Titanium Is A Currently Used Biomaterial

Published Date: 02 Nov 2017

1. Introduction

Surface modification of titanium can significantly affect the osseointegration of prosthetic implants. In particular, micro roughness [2] is a key factor in adhesion and colonization osteoblasts during bone formation around the implant.

In the last years, the research has been directed on the application of titanium surface functional groups able to induce and accelerate the deposition of bone (osteoblasts adhesion and nucleation of hydroxyapatite cristals). One method is the titanium surface bioactivation by making nanostructured surfaces consisting of TiO2 nanotubes.

Titanium dioxide can be prepared in the form of tubes with diameter in nanometres and lengths ranging from several nanometres to micrometers. Such nanostructure may be created on titanium by its electrochemical - anodic oxidation in fluoride containing electrolytes. Growth of nanotubes is in this case result of two simultaneous processes. First of them is anodic oxidation of the surface and the second one is a local dissolution of the growing titanium dioxide by fluoride ions.

Titanium was a laboratory curiosity until 1946, when Kroll developed a process for commercial production of titanium by reducing titanium tetrachloride. Since that time the availability of titanium has prompted much work on the development of new and improved alloys as well as extensive evaluations of the properties of these materials. Rapid progress has been made in the development of medical instrumentation and surgical implants. The clinical success of titanium and its alloys (particularly the alloy with 6 wt% aluminium and 4 wt% vanadium) is due to some outstanding properties.

Titanium is a reactive metal: in air, water, or arbitrary electrolytes a tenacious oxide layer is formed on the surface of the material. This oxide belongs to one of the most resistant compounds in the mineral world. As a dense film it protects the metal to chemical attack, also in the agressive biological environment. In biological tissue titanium is inert: the oxide layer, that is in contact with the tissue, is hardly soluble and in particular no ions are released that could react with other molecules. The mechanical properties of titanium compare favorably with those of other implantable metals and alloys. The yield strength is approximately the same as that of surgical quality 316L stainless steel and almost twice that of the familiar cast Co-Cr-Mo alloy used in orthopedic implants. The elastic modulus is approximately half that of the other common metal alloys used in surgery. This low modulus results in a material that is less rigid and deforms elastically under applied loads. This is important in the development of orthopedic products where a close match is desired between the elastic properties of long bone and the surgical implant. The fatique strength is about twice that of stainless steel.

The outstanding biocompatibility of titanium was already recognized by early researchers. Titanium has an extreme low toxicity and is well tolerated by both bone and soft tissue. Animal experiments have revealed that the material may be implanted for an extensive length of time; fibrous encapsulation of the implants is minimal to nonexistant. Histopathological examinations have failed to reveal any cellular changes adjacent to titanium implants. Careful examination of tissues adjacent to titanium have revealed neither giant cells nor macrophages, nor any other signs of inflammation. The material has been found to be safe in intravascular applications, owing to its high electronegativity and passive surface. For the same reason titanium does not cause hypersensitivity, which makes it the metal of choice in patients suspected of being sensitive to metals. For several decades, special titanium implants have been used with outstanding success in patients with histories of severe allergic reactions. Titanium implants are extensively used in cardiovascular, spinal surgery, orthopedic and dental surgery as well as in reconstructive and plastic surgery.

2. Materials and methods

2.1 TiO2-based nanotubes synthesis

The metallic samples, 10 x 10 mm, were Ti foil (Ti=base; Al=0.30; Cd=0.003; Cr=0.010; Cu=0.020; Fe=0.040; Mg=0.05, Mn=0.005; Mo=0.005; Ni=0.009, Pb=0.40; Sb=0.020; Si=0.05; Zn=0.005), Ti6Al4V alloy foil (Ti=base; N=0.0051; C=0.030; Al=5.53; V=3.90; Fe=0.13; Si=0.05-0.1; Ni=0.01-0.05; Cr=0.005-0.01; Coï€¼0.005; Cuï€0.001; Pbï€¼0.005), provided by National Institute for Nonferrous and Rare Metals, Bucharest, and Ti6Al7Nb alloy plate (Ti=base; C < 0.08; N < 0.05; Fe < 0.25; H < 0.009; O < 0.20; Ta < 0.5; Al 5.5 â€“ 6.5; Nb 6.5 â€“ 7.5) provided by Zielona Gora University, Poland.

