6C) and ten weeks (Figures 6D and 6E) in which signs of osteoid or new bone formation was observed in accordance with embodiments of the disclosed technology. The images in Figures 6A-6E show that titanium oxide nanotubes with 1 wt% dopants having good cell spreading as compared to samples without nanotubes and nanotube only samples without dopants. Figures 7A-7D are SEM images of example stained porous titanium nanotube samples after four (Figures 7A and 7B) and ten (Figures 7C and 7D) weeks showing interfacial bonding between example biomedical device and bone tissue in accordance with embodiments of the disclosed technology. As shown in Figures 7A-7D, improved osseointegration was observed between the bone tissue and the implant interface.

[0053] TNT fabricated using ethylene glycol based electrolyte resulted in nanotubes of about 1 pm in length and 105 nm in diameter. Electrothermal polarization for all samples were carried at 300°C by passing a constant electric field of 2kV cm-1 and depolarized at 450°C. Table 1 below summarizes the charge storage values along with the maximum current densities and the temperatures corresponding to the maximum current density. Sample Maximum Current Temperature at Stored Charge Density (mA/cm ² ) maximum current (mC/cm ² ) density (°C)

TNT 0.072±0.054 390.69±30.46 37.15±14.4

[0054] Table 2 below shows a comparison between the specific capacitance for electrothermally polarized TNT with the maximum stored charge calculated using capacitance formula with literature values for specific capacitance calculated for TNT used for supercapacitor studies.

TNT sample was characterized before and after electrothermal polarization to ensure the heat treatment did not damage or thermally degrade the nanotubes. Figures 8A and 8B respectively show SEM images of TNT before and after electrothermal polarization indicating no signs of thermal degradation.

[0055] Table 3 below shows contact angle values for measured samples. It can be seen that contact angle values for TNT-P samples have excellent surface wettability with contact angle value less than 1° as compared to other samples which have higher contact angles. The low contact angle values indicate electrothermal polarization improves the surface wettability properties and good biocompatibility.

[0056] MTT assay was performed for all samples after three days of incubation of the samples with hFOB and cell media. As shown in Figures 9A-10, higher vitality of cells in contact with TNT-P was observed in comparison to TNT and the control sample without any nanotubes. The SEM images further show more uniform flattening of osteoblasts on the TNT-P samples in contrast to TNT and control samples. The high values of viable densities with better cell morphology and adherence on the TNT-P surfaces indicate that the TNT-p are biocompatible with no inhibition to osteoblast growth.

[0057] CT scans were performed for all samples after five weeks. As shown in Figures 1 1 A-1 1 C, the samples were properly lodged in the femur bone of the rats. The interfacial shear modulus values resulting from the push out experiments are shown in Table 5 below.

As shown above, after five weeks, the interfacial shear modulus for TNT-P (123.26 ± 17.47) is higher than that of the TNT (83.28) and CpTi (39.21 ± 6.5) samples. Such high interfacial shear modulus indicates good interfacial bonding between the TNT-P samples and the tissue at early stages.

[0058] Histological evaluation at the bone-implant interface was performed to study the effect of polarization on TNT surface for biocompatibility and new bone formation at an early stage of five weeks. As shown in Figures 12A-12C, osteoid like new bone formation can be observed as early as five weeks. The orange-red region surrounding the implant area represents the osteoid formation which indicates no cytotoxic effects due to implantation. The greenish area indicates the mineralized bone and the bluish black spots indicate the nuclei. Polarized nanotube surface showed better signs of new bone formation as compared to other samples.

[0059] SEM characterization was performed for the stained samples using SEM. Figures 13A-13C show SEM images of all samples indicating interfacial bonding. It can be observed from Figures 13A-13C that the control CpTi samples show poor bonding with presence of significant gaps. The TNT-P samples showed complete bonding as early as five weeks indicative of early stage osseointegration. [0060] Histomorphometric evaluation was performed for a percentage of osteoid (new bone) formation around the samples. TNT-P samples show the highest osteoid formation >80% around the implanted samples as compared to TNT (~ 8%) and CpTi control (~ 2%) samples, indicating significantly higher biocompatibility, as shown in Figure 14.

[0061] Figures 15A-15D are SEM images of example titanium oxide nanotubes without any dopants, doped with Strontium (Sr), doped with Magnesium (Mg), and doped with Zinc (Zn) in accordance with embodiments of the disclosed technology. The resulting un-doped, Sr, Mg, and Zn doped Ti02 nanotubes were about 1 pm long and about 1 10 nm in diameter.

