**3. Ultraviolet photofunctionalization**

Ultraviolet (UV) - mediated photofunctionalization is a method of surface modification for titanium that alters its physiochemical properties and enhances its biologic capability. It is characterized by remarkable efficacy, unique mechanisms, and a simple delivery method [56]. The effectiveness of UV treatment has been proven for all surface topographies tested. One of its unique features that set it apart from previously discussed surface modification techniques is that it does not alter the existing topography, roughness, or other morphologic features of miniscrews and is therefore categorized as neither an additive nor a subtractive method.

#### **3.1 Physiochemical properties**

For a very long time, it was assumed that the biologic properties of implant surfaces remained stable over time. It was later noted that over time, these surfaces underwent biologic degradation even when kept sterilized under optimal storage conditions. This is known as the time-dependent biologic degradation or biological aging of implant surfaces [57]. UV photofunctionalization affects these physiochemical changes via three key surface properties: i) the generation of superhydrophilicity; ii) a significant reduction of surface carbon, which unavoidably and unexceptionally accumulates on titanium surfaces; and iii) electrostatic conversion of surface charge from negative to positive.

#### *3.1.1 Hydrophilic conversion*

Titanium surfaces that have been sufficiently aged (i.e., more than 1 month after surface preparation) are hydrophobic; that is, the contact angle of water is greater than 60 degrees and close to or above 90 degrees on most surface types. Such a hydrophobic nature is common to all surface topographies of titanium and has been reported extensively [58, 59]. Water dropped on these surfaces does not spread and stays in a hemispherical form. Very intriguingly, after treatment with UV light, these titanium surfaces become remarkably wettable to water, with a contact angle of 0 degrees, which is referred to as being superhydrophilic (**Figures 8** and **9**) [56, 58, 59–63]. The superhydrophilic surfaces were obtained after UV treatment at

*Surface Modification of Titanium Orthodontic Implants DOI: http://dx.doi.org/10.5772/intechopen.100038*

#### **Figure 8.**

 *(A) Untreated titanium surface showing lack of droplet spread. (B) UV-treated titanium surface showing complete spread of water droplet. (Taken from: Rampurawala et al. [ 105 ].)* 

 **Figure 9.**

 *Hydrophilic conversion by photofunctionalization: (A) miniscrew; (B) untreated miniscrew with 2 drops (1* μ*L each); (C) photofunctionalized miniscrew with 2 drops (Taken from: Tabuchi et al. [ 103 ].)* 

an intensity of 0.1 mW/cm2 (λ = 360 ± 20 nm) and 2 mW/cm2 (λ = 250 ± 20 nm) for varying durations of time ranging from as little as 20 minutes to as much as 48 hours.

#### *3.1.2 Carbon reduction*

 Another notable change affected by UV modification is seen in the chemical composition of implant materials. Titanium surfaces, which become titanium dioxide surfaces as soon as they are exposed to the atmosphere, are covered by carbon-containing molecules to a significant degree because of the unavoidable constant accumulation of carbonyl moiety, particularly hydrocarbons, from the atmosphere and surrounding environment during surface preparation and storage [ 56 , 57 ]. Similarly, presently used titanium implants are also contaminated with hydrocarbons. The amount of carbon varies depending upon the age of the surface. X-ray photoelectron spectroscopy (XPS) studies have revealed that the atomic percentage of carbon increases from 20% up to 60% after 4 weeks of aging. UV photofunctionalization of these surfaces reduces the atomic carbon percentage to 20–35% depending on the wavelength of UV light used [ 58 ]. Thus, photofunctionalization of titanium has proven to be effective in reducing the atomic percentage of carbon, thereby cleaning such carbon-contaminated surfaces [ 56 , 57 , 60 – 62 , 64 – 66 ].

#### *3.1.3 Electrostatic conversion*

At an ionic level, ordinary titanium surfaces, viz. titanium surfaces without UV treatment, require inorganic bridges for protein adsorption and cell surface interaction, thus making titanium a bioinert material. In contrast, UV-treated titanium enables a direct cell-surface protein-titanium interaction without the aid of any inorganic bridges, thereby converting it into a bioactive surface (**Figure 10**). Albumin adsorption examined under different electrostatic environments revealed that adsorption on UV-treated surfaces at pH 7.0 was considerably greater than that on untreated surfaces (6-fold after 3 hrs of incubation and 2.5-fold after 24 hrs). Albumin adsorption on untreated control titanium surfaces increased after treating these surfaces with divalent cations but not after treating them with monovalent cations [66]. These findings suggest that the distinctly induced electropositive charge on UV-photofunctionalized titanium surfaces was responsible for the substantially increased efficiency of and capacity for protein adsorption on these titanium surfaces. Conversely, UV-enhanced cell adhesion was eliminated when the UV-treated titanium surfaces were electrostatically neutralized by either removing the electric charge or masking with monovalent anions, while the surfaces maintained their superhydrophilicity [67]. This unique electrostatic status of UV-treated titanium surfaces serves as a chemo-attractant for proteins, superseding the effect of the hydrophilic status, and may, therefore, be a critical regulatory factor in determining its subsequent bioactivity.

