Ultraviolet Photofunctionalization of Titanium Implants Takahiro Ogawa, DDS, PhD1 In the face of growing demands and challenges in implant therapy, implant surfaces with improved biologic capabilities are required. This review paper summarizes the findings of recent in vitro and in vivo studies related to ultraviolet (UV) photofunctionalization of titanium. UV photofunctionalization is defined as an overall phenomenon of modification of titanium surfaces occuring after UV treatment, including the alteration of physicochemical properties and the enhancement of biologic capabilities. Bone morphogenesis around UV-treated titanium implants is distinctly improved compared with that seen around untreated control implants, leading to rapid and complete establishment of osseointegration with nearly 100% bone-toimplant contact in an animal model, as opposed to less than 55% for untreated implants. A series of in vitro studies demonstrated considerable enhancement of attachment, retention, and subsequent functional cascades of osteogenic cells derived from animals and humans after UV treatment. UV treatment converts titanium surfaces from hydrophobic to superhydrophilic and removes unavoidably contaminated hydrocarbons. UV-treated titanium surfaces also manifest a unique electrostatic status and act as direct cell attractants without the aid of ionic and organic bridges, which imparts a novel physicochemical functionality to titanium, which has long been understood as a bioinert material. UV treatment is simple and low in cost, and it has been proven effective for all types of titanium surfaces tested. These data suggest that UV photofunctionalization can be a novel, effective measure to improve implant therapy in the dental and orthopedic fields. Future research will focus on validating these findings in clinical studies. Int J Oral Maxillofac Implants 2014;29:e95–e102. doi: 10.11607/jomi.te47 Key words: bioactivity, biologic aging, bone-titanium integration, hydrocarbon, osseointegration, superhydrophilic, ultraviolet treatment
P
rosthetic replacement of missing teeth via dental implants has a considerable effect on oral health; masticatory function,1,2 speech,3 and quality of 4 life are improved in comparison to conventional removable dentures. However, despite the success of modern implant therapy and science, the application of implants is still limited because of various risk factors, including insufficient quality and quantity of
1P rofessor
and Director, Laboratory for Bone and Implant Sciences, The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics, Biomaterials and Hospital Dentistry, UCLA School of Dentistry, Los Angeles, California, USA.
Correspondence to: Dr Takahiro Ogawa, UCLA School of Dentistry, 10833 Le Conte Avenue (B3-081 CHS), Box 951668, Los Angeles, CA 90095-1668, USA. Fax: +310-825-6345. Email: [email protected] ©2014 by Quintessence Publishing Co Inc.
host bone,5 systemic conditions,6,7 and age.8,9 The protracted healing time (3 to 8 months) required for implants to integrate with the bone also limits their application. In the United States, 10% of adults and one-third of adults older than 65 years are completely edentulous.10,11 Despite the increasing need in an aging society, dental implant therapy has been employed in only 2% to 5% of potential patients.12 Challenges in orthopedic treatment cannot be ignored because of a close link and mutual endeavor between dental and orthopedic implants in exploring an improved biologic capability of implants as load-bearing devices. Osteoporotic fractures and degenerative changes in joints are common. Annual expenditures for osteoporotic fractures alone are estimated at $13.8 billion in the United States.13 More than 500,000 procedures are performed annually in the United States for hip and knee reconstruction. Although titanium implants are used for these procedures as endosseous anchors, there are differences The International Journal of Oral & Maxillofacial Implants e95
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UV photofunctionalized
Sandblasted surface
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Fig 1 Conversion from hydrophobic to hydrophilic surfaces of titanium by UV treatment. Top and side view images are shown of 10 µL of water on acid-etched and sandblasted titanium disks before and after UV treatment.
versus the therapeutic and biologic demands of dental implants. Bone fracture in these regions often does not permit postoperative immobilization of the implant (eg, cast splinting), and the implants are immediately subjected to the constant loading caused by gravity and activities of daily living (eg, walking). The metabolic potential of host bone in these patients is already compromised, as represented by osteoporotic and aged patients. Unlike the option of removable dentures, most orthopedic patients have limited therapeutic alternatives if the implants fail, and the resulting complications of orthopedic implant treatment may include disability14 and long-term dependence.15 Rapid and firm establishment of osseointegration has, therefore, been a persistent challenge.16–18 Successful implant anchorage is dependent upon the amount of bone directly deposited onto titanium surfaces without soft/connective tissue intervention. Current dental and orthopedic titanium implants have been developed based on this concept and are called “osseointegrated implants.” However, the total implant area covered by bone (bone-to-implant contact [BIC]) remains at 45% ± 16%19 or 50% to 75%20–22—far below the ideal 100% BIC. Most implants fail because of
an incomplete establishment, or early or late destructive changes to, the bone-to-implant interface.23–25 A crucial problem is the unknown cause of a lack of bone tissue formation entirely around the implant. Ultraviolet (UV)-mediated photofunctionalization is a recently reported method of surface modification for titanium to increase its biologic capacity; it is characterized by remarkable efficacy, unique mechanisms, and a simple delivery method.26,27 The biomechanical strength of in vivo osseointegration for UV-treated implants was three times greater than that for untreated implants at the early healing stage in an animal model,26 and nearly 100% BIC was achieved, as opposed to less than 55% BIC for untreated implants.26 The effectiveness of UV treatment has been proven for all surface topographies tested.26,28–32 The technology does not alter the existing topography, roughness, or other morphologic features of the implants and is categorized as neither an additive nor a subtractive method. This review paper summarizes the findings from recent in vitro and in vivo studies related to UV photofunctionalization of titanium implants.