[0062] Figure 16 is a graph showing optical density values measured using (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) ("MTT") Assay after three and seven days of culture at a wavelength of 570 nm in accordance with embodiments of the disclosed technology. It can be seen from Figure 16 that the cell density for Sr, Mg and Zn doped nanotube samples is higher compared to both bare cp-Ti and un-doped nanotube samples. Also, the cell densities for the samples after day 7 increased as compared to the control samples. Such an increase indicates improved cell-material interactions of doped nanotube samples as compared to the control sample without any cytotoxic effects from the dopants.

[0063] Figures 17A and 17B are SEM images of osteoblast cell proliferation after three and seven days on un-doped, Mg doped, Sr doped, and Zn doped nanotube samples. As shown in Figures 17A and 17B, cell proliferation was better for doped nanotube samples than the control samples. Also, dopants with 1 wt.% concentration showed better results in terms of cell density and cell proliferation with excellent cell adhesion.

Discussion of Experiment Results

[0064] Nanostructural surface modification such as forming TNT helps to achieve a high surface to volume ratio. Due to the high surface area to volume ratio, it is believed that TNT possesses charge transport/storage ability. However, as TNT are amorphous in nature and offer poor electrical conductivity, the simplest way to improve conductivity is to make TNT crystalline via, for example, annealing. In particular, loosely packed nanotube arrays fabricated using a diethylene glycol electrolyte contained higher presence of anatase crystal phase and were subsequently more conductive in comparison to water based electrolyte. Annealing titanium oxide samples at 300°C results in anatase in nature. Presence of anatase upon annealing is believed to facilitate charge transfer through transfer of electrons thus reducing the band gap of ΤΊ02. The improvement in conductivity upon heat treatment is also believed to be a result of introduction of oxygen vacancies into ΤΊ02 lattices.

[0065] It is believed that a negatively poled ceramic surface had better cell adhesion and mineralization as compared to un-poled and positively poled surfaces. Polarization charge between 0.08 pC/cm ² to 1 .2 mC/cm ² can be stored within sintered hydroxyapatite (Hap) implants by electrothermal polarization. The microstructural effects on polarizability of ceramic hydroxyapatites have also been explored under varying poling temperatures of 250°C to 500°C which increased the stored charge values to about 0.5 to 45 pC/cm ² In contrast, as discussed above, the conducted experiments showed that electrothermal polarization of TNT at 300°C resulted in an average charge storage around 40 mC/cm ² .

[0066] The conducted experiments also showed that the increased charge storage of TNT-P significantly improved in vitro osteogenic and early stage osseointegration. Electrical charge or stimulation has been shown to promote the adsorption of osteoblast cells by releasing various proteins, cytokines and growth factors which leads to enhanced osteoblast density. Also, better cell spreading was observed due to electrical stimulation with a more flattened and well-spread morphology of osteoblast cells. On both the TNT and TNT-P sample surfaces, cells were seen to adhere to each other with cellular micro-extensions called filopodia, and were connected to the substrate in addition to neighboring cells.

[0067] Moreover, on the TNT-P samples, cells were observed to grow following a more flattened architecture. In contrast, on the CpTi surface cell growth was less pronounced, with limited establishment of cell spreading being seen after three days of culture. The cells assumed a more rounded cylindrical shape, with diffuse or underdeveloped filopodia on the TNT surface compared to the TNT-P surface. The present in vitro bone cell-material interaction results showed that the TNT-P samples have superior biocompatibility over TNT samples with identical surface topography.

[0068] The experiments also showed that doping the TNT before, during, or after polarization also improve bone healing when compared to un-doped TNT samples. As the bone matrix include trace metallic elements such as Sr, Mg, Zn, Si, etc. along with calcium phosphate, the experiments were conducted to use these trace metallic elements in the form of dopants in ΤΊ02 nanotubes for further improving biological response of the ΤΊ02 nanotubes surface.

[0069] From the foregoing, it will be appreciated that specific embodiments of the disclosure have been described herein for purposes of illustration, but that various modifications may be made without deviating from the disclosure. In addition, many of the elements of one embodiment may be combined with other embodiments in addition to or in lieu of the elements of the other embodiments. Accordingly, the technology is not limited except as by the appended claims.