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TiO2 nanotubes for bone regeneration
Review
TiO2 nanotubes for bone regeneration Karla S. Brammer, Christine J. Frandsen and Sungho Jin Materials Science and Engineering, University of California, San Diego, La Jolla, CA 92093-0411, USA
Nanostructured materials are believed to play a fundamental role in orthopedic research because bone itself has a structural hierarchy at the first level in the nanometer regime. Here, we report on titanium oxide (TiO2) surface nanostructures utilized for orthopedic implant considerations. Specifically, the effects of TiO2 nanotube surfaces for bone regeneration will be discussed. This unique 3D tube shaped nanostructure created by electrochemical anodization has profound effects on osteogenic cells and is stimulating new avenues for orthopedic material surface designs. There is a growing body of data elucidating the benefits of using TiO2 nanotubes for enhanced orthopedic implant surfaces. The current trends discussed within foreshadow the great potential of TiO2 nanotubes for clinical use. Nanostructures for bone regeneration For optimal functioning and support, cells in our body rely on their interaction with an extracellular matrix (ECM) [1]. The ECM is a complex network of substrates with intricate features that are nanoscale in dimension. The ECM stimulates and interacts with cell physiology on a nanoscale level. Recent in vitro studies also reveal that nanofeatures on artificial substrate surfaces can impact cell physiology in the same way as the natural ECM. Artificial nanostructures influence how the cell adheres to the surface, cell spreading on the surface, cell biochemical functioning and even cell differentiation [2]. New fabrication techniques and new nanotechnologies have allowed precise control and manipulation of nanoscale features on artificial surfaces. The development of nanostructures has allowed unprecedented access to and intimate interaction with the fundamental subunits of biology. For bone regeneration and repair, recreating substrates on a nanoscale allows scientists to probe the first level of the bone structural hierarchy. This knowledge is used to present host cells with an innate ECM interface where they can repopulate and resynthesize a new matrix for creating new bone [3]. Most orthopedic materials research has focused on the nanometer level of precision for cell–material interactions for enhanced bioactivity. A rapidly established, strong and long lasting connection at the interface of an implant and bone is essential for the clinical success of orthopedic implants. A better understanding of how osteogenic cells interact with nanostructured materials in vitro has helped to design better implants for bone regeneration and implant integration in vivo. For orthopedic implant success, both osteoblast cells and mesenchymal stem cells (MSCs) play a critical role in Corresponding author: Jin, S. ([email protected]). Keywords: nanostructure; TiO2 nanotube; osteoblast; mesenchymal stem cell.
osteointegration, the direct physical and chemical bond at the implant–bone interface. There is little doubt that improving osseointegration for biomaterials and prosthesis will decrease implant failures and have a major impact on the quality of life for millions of people in the USA [4]. In terms of nanotopographic effects on osteoblast (bone cell) behavior, it has been shown that ceramics, such as titanium oxide (TiO2), with different nanostructures exhibit enhanced effects on growth rates and bone forming ability [5–9]. Many researchers utilize the common Ti surface nanostructuring technique of electrochemical anodization for creating nanotubular structures on the surface to stimulate rapid cell growth and mineralization of osteoblast cells. Anodized TiO2 nanotubes also show enhanced growth and accelerated osteo-differentiation of MSCs [10–13]. Direct efforts have been made to optimize the features of the nanotubes to (i) improve the bone forming function and mineralization of bone cells and (ii) induce differentiation of MSCs into mature bone cells for rapid bone regeneration and integration in vivo. The potential to modulate cellular response with nanostructured materials has generated a landslide of research in the orthopedic domain [1,14]. A comprehensive review of all the applications within orthopedics is beyond the scope of this work. We focused our perspective on TiO2 nanotube surface structuring in bone regeneration research. Examples of the current design strategies utilizing TiO2 nanotubes will be discussed. Orthopedic cell–surface interactions Manipulation of cell–surface interactions can improve the design and integration of orthopedic implants. When a material is initially exposed to in vitro or in vivo culture conditions, proteins in the cell culture media or bodily fluids adsorb to the material’s surface in less than 1 s [15]. There are several material factors that affect how the proteins will adhere, unfold and present different functional groups that impact how the surface is perceived by the cell. These include and are not limited to surface chemistry, surface energy/tension/wettability, surface roughness, crystallinity, surface charge, feature size (nano vs micro), feature geometry and other mechanical properties such as elasticity. The cellular events that occur at the surface post protein adsorption include the formation of bonds between cell surface receptors (integrins) and the protein functional groups (ligands) [15]. Subsequently, cytoskeletal reorganization with progressive spreading of the cell on the substrate takes place. The critical step in this process occurs during the formation of bonds between the cell integrins and the ligands on the surface [15]. The complex mechanisms and receptors involved in the cell
0167-7799/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2012.02.005 Trends in Biotechnology, June 2012, Vol. 30, No. 6
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interactions with the surface have been a major focus in optimizing the biocompatibility and bioactivity of biomaterials. New nanotechnologies have been used to construct defined microenvironments for cells at the nanoscale in order to further investigate nanotopography-induced mechanisms of integrin-mediated cell signaling [11]. Hypothetically, cell behavior can be directly controlled by a surface. The incorporation of nanotopographic features that mimic the nanostructure of natural bone is becoming an interesting area of research in tissue engineering [16]. A number of studies suggest nanostructured biomaterials may offer surface and/or chemical properties closer to native bone, and therefore they might represent ideal substrates to support bone regeneration [17]. The two types of cells that orthopedic implants encounter are osteoblast cells (bone cells) and osteo-progenitor (mesenchymal) stem cells. Osteoblast cells are mature adult cells specific to bone tissue. Osteoblasts are responsible for building bone and depositing minerals to make up bone matrix components. MSCs are bone marrow derived, pluripotent cells with the capacity to differentiate into different cell types including osteoblasts, chondrocytes and adipocytes. One of the major challenges in bone implant materials is the surface design. A surface needs to stimulate osteoblast cells to re-synthesize the mineralized matrix and allow MSCs to attach and differentiate so that they deliver a mature population of bone-building cells for osseointegration and rapid bone regeneration.