DEFINITION OF UV PHOTOFUNCTIONALIZATION UV photofunctionalization is defined as an overall phenomenon of surface modification on titanium surfaces occurring after UV treatment, including the alteration of physicochemical properties and the enhancement in biologic capability.26,27,31,33–35 Titanium surfaces that have been sufficiently aged (ie, 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. The hydrophobic nature is common to all surface topographies of titanium, as shown in Fig 1 and reported extensively.28,29,31,35–37 On these surfaces, water dropped on the surfaces does not spread and stays in a hemispherical form. Very intriguingly, after treatment with UV light, these surfaces become remarkably wettable to water, with a contact angle of 0 degrees, which is referred to as super hydrophilic (Fig 1). The superhydrophilic surfaces shown in Fig 1 were obtained after 48 hours of UV treatment at an intensity of 0.1 mW/cm2 (λ= 360 ± 20 nm) and 2 mW/cm2 (λ= 250 ± 20 nm). Another notable change is in the chemical composition of the surfaces. Titanium surfaces, which actually become titanium dioxide surfaces as soon as they are exposed to the atmosphere, are covered by carboncontaining molecules to a significant degree because of the unavoidable constant accumulation of carbonyl moiety, particularly hydrocarbons, on titanium surfaces
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Fig 2 Evidence of increased peri-implant bone generation promoted by UV functionalization. These histologic images show peri-implant tissue at 2 weeks postimplantation in a rat femur model with and without UV treatment (Goldner trichrome).
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from the atmosphere and surrounding environments during surface preparation and storage.26,28,37–40 Currently used titanium implants are also contaminated with hydrocarbons.41–46 The amount of surface carbon is known to vary depending upon the age of the surface and reportedly can increase to approximately 60% to 75% of surface atomic components.45 UV treatment cleans such carbon-contaminated titanium surfaces, reducing the carbon percentage to less than 20%.26,28
IN VIVO EFFECTS OF UV PHOTOFUNCTIONALIZATION In vivo establishment of implant fixation in bone is a pertinent variable that reflects the clinical capacity of implants to bear loading. Via biomechanical testing in a rat model, extensive documentation has been established. UV-treated acid-etched titanium implants showed three times greater strength of osseointegration compared with untreated control implants in an early healing stage (2 weeks).26 The level of osseointegration seen at week 2 around UV-treated implants was equivalent to that seen around the untreated implants at week 8, indicating that UV treatment may have the potential to accelerate the process of osseointegration fourfold.26 Likewise, UV treatment was effective in enhancing the strength of osseointegration of sandblasted implants by a factor of 2.5.28,29 A clear distinction has been observed in osteomorphogenesis around UV-treated implants, as shown in Fig 2. New bone formation occurred extensively around UV-treated implants, with little intervention by soft tissue, while the bone tissues around untreated implants were fragmentary and localized.26 At the early healing stage (week 2) in a rat model, the BIC around UV-treated implants was 72%, which was 2.5 times greater than that seen around untreated implants. At week 4, the BIC was 98.2% for UV-treated implants,
while it was 53% for untreated implants. Peri-implant bone volume was also increased by approximately twofold after UV treatment at the early and late stages.26 It was noteworthy that UV photofunctionalization maintained its advantage during later stages of healing, unlike other surface modification technologies, indicating that UV photofunctionalization does not merely accelerate the process of osseointegration but also increases the level of osseointegration. The percentage of soft tissue intervention between the de novo bone and implant surface was less than 1% around UV-treated surfaces, as opposed to 21% around untreated implants.26 The effects of UV photofunctionalization of implants have been studied in challenging host conditions for osseointegration to simulate clinical situations. One study sought to determine whether UV treatment could enhance the process of osseointegration sufficiently to overcome the limitations of short implants.47 The use of short implants results in less invasive surgery and may confer a lower risk of surgical and postsurgical complications. It could also reduce the cost of treatment by avoiding the necessity of preimplantation site-development surgery, such as sinus elevation and bone augmentation procedures. Acid-etched titanium implants of 2-mm (regular) and 1.2-mm (short) lengths were prepared for an animal model.47 The reduced capacity for anchorage of short implants was demonstrated by the finding that the strength of osseointegration for regular-length implants was 80% and 100% greater than that achieved by the short implants at weeks 4 and 8 of healing, respectively. However, UV treatment of the short implants significantly increased the strength of osseointegration throughout the healing time. The strength of osseointegration at week 2 for UV-treated short implants was 100% greater than that measured around the regular-length implants without UV treatment. At later healing stages (weeks 4 and 8), UV-treated short implants showed a strength of osseointegration equivalent to that of The International Journal of Oral & Maxillofacial Implants e97
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untreated regular-length implants. The aforementioned substantial increases in BIC and bone volume conferred by UV photofunctionalization may theoretically explain this successful negation of the disadvantage of short implants. These results indicated that UV photo functionalization overcame the deficiency of 40% shorter implants in the animal model, which warrants future clinical research. Another compromised condition of osseointegration was tested using a gap healing model.48 Implant healing without bony support is a challenge in modern implant dentistry, as represented by implant placement in fresh extraction sockets and situations with simultaneous guided bone generation. Titanium implants with and without UV treatment were placed into a rat femur with contact (contact healing) or without contact (gap healing) with the native cortical bone.48 The strength of osseointegration in the gap healing model was onethird of that in the contact healing model. However, when UV-treated implants were placed in the gap healing model, the strength of osseointegration was equivalent to that of untreated implants in the contact healing sites. Microcomputed tomography analyses revealed that the bone volume around UV-treated implants was two to three times greater than that around the untreated implants in the gap healing model.48 The dynamic of bone formation was clearly distinguishable around implants with and without UV treatment. Bone formation around UV-photofunctionalized implants was characterized by connection osteogenesis, where new bone formation had been initiated at the implant interface, and most de novo bone tissues were located in the adjacent circumference of the implant surface, while bone formation around untreated implants consisted of outlying osteogenesis, in which bone formation occurred in the outer area of the implant surfaces, leaving the interface bone-free.