Glycerol and ethylene glycol - based electrolytes (glycerol, 9.3% H2O, 0.7% NH4F and ethylene glycol, 1% H2O, 0.55% NH4F, respectively, pro analysis reagents) were used, due to their high viscosity which influences ionic species diffusion and the nanotubes formation kinetics and morphology [3]. Prior to anodization, metallic foils and plates were ultrasonic degreased in acetone followed by rinsing with deionised water, and drying in hot air stream. Anodization was performed in a standard two-electrode bath with circular platinum mesh cathode and MLW DC power source, of 150V and 10A. The anodization temperature was fixed to room temperature 25Â°C. The resulting TiO2 nanostructures were then rinsed and subsequently heat-treated in a VULCAN 3-350 furnace, in air, at 5500C for 2h.

with 1.5 mM L-glutamine and 2.2 g/L sodium bicarbonate,

Effects of titanium-based nanotube films on osteoblast behaviour in vitro

Miruna-Silvia Stan1, Cornel Fratila2, Ioan Roman3, Anca Dinischiotu1

1 Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Bucharest, 91-95 Splaiul

Independentei, 050095, Bucharest, Romania

2 Research & Development National Institute for Nonferrous and Rare Metals, 102 Biruintei Blvd, Pantelimon, 077145, Romania

3 METAV Research & Development, 31 C.A. Rosetti, 020011, Bucharest, Romania

Email: [email protected]

Nowadays, one of the nanotoxicological research efforts is focused on designing harmless and biocompatible medical devices. Geometry, surface and chemical treatment of nanomaterials can control their cytotoxic action and biological response. It is well known that Titanium and Ti-rich alloys exhibit a good ability to interface with human tissues, but they cannot provide the right surface required for implant longevity. In order to improve the features of Ti surface, TiO2 based nanotube (TNT) films (50 nm pore size) achieved by anodic oxidation and hydrothermal treatment were grown on titanium and on Ti6Al4V and Ti6Al7Nb alloys. In vitro toxicity and biocompatibility of TNT layers deposited on different substrates was investigated by using G292 osteoblast cell line. The cell viability was evaluated by LDH test and intracellular organization of the actin cytoskeleton was visualized by immunofluorescence Phalloidin-FITC assay. Measurement of LDH release after 24 and 48 h exposure demonstrated the absence of any cytotoxic effect of TNT layers. After 24 hours, cells began to adhere and spread on the nanotube films displaying small cell density and short cytoplasmatic extension. The attachment and proliferation of osteoblasts were substantially increased after 48 hours of growth on TNT films due to the propagation of filopodia that produced an interlocked cell structure. F-actin labeling showed that osteoblast cell growth on TiO2 nanotube layers deposited on titanium and Ti6Al4V was significantly increased compared with Ti6Al7Nb alloy, indicating that Ti6Al4V substrate provided best conditions that favored osteoblasts attachment, spreading and actin cytoskeleton organization. These differences suggest that changes in TiO2 nanotube substrates can have a huge impact on cell morphology, cell shape and fate. In conclusion, titanium-based nanotube films tested are toxicity-free and can provide the proper nanostructure as an implant material for a positive cell response in tissue engineering and regenerative medicine applications.

Keywords: TiO2 based nanotube; osteoblast; cytotoxicity; biocompatibility; actin cytoskeleton.

Highlights

In vitro toxicity and biocompatibility of TNT layers deposited on different substrates was investigated.

The attachment and proliferation of osteoblasts were substantially increased after 48 hours of growth on TNT films.

Measurement of LDH release demonstrated the absence of any cytotoxic effect of TNT layers.

All TiO2 based nanotube tested are promising nanomaterials for future in vivo investigations.

1. Introduction

Titanium is a currently used biomaterial for oral implantology and orthopedics due to its outstanding physico-chemical propreties and inertia in biological environments [1]. Having an extreme low toxicity and being well tolerated by both bone and soft tissue, Ti-based implants are extensively used in cardiovascular, spinal surgery, orthopedic and dental surgery, as well as in reconstructive and plastic surgery.

The most widely accepted and successful is the osseointegrated implant, based on the fact that it can be successfully incorporated into bone when osteoblasts grow on and into the rough surface of it (Swedish Professor Per-Ingvar BrÃ¥nemark discovery). This forms a structural and functional connection between living bone and dental implant. Surface modification of titanium can significantly affect the osseointegration of prosthetic implants. In particular, micro roughness [2] is a key factor in adhesion and colonization osteoblasts during bone formation around the implant.