Orthopedic biomaterials utilizing nanostructures Many studies have demonstrated better osteoblast functionality, including alkaline phosphatase (ALP) activity or bone forming ability and increased mineral deposition on various nanostructured materials. These include nanocrystalline and hydrothermal hydroxyapatite [18,19], electrospun nanofiber silk [20,21], anodized titanium [5,22–24] and nanostructured titanium surfaces [25]. Osteoblasts are very sensitive to the different material topographies and respond differently on submicron and nanometer sized surfaces despite even minimal size differences between two surface topographies [25]. Thus, bone cell behavior is highly dependent on the size and structure of the surface. As a result, research on bone regeneration has focused on investigating small changes in features to determine osteoblast cell functions. Another approach to further advance orthopedics implant technology is to introduce a nanostructured surface capable of controlling stem cell fate. MSCs need to be guided to selectively differentiate to osteoblasts, rather than differentiating into other types of cells such as myocytes, adipocytes or neural cells. Most previous studies have used the combination of osteogenic inducing media and nanostructured culture substrates to accelerate osteogenesis and bone formation of MSCs in vitro, but it is difficult to truly determine if the nanostructure itself induces MSC differentiation. Moreover, introducing biochemical factors into the media is very risky because they are usually synthetic
Table 1. Summary of in vitro studies of osteoblast and osteo-progenitor cells on various nanostructures Type of nanostructure
Size of feature Material
Nanophase
200–500
Ti, Ti6Al4V and CoCrMo
Nanopowder sintering
Nanopits Nanopores Nanotubes
120 – 15–100
Polymer demixed Alumina TiO2
E-beam lithography Anodization Anodization
Nanotubes
30–100
TiO2
Anodization
Nanotubes
20–120
TiO2
Anodization
Nanopillars
15, 55, 100
TiO2
Anodization through alumina mask
Nanogratings
350
PDMS (polydimethylsiloxane)
Soft lithography, embossing
Nanofibers
7.6
Peptide-amphiphile
Self-assembly
Nanofiber and Nanotexture
Various
Nano-roughened, Nanodomes Nanodots/lines
335 1352 150, 400, 600
PEOT/PBT [poly(ethylene Electrospinning oxide terephthalate)– poly(butylene terephthalate)] copolymer Tantalum Sputter coating
Complex
Various
Various Combinatory micro/nano-topography, nanoroughened 316
Method
Cell type
Assessment of cell behavior Osteoblast Increased metabolic activities, calcium deposition MSC Osteo-differentiation MSC Osteo-differentiation MSC Proliferation, osteo-differentiation Osteoblast, ALP activity, MSC osteo-differentiation Osteoblast Proliferation, differentiation Skeletal cells Adhesion, spreading, cytoskeletal formation, differentiation MSC Focal adhesions, cytoskeletal organization, mechanical properties MSC Attachment, proliferation, osteo-differentiation MSC Proliferation, morphology
Refs
MSC
Adhesion, proliferation
[35]
MSC
Synergistically enhanced [36] osteo-differentiation Cell shape, cytoskeleton [37] organization, osteogenic differentiation Osteogenic gene expression [38]
UV curable polyurethane polymer Chitosan-hydroxyapatite
UV-assisted capillary force lithography Crosslinkage and freeze-drying
Ti
Grit-blast + acid etched MSC
MSC
[28]
[29] [30] [12] [31] [40] [32]
[25]
[33] [39]
Review
Vertically aligned TiO2 nanotubes for bone regeneration The most common and successful biomaterial being used for bone implants is titanium, which does not elicit an inflammatory response in vivo. The bone bonding generally occurs without the common connective tissue layer that forms from the body’s immune response (foreign body reaction) between the implant metal and the underlying bone surface [42]. The majority of joint implants fail because they become loose at the biomaterial/bone interface, suggesting that poor osseointegration contributes to the failure [42]. Attempts are being made to replicate the porous structure of bone for implant designs. However, porous structures at present lack the mechanical strength needed for load bearing. There are a variety of modified titanium implants on the market that focus on incorporating rough structures and chemical coatings to improve osseointegration for bone implants, however, the implant coating materials are far from being optimal. Ti and its alloys have long been used as implantable biomaterials because (i) they have high-quality mechanical properties with a tensile strength and a modulus (110 GPa [43]) adequate for transferring stress between implant and bone; (ii) they exhibit a surface native oxide layer (TiO2) that is resistant to corrosion; and (iii) they are biocompatible and bioactively react with native human plasma and tissue. To further optimize the Ti surface for enhancing orthopedic implants, research groups have been modifying the Ti oxide surface structuring by electrochemical means to impart even better osseointegration by forming nanostructures on the oxide surface. Vertically aligned and laterally spaced TiO2 nanotubes created by electrochemical anodization have become increasingly popular for achieving superior osteoblast cell growth and directed osteogenic differentiation of MSCs because the modified topography, with nanometer thickness, increased surface area, porous structure and overall impact of the nanofeatures significantly accelerates cell adhesion and bone growth capabilities [5,6,10,11,13,31,40,44]. Formation mechanism and characterization of TiO2 nanotubes In this review, we describe TiO2 nanotubes prepared by anodization in fluorine-ion containing electrolytes. In general, the mechanism of TiO2 nanotube formation in fluoride-based electrolytes is said to occur as a result of three simultaneous processes: (i) the field assisted oxidation of Ti metal to form titanium dioxide; (ii) the field assisted dissolution of Ti metal ions in the electrolyte; and (iii) the chemical dissolution of Ti and TiO2 due to etching by fluoride ions [45]. TiO2 nanotubes are not formed on the
e-
TiO2 nanotube
MOx
{
molecules, foreign to the human body and their use in vivo would be unpredictable [26]. Consequently, it is important to optimize the nanostructure of the surface for enhanced orthopedic materials. Table 1 highlights some of the recent studies revealing how different nanostructures have influenced osteoblast and osteo-progenitor stem cells. Not only have the dimensions of the nanostructures been shown to affect cell response, but the geometry, spacing, pattern and symmetry of the surface features have been shown to be equally important [12,27–40] (for a recent review, see [41]).
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Ti Anode
Ti sheet
Pt Cathode
Diluted hydrofluoric acid
+
–
Power supply (Voltage applied) TRENDS in Biotechnology
Figure 1. This schematic illustrates the electrochemical anodization process used to fabricate the titanium oxide (TiO2) nanotube surface on titanium (Ti) metal. A Ti sheet is used as the anode and a platinum (Pt) sheet is used as the cathode submersed in a fluoride-based electrolyte solution. The duration and magnitude of power supplied to the system determines the height and diameter of the nanotubes.
pure Ti surface but on the thin TiO2 oxide layer naturally present on the Ti surface. The two-electrode electrochemical anodization set-up is schematically shown in Figure 1. An optional heat treatment step at 500 8C for 2 h is often employed to crystallize the as-fabricated amorphous structure into anatase structure. The anatase phase plays an important role in cell proliferation [6,46]. The final fabricated structure is shown in Figure 2 [40]. The nanotube surface on the anodized Ti substrate has a well defined and structurally sturdy morphology. The self assembled layers are highly ordered and vertically aligned. Different pore sizes and heights can be achieved by changing the voltage and electrolyte during the anodization setup. Table 2 [40] highlights the surface properties of the TiO2 nanotube layers. Enhanced surface roughness (such as by sand blasting the Ti or Ti alloy implant surface) is one of the important factors in providing the proper cues for a positive osteoblast response to implanted materials. However, much of the research related to the effect of macro- and micro-roughness on cellular responses and bone formation are inconclusive due to the non-uniformity of macro- and microroughness stemming from crude fabrication methods like polishing, sand blasting, chemical etching and so on. An important aspect of the nanotube system shown in the scanning electron microscopy (SEM) images in Figure 2 is that the nanotopography can feature a more defined, reproducible and reliable roughness than micro- and macrotopography for enhanced bone cell function in vivo. The TiO2 nanotube surfaces with diameters between 20 and 120 nm have surface roughness values between 9 and 46 nm, respectively. Another feature of the nanotube system is the superhydrophilicity. It is reasonable to hypothesize that TiO2 nanotubes promote greater cell adhesion over flat Ti partly due to nanotopography, and partly due to the significantly enhanced hydrophilic surface characteristics of the TiO2 nanotubes. The nanotube surfaces possess contact angles 317
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(a)
(b)
(c) m
.8 n
10
3. 0
nm
297
250 nm
300 nm
(e) .7nm
(d)
(f)
379
398
.9n
m
2 um
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Figure 2. Scanning electron microscopy (SEM) micrographs of titanium oxide (TiO2) nanotube layers by anodization under (a) 5 V, (b) 10 V, (c) 15 V, (d) 20 V and (e) 25 V. (f) is unanodized titanium (cross-sections are shown on the top left). Reprinted, with permission, from [40].
with water of 08, whereas Ti is more hydrophobic in nature with a contact angle of 808 (Table 2). Although it has not been directly measured, it is also interesting to note that there are inter connecting spaces between the nanotube walls {10 nm observed by transmission electron microscopy (TEM) for 100 nm diameter nanotubes [6]} which, even after cell adhesion or confluency, can possibly allow for continued fluid flow of culture media and increased exchange spaces for gas, nutrients and cell signaling molecules for an overall enhanced cell environment [5]. Fluid flow occurs naturally in the interstitial spaces around bone cells in the human body due to circulation and repetitive loading and unloading of bones during activity [47]. TiO2 nanotubes may provide increased channeling for proper fluid exchange for improving bone remodeling signaling molecules and functioning characteristics.