IN VITRO EFFECTS OF UV PHOTOFUNCTIONALIZATION It is important to correlate the results from in vitro studies with the results of in vivo studies to help understand the biologic process and mechanisms behind them. The in vitro effects of UV-treated titanium surfaces demonstrated in the literature include (1) increased adsorption of proteins, (2) increased osteogenic cell attachment, (3) increased retention of cells, (4) facilitated cell spread, (5) increased cell proliferation, and (6) enhanced osteoblastic differentiation. Affinity between biomaterials and cells is determined initially by the interaction between cells and proteins adsorbed on material surfaces. Protein adsorption to titanium implant surfaces is not the exception; it plays
a crucial role in cell attachment and subsequently regulates the spread, proliferation, and other functions of cells. Different surface topographies (machined, sandblasted, acid-etched, and nanofeatured) were tested in a series of studies for their potential enhancement after UV treatment. The amount of albumin and fibronectin adsorbed to titanium surfaces during 6 hours of incubation was greater for UV-treated surfaces by 80% to 300% compared to respective untreated surfaces.35,36 This overwhelming difference in protein adsorption capability between UV-treated and untreated surfaces was not diminished, even after 24 hours of incubation,26,33,35 suggesting that the effect of UV treatment may last beyond the initial stage of cell-titanium interaction. Various behaviors and responses of osteogenic/ osteoblastic cells have been compared in cultures on untreated and UV-treated titanium surfaces. Different surface topographies, including but not limited to acid-etched, sandblasted, machined, and nanofeatured surfaces, have been investigated. The number of osteoblasts attached to UV-treated surfaces during a certain period of incubation was substantially greater than that attached to untreated surfaces (three- to fivefold and two- to threefold after 3 and 24 hours of incubation, respectively).26,27,32 Typical examples of culture images are presented in Fig 3, in which an obviously greater number of cells had attached to and spread on UV-treated surfaces. The degree and nature of osteogenic cell settlement on implant surfaces are important. For instance, unsuccessful attachment and spread of osteogenic cells, which leaves the cells in a suspension and circular form, barely induces osteogenic phenotypes or even induces their differentiation.49,50 Further, in light of the fact that implant materials are subjected to functional loading, leading to mechanical stress and physical motion and friction at the interface, the initial settlement and retention of osteogenic cells are crucial.51–53 Osteoblasts cultured for 3 hours and 24 hours on titanium surfaces were detached mechanically by vibrational force and enzymatically by trypsin treatment.27 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 titanium surfaces (110% to 120% greater for cells incubated for 3 hours and 50% to 60% greater for cells incubated for 24 hours). The increased retention of the cells may be caused by the expedited settlement and reinforced adhesion of cells on UV-treated surfaces.27 During the initial stage of culture, osteoblasts on UV-treated surfaces are larger, with elongated cytoplasmic projections (filopodia and lamellipodia) and increased formation of cytoskeleton.27,36 They
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Fig 3 Evidence of enhanced osteoblast attachment and spread following UV photofunctionalization. Confocal laser microscopic images of bone marrow–derived osteoblasts at 3 hours after seeding on acid-etched titanium disks with and without UV treatment are shown. The cells were stained with rhodamine phalloidin for cytoskeletal actin filaments (red) and anti vinculin antibody for vinculin, a focal adhesion protein (green).
Untreated control
UV photofunctionalized
50 µm
also showed stronger expression of vinculin, a focal adhesion protein, indicating that the cells settled faster and more firmly on UV-treated surfaces.27 These unique cellular behaviors are exemplarily seen in Fig 3. The distinctive behaviors of cells on UV-treated surfaces may bring about additional biologic advantages in enabling the expedited and efficient initiation of peri-implant osteogenesis, particularly in immediate and early loading conditions. The proliferation and differentiation of osteogenic cells determine the amount and speed of bone formation, respectively. The rate of proliferation, normalized by the number of cells, increased by 20% to 50% on UV-treated surfaces,26 which supports the findings of an increased number of cells on UV-treated surfaces at later stages of cell culture, along with the increased cell attachment at the initial stage.26 The rate of osteogenic differentiation was examined using multiple assays. Levels of biologic markers, such as alkaline phosphatase activity and calcium ion deposition, and the expression of osteoblastic genes were consistently greater in cells cultured on UV-treated surfaces compared with those cultured on untreated surfaces.26,28,32,35,54 These in vitro effects have been confirmed with both animal- and human-derived osteoblasts, as well as periosteum-derived osteogenic cells.29,35,36,48
MECHANISMS BEHIND UV PHOTOFUNCTIONALIZATION The most significant link found between physicochemical factors and the biologic capability of titanium surfaces was the role of carbon contaminants on titanium surfaces.26,37 An inverse correlation was found between the amount of carbon element on titanium and its osteoblast attractiveness. Chemical analysis of acid-etched titanium surfaces was performed with
x-ray photoelectron spectroscopy. Prior to UV treatment, the titanium surfaces were characterized by a strong peak of atomic carbon ascribed to hydrocarbon contaminants. The atomic percentage of carbon increased up to more than 55%, depending upon the age of the titanium.26,37 The percentage of carbon was found to decrease to less than 20% after UV treatment, depending on the duration.26 A leastmean-squares approximation revealed that the rate of osteoblast attachment increased exponentially with UV-mediated progressive removal of carbon,26 suggesting a significant negative impact of surface carbon on osseointegration capability. Interestingly, the contact angle of water did not correlate with the rate of osteoblast attachment, although there seemed to be a positive effect of higher hydrophilicity on its cell attractiveness.26,35 The role of the surface hydrophilicity of biomaterials in determining their bioactivity is contentious. It is not a universal principle that the more hydrophilic the surface, the more biocompatible the material. For instance, under certain conditions, fewer cells attach to hydrophilic titanium surfaces than on hydrophobic titanium surfaces.55 Poly(lactide-co-beta-benzyl malolactonate) with improved hydrophilicity promotes NIH3T3 fibroblast attachment and proliferation,56 whereas poly hydroxyalkanoates with improved hydrophilicity showed reduced proliferation.57 Biofilms coated with calcium phosphate, which exhibit improved hydrophilicity, were associated with increased osteoblast proliferation.58 In contrast, the more hydrophobic polymer scaffold materials are effective in promoting bone regeneration.59 Although the conversion from hydrophobicity to hydrophilicity is a concomitant phenomenon during UV photofunctionalization, there is not sufficient evidence to support a cause-and-effect relationship between the degree of hydrophilicity and osseointegration capability. The International Journal of Oral & Maxillofacial Implants e99
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Fig 4 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.