#### **3.2** *In vitro* **effects**

The *in vitro* effects of UV-treated titanium have been studied extensively. A majority of these studies have been aimed at the discovery and explanation of the interaction between living cells and implant material after UV treatment. The key findings of these studies are that UV photofunctionalization leads to: i) increased protein adsorption, ii) increased osteogenic cell attachment and facilitated cell spread, iii) increased retention of cells, iv) increased cell proliferation, and v) enhanced osteoblastic differentiation.

The affinity between biomaterials and cells is determined initially by the interaction between cells as well as proteins adsorbed on material surfaces. Protein adsorption to titanium implant surfaces plays a crucial role in cell attachment and subsequently regulates the spread, proliferation, and other cell functions [56, 60, 63, 66, 68–70]. UV-mediated enhancement of protein adsorption has been reported with different surface topographies of titanium as well as with different proteins. The amount of albumin and fibronectin adsorbed to titanium surfaces after 3 to 6 hrs of incubation was 6-fold greater for UV-treated surfaces in the initial few hours and remained up to 3-fold greater after 24 hrs [56, 66]. Iwasa et al. reported that the protein adsorption levels on the UV-treated 4-week-old titanium surface were equivalent to that on the new surfaces after 3 and 24 hrs of incubation [63]. Qin *et al.* reported that UV photofunctionalization increased adsorption of fibrinogen along with albumin but had no influence on competition between the two proteins [68]. Even though most studies have reported increased protein adsorption following UV treatment, Areid et al. found no qualitative differences in protein adsorption between UV and non-UV treated surfaces, but found that platelet adhesion was increased after UV treatment and that might suggest UV-enhanced thrombogenicity of nanostructured titanium [70].

Various behaviors and responses of osteogenic/osteoblastic cells have been compared in cultures on UV-treated and untreated titanium surfaces. Osteogenic cell

*Surface Modification of Titanium Orthodontic Implants DOI: http://dx.doi.org/10.5772/intechopen.100038*

#### **Figure 10.**

*Schematic description of the proposed mechanism of electrostatic interactions underlying the UV-photofunctionalization of titanium dioxide surfaces: UV-mediated conversion of titanium surfaces from bioinert to bioactive. (A) Hypothetical electric status of untreated and UV-treated TiO2 surfaces. As known and understood, ordinary TiO2 surfaces are electronegative, whereas UV-treated TiO2 surfaces are electropositively charged because of exited electrons from valence bands to conduction bands. (B) Electrostatic interaction of TiO2 surfaces with ions, proteins and cells. The untreated titanium surface (left) largely involves cell-inert terminals consisting of competitive binding of monovalent cations to negatively charged TiO2 surface. When cations are insufficient, this titanium surface remains electronegative and protein- and cell-repellent. The surface attracts proteins and cells only with an aid of divalent cations, such as Ca2+. In contrast, the UV-treated titanium surface (right) is full of cell-attracting terminals consisting of the RGD sequence of proteins or positively charged TiO2 surface, which serve as direct chemo-attractants to cells without divalent cations such as Ca2+. Proteins, that are negatively charged, adsorb directly to the positively charged the TiO2 surface. Cells, that are negatively charged, also attach directly to the positively charged the TiO2 surface. (C) A distinct interfacial layer formation at UV-photofunctionalized titanium surfaces. Based on the mechanisms in panel B, UV-induced bioactive titanium surfaces enable direct titanium–cell interaction, as opposed to untreated titanium surfaces that are bioinert and require inorganic and biological bridges for cell attachment and adhesion. (Taken from: Iwasa et al. [67].)*