Cell adhesion is an important parameter in evaluation of implant materials and to establish if they are suitable as medical devices. In vitro assessment of biomaterialsâ€™ toxicity is the first step in biocompatibility studies, and is usually performed using immortalized cell lines, being a qualitative analysis based on the examination of cell injury and cell growth after a direct or indirect contact with the materials. Toxicity involves impaired cellular homeostasis which further disturbs cellular functions and leads to a variety of biochemical changes. Loss of cell viability is a critical consequence of biomaterial toxicity (Allen et al., 1994). Qualitative evaluation of cell morphology in the first stage is based on inverted microscope examination. Typical characteristics of cellular alterations caused by toxic materials include cell membrane perforation, shrinking of the nucleus, cytoplasm fragmentation, granulation formation, rounding up and detachment of the cells from the support (Dekker et al., 1994, Schaeffer WI, 1990).

Cell attachment to the material tested involves the production of extracellular matrix proteins and reorganization of cytoskeleton proteins in order to stabilize the cell-material interface. Morphological characteristics specific to cell adhesion, highlighted by fluorescence microscopy, involve the aspect of filopodia, protrusions of the plasma-membrane with finger shape formed as a result of actin filaments polimerization in long fibers, or that of lamelipodia, if the actin is assembled in the form of a network that supports extensions (Anselme K, 2000).

In order to investigate the interactions between osteoblasts and nanotube films grown on titanium and Ti6Al4V and Ti6Al7Nb alloys, was evaluated the degree of in vitro toxicity, and were established the morphology, cell adhesion and spreading on the surface of nanotubes.

2. Materials and methods

2.1 TiO2-based nanotube synthesis

The Ti samples (1.27 x 1.27 cm2) were sterilized by autoclaving at 1800C for 30 minutes before use in biological experiments. An identically sized flat Ti sample (without TNT film) was used as a control after being chemically cleaned by acetone and isopropyl alcohol, dried, and autoclaved.

2.2 TNT surface analysis

The nanostructural films were investigated by scanning electron microscopy (SEM, QUANTA INSPECT F) and by high resolution transmission electron microscopy (HRTEM, TECNAI F30 G2) with a line resolution of 1Ã….

2.3 Cell culture and exposure to TNT films

G292 osteoblastic cells (ATCC CRL-1423), originally isolated from human osteosarcoma, were cultured in McCoyâ€™s 5a medium (Gibco, USA) supplemented 10% fetal bovine serum (Gibco, USA), 100 U/mL penicillin and 100 Î¼g/mL streptomycin, in a humidified atmosphere (5% CO2) at 37Â°C. The culture medium was changed every 2 days until cells reached confluence and then were trypsinized with 0.25% trypsin â€“ 0.03% EDTA (Sigma-Aldrich). The cells were seeded onto TiO2 nanotube substrates or cultured directly on tissue culture polystyrene (control cells) in a six-well plate at a density of 2 x 104 cells/ well, for 24 or 48 hours. The Ti samples were sterilized at 1800C for 30 minutes prior to biological experiments.

2.4 LDH release assay

LDH leakage into the culture medium as a result of plasma membrane lysis was evaluated with a cytotoxicity detection kit (TOX-7, Sigma-Aldrich Co.) according to the manufacturerâ€™s protocol. The LDH activity was determined spectrophotometrically after 30 min incubation at 250C of 50Î¼l of culture and 100Î¼l of the reaction mixture by measuring the oxidation of NADH at 490 nm in the presence of pyruvate, according to the manufacturerâ€™s kit instructions. Results were presented relative to the LDH activity in the medium of cells seeded on tissue culture plastic (0% of cell death) and on TCP adding triton X-100 1% (high control, 100% of death), using the equation

Cytotoxicity (%) = (exp . value âˆ’ low control)/(high control âˆ’ low control) âˆ— 100.

Osteogenic functionality depends on nanotube diameter

In terms of the dimensions of the nanotubes in our osteoblast (bone cell) and mesenchymal

stem cell (osteo-progenitor cell) studies, we have reported a unique variation in cell

behavior even within a narrow range of nanotube diameters from 30-100nm (Brammer et al.

2009; Oh et al. 2009) and it seems that a similar trend is established for both cell types.

When cells were grown on four different diameter nanotubes, shown in Figure 2, osteoblast

functionality in terms of bone forming ability, or alkaline phosphatase activity (ALP), and

mesenchymal stem cell (MSC) osteogenesis (bone cell differentiation) in terms of osteogenic

gene expression (osteopontin (OPN), osteocalcin (OCN), and alkaline phosphatase (ALP))

were most prominent on the large 100 nm diameter nanotubes, Figure 9 (a and b). During

periods of active bone growth, ALP activity levels are elevated in osteoblast cells so it is

beneficial to design an implant surface that would enhance the ALP activity to initiate the

formation of new bone. As well, it is critical to design a surface that is capable of allowing

the attachment of MSCs and promote osteogenic differentiation of cells for delivering a

mature osteoblastic cell population capable of rapidly forming bone. On the nanotube

surfaces, a reoccurring trend was revealed that as we increased the diameter of the

nanotubes, there was an increase in osteogenic biochemical activity and relative gene

expression, Figure 9 (c).