Ti
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Fig 4a Electrostatic interaction of untreated titanium oxide (TiO2) surfaces with ions, proteins (green), and cells (blue). As long understood, ordinary TiO2 surfaces are electronegative. Proteins and cells are also electronegative. Therefore, the surface attracts proteins only with the aid of divalent cations, such as Ca2+, which in turn attracts cells via RGD-integrin interaction. There is only one biochemical tactic to successful migration and attachment of osteoblasts.
Fig 4b Direct electrostatic interaction of UV-treated TiO2 surfaces with cells (blue). The UV-treated titanium surface is full of cellattracting terminals consisting of the RGD sequence of proteins (green) or a positively charged TiO2 surface, which serve as direct chemoattractants to cells without divalent cations such as Ca2+. Proteins, which are negatively charged, adsorb directly to the positively charged TiO2 surface. Cells, which are negatively charged, also attach directly to the positively charged TiO2 surface.
Titanium has been used as a bioinert implantable material because of its good anticorrosive properties.60,61 There seems to be no direct interaction of titanium surfaces with biologic molecules and cells as a result of their bioinert nature. In fact, it has been a common understanding that titanium surfaces require ionic bridges (in particular, divalent cations) to attract necessary proteins and cells,60–62 as illustrated in Fig 4. However, as mentioned earlier, UV-treated titanium surfaces aggressively attract the proteins and cells necessary for osseointegration. Moreover, recent studies found that UV-treated titanium surfaces act as a direct attractant for cells because of their improved electrostatic status and no longer require the ionic bridges.27,31,33 The electrostatic differences between untreated and UV-treated titanium surfaces are schematically described in Fig 4. These known facts and new findings have convinced the authors to redefine the UV-treated titanium surface as a bioactive surface to distinguish it from the known bioinert titanium surface.31–34,37 Along with the remarkably increased level of osseointegration—nearly 100%—the biologic phenomenon newly induced around UV-treated bio active titanium surfaces can be defined as super osseointegration.31,33,34,54
CONCLUSION AND FUTURE PERSPECTIVES Bone morphogenesis induced around ultraviolet (UV)treated titanium implants was found to be distinctly improved, leading to a rapid and complete establishment of osseointegration with nearly 100% bone-toimplant contact in an animal model. A series of in vitro studies demonstrated a remarkable enhancement of attraction and subsequent functional cascades in osteogenic cells derived from animals and humans. UV treatment is simple and inexpensive and has been proven effective in all types of titanium surfaces tested. These data suggest that UV photofunctionalization can be a novel effective means to improve current implant therapies and to meet its growing demands in the dental and orthopedic fields. Future research will focus on validating the current and future findings in clinical analyses.
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37. Att W, Hori N, Takeuchi M, et al. Time-dependent degradation of titanium osteoconductivity: An implication of biological aging of implant materials. Biomaterials 2009;30: 5352–5363. 38. Kasemo B, Lausmaa J. Biomaterial and implant surfaces: On the role of cleanliness, contamination, and preparation procedures. J Biomed Mater Res 1988;22:145–158. 39. Kilpadi DV, Lemons JE, Liu J, Raikar GN, Weimer JJ, Vohra Y. Cleaning and heat-treatment effects on unalloyed titanium implant surfaces. Int J Oral Maxillofac Implants 2000;15: 219–230. 40. Serro AP, Saramago B. Influence of sterilization on the mineralization of titanium implants induced by incubation in various biological model fluids. Biomaterials 2003;24: 4749–4760. 41. Buser D, Broggini N, Wieland M, et al. Enhanced bone apposition to a chemically modified SLA titanium surface. J Dent Res 2004;83:529–533. 42. Massaro C, Rotolo P, De Riccardis F, et al. Comparative investigation of the surface properties of commercial titanium dental implants. Part I: Chemical composition. J Mater Sci Mater Med 2002;13:535–548. 43. Takeuchi M, Sakamoto K, Martra G, Coluccia S, Anpo M. Mechanism of photoinduced superhydrophilicity on the TiO2 photocatalyst surface. J Phys Chem B 2005;109: 15422–15428. 44. Kikuchi L, Park JY, Victor C, Davies JE. Platelet interactions with calcium-phosphate-coated surfaces. Biomaterials 2005;26:5285–5295. 45. Morra M, Cassinelli C, Bruzzone G, et al. Surface chemistry effects of topographic modification of titanium dental implant surfaces: 1. Surface analysis. Int J Oral Maxillofac Implants 2003;18:40–45. 46. Pae A, Kim SS, Kim HS, Woo YH. Osteoblast-like cell attachment and proliferation on turned, blasted, and anodized titanium surfaces. Int J Oral Maxillofac Implants 2011;26: 475–481. 47. Ueno T, Yamada M, Hori N, Suzuki T, Ogawa T. Effect of ultraviolet photoactivation of titanium on osseointegration in a rat model. Int J Oral Maxillofac Implants 2010;25:287–294. 48. Ueno T, Yamada M, Suzuki T, et al. Enhancement of bonetitanium integration profile with UV-photofunctionalized titanium in a gap healing model. Biomaterials 2010;31: 1546–1557.