#### **Figure 11.**

*Initial morphologies of the MG-63 osteoblasts on the titanium surface. (3000X, bar = 10 mm) SEM images of cells on the micro-arc oxidized (MAO), UVA-treated and UVC-treated surfaces after (A–C) 1 h and (D–F) 4 h incubation; (400X, bar = 50 mm) Fluorescence microscopy images of cells on the MAO, UVA-treated and UVCtreated surfaces after (G–I) 24 h incubation. (Taken from: Gao et al. [60].)*

attachment and spread is one such behavior that may indicate the responsiveness of implant materials towards UV pre-treatment. Different surface topographies, including but not limited to acid-etched, sandblasted, machined, and nano-featured surfaces, have been investigated [56, 57, 59, 60, 63, 67, 69, 71–76]. The number of osteoblasts attached to UV-treated surfaces was reported to be 3 to 5-fold higher after 3 hrs of incubation, and 2 to 3-fold higher after 24 hrs of incubation [56, 67, 74]. It is evident from these studies that UV photofunctionalization increases the capacity of osteoblastic cells to attach to and spread along titanium surfaces (**Figure 11**).

The degree and nature of osteogenic cell settlement on implant surfaces is important. For instance, lack of adequate attachment and spread of osteogenic cells fails to induce their functional phenotypes or even their differentiation [69, 71]. Further, considering that implant materials are subjected to functional loading which causes mechanical stress and friction at the interface, the initial settlement and retention of osteogenic cells is crucial. Iwasa et al. studied the retentive capacity of osteoblasts cultured on titanium surfaces for 3 and 24 hrs [67]. Cell detachment was attempted mechanically by vibrational force and enzymatically by trypsin treatment. Retention of the cells, as evaluated by the percentage of cells remaining after the detachment procedures, was substantially enhanced on UV-treated titanium surfaces compared to untreated surfaces (110–120% greater for cells incubated for 3 hrs and 50–60% greater for cells incubated for 24 hrs). Miyauchi et al. and Yamada et al. used a special biomechanical setup monitored under phase-contrast microscopy to assess the retention capacity of cultured osteoblasts [73, 77]. Their results showed that after incubation

#### *Surface Modification of Titanium Orthodontic Implants DOI: http://dx.doi.org/10.5772/intechopen.100038*

of 3 hrs, the mean critical shear force required to initiate detachment of a single osteoblast and the total energy required to complete the detachment was much greater for UV-treated TiO2 surfaces as compared to untreated surfaces. Such substantial increases in single-cell adhesion were also observed for osteoblasts cultured for 24 hrs.

Cell retention and adhesion can also be assessed by studying the cytoskeletal structure and proteins on the osteoblasts. It was observed by Iwasa et al. that during the initial stage of cell culture, osteoblasts on UV-treated surfaces were larger, with elongated cytoplasmic projections (filopodia and lamellipodia) and increased formation of cytoskeleton [67]. Vinculin, a focal adhesion protein involved in cell linkage serving a key role in initiating and establishing cell adhesion, has also been used to evaluate cell retention capacity. Studies using image-based densitometry as well as western blot test revealed that the extent of vinculin expression in an individual osteoblast was substantially higher on UV-treated surfaces than on untreated surfaces after incubation with rat-derived osteoblasts (up to 5-fold higher at 3 hrs and 2.5-fold higher at 24 hrs). However, the increased vinculin expression was observed only when standardized with the total protein and not when standardized with the cell area [63, 67, 69, 73, 77]. Iwasa et al. found that expression of other focal adhesion proteins such as paxillin and phosphorylated paxillin was higher on UV-treated surfaces [63]. Thus, the increased retention of the cells may be caused by the expedited and efficient settlement as well as reinforced adhesion of cells on UV-treated titanium surfaces.

The proliferation and differentiation of osteogenic cells determine the amount and speed of bone formation, respectively. The rate of proliferation of osteoblasts evaluated by BrdU incorporation assay, which targets the S phase of the cell cycle, has been reported to increase by up to 50–80% after UV-treatment of titanium [71]. The rate of osteogenic differentiation can be examined using multiple assays for various biologic markers. Alkaline phosphatase activity, calcium ion deposition, expression of collagen I, osteopontin, osteocalcin, and expression of other osteoblastic genes are some parameters which have been consistently evaluated on UV-treated titanium surfaces [67, 71, 72, 77]. Cell mineralization assays have reported increased alkaline phosphatase activity as well as increased calcium ion deposition for all UV-treated surfaces with different topographies. Studies with RT-PCR analysis showed an upregulation of the expression of collagen I, osteopontin and osteocalcin by up to 70%. As much as adhesion behavior varies with surface properties of implant materials, it is also regulated by the Rho-family GTPase enzymes. These enzymes are controlled by the Rac, Rho and Cdc42 genes. Gene expression analysis by Iwasa et al. revealed that for UV-treated titanium surfaces cultured with rat-derived osteoblasts, expression of Rac was upregulated by 1.5-fold after 3 hrs and 1.7-fold after 24 hrs of incubation, expression of Cdc42 was upregulated by 2-fold after 3 hrs and 1.5-fold after 24 hrs, but expression of Rho was not altered significantly [63]. Harder et al. studied the changes in pro-inflammatory gene expression in human whole blood after initial contact with UV-conditioned implant surfaces and found that there was suppression of IL-1β expression whereas there was no change in TNF-α expression [78]. All of the above *in vitro* studies have been confirmed with both animal and human-derived osteoblasts, as well as periosteum-derived osteogenic cells [63, 67, 68].