TiO2 nanotube lateral spacing effects Evidence that the lateral spacing of the features and the size of features on a nanoscale can impact cell behavior is rapidly increasing [7,48]. Therefore, in order to optimize the lateral spacing of TiO2 nanotubes for orthopedic implant surfaces, research groups have investigated comparative cell behavior cultured on various inner pore (15–300 nm) diameters of vertically aligned TiO2 nanotubes [10–12,31,49–51]. Based on the anodization mechanism of TiO2 nanotube formation, it is inherent that the nanotubular formation depends on the intensity of the voltage, where increasing voltage induces larger diameter nanotubes to be formed [52]. We will further discuss the effects of the diameter pore size by changing the voltage during anodization for optimizing the nanotube size for tuning the effects on bone regeneration. There is a unique variation in osteogenic cell behavior even within a narrow range of nanotube diameters
Table 2. Surface properties of unanodized titanium and anodized titanium under different voltagesa Voltage (V) 0 5 10 15 20 25 a
Diameter (nm) 0 20 50 70 100 120
Reprinted, with permission, from [40].
318
Thickness (nm) 0 100 250 300 400 400
Surface roughness (nm) 4.63 9.29 11.84 18.02 23.56 45.56
Contact angle (deg) 80.3 0 0 0 0 0
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[5,10–12,31]. In several studies where osteoblast cells [5,40] and MSCs [31] were grown on different diameter nanotubes (20, 30, 50, 70 and 100 nm), larger 70–100 nm diameter nanotubes had the highest osteoblast functionality and MSC osteo-differentiation compared to smaller diameters [5,31]. As the nanotube diameter increased, the osteogenic biochemical activity also increases, reaching the best values on 100 nm diameter nanotubes. Additional diameterdependent nanotube studies have determined that on a cellular level, functioning is related to the lateral spacing between cell integrins and adhesion points on the nanotube surfaces [12]. Because changing the diameter sizes also changed the location and spacing of transmembrane integrins, cytoskeletal tension in the actin filaments in the adhered cells are effected differently. Figure 3 illustrates the proposed mechanism of the assembly of the nanotubeintegrin-cytoskeleton linkage, which is highly sensitive to force [53]. The force of an adhering cell can be transmitted between the surface through the integrin and transferred to the cytoskeleton. The resultant stress on the cytoskeleton may trigger a cascade of reactions that alter the balance of anabolic/catabolic events within the cell. Ultimately, the modulation of focal adhesions, by changing the nanotube diameter, will alter cellular stress and signaling [53]. A nanotube diameter of 100 nm was large enough to increase cytoskeletal stress by inducing cell stretching across the pores and subsequently induce differentiation. Although the nature of cell adhesion and the degree of cytoskeletal tension affect cell response, the precise role of nanotopography on the adhesion, morphology and differentiation of cells has not yet been established. The mechanism by which such nanosurfaces direct MSC osteogenesis needs to be better understood, and the culture conditions need to be optimized to maximize MSC expansion and differentiation. The full potential of MSCs in regenerative medicine, in particular bone growth, requires cell proliferation and selective differentiation, and these processes occur at different but discrete nanosurface topography conditions such as variations in nanotube diameter. A concept developed based on nanopit topography [9] suggested that MSC differentiation is determined by mechanotransductive pathways that
Nucleus Fcell Cell membrane
Cytoskeleton Focal adhesion complex
FECM Integrins (brown)
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Figure 3. Effects of surface-induced stress by the nanotube surface. The focal adhesion complex experiences stress from a cell generated contractile force (Fcell) pulling against the extracellular matrix (ECM); in this case, the nanotube surface is the acting ECM. Stress generation is dependent on nanotube diameter. This stress is transformed through the cytoskeleton to the nucleus in the form of signaling molecules.
change depending on tension as the cell adheres to the underlying nanostructure surface. Moreover, mechanical forces from the topography are transduced to the cell nucleus [54]. Nanostructures act as an ECM, which imposes physical forces and morphological changes on the cell [53]. Furthermore, cells are exquisitely sensitive to forces of varying magnitudes, and they convert mechanical stimuli into a biochemical response. This phenomenon known as mechanotransduction provides a simple means by which cells and organisms can ensure structural stability, as well as a way to regulate morphogenetic movements to generate precise functionality; for instance, bone is shaped by forces of gravity and muscle contraction [55]. A recent review addresses the nanotopography/mechanical induction of stem cell differentiation based on changes of cell stiffness while under stress [56]. Different responses to TiO2 nanotube feature size Recently, more and more repetitive topographies at the nanoscale are being investigated to study cellular interactions with extracellular surfaces. The need for more extensive and repetitive studies is becoming more prominent because different research groups have observed varied responses. For instance, it has been found that MSCs behave differently on TiO2 nanotubes with diameters 15– 100 nm [11,12]. One study indicated that a spacing of 15– 30 nm was the optimal length scale for integrin clustering and focal contact formation for inducing differentiation and cells underwent apoptosis on 100 nm nanotubes. The results were seemingly different from the study where 100 nm diameters were found to be most optimal [33]. In yet another MSC study on 30, 150 and 300 nm Ti nanopores, the most potent nanostructure for osteogenic differentiation consisted of both Ti30 and Ti150, whereas Ti300 had limited effects [49]. In a slightly different study, 200 nm was the optimal feature size for MSC adhesion and proliferation [57]. The different results from these in vitro studies show that MSC osteogenesis is variable, and introduces several discussion points [57,58]. Such discussion points include: (i) the variation in cell types and/or cell species; (ii) the disparity in nanoscale features and physical properties; and (iii) the different experimental protocols, which all render in-depth analysis difficult and sometimes produce conflicting results even with similar nanostructures. Modification of the TiO2 nanotube surface To further improve the benefits of TiO2 nanotubes, several research groups are interested in modifying the surface of TiO2 nanotubes to investigate the influence of surface functionalization, surface chemistry and surface properties of the nanostructures on MSC osteogenesis. In one study, bone morphogenetic protein 2 (BMP2) was functionalized on the surface of nanotubes and it was found that the BMP2-coated surfaces were beneficial for cell proliferation and differentiation, with significantly higher differentiation levels than uncoated nanotubes [58]. Another study demonstrated that changing the wetting behavior of the nanotubes from superhydrophilic (as prepared) to superhydrophobic after the addition of a self-assembled monolayer (octadecylphosphonic acid) altered the adsorption of ECM proteins such as fibronectin, collagen type I, laminin and bovine serum 319
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Anodization
BMP2 loading
TiO2 nanotube
Ti substrate
Gel/Chi
Cells uptake
Layer by layer assembly
Cells adhesion
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Figure 4. Schematic illustration of the fabrication of bone morphogenetic protein 2 (BMP2)-loaded titanium nanotubes and cellular response. Reprinted, with permission, from [72].