49. Luo Y, Shoichet MS. A photolabile hydrogel for guided threedimensional cell growth and migration. Nat Mater 2004;3: 249–253. 50. Benya PD, Shaffer JD. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 1982;30:215–224. 51. Wang C, Gong Y, Zhong Y, Yao Y, Su K, Wang DA. The control of anchorage-dependent cell behavior within a hydrogel/microcarrier system in an osteogenic model. Biomaterials 2009;30:2259–2269. 52. Ruoslahti E, Reed JC. Anchorage dependence, integrins, and apoptosis. Cell 1994;77:477–478. 53. Grigoriou V, Shapiro IM, Cavalcanti-Adam EA, Composto RJ, Ducheyne P, Adams CS. Apoptosis and survival of osteoblastlike cells are regulated by surface attachment. J Biol Chem 2005;280:1733–1739. 54. Hori N, Iwasa F, Tsukimura N, et al. Effects of UV photofunctionalization on the nanotopography enhanced initial bioactivity of titanium. Acta Biomater 2011;7:3679–3691. 55. Zhao G, Schwartz Z, Wieland M, et al. High surface energy enhances cell response to titanium substrate microstructure. J Biomed Mater Res A 2005;74:49–58. 56. He B, Wan Y, Bei J, Wang S. Synthesis and cell affinity of functionalized poly(L-lactide-co-beta-malic acid) with high molecular weight. Biomaterials 2004;25:5239–5247. 57. Wang YW, Wu Q, Chen GQ. Reduced mouse fibroblast cell growth by increased hydrophilicity of microbial polyhydroxyalkanoates via hyaluronan coating. Biomaterials 2003;24: 4621–4629. 58. Ber S, Torun Kose G, Hasirci V. Bone tissue engineering on patterned collagen films: An in vitro study. Biomaterials 2005; 26:1977–1986. 59. Jansen EJ, Sladek RE, Bahar H, et al. Hydrophobicity as a design criterion for polymer scaffolds in bone tissue engineering. Biomaterials 2005;26:4423–4431. 60. Klinger A, Steinberg D, Kohavi D, Sela MN. Mechanism of adsorption of human albumin to titanium in vitro. J Biomed Mater Res 1997;36:387–392. 61. Ellingsen JE. A study on the mechanism of protein adsorption to TiO2. Biomaterials 1991;12:593–596. 62. Steinberg D, Klinger A, Kohavi D, Sela MN. Adsorption of human salivary proteins to titanium powder. I. Adsorption of human salivary albumin. Biomaterials 1995;16:1339–1343.
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P
rosthetic replacement of missing teeth via dental implants has a considerable effect on oral health; masticatory function,1,2 speech,3 and quality of 4 life are improved in comparison to conventional removable dentures. However, despite the success of modern implant therapy and science, the application of implants is still limited because of various risk factors, including insufficient quality and quantity of
1P rofessor
and Director, Laboratory for Bone and Implant Sciences, The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics, Biomaterials and Hospital Dentistry, UCLA School of Dentistry, Los Angeles, California, USA.
Correspondence to: Dr Takahiro Ogawa, UCLA School of Dentistry, 10833 Le Conte Avenue (B3-081 CHS), Box 951668, Los Angeles, CA 90095-1668, USA. Fax: +310-825-6345. Email: [email protected] ©2014 by Quintessence Publishing Co Inc.
host bone,5 systemic conditions,6,7 and age.8,9 The protracted healing time (3 to 8 months) required for implants to integrate with the bone also limits their application. In the United States, 10% of adults and one-third of adults older than 65 years are completely edentulous.10,11 Despite the increasing need in an aging society, dental implant therapy has been employed in only 2% to 5% of potential patients.12 Challenges in orthopedic treatment cannot be ignored because of a close link and mutual endeavor between dental and orthopedic implants in exploring an improved biologic capability of implants as load-bearing devices. Osteoporotic fractures and degenerative changes in joints are common. Annual expenditures for osteoporotic fractures alone are estimated at $13.8 billion in the United States.13 More than 500,000 procedures are performed annually in the United States for hip and knee reconstruction. Although titanium implants are used for these procedures as endosseous anchors, there are differences The International Journal of Oral & Maxillofacial Implants e95
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UV photofunctionalized
Sandblasted surface
Acid-etched surface
Untreated control
Fig 1 Conversion from hydrophobic to hydrophilic surfaces of titanium by UV treatment. Top and side view images are shown of 10 µL of water on acid-etched and sandblasted titanium disks before and after UV treatment.