Microbial attachment on implant surfaces, especially at the implant-tissue interface is the primary cause of peri-implant inflammation and subsequent implant failure. UV photofunctionalization has been shown to have a considerable effect on bacterial accumulation around implants. The UV-induced physiochemical changes in titanium surfaces were reported to be responsible for the reduced bacterial attachment and biofilm formation. Yamada et al. reported via fluorescence microscopic

quantification that attachment of bacterial pathogens such as *Staphylococcus aureus* or *Streptococcus pyogenes* on titanium surfaces (irrespective of their topography) was reduced following UV treatment [61]. Denaturing gradient gel-electrophoresis (DGGE) and DNA sequencing analyses by de Avila et al. revealed that while bacterial community profiles appeared different between UV-treated and untreated titanium in the initial attachment phase, this difference vanished as biofilm formation progressed [79]. Jain et al. reported that despite the reductive effect of UV pre-irradiation on bacterial attachment, cell viability was not affected adversely as 50% of bacterial killing capacity was maintained [80]. This suggests that UV-photofunctionalization of titanium has a strong potential to improve the outcome of implant placement by creating and maintaining antimicrobial surfaces.

A few authors sought to explain the effect of implant photofunctionalization from a technical perspective. Ohyama et al. carried out finite element analyses to understand how the photofunctionalization-led increase in BSC affected the peri-implant mechanical stress distribution. They reported that the simulated increase in BSC from 53–98% improved distribution and diffusion of peri-implant stress more effectively than using longer implants [81]. Another such study by Ohyama et al. concluded that under vertical loading, photofunctionalization had a greater effect than increased implant diameter on stress reduction [82]. Thus, UV treatment of implants may potentially reduce periimplant stress and counteract the stress-induced marginal bone loss.

#### **3.3** *In vivo* **effects**

It is important to correlate the results from *in vitro* studies with the results of *in vivo* studies to help understand and validate the biologic processes and mechanisms behind them. *In vivo* establishment of implant fixation in bone is a pertinent variable that reflects the clinical capacity of implants to bear loading. There has been extensive documentation regarding the strength of osseointegration and implant stability as determined by the histomorphometric assessment of BSC, biomechanical testing, and ISQ measurements.

Photofunctionalization substantially increases the strength of bone-implant integration by enabling near-complete coverage of bone around the implant. Various studies have reported the degree of osseointegration as evaluated by micro-CT, SEM and EDS analyses to be considerably higher when implants were pre-treated with UV light [56, 64, 71, 72, 83–85]. Pyo et al. evaluated the bone-implant interface of UV-treated implants using static and dynamic histological techniques, and when compared to UV-untreated implants, they reported an intensive mineralized layer in marginal bone which improved marginal bone seal and support, and expedited robust interfacial bone deposition (**Figure 12**) [83]. Studies have also shown that new bone formation occurs extensively around UV-treated implants, with little intervention by soft tissue (less than 1%), while the bone tissues around untreated implants are fragmentary and localized with intervening soft tissue (up to 21%) [71]. Yamazaki et al. reported increased peri-implant bone volume (1.5–2 fold) after UV treatment at the early and late stages without deterioration of bone mineral density [84]. In addition to bone volume studies, Hirota et al. used EDS mapping to determine the mineral content of new bone. Their results showed elemental peaks of calcium and phosphorus on various parts of UV-treated implants but the treated, as well as untreated implants, comprised the same Ca/P ratio, indicating bone tissue. However, the Ca/Ti ratios of the UV-treated implant surfaces were approximately 20 times greater than those of the control group (**Figure 13**) [72]. It is noteworthy that UV photofunctionalization maintains its advantage during later stages of healing, unlike other surface modification