In a recent review article, Bettinger et al. claimed that the most palpable effect of

nanotopography on cells is the distinct changes in cell geometry or shape (Bettinger, Langer,

and Borenstein 2009). In fact, on the nanotube substrates with varying diameters, it was

revealed that the osteoblast and mesenchymal stem cells have reacted to the nanostructures

by changing shape. As the nanotube diameter increases we found a clear trend of increasing

cell elongation, Figure 10. The stretching aspect ratio was as great as 12:1 (length:width) on

the 100 nm diameter nanotubes. It can be assumed that the initial cell stretching and

elongated shape of the adhering cells on the large nanotubes impacted the cytoskeletal

(actin) stress. This is supported by the general notion that nanostructures (and the adhered

protein configuration for example, Figure 3) act as an extracellular matrix which imposes

physical forces and morphological changes to the cell (Chen 2008). It is probable that cells

must elongate their bodies to find a protein deposited surface, extending across larger areas

and thus eventually forming an exceedingly elongated shape on the 100nm diameter

nanotubes (because of the sparse distribution, Figure 3). Thus altering the density of

extracellular matrix (ECM) attachment sites or initial protein adhesion density affects the

shape of adhered cells. It has been reported that the focal attachments made by the cells with

their substrate determine cell shape which, when transduced via the cytoskeleton to the

nucleus, result in expression of specific phenotypes (Boyan et al. 1996). In our results, we

hypothesize that increasing nanotube diameters, changes the focal adhesion sites of the

osteoblast and mesenchymal stem cells, increases the cell elongation, and increases

osteogenic potential.

Interestingly, the cell nuclei also exhibit a somewhat similar trend of increased elongation

with increasing nanotube diameter, with the 100nm TiO2 nanotubes showing the most

significantly elongated nuclear shape (by ~20-25%) (data not shown). It can be hypothesized

that the nucleus organelle elongation on the TiO2 nanotube surfaces is in part due to the

It is generally accepted that the cytotoxicity effect of the materials in investigation can manifest

itself in different forms such as cell death, loss of membrane integrity, reduced cell adhesion, altered

cell morphology, reduced cell proliferation, and reduced biosynthetic activity.

9,16,49

The numerous

techniques currently used can be grouped according to the parameters evaluated â€” morphological,

biochemical, and genetic â€” even though a parameter prevails: the assessment of cell death, which In terms of current biologically active implants, enhanced surface roughness is one of the

important factors in providing the proper cues for a positive cell response to implanted

materials. However, much of the research related to the effect of macro and microroughness

on cellular responses and tissue formation are inconclusive due to the nonuniformity

of macro and micro-roughness stemming from crude fabrication methods like

polishing, sand blasting, chemical etching and so on. An important aspect of our nanotube

system shown in the SEM images (Figure 2) is that the nano-topography can feature a more

defined, reproducible and reliable roughness than micro and macro-topography for

enhanced bone cell function in vivo. Although, the heights of the nanotube walls increase

proportionally to the increasing diameter, there is no evidence of changes in surface

roughness between the different sized nanotubes based on atomic force microscopy (AFM)

data (Figure 2 (b)). As expected, the nanotube surfaces have a slightly higher roughness over

flat Ti, but between the nanotubes, there appears to be no difference. The AFM data was

performed because it is a somewhat standard surface analysis technique as it is useful for

coarser or microscale roughness measurements, say for other convential coatings, but for

the nanotube dimensions it may not always represent the true roughness when the probe tip

radius is not substantially finer than the nanotube dimensions such as in the TiO2 nanotube

case. The wall thickness, pore diameter, nanotube spacing, etc can be as small as ~10 nm,

while the AFM probe tip diameter can be as large as 30 - 50 nm.

Furthermore, it can be assumed that the surface area on the nano-scale may be affected

based on the various sizes and the surface area probably increases proportionally with

196 Biomaterials Science and Engineering

increasing nanotube size. It is expected that the surface area to be 3 times higher on the

100nm diameter nanotubes compared to the 30nm diameter nanotubes, respectively.