versus the therapeutic and biologic demands of dental implants. Bone fracture in these regions often does not permit postoperative immobilization of the implant (eg, cast splinting), and the implants are immediately subjected to the constant loading caused by gravity and activities of daily living (eg, walking). The metabolic potential of host bone in these patients is already compromised, as represented by osteoporotic and aged patients. Unlike the option of removable dentures, most orthopedic patients have limited therapeutic alternatives if the implants fail, and the resulting complications of orthopedic implant treatment may include disability14 and long-term dependence.15 Rapid and firm establishment of osseointegration has, therefore, been a persistent challenge.16–18 Successful implant anchorage is dependent upon the amount of bone directly deposited onto titanium surfaces without soft/connective tissue intervention. Current dental and orthopedic titanium implants have been developed based on this concept and are called “osseointegrated implants.” However, the total implant area covered by bone (bone-to-implant contact [BIC]) remains at 45% ± 16%19 or 50% to 75%20–22—far below the ideal 100% BIC. Most implants fail because of
an incomplete establishment, or early or late destructive changes to, the bone-to-implant interface.23–25 A crucial problem is the unknown cause of a lack of bone tissue formation entirely around the implant. Ultraviolet (UV)-mediated photofunctionalization is a recently reported method of surface modification for titanium to increase its biologic capacity; it is characterized by remarkable efficacy, unique mechanisms, and a simple delivery method.26,27 The biomechanical strength of in vivo osseointegration for UV-treated implants was three times greater than that for untreated implants at the early healing stage in an animal model,26 and nearly 100% BIC was achieved, as opposed to less than 55% BIC for untreated implants.26 The effectiveness of UV treatment has been proven for all surface topographies tested.26,28–32 The technology does not alter the existing topography, roughness, or other morphologic features of the implants and is categorized as neither an additive nor a subtractive method. This review paper summarizes the findings from recent in vitro and in vivo studies related to UV photofunctionalization of titanium implants.
DEFINITION OF UV PHOTOFUNCTIONALIZATION UV photofunctionalization is defined as an overall phenomenon of surface modification on titanium surfaces occurring after UV treatment, including the alteration of physicochemical properties and the enhancement in biologic capability.26,27,31,33–35 Titanium surfaces that have been sufficiently aged (ie, 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. The hydrophobic nature is common to all surface topographies of titanium, as shown in Fig 1 and reported extensively.28,29,31,35–37 On these surfaces, water dropped on the surfaces does not spread and stays in a hemispherical form. Very intriguingly, after treatment with UV light, these surfaces become remarkably wettable to water, with a contact angle of 0 degrees, which is referred to as super hydrophilic (Fig 1). The superhydrophilic surfaces shown in Fig 1 were obtained after 48 hours of UV treatment at an intensity of 0.1 mW/cm2 (λ= 360 ± 20 nm) and 2 mW/cm2 (λ= 250 ± 20 nm). Another notable change is in the chemical composition of the surfaces. Titanium surfaces, which actually become titanium dioxide surfaces as soon as they are exposed to the atmosphere, are covered by carboncontaining molecules to a significant degree because of the unavoidable constant accumulation of carbonyl moiety, particularly hydrocarbons, on titanium surfaces
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Fig 2 Evidence of increased peri-implant bone generation promoted by UV functionalization. These histologic images show peri-implant tissue at 2 weeks postimplantation in a rat femur model with and without UV treatment (Goldner trichrome).
Untreated control
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100 µm
from the atmosphere and surrounding environments during surface preparation and storage.26,28,37–40 Currently used titanium implants are also contaminated with hydrocarbons.41–46 The amount of surface carbon is known to vary depending upon the age of the surface and reportedly can increase to approximately 60% to 75% of surface atomic components.45 UV treatment cleans such carbon-contaminated titanium surfaces, reducing the carbon percentage to less than 20%.26,28
IN VIVO EFFECTS OF UV PHOTOFUNCTIONALIZATION In vivo establishment of implant fixation in bone is a pertinent variable that reflects the clinical capacity of implants to bear loading. Via biomechanical testing in a rat model, extensive documentation has been established. UV-treated acid-etched titanium implants showed three times greater strength of osseointegration compared with untreated control implants in an early healing stage (2 weeks).26 The level of osseointegration seen at week 2 around UV-treated implants was equivalent to that seen around the untreated implants at week 8, indicating that UV treatment may have the potential to accelerate the process of osseointegration fourfold.26 Likewise, UV treatment was effective in enhancing the strength of osseointegration of sandblasted implants by a factor of 2.5.28,29 A clear distinction has been observed in osteomorphogenesis around UV-treated implants, as shown in Fig 2. New bone formation occurred extensively around UV-treated implants, with little intervention by soft tissue, while the bone tissues around untreated implants were fragmentary and localized.26 At the early healing stage (week 2) in a rat model, the BIC around UV-treated implants was 72%, which was 2.5 times greater than that seen around untreated implants. At week 4, the BIC was 98.2% for UV-treated implants,