albumin (BSA) and improved the attachment of MSCs [51]. Differences in hydrophobicity were found to be diameter dependent. In yet another study, epidermal growth factor (EGF) was coated on the surface of the different diameter nanotubes [59]. Here, EGF coating improved cell activity and cell number on 100 nm nanotubes. Another study preloaded the nanotubes with a synthetic hydroxyapatite on the tops and insides of the nanotubes using an alternative immersion method in order to stimulate enhanced apatite formation in simulated body fluids [60]. Mineral formation on the surface of implants is important for developing the natural integration with bone. MSCs have also been investigated on nanotubes coated with different materials such as carbon [44] and AuPd [10]. Although the different chemistries do not affect the diameter dependence on cell growth or function, the different chemistries do affect different cell types. Interestingly, carbon coated nanotubes enhance MSC differentiation over TiO2 nanotubes, but TiO2 nanotubes are better for osteoblast bone forming ability. Thus, it can be concluded that different surface properties are optimal for different cell types. Another interesting approach for bone regeneration materials has been to make nanostructured metal coatings, such as TiO2 nanotubes, on other non-metal materials, i.e. polymeric materials [61]. An in vitro study was carried out to characterize osteoblast (bone-forming cell) adhesion on several potential orthopedic polymeric materials (specifically, polyetheretherketone, ultra-high molecular weight polyethylene and polytetrafluoroethylene) coated with either titanium or gold using a novel ionic plasma deposition process that creates a surface-engineered nanostructure (with features below 100 nm). The result was improved osteoblast adhesion over flat Ti and uncoated polymers. To date, a large part of the interest has remained on modifying the existing TiO2 nanotubes, however little notice has been given to zirconium oxide (ZrO2) nanotubes, which are formed via a similar self-assembly mechanism to TiO2 nanotubes, via electrochemical anodization [62]. Zirconium (Zr) is similar to titanium: it possesses a thin passivation oxide layer which makes it highly resistant to corrosion in bodily fluids [63]. It was found that MSC growth and vitality depends on the diameter of the ZrO2 nanotubes in the same trend as TiO2 nanotubes 320
[10]. Alumina nanotubes [64] and tantalum oxide nanotubes [65], also fabricated by anodization, could also be potential osteogenic nanotube materials for orthopedic considerations. TiO2 nanotubes for nanodelivery Surface nanostructures could also be used for more efficient and precise nanodelivery than conventional approaches. Nanoporous materials with ordered and controlled pore structures have attracted great attention, particularly for implantable drug delivery systems [66]. For bone healing, both genes and proteins could be delivered to promote osteoblast bone forming ability, MSC osteogenesis and mineral formation. Although nanopore configuration has been studied greatly for potential biomedical and drug delivery type applications, 10–20 nm spacing between neighboring nanotubes provides increased volume and surface area. Long term small molecule and protein release has been reported for up to 1 month from TiO2 nanotubes by several research groups [67–71]. Loading the nanotubes with antibiotics was also found to increase osteoblast functionality [70]. Changing the nanotube diameter, wall thickness and length can alter the release kinetics for each specific drug to achieve a sustained release [69]. Arrays of 110 nm diameter TiO2 nanotubes on the surface of Ti films have been used as nanoreservoirs for growth factor (BMP2) using gelatin/chitosan (Gel/Chi) multilayers to control the release of the functional molecule and maintain its bioactivity. The arrays display great potential for retaining the bioactivity of the drug and regulating the motility and differentiation of MSCs [72] (Figure 4). Concluding remarks Material design of the substrate or implant topography to influence cell behavior, from proliferation to differentiation, is desirable for many regenerative medicine applications [4]. This approach utilizes the physical properties of nanostructured surfaces to establish a method for optimal control of cell physiology. By changing the dimensions or geometry of the nanofeatures, and the chemical properties, or by adding a nanometer thick film, a highly active surface can be developed to provide a solid and practical foundation of effective substrates for implant designs.
Review Although the careful analysis of numerous studies on cell and nanotopography interactions sheds light on some of the aspects of the underlying mechanism, many questions remain unanswered. For example, the optimal dimension of the anodized nanotubes varies between different research groups. This is why it is especially beneficial to try to translate the in vitro results to in vivo experiments using animal models and eventually used in clinical trials. For example, 30 nm TiO2 nanotubes have been shown to promote optimal growth in vitro [12], but in vivo, do not stimulate bone growth as expected [73]. However, another group showed 100 nm TiO2 nanotubes enhanced osseointegration in vitro and in vivo over both control and microtopographic surface features in rabbit tibia [74]. In another in vivo study in minipigs, the differences in osteogenesis response among 30, 70 and 100 nm nanotubes during the whole test period suggest that 70 nm were optimal [75]. Altogether, it is clear that TiO2 nanotube implants demonstrated a significant increase in new bone formation and gene expression associated with bone formation and remodeling over control surfaces; however, these studies are a workin-progress that can be utilized to improve and control bone forming functionality for advanced orthopedic implant technologies. Clinical studies with nanomaterials for bone repair are currently lacking. MSCs represent a unique new approach for bone regeneration. There is plenty of evidence that human stem cells can be seeded onto nanomaterials for growth and amplification as emphasized in this report and others [76,77]. This work helps to enable the design of improved nano-surfaces for selective MSC osteogenesis, thus providing a novel platform for MSC-based advanced implants. Engineering new materials to conveniently incorporate stem cells into implant design for accelerated bone repair and to enable new prostheses that integrate directly into bone is clinically desirable. An additional approach to fabricating bone regeneration materials is to combine both nanostructures and microstructures [38] to create 3D complex shaped tissue engineering scaffolds with cell controlled architecture and pore structures. This would better mimic the 3D nano-micromacro hierarchical structures. A novel aerosol-based 3D printing technique developed by OPTOMEC1 has been proposed recently to process nanophase ceramic/polymer scaffolds for bone tissue engineering applications created layer-by-layer from a pre-defined computer-aided design (CAD) model. Osteoblast adhesion tests were conducted on the 3D titania/poly(lactide-co-glycolide) (PLGA) nanocomposite scaffolds created by this technique and the results demonstrated that these 3D scaffolds further promoted osteoblast infiltration into porous structures compared to previous nanostructured surfaces [78]. In summary, there is a vast parameter space within which the size, geometry and pattern of the topographical features should be investigated for bone regeneration. The potential gain of these studies would probably be enormous both in the fundamental understanding of the central processes in cell biology and in application of a new approach to advance biomaterials and orthopedic implant design.
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Acknowledgments This work was supported by K. Iwama Endowed Chair fund, the von Liebig Grant at UC San Diego, and UC Discovery Grant No. ele08-128656/Jin.
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52 Rani, S. et al. (2010) Synthesis and applications of electrochemically self-assembled titania nanotube arrays. Phys. Chem. Chem. Phys. 12, 2780–2800 53 Chen, C.S. (2008) Mechanotransduction – a field pulling together? J. Cell Sci. 121, 3285–3292 54 Dalby, M.J. (2005) Topographically induced direct cell mechanotransduction. Med. Eng. Phys. 27, 730–742 55 Orr, A.W. et al. (2006) Mechanisms of mechanotransduction. Dev. Cell 10, 11–20 56 Teo, B.K. et al. (2010) Nanotopography/mechanical induction of stemcell differentiation. Methods Cell Biol. 98, 241–294 57 Dulgar-Tulloch, A.J. et al. (2009) Human mesenchymal stem cell adhesion and proliferation in response to ceramic chemistry and nanoscale topography. J. Biomed. Mater. Res. A 90, 586–594 58 Lai, M. et al. (2011) Surface functionalization of TiO2 nanotubes with bone morphogenetic protein 2 and its synergistic effect on the differentiation of mesenchymal stem cells. Biomacromolecules 12, 1097–1105 59 Bauer, S. et al. (2011) Covalent functionalization of TiO2 nanotube arrays with EGF and BMP-2 for modified behavior towards mesenchymal stem cells. Integr. Biol. 3, 927–936 60 Kodama, A. et al. (2009) Bioactivation of titanium surfaces using coatings of TiO(2) nanotubes rapidly pre-loaded with synthetic hydroxyapatite. Acta Biomater. 5, 2322–2330 61 Yao, C. et al. (2007) Nanostructured metal coatings on polymers increase osteoblast attachment. Int. J. Nanomedicine 2, 487–492 62 Berger, S. et al. (2008) Enhanced self-ordering of anodic ZrO2 nanotubes in inorganic and organic electrolytes using two-step anodization. Physica Status Solidi-Rapid Res. Lett. 2, 102–104 63 Oliveira, N.T. et al. (2005) Electrochemical studies on zirconium and its biocompatible alloys Ti-50Zr at.