Additionally, the contact angle describing the wettability of the surface is enhanced, more

hydrophilic, on the nanotube surfaces (showing contact angles between 4-11Â°), which can

been advantageous for enhancing protein adsorption and cell adhesion.

3.1 Protein adhesion properties based on pore size

Cells respond to the amount and area of proteins that are available for binding. In fact, cells

do not see a naked material, in vivo or in in vitro culture. At all times, the material is

conditioned by the components of the fluid in which the material is immersed, whether it is

serum, saliva, cervicular fluid or cell culture media. As the cell begins to adhere and spread

on the nanotubes, there will be a dissimilar protein density and extra cellular configuration

based on the nanotube diameter. The behaviour of protein adsorption on the nanotube

surfaces are shown in Figure 3. On the 30nm diameter nanotubes there is a large number

and thorough distribution of protein nanoparticles covering the whole surface of the

nanotubes after just 2 hours of incubation in culture media. However, proteins on 100 nm

TiO2 nanotubes can only adhered sparsely at the top wall surface owing to the presence of

large empty nanotube pore spaces. This inherent protein adsorption property of the

nanotubes based on poresize is hypothesized to influence cell shape and fate. It is shown in

the next sections that the changes in poresize even in such a small range of dimensions (30-

100nm) will have huge impacts on downstream cell morphology and behavior. As mentioned earlier, while a thin TiO2 passivation layer on the Ti surface can impart

improved bioactivity and better chemical bonding to the bone (Feng et al. 2003), other

techniques have been developed to further enhance the bioactivity of a pure Ti surface, such

as direct coating of bioactive materials like hydroxyapatite and calcium phosphate (Puleo et

al. 1991; Salata 2004; Satsangi et al. 2003). However, even though these surface modified

layers have good bioactivity and high surface area, they tend to delaminate at the interface

between the implant and the bone due to the relatively large, micrometer-regime thickness

of the coated layer on Ti (Ong et al. 1992), presumably due to the stress accumulation

commonly seen in a thick coating of foreign material. This ultimately leads to implant

failure. In order to overcome this problem, some plasma spray Ca-Si based ceramic coatings

have been developed but still have roughness and layer thickness in the micrometer range.

For the purposes of this study, the focus is on nanoscale thickness surface coatings.

Therefore, developing an implant bioactive surface layer having high surface area for

enhanced bonding yet thin enough, in the nanometer range per se, to minimize

delamination would be desirable.

Recent reports indicate that modifying Ti surfaces with TiO2 nanotubes for orthopedic

applications significantly enhances the mineral formation (Oh et al. 2005), adhesion of

osteoblasts in vitro (Oh et al. 2006), and strongly adherent bone growth in vivo (Bjursten

2009), showing better bone bonding characteristics than conventional micro-roughened Ti

surfaces by sandblasting. One physical advantage of the TiO2 nanotube surface system is

that it is composed of and created directly from the native underlying Ti constituent, unlike

the foreign ceramic and spray coatings on Ti or Ti alloyed surfaces mentioned previously.

As well, the nanotube layer is at most ~300nm tall (for the purposes of this work) which in

the scheme of things is a much thinner layer and this nanometer length scale eliminates the

tendency of delamination prevalent in thick micrometer layers.

Because orthopedic implants encounter two types of cells, osteoblast cells in bone tissue and

osteo-progenitor cells also know as mesenchymal stem cells (MSCs) present in bone

marrow, it is advantageous to look at the differentiation potential of orthepeadic implant

surfaces in order to initiate a mature population of bone building cells for enhanced

osseointegration. One key principle in terms of orthepaedic implant technologies, to initiate

the differentiation of bone in the absence of chemical factors, hormones, or any other

synthetic, possibly toxic chemicals traditionally used by biochemists in in vitro

differentiation. The two cell types are illustrated and described in Figure 8. Stem cells, which have the potential to differentiate into multiple cell types, provide great

promises in advances in regenerate medicine. The differentiation of stem cells into the

appropriated lineages is temporal- and spatial-specific, with the surrounding

microenvironment playing critical roles in governing the stem cell fate. For generation of

osteoblasts, the MSCs need to be guided to selectively differentiate to osteoblasts, rather

than differentiating into other types of cells. The use of nanostructures and surface

topographical features have recently been shown to have positive effects on specific

differentiation of mesenchymal stem cells (Dalby, Andar et al. 2008), illustrating that the

surface topography alone can stimulated osteogenic differentiation.

5.1 Osteogenic functionality depends on nanotube diameter