while it was 53% for untreated implants. Peri-implant bone volume was also increased by approximately twofold after UV treatment at the early and late stages.26 It was noteworthy that UV photofunctionalization maintained its advantage during later stages of healing, unlike other surface modification technologies, indicating that UV photofunctionalization does not merely accelerate the process of osseointegration but also increases the level of osseointegration. The percentage of soft tissue intervention between the de novo bone and implant surface was less than 1% around UV-treated surfaces, as opposed to 21% around untreated implants.26 The effects of UV photofunctionalization of implants have been studied in challenging host conditions for osseointegration to simulate clinical situations. One study sought to determine whether UV treatment could enhance the process of osseointegration sufficiently to overcome the limitations of short implants.47 The use of short implants results in less invasive surgery and may confer a lower risk of surgical and postsurgical complications. It could also reduce the cost of treatment by avoiding the necessity of preimplantation site-development surgery, such as sinus elevation and bone augmentation procedures. Acid-etched titanium implants of 2-mm (regular) and 1.2-mm (short) lengths were prepared for an animal model.47 The reduced capacity for anchorage of short implants was demonstrated by the finding that the strength of osseointegration for regular-length implants was 80% and 100% greater than that achieved by the short implants at weeks 4 and 8 of healing, respectively. However, UV treatment of the short implants significantly increased the strength of osseointegration throughout the healing time. The strength of osseointegration at week 2 for UV-treated short implants was 100% greater than that measured around the regular-length implants without UV treatment. At later healing stages (weeks 4 and 8), UV-treated short implants showed a strength of osseointegration equivalent to that of The International Journal of Oral & Maxillofacial Implants e97
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untreated regular-length implants. The aforementioned substantial increases in BIC and bone volume conferred by UV photofunctionalization may theoretically explain this successful negation of the disadvantage of short implants. These results indicated that UV photo functionalization overcame the deficiency of 40% shorter implants in the animal model, which warrants future clinical research. Another compromised condition of osseointegration was tested using a gap healing model.48 Implant healing without bony support is a challenge in modern implant dentistry, as represented by implant placement in fresh extraction sockets and situations with simultaneous guided bone generation. Titanium implants with and without UV treatment were placed into a rat femur with contact (contact healing) or without contact (gap healing) with the native cortical bone.48 The strength of osseointegration in the gap healing model was onethird of that in the contact healing model. However, when UV-treated implants were placed in the gap healing model, the strength of osseointegration was equivalent to that of untreated implants in the contact healing sites. Microcomputed tomography analyses revealed that the bone volume around UV-treated implants was two to three times greater than that around the untreated implants in the gap healing model.48 The dynamic of bone formation was clearly distinguishable around implants with and without UV treatment. Bone formation around UV-photofunctionalized implants was characterized by connection osteogenesis, where new bone formation had been initiated at the implant interface, and most de novo bone tissues were located in the adjacent circumference of the implant surface, while bone formation around untreated implants consisted of outlying osteogenesis, in which bone formation occurred in the outer area of the implant surfaces, leaving the interface bone-free.
IN VITRO EFFECTS OF UV PHOTOFUNCTIONALIZATION It is important to correlate the results from in vitro studies with the results of in vivo studies to help understand the biologic process and mechanisms behind them. The in vitro effects of UV-treated titanium surfaces demonstrated in the literature include (1) increased adsorption of proteins, (2) increased osteogenic cell attachment, (3) increased retention of cells, (4) facilitated cell spread, (5) increased cell proliferation, and (6) enhanced osteoblastic differentiation. Affinity between biomaterials and cells is determined initially by the interaction between cells and proteins adsorbed on material surfaces. Protein adsorption to titanium implant surfaces is not the exception; it plays
a crucial role in cell attachment and subsequently regulates the spread, proliferation, and other functions of cells. Different surface topographies (machined, sandblasted, acid-etched, and nanofeatured) were tested in a series of studies for their potential enhancement after UV treatment. The amount of albumin and fibronectin adsorbed to titanium surfaces during 6 hours of incubation was greater for UV-treated surfaces by 80% to 300% compared to respective untreated surfaces.35,36 This overwhelming difference in protein adsorption capability between UV-treated and untreated surfaces was not diminished, even after 24 hours of incubation,26,33,35 suggesting that the effect of UV treatment may last beyond the initial stage of cell-titanium interaction. Various behaviors and responses of osteogenic/ osteoblastic cells have been compared in cultures on untreated and UV-treated titanium surfaces. Different surface topographies, including but not limited to acid-etched, sandblasted, machined, and nanofeatured surfaces, have been investigated. The number of osteoblasts attached to UV-treated surfaces during a certain period of incubation was substantially greater than that attached to untreated surfaces (three- to fivefold and two- to threefold after 3 and 24 hours of incubation, respectively).26,27,32 Typical examples of culture images are presented in Fig 3, in which an obviously greater number of cells had attached to and spread on UV-treated surfaces. The degree and nature of osteogenic cell settlement on implant surfaces are important. For instance, unsuccessful attachment and spread of osteogenic cells, which leaves the cells in a suspension and circular form, barely induces osteogenic phenotypes or even induces their differentiation.49,50 Further, in light of the fact that implant materials are subjected to functional loading, leading to mechanical stress and physical motion and friction at the interface, the initial settlement and retention of osteogenic cells are crucial.51–53 Osteoblasts cultured for 3 hours and 24 hours on titanium surfaces were detached mechanically by vibrational force and enzymatically by trypsin treatment.27 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 titanium surfaces (110% to 120% greater for cells incubated for 3 hours and 50% to 60% greater for cells incubated for 24 hours). The increased retention of the cells may be caused by the expedited settlement and reinforced adhesion of cells on UV-treated surfaces.27 During the initial stage of culture, osteoblasts on UV-treated surfaces are larger, with elongated cytoplasmic projections (filopodia and lamellipodia) and increased formation of cytoskeleton.27,36 They
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Fig 3 Evidence of enhanced osteoblast attachment and spread following UV photofunctionalization. Confocal laser microscopic images of bone marrow–derived osteoblasts at 3 hours after seeding on acid-etched titanium disks with and without UV treatment are shown. The cells were stained with rhodamine phalloidin for cytoskeletal actin filaments (red) and anti vinculin antibody for vinculin, a focal adhesion protein (green).