% and Zr-2.5Nb wt.% in simulated physiologic media. J. Biomed. Materials Res. A 74, 397–407 64 Feil, A.F. et al. (2011) From alumina nanopores to nanotubes: dependence on the geometry of anodization system. J. Nanosci. Nanotechnol. 11, 2330–2335 65 El-Sayed, H.A. and Birss, V.I. (2010) Controlled growth and monitoring of tantalum oxide nanostructures. Nanoscale 2, 793–798 66 Losic, D. and Simovic, S. (2009) Self-ordered nanopore and nanotube platforms for drug delivery applications. Expert Opin. Drug Deliv. 6, 1363–1381 67 Song, Y.Y. et al. (2009) Amphiphilic TiO2 nanotube arrays: an actively controllable drug delivery system. J. Am. Chem. Soc. 131, 4230–4232 68 Peng, L. et al. (2009) Long-term small molecule and protein elution from TiO2 nanotubes. Nano Lett. 9, 1932–1936 69 Popat, K.C. et al. (2007) Titania nanotubes: a novel platform for drugeluting coatings for medical implants? Small 3, 1878–1881 70 Popat, K.C. et al. (2007) Decreased Staphylococcus epidermis adhesion and increased osteoblast functionality on antibiotic-loaded titania nanotubes. Biomaterials 28, 4880–4888 71 Yao, C. and Webster, T.J. (2009) Prolonged antibiotic delivery from anodized nanotubular titanium using a co-precipitation drug loading method. J. Biomed. Mater. Res. B Appl. Biomater. 91, 587–595 72 Hu, Y. et al. (2012) TiO2 nanotubes as drug nanoreservoirs for the regulation of mobility and differentiation of mesenchymal stem cells. Acta Biomater 8, 439–448 73 von Wilmowsky, C. et al. (2009) In vivo evaluation of anodic TiO2 nanotubes: an experimental study in the pig. J. Biomed. Mater. Res. B Appl. Biomater. 89, 165–171 74 Bjursten, L.M. et al. (2010) Titanium dioxide nanotubes enhance bone bonding in vivo. J. Biomed. Mater. Res. 92, 1218–1224 75 Wang, N. et al. (2011) Effects of TiO2 nanotubes with different diameters on gene expression and osseointegration of implants in minipigs. Biomaterials 32, 6900–6911 76 Soumetz, F.C. et al. (2008) Human osteoblast-like cells response to nanofunctionalized surfaces for tissue engineering. J. Biomed. Mater. Res. B Appl. Biomater. 84, 249–255 77 Sundelacruz, S. and Kaplan, D.L. (2009) Stem cell- and scaffold-based tissue engineering approaches to osteochondral regenerative medicine. Semin. Cell Dev. Biol. 20, 646–655 78 Liu, H. and Webster, T.J. (2006) Ceramic/polymer nanocomposite tissue engineering scaffolds for more effective orthopedic applications: from 2D surfaces to novel 3D architectures. MRS Proc. 950, 0950-D10-03, DOI:10.1557/PROC-0950-D10-03
TiO2 nanotubes for bone regeneration Karla S. Brammer, Christine J. Frandsen and Sungho Jin Materials Science and Engineering, University of California, San Diego, La Jolla, CA 92093-0411, USA
Nanostructured materials are believed to play a fundamental role in orthopedic research because bone itself has a structural hierarchy at the first level in the nanometer regime. Here, we report on titanium oxide (TiO2) surface nanostructures utilized for orthopedic implant considerations. Specifically, the effects of TiO2 nanotube surfaces for bone regeneration will be discussed. This unique 3D tube shaped nanostructure created by electrochemical anodization has profound effects on osteogenic cells and is stimulating new avenues for orthopedic material surface designs. There is a growing body of data elucidating the benefits of using TiO2 nanotubes for enhanced orthopedic implant surfaces. The current trends discussed within foreshadow the great potential of TiO2 nanotubes for clinical use. Nanostructures for bone regeneration For optimal functioning and support, cells in our body rely on their interaction with an extracellular matrix (ECM) [1]. The ECM is a complex network of substrates with intricate features that are nanoscale in dimension. The ECM stimulates and interacts with cell physiology on a nanoscale level. Recent in vitro studies also reveal that nanofeatures on artificial substrate surfaces can impact cell physiology in the same way as the natural ECM. Artificial nanostructures influence how the cell adheres to the surface, cell spreading on the surface, cell biochemical functioning and even cell differentiation [2]. New fabrication techniques and new nanotechnologies have allowed precise control and manipulation of nanoscale features on artificial surfaces. The development of nanostructures has allowed unprecedented access to and intimate interaction with the fundamental subunits of biology. For bone regeneration and repair, recreating substrates on a nanoscale allows scientists to probe the first level of the bone structural hierarchy. This knowledge is used to present host cells with an innate ECM interface where they can repopulate and resynthesize a new matrix for creating new bone [3]. Most orthopedic materials research has focused on the nanometer level of precision for cell–material interactions for enhanced bioactivity. A rapidly established, strong and long lasting connection at the interface of an implant and bone is essential for the clinical success of orthopedic implants. A better understanding of how osteogenic cells interact with nanostructured materials in vitro has helped to design better implants for bone regeneration and implant integration in vivo. For orthopedic implant success, both osteoblast cells and mesenchymal stem cells (MSCs) play a critical role in Corresponding author: Jin, S. ([email protected]). Keywords: nanostructure; TiO2 nanotube; osteoblast; mesenchymal stem cell.
osteointegration, the direct physical and chemical bond at the implant–bone interface. There is little doubt that improving osseointegration for biomaterials and prosthesis will decrease implant failures and have a major impact on the quality of life for millions of people in the USA [4]. In terms of nanotopographic effects on osteoblast (bone cell) behavior, it has been shown that ceramics, such as titanium oxide (TiO2), with different nanostructures exhibit enhanced effects on growth rates and bone forming ability [5–9]. Many researchers utilize the common Ti surface nanostructuring technique of electrochemical anodization for creating nanotubular structures on the surface to stimulate rapid cell growth and mineralization of osteoblast cells. Anodized TiO2 nanotubes also show enhanced growth and accelerated osteo-differentiation of MSCs [10–13]. Direct efforts have been made to optimize the features of the nanotubes to (i) improve the bone forming function and mineralization of bone cells and (ii) induce differentiation of MSCs into mature bone cells for rapid bone regeneration and integration in vivo. The potential to modulate cellular response with nanostructured materials has generated a landslide of research in the orthopedic domain [1,14]. A comprehensive review of all the applications within orthopedics is beyond the scope of this work. We focused our perspective on TiO2 nanotube surface structuring in bone regeneration research. Examples of the current design strategies utilizing TiO2 nanotubes will be discussed. Orthopedic cell–surface interactions Manipulation of cell–surface interactions can improve the design and integration of orthopedic implants. When a material is initially exposed to in vitro or in vivo culture conditions, proteins in the cell culture media or bodily fluids adsorb to the material’s surface in less than 1 s [15]. There are several material factors that affect how the proteins will adhere, unfold and present different functional groups that impact how the surface is perceived by the cell. These include and are not limited to surface chemistry, surface energy/tension/wettability, surface roughness, crystallinity, surface charge, feature size (nano vs micro), feature geometry and other mechanical properties such as elasticity. The cellular events that occur at the surface post protein adsorption include the formation of bonds between cell surface receptors (integrins) and the protein functional groups (ligands) [15]. Subsequently, cytoskeletal reorganization with progressive spreading of the cell on the substrate takes place. The critical step in this process occurs during the formation of bonds between the cell integrins and the ligands on the surface [15]. The complex mechanisms and receptors involved in the cell
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interactions with the surface have been a major focus in optimizing the biocompatibility and bioactivity of biomaterials. New nanotechnologies have been used to construct defined microenvironments for cells at the nanoscale in order to further investigate nanotopography-induced mechanisms of integrin-mediated cell signaling [11]. Hypothetically, cell behavior can be directly controlled by a surface. The incorporation of nanotopographic features that mimic the nanostructure of natural bone is becoming an interesting area of research in tissue engineering [16]. A number of studies suggest nanostructured biomaterials may offer surface and/or chemical properties closer to native bone, and therefore they might represent ideal substrates to support bone regeneration [17]. The two types of cells that orthopedic implants encounter are osteoblast cells (bone cells) and osteo-progenitor (mesenchymal) stem cells. Osteoblast cells are mature adult cells specific to bone tissue. Osteoblasts are responsible for building bone and depositing minerals to make up bone matrix components. MSCs are bone marrow derived, pluripotent cells with the capacity to differentiate into different cell types including osteoblasts, chondrocytes and adipocytes. One of the major challenges in bone implant materials is the surface design. A surface needs to stimulate osteoblast cells to re-synthesize the mineralized matrix and allow MSCs to attach and differentiate so that they deliver a mature population of bone-building cells for osseointegration and rapid bone regeneration.
Orthopedic biomaterials utilizing nanostructures Many studies have demonstrated better osteoblast functionality, including alkaline phosphatase (ALP) activity or bone forming ability and increased mineral deposition on various nanostructured materials. These include nanocrystalline and hydrothermal hydroxyapatite [18,19], electrospun nanofiber silk [20,21], anodized titanium [5,22–24] and nanostructured titanium surfaces [25]. Osteoblasts are very sensitive to the different material topographies and respond differently on submicron and nanometer sized surfaces despite even minimal size differences between two surface topographies [25]. Thus, bone cell behavior is highly dependent on the size and structure of the surface. As a result, research on bone regeneration has focused on investigating small changes in features to determine osteoblast cell functions. Another approach to further advance orthopedics implant technology is to introduce a nanostructured surface capable of controlling stem cell fate. MSCs need to be guided to selectively differentiate to osteoblasts, rather than differentiating into other types of cells such as myocytes, adipocytes or neural cells. Most previous studies have used the combination of osteogenic inducing media and nanostructured culture substrates to accelerate osteogenesis and bone formation of MSCs in vitro, but it is difficult to truly determine if the nanostructure itself induces MSC differentiation. Moreover, introducing biochemical factors into the media is very risky because they are usually synthetic
Table 1. Summary of in vitro studies of osteoblast and osteo-progenitor cells on various nanostructures Type of nanostructure
Size of feature Material
Nanophase
200–500
Ti, Ti6Al4V and CoCrMo
Nanopowder sintering
Nanopits Nanopores Nanotubes
120 – 15–100
Polymer demixed Alumina TiO2
E-beam lithography Anodization Anodization
Nanotubes
30–100
TiO2
Anodization
Nanotubes
20–120
TiO2
Anodization
Nanopillars
15, 55, 100
TiO2
Anodization through alumina mask
Nanogratings
350
PDMS (polydimethylsiloxane)
Soft lithography, embossing
Nanofibers
7.6
Peptide-amphiphile
Self-assembly
Nanofiber and Nanotexture
Various
Nano-roughened, Nanodomes Nanodots/lines
335 1352 150, 400, 600
PEOT/PBT [poly(ethylene Electrospinning oxide terephthalate)– poly(butylene terephthalate)] copolymer Tantalum Sputter coating
Complex
Various
Various Combinatory micro/nano-topography, nanoroughened 316
Method
Cell type
Assessment of cell behavior Osteoblast Increased metabolic activities, calcium deposition MSC Osteo-differentiation MSC Osteo-differentiation MSC Proliferation, osteo-differentiation Osteoblast, ALP activity, MSC osteo-differentiation Osteoblast Proliferation, differentiation Skeletal cells Adhesion, spreading, cytoskeletal formation, differentiation MSC Focal adhesions, cytoskeletal organization, mechanical properties MSC Attachment, proliferation, osteo-differentiation MSC Proliferation, morphology
Refs
MSC
Adhesion, proliferation
[35]
MSC
Synergistically enhanced [36] osteo-differentiation Cell shape, cytoskeleton [37] organization, osteogenic differentiation Osteogenic gene expression [38]
UV curable polyurethane polymer Chitosan-hydroxyapatite
UV-assisted capillary force lithography Crosslinkage and freeze-drying
Ti
Grit-blast + acid etched MSC
MSC
[28]
[29] [30] [12] [31] [40] [32]
[25]
[33] [39]
Review
Vertically aligned TiO2 nanotubes for bone regeneration The most common and successful biomaterial being used for bone implants is titanium, which does not elicit an inflammatory response in vivo. The bone bonding generally occurs without the common connective tissue layer that forms from the body’s immune response (foreign body reaction) between the implant metal and the underlying bone surface [42]. The majority of joint implants fail because they become loose at the biomaterial/bone interface, suggesting that poor osseointegration contributes to the failure [42]. Attempts are being made to replicate the porous structure of bone for implant designs. However, porous structures at present lack the mechanical strength needed for load bearing. There are a variety of modified titanium implants on the market that focus on incorporating rough structures and chemical coatings to improve osseointegration for bone implants, however, the implant coating materials are far from being optimal. Ti and its alloys have long been used as implantable biomaterials because (i) they have high-quality mechanical properties with a tensile strength and a modulus (110 GPa [43]) adequate for transferring stress between implant and bone; (ii) they exhibit a surface native oxide layer (TiO2) that is resistant to corrosion; and (iii) they are biocompatible and bioactively react with native human plasma and tissue. To further optimize the Ti surface for enhancing orthopedic implants, research groups have been modifying the Ti oxide surface structuring by electrochemical means to impart even better osseointegration by forming nanostructures on the oxide surface. Vertically aligned and laterally spaced TiO2 nanotubes created by electrochemical anodization have become increasingly popular for achieving superior osteoblast cell growth and directed osteogenic differentiation of MSCs because the modified topography, with nanometer thickness, increased surface area, porous structure and overall impact of the nanofeatures significantly accelerates cell adhesion and bone growth capabilities [5,6,10,11,13,31,40,44]. Formation mechanism and characterization of TiO2 nanotubes In this review, we describe TiO2 nanotubes prepared by anodization in fluorine-ion containing electrolytes. In general, the mechanism of TiO2 nanotube formation in fluoride-based electrolytes is said to occur as a result of three simultaneous processes: (i) the field assisted oxidation of Ti metal to form titanium dioxide; (ii) the field assisted dissolution of Ti metal ions in the electrolyte; and (iii) the chemical dissolution of Ti and TiO2 due to etching by fluoride ions [45]. TiO2 nanotubes are not formed on the
e-
TiO2 nanotube
MOx
{
molecules, foreign to the human body and their use in vivo would be unpredictable [26]. Consequently, it is important to optimize the nanostructure of the surface for enhanced orthopedic materials. Table 1 highlights some of the recent studies revealing how different nanostructures have influenced osteoblast and osteo-progenitor stem cells. Not only have the dimensions of the nanostructures been shown to affect cell response, but the geometry, spacing, pattern and symmetry of the surface features have been shown to be equally important [12,27–40] (for a recent review, see [41]).
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Ti Anode
Ti sheet
Pt Cathode
Diluted hydrofluoric acid
+
–
Power supply (Voltage applied) TRENDS in Biotechnology
Figure 1. This schematic illustrates the electrochemical anodization process used to fabricate the titanium oxide (TiO2) nanotube surface on titanium (Ti) metal. A Ti sheet is used as the anode and a platinum (Pt) sheet is used as the cathode submersed in a fluoride-based electrolyte solution. The duration and magnitude of power supplied to the system determines the height and diameter of the nanotubes.