Untreated control
UV photofunctionalized
50 µm
also showed stronger expression of vinculin, a focal adhesion protein, indicating that the cells settled faster and more firmly on UV-treated surfaces.27 These unique cellular behaviors are exemplarily seen in Fig 3. The distinctive behaviors of cells on UV-treated surfaces may bring about additional biologic advantages in enabling the expedited and efficient initiation of peri-implant osteogenesis, particularly in immediate and early loading conditions. The proliferation and differentiation of osteogenic cells determine the amount and speed of bone formation, respectively. The rate of proliferation, normalized by the number of cells, increased by 20% to 50% on UV-treated surfaces,26 which supports the findings of an increased number of cells on UV-treated surfaces at later stages of cell culture, along with the increased cell attachment at the initial stage.26 The rate of osteogenic differentiation was examined using multiple assays. Levels of biologic markers, such as alkaline phosphatase activity and calcium ion deposition, and the expression of osteoblastic genes were consistently greater in cells cultured on UV-treated surfaces compared with those cultured on untreated surfaces.26,28,32,35,54 These in vitro effects have been confirmed with both animal- and human-derived osteoblasts, as well as periosteum-derived osteogenic cells.29,35,36,48
MECHANISMS BEHIND UV PHOTOFUNCTIONALIZATION The most significant link found between physicochemical factors and the biologic capability of titanium surfaces was the role of carbon contaminants on titanium surfaces.26,37 An inverse correlation was found between the amount of carbon element on titanium and its osteoblast attractiveness. Chemical analysis of acid-etched titanium surfaces was performed with
x-ray photoelectron spectroscopy. Prior to UV treatment, the titanium surfaces were characterized by a strong peak of atomic carbon ascribed to hydrocarbon contaminants. The atomic percentage of carbon increased up to more than 55%, depending upon the age of the titanium.26,37 The percentage of carbon was found to decrease to less than 20% after UV treatment, depending on the duration.26 A leastmean-squares approximation revealed that the rate of osteoblast attachment increased exponentially with UV-mediated progressive removal of carbon,26 suggesting a significant negative impact of surface carbon on osseointegration capability. Interestingly, the contact angle of water did not correlate with the rate of osteoblast attachment, although there seemed to be a positive effect of higher hydrophilicity on its cell attractiveness.26,35 The role of the surface hydrophilicity of biomaterials in determining their bioactivity is contentious. It is not a universal principle that the more hydrophilic the surface, the more biocompatible the material. For instance, under certain conditions, fewer cells attach to hydrophilic titanium surfaces than on hydrophobic titanium surfaces.55 Poly(lactide-co-beta-benzyl malolactonate) with improved hydrophilicity promotes NIH3T3 fibroblast attachment and proliferation,56 whereas poly hydroxyalkanoates with improved hydrophilicity showed reduced proliferation.57 Biofilms coated with calcium phosphate, which exhibit improved hydrophilicity, were associated with increased osteoblast proliferation.58 In contrast, the more hydrophobic polymer scaffold materials are effective in promoting bone regeneration.59 Although the conversion from hydrophobicity to hydrophilicity is a concomitant phenomenon during UV photofunctionalization, there is not sufficient evidence to support a cause-and-effect relationship between the degree of hydrophilicity and osseointegration capability. The International Journal of Oral & Maxillofacial Implants e99
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Fig 4 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.
Ti
Ti
Ti
Ti
O
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O O O
RGD
Ti
Ca
O
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O O O O O
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Fig 4a Electrostatic interaction of untreated titanium oxide (TiO2) surfaces with ions, proteins (green), and cells (blue). As long understood, ordinary TiO2 surfaces are electronegative. Proteins and cells are also electronegative. Therefore, the surface attracts proteins only with the aid of divalent cations, such as Ca2+, which in turn attracts cells via RGD-integrin interaction. There is only one biochemical tactic to successful migration and attachment of osteoblasts.
Fig 4b Direct electrostatic interaction of UV-treated TiO2 surfaces with cells (blue). The UV-treated titanium surface is full of cellattracting terminals consisting of the RGD sequence of proteins (green) or a positively charged TiO2 surface, which serve as direct chemoattractants to cells without divalent cations such as Ca2+. Proteins, which are negatively charged, adsorb directly to the positively charged TiO2 surface. Cells, which are negatively charged, also attach directly to the positively charged TiO2 surface.
Titanium has been used as a bioinert implantable material because of its good anticorrosive properties.60,61 There seems to be no direct interaction of titanium surfaces with biologic molecules and cells as a result of their bioinert nature. In fact, it has been a common understanding that titanium surfaces require ionic bridges (in particular, divalent cations) to attract necessary proteins and cells,60–62 as illustrated in Fig 4. However, as mentioned earlier, UV-treated titanium surfaces aggressively attract the proteins and cells necessary for osseointegration. Moreover, recent studies found that UV-treated titanium surfaces act as a direct attractant for cells because of their improved electrostatic status and no longer require the ionic bridges.27,31,33 The electrostatic differences between untreated and UV-treated titanium surfaces are schematically described in Fig 4. These known facts and new findings have convinced the authors to redefine the UV-treated titanium surface as a bioactive surface to distinguish it from the known bioinert titanium surface.31–34,37 Along with the remarkably increased level of osseointegration—nearly 100%—the biologic phenomenon newly induced around UV-treated bio active titanium surfaces can be defined as super osseointegration.31,33,34,54
CONCLUSION AND FUTURE PERSPECTIVES Bone morphogenesis induced around ultraviolet (UV)treated titanium implants was found to be distinctly improved, leading to a rapid and complete establishment of osseointegration with nearly 100% bone-toimplant contact in an animal model. A series of in vitro studies demonstrated a remarkable enhancement of attraction and subsequent functional cascades in osteogenic cells derived from animals and humans. UV treatment is simple and inexpensive and has been proven effective in all types of titanium surfaces tested. These data suggest that UV photofunctionalization can be a novel effective means to improve current implant therapies and to meet its growing demands in the dental and orthopedic fields. Future research will focus on validating the current and future findings in clinical analyses.
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