pure Ti surface but on the thin TiO2 oxide layer naturally present on the Ti surface. The two-electrode electrochemical anodization set-up is schematically shown in Figure 1. An optional heat treatment step at 500 8C for 2 h is often employed to crystallize the as-fabricated amorphous structure into anatase structure. The anatase phase plays an important role in cell proliferation [6,46]. The final fabricated structure is shown in Figure 2 [40]. The nanotube surface on the anodized Ti substrate has a well defined and structurally sturdy morphology. The self assembled layers are highly ordered and vertically aligned. Different pore sizes and heights can be achieved by changing the voltage and electrolyte during the anodization setup. Table 2 [40] highlights the surface properties of the TiO2 nanotube layers. Enhanced surface roughness (such as by sand blasting the Ti or Ti alloy implant surface) is one of the important factors in providing the proper cues for a positive osteoblast response to implanted materials. However, much of the research related to the effect of macro- and micro-roughness on cellular responses and bone formation are inconclusive due to the non-uniformity of macro- and microroughness stemming from crude fabrication methods like polishing, sand blasting, chemical etching and so on. An important aspect of the nanotube system shown in the scanning electron microscopy (SEM) images in Figure 2 is that the nanotopography can feature a more defined, reproducible and reliable roughness than micro- and macrotopography for enhanced bone cell function in vivo. The TiO2 nanotube surfaces with diameters between 20 and 120 nm have surface roughness values between 9 and 46 nm, respectively. Another feature of the nanotube system is the superhydrophilicity. It is reasonable to hypothesize that TiO2 nanotubes promote greater cell adhesion over flat Ti partly due to nanotopography, and partly due to the significantly enhanced hydrophilic surface characteristics of the TiO2 nanotubes. The nanotube surfaces possess contact angles 317
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(a)
(b)
(c) m
.8 n
10
3. 0
nm
297
250 nm
300 nm
(e) .7nm
(d)
(f)
379
398
.9n
m
2 um
TRENDS in Biotechnology
Figure 2. Scanning electron microscopy (SEM) micrographs of titanium oxide (TiO2) nanotube layers by anodization under (a) 5 V, (b) 10 V, (c) 15 V, (d) 20 V and (e) 25 V. (f) is unanodized titanium (cross-sections are shown on the top left). Reprinted, with permission, from [40].
with water of 08, whereas Ti is more hydrophobic in nature with a contact angle of 808 (Table 2). Although it has not been directly measured, it is also interesting to note that there are inter connecting spaces between the nanotube walls {10 nm observed by transmission electron microscopy (TEM) for 100 nm diameter nanotubes [6]} which, even after cell adhesion or confluency, can possibly allow for continued fluid flow of culture media and increased exchange spaces for gas, nutrients and cell signaling molecules for an overall enhanced cell environment [5]. Fluid flow occurs naturally in the interstitial spaces around bone cells in the human body due to circulation and repetitive loading and unloading of bones during activity [47]. TiO2 nanotubes may provide increased channeling for proper fluid exchange for improving bone remodeling signaling molecules and functioning characteristics.
TiO2 nanotube lateral spacing effects Evidence that the lateral spacing of the features and the size of features on a nanoscale can impact cell behavior is rapidly increasing [7,48]. Therefore, in order to optimize the lateral spacing of TiO2 nanotubes for orthopedic implant surfaces, research groups have investigated comparative cell behavior cultured on various inner pore (15–300 nm) diameters of vertically aligned TiO2 nanotubes [10–12,31,49–51]. Based on the anodization mechanism of TiO2 nanotube formation, it is inherent that the nanotubular formation depends on the intensity of the voltage, where increasing voltage induces larger diameter nanotubes to be formed [52]. We will further discuss the effects of the diameter pore size by changing the voltage during anodization for optimizing the nanotube size for tuning the effects on bone regeneration. There is a unique variation in osteogenic cell behavior even within a narrow range of nanotube diameters
Table 2. Surface properties of unanodized titanium and anodized titanium under different voltagesa Voltage (V) 0 5 10 15 20 25 a
Diameter (nm) 0 20 50 70 100 120
Reprinted, with permission, from [40].
318
Thickness (nm) 0 100 250 300 400 400
Surface roughness (nm) 4.63 9.29 11.84 18.02 23.56 45.56
Contact angle (deg) 80.3 0 0 0 0 0
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[5,10–12,31]. In several studies where osteoblast cells [5,40] and MSCs [31] were grown on different diameter nanotubes (20, 30, 50, 70 and 100 nm), larger 70–100 nm diameter nanotubes had the highest osteoblast functionality and MSC osteo-differentiation compared to smaller diameters [5,31]. As the nanotube diameter increased, the osteogenic biochemical activity also increases, reaching the best values on 100 nm diameter nanotubes. Additional diameterdependent nanotube studies have determined that on a cellular level, functioning is related to the lateral spacing between cell integrins and adhesion points on the nanotube surfaces [12]. Because changing the diameter sizes also changed the location and spacing of transmembrane integrins, cytoskeletal tension in the actin filaments in the adhered cells are effected differently. Figure 3 illustrates the proposed mechanism of the assembly of the nanotubeintegrin-cytoskeleton linkage, which is highly sensitive to force [53]. The force of an adhering cell can be transmitted between the surface through the integrin and transferred to the cytoskeleton. The resultant stress on the cytoskeleton may trigger a cascade of reactions that alter the balance of anabolic/catabolic events within the cell. Ultimately, the modulation of focal adhesions, by changing the nanotube diameter, will alter cellular stress and signaling [53]. A nanotube diameter of 100 nm was large enough to increase cytoskeletal stress by inducing cell stretching across the pores and subsequently induce differentiation. Although the nature of cell adhesion and the degree of cytoskeletal tension affect cell response, the precise role of nanotopography on the adhesion, morphology and differentiation of cells has not yet been established. The mechanism by which such nanosurfaces direct MSC osteogenesis needs to be better understood, and the culture conditions need to be optimized to maximize MSC expansion and differentiation. The full potential of MSCs in regenerative medicine, in particular bone growth, requires cell proliferation and selective differentiation, and these processes occur at different but discrete nanosurface topography conditions such as variations in nanotube diameter. A concept developed based on nanopit topography [9] suggested that MSC differentiation is determined by mechanotransductive pathways that
Nucleus Fcell Cell membrane
Cytoskeleton Focal adhesion complex
FECM Integrins (brown)
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Figure 3. Effects of surface-induced stress by the nanotube surface. The focal adhesion complex experiences stress from a cell generated contractile force (Fcell) pulling against the extracellular matrix (ECM); in this case, the nanotube surface is the acting ECM. Stress generation is dependent on nanotube diameter. This stress is transformed through the cytoskeleton to the nucleus in the form of signaling molecules.
change depending on tension as the cell adheres to the underlying nanostructure surface. Moreover, mechanical forces from the topography are transduced to the cell nucleus [54]. Nanostructures act as an ECM, which imposes physical forces and morphological changes on the cell [53]. Furthermore, cells are exquisitely sensitive to forces of varying magnitudes, and they convert mechanical stimuli into a biochemical response. This phenomenon known as mechanotransduction provides a simple means by which cells and organisms can ensure structural stability, as well as a way to regulate morphogenetic movements to generate precise functionality; for instance, bone is shaped by forces of gravity and muscle contraction [55]. A recent review addresses the nanotopography/mechanical induction of stem cell differentiation based on changes of cell stiffness while under stress [56]. Different responses to TiO2 nanotube feature size Recently, more and more repetitive topographies at the nanoscale are being investigated to study cellular interactions with extracellular surfaces. The need for more extensive and repetitive studies is becoming more prominent because different research groups have observed varied responses. For instance, it has been found that MSCs behave differently on TiO2 nanotubes with diameters 15– 100 nm [11,12]. One study indicated that a spacing of 15– 30 nm was the optimal length scale for integrin clustering and focal contact formation for inducing differentiation and cells underwent apoptosis on 100 nm nanotubes. The results were seemingly different from the study where 100 nm diameters were found to be most optimal [33]. In yet another MSC study on 30, 150 and 300 nm Ti nanopores, the most potent nanostructure for osteogenic differentiation consisted of both Ti30 and Ti150, whereas Ti300 had limited effects [49]. In a slightly different study, 200 nm was the optimal feature size for MSC adhesion and proliferation [57]. The different results from these in vitro studies show that MSC osteogenesis is variable, and introduces several discussion points [57,58]. Such discussion points include: (i) the variation in cell types and/or cell species; (ii) the disparity in nanoscale features and physical properties; and (iii) the different experimental protocols, which all render in-depth analysis difficult and sometimes produce conflicting results even with similar nanostructures. Modification of the TiO2 nanotube surface To further improve the benefits of TiO2 nanotubes, several research groups are interested in modifying the surface of TiO2 nanotubes to investigate the influence of surface functionalization, surface chemistry and surface properties of the nanostructures on MSC osteogenesis. In one study, bone morphogenetic protein 2 (BMP2) was functionalized on the surface of nanotubes and it was found that the BMP2-coated surfaces were beneficial for cell proliferation and differentiation, with significantly higher differentiation levels than uncoated nanotubes [58]. Another study demonstrated that changing the wetting behavior of the nanotubes from superhydrophilic (as prepared) to superhydrophobic after the addition of a self-assembled monolayer (octadecylphosphonic acid) altered the adsorption of ECM proteins such as fibronectin, collagen type I, laminin and bovine serum 319
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Anodization
BMP2 loading
TiO2 nanotube
Ti substrate
Gel/Chi
Cells uptake
Layer by layer assembly
Cells adhesion
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Figure 4. Schematic illustration of the fabrication of bone morphogenetic protein 2 (BMP2)-loaded titanium nanotubes and cellular response. Reprinted, with permission, from [72].
albumin (BSA) and improved the attachment of MSCs [51]. Differences in hydrophobicity were found to be diameter dependent. In yet another study, epidermal growth factor (EGF) was coated on the surface of the different diameter nanotubes [59]. Here, EGF coating improved cell activity and cell number on 100 nm nanotubes. Another study preloaded the nanotubes with a synthetic hydroxyapatite on the tops and insides of the nanotubes using an alternative immersion method in order to stimulate enhanced apatite formation in simulated body fluids [60]. Mineral formation on the surface of implants is important for developing the natural integration with bone. MSCs have also been investigated on nanotubes coated with different materials such as carbon [44] and AuPd [10]. Although the different chemistries do not affect the diameter dependence on cell growth or function, the different chemistries do affect different cell types. Interestingly, carbon coated nanotubes enhance MSC differentiation over TiO2 nanotubes, but TiO2 nanotubes are better for osteoblast bone forming ability. Thus, it can be concluded that different surface properties are optimal for different cell types. Another interesting approach for bone regeneration materials has been to make nanostructured metal coatings, such as TiO2 nanotubes, on other non-metal materials, i.e. polymeric materials [61]. An in vitro study was carried out to characterize osteoblast (bone-forming cell) adhesion on several potential orthopedic polymeric materials (specifically, polyetheretherketone, ultra-high molecular weight polyethylene and polytetrafluoroethylene) coated with either titanium or gold using a novel ionic plasma deposition process that creates a surface-engineered nanostructure (with features below 100 nm). The result was improved osteoblast adhesion over flat Ti and uncoated polymers. To date, a large part of the interest has remained on modifying the existing TiO2 nanotubes, however little notice has been given to zirconium oxide (ZrO2) nanotubes, which are formed via a similar self-assembly mechanism to TiO2 nanotubes, via electrochemical anodization [62]. Zirconium (Zr) is similar to titanium: it possesses a thin passivation oxide layer which makes it highly resistant to corrosion in bodily fluids [63]. It was found that MSC growth and vitality depends on the diameter of the ZrO2 nanotubes in the same trend as TiO2 nanotubes 320
[10]. Alumina nanotubes [64] and tantalum oxide nanotubes [65], also fabricated by anodization, could also be potential osteogenic nanotube materials for orthopedic considerations. TiO2 nanotubes for nanodelivery Surface nanostructures could also be used for more efficient and precise nanodelivery than conventional approaches. Nanoporous materials with ordered and controlled pore structures have attracted great attention, particularly for implantable drug delivery systems [66]. For bone healing, both genes and proteins could be delivered to promote osteoblast bone forming ability, MSC osteogenesis and mineral formation. Although nanopore configuration has been studied greatly for potential biomedical and drug delivery type applications, 10–20 nm spacing between neighboring nanotubes provides increased volume and surface area. Long term small molecule and protein release has been reported for up to 1 month from TiO2 nanotubes by several research groups [67–71]. Loading the nanotubes with antibiotics was also found to increase osteoblast functionality [70]. Changing the nanotube diameter, wall thickness and length can alter the release kinetics for each specific drug to achieve a sustained release [69]. Arrays of 110 nm diameter TiO2 nanotubes on the surface of Ti films have been used as nanoreservoirs for growth factor (BMP2) using gelatin/chitosan (Gel/Chi) multilayers to control the release of the functional molecule and maintain its bioactivity. The arrays display great potential for retaining the bioactivity of the drug and regulating the motility and differentiation of MSCs [72] (Figure 4). Concluding remarks Material design of the substrate or implant topography to influence cell behavior, from proliferation to differentiation, is desirable for many regenerative medicine applications [4]. This approach utilizes the physical properties of nanostructured surfaces to establish a method for optimal control of cell physiology. By changing the dimensions or geometry of the nanofeatures, and the chemical properties, or by adding a nanometer thick film, a highly active surface can be developed to provide a solid and practical foundation of effective substrates for implant designs.
Review Although the careful analysis of numerous studies on cell and nanotopography interactions sheds light on some of the aspects of the underlying mechanism, many questions remain unanswered. For example, the optimal dimension of the anodized nanotubes varies between different research groups. This is why it is especially beneficial to try to translate the in vitro results to in vivo experiments using animal models and eventually used in clinical trials. For example, 30 nm TiO2 nanotubes have been shown to promote optimal growth in vitro [12], but in vivo, do not stimulate bone growth as expected [73]. However, another group showed 100 nm TiO2 nanotubes enhanced osseointegration in vitro and in vivo over both control and microtopographic surface features in rabbit tibia [74]. In another in vivo study in minipigs, the differences in osteogenesis response among 30, 70 and 100 nm nanotubes during the whole test period suggest that 70 nm were optimal [75]. Altogether, it is clear that TiO2 nanotube implants demonstrated a significant increase in new bone formation and gene expression associated with bone formation and remodeling over control surfaces; however, these studies are a workin-progress that can be utilized to improve and control bone forming functionality for advanced orthopedic implant technologies. Clinical studies with nanomaterials for bone repair are currently lacking. MSCs represent a unique new approach for bone regeneration. There is plenty of evidence that human stem cells can be seeded onto nanomaterials for growth and amplification as emphasized in this report and others [76,77]. This work helps to enable the design of improved nano-surfaces for selective MSC osteogenesis, thus providing a novel platform for MSC-based advanced implants. Engineering new materials to conveniently incorporate stem cells into implant design for accelerated bone repair and to enable new prostheses that integrate directly into bone is clinically desirable. An additional approach to fabricating bone regeneration materials is to combine both nanostructures and microstructures [38] to create 3D complex shaped tissue engineering scaffolds with cell controlled architecture and pore structures. This would better mimic the 3D nano-micromacro hierarchical structures. A novel aerosol-based 3D printing technique developed by OPTOMEC1 has been proposed recently to process nanophase ceramic/polymer scaffolds for bone tissue engineering applications created layer-by-layer from a pre-defined computer-aided design (CAD) model. Osteoblast adhesion tests were conducted on the 3D titania/poly(lactide-co-glycolide) (PLGA) nanocomposite scaffolds created by this technique and the results demonstrated that these 3D scaffolds further promoted osteoblast infiltration into porous structures compared to previous nanostructured surfaces [78]. In summary, there is a vast parameter space within which the size, geometry and pattern of the topographical features should be investigated for bone regeneration. The potential gain of these studies would probably be enormous both in the fundamental understanding of the central processes in cell biology and in application of a new approach to advance biomaterials and orthopedic implant design.
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Acknowledgments This work was supported by K. Iwama Endowed Chair fund, the von Liebig Grant at UC San Diego, and UC Discovery Grant No. ele08-128656/Jin.
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