Silk electrogel coatings for titanium dental implants.

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Silk electrogel coatings for titanium dental implants Roberto Elia, Courtney D Michelson, Austin L Perera, Masly Harsono, Gray G Leisk, Gerard Kugel and David L Kaplan J Biomater Appl published online 25 November 2014 DOI: 10.1177/0885328214561536 The online version of this article can be found at: http://jba.sagepub.com/content/early/2014/11/25/0885328214561536

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Functional biomaterials surfaces

Silk electrogel coatings for titanium dental implants

Journal of Biomaterials Applications 0(0) 1–9 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328214561536 jba.sagepub.com

Roberto Elia1, Courtney D Michelson2, Austin L Perera2, Masly Harsono2, Gray G Leisk3, Gerard Kugel2 and David L Kaplan1

Abstract The aim of this study was to develop biocompatible, biodegradable dental implant coatings capable of withstanding the mechanical stresses imparted during implant placement. Two techniques were developed to deposit uniform silk fibroin protein coatings onto dental implants. Two novel coating techniques were implemented to coat titanium shims, studs, and implants. One technique involved electrodeposition of the silk directly onto the titanium substrates. The second technique consisted of melting electrogels and dispensing the melted gels onto the titanium to form the coatings. Both techniques were tested for coating reproducibility using a stylus profilometer and a dial thickness gauge. The mechanical strength of adhered titanium studs was assessed using a universal mechanical testing machine. Uniform, controllable coatings were obtained from both the electrodeposition and melted electrogel coating techniques, tunable from 35 to 1654 mm thick under the conditions studied, and able to withstand delamination during implantation into implant socket mimics. Mechanical testing revealed that the adhesive strength of electrogel coatings, 0.369 0.09 MPa, rivaled other biologically derived coating systems such as collagen, hydroxyapatite, and chitosan (0.07–4.83 MPa). These novel silkbased techniques offer a unique approach to the deposition of safe, simple, mechanically robust, biocompatible, and degradable implant coatings. Keywords Electrogelation, adhesive silk coating, dental implant coating, electrodeposition, bioactive coating

Introduction There has been an increase in dental implant use for restoration of normal function, speech, and aesthetics over the past 50 years.1 The success rate for these implants depends on implant location2 but generally exceeds 90% in healthy individuals.2–4 However, there remains a subset of the population with one or more risk factors that greatly reduces implant success. Patients who smoke, have diabetes or osteoporosis, or have had head/neck radiation from past cancer treatments are all considered high risk for dental implant failure.5–8 For these patients, bone quality, density, and healing abilities are often compromised and implant integration is limited, leading to implant failures.9,10 Osseointegration is generally accepted as essential for implant stability and eventual clinical success.10 Wound healing and osseointegration following dental implant placement is a complex process involving a

number of biological events.10 Titanium implants have been widely used because of their bio-inertness and biocompatibility, mechanical characteristics including proper transfer of biting forces, and osteoconductive and osteoinductive properties.11–14 Depending on surface texture, roughness, and chemical treatments, titanium implants promote cellular growth, adhesion, and bone integration.9,15–19

1

Department of Biomedical Engineering, Tufts University Medford, Massachusetts, USA 2 School of Dental Medicine, Tufts University Boston, Massachusetts, USA 3 Department of Mechanical Engineering, Tufts University Medford, Massachusetts, USA Corresponding author: David L Kaplan, Department of Biomedical Engineering, Tufts University Medford, 4 Colby Street, Science and Technology Center, MA 02155, USA. Email: [email protected]



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The incorporation of biological agents onto dental implant surfaces or within coatings has been increasing over the last 10 years to improve implant osseointegration and reduce implant site inflammation. Although promising, implant surface modifications and the incorporation of bioactive molecules is complex. During implant preparation, treatments may include expensive processes or extreme temperatures and pHs, which can interfere with the bioactivity of many incorporated molecules,11,20 thereby limiting options. The external coatings on titanium implants are also subjected to coating delamination which can result in poor osseointegration and ultimately failure.21 Regenerated silk fibroin protein derived from the silkworm Bombyx mori cocoons has a unique combination of mechanical and physical properties, including tunable degradation, biocompatibility, drug stabilizing capabilities, and impressive mechanical properties.22–25 The fibroin protein is versatile and depending on processing conditions can be formed into coatings, films, mats, gels, and foams.26–29 Most recently, an electrogelation technique was developed which allows the direct deposition of silk gels on conductive materials with associated adhesion.30–32 Upon drying, these electrogels (egels) form a uniform film coating of controlled thickness that conforms to the topography of the underlying substrate. Like other silk formats, silk egel degradation can be tuned based on annealing conditions, and silk has demonstrated good in vitro cell adhesion and proliferation, with minimal inflammation when implanted in vivo. The aim of this work was to study the utility of silk egels for dental implant coatings as a route to expand biofunctionalization options for implants by optimizing implant-coating adherence and reducing exposure to the harsh processing conditions required by current coating technologies. Longer term, the goal is for more cost-effective coating options with improved integration for both healthy as well as high-risk patients.

Materials and methods Silk extraction Silk fibroin protein was purified from B. mori silkworm cocoons resulting in an aqueous silk fibroin solution.24 Cocoons were cut into pieces and boiled for 10 min in an aqueous sodium carbonate solution (0.02 M) (Sigma-Aldrich, St. Louis, MO). Fibers were rinsed three times for 20 min in deionized water. After drying, the extracted silk fibers were dissolved in lithium bromide (9.3 M) (Sigma-Aldrich) solution at 60 C for 2–3 h. Solution was placed into dialysis cassettes (Thermo Fisher, Rockford, IL) and dialyzed in deionized water for 48 h. The resulting 5–8% w/v silk

fibroin solution was then concentrated to 10% via dialysis against 15% (w/v) polyethylene glycol and stored at 4 C until needed.

Electrogelation: Adhering titanium studs with electrogels To form egels on titanium studs, 10% silk solution (20 mL) was dispensed onto the stud surface (diameter ¼ 7 mm). Using a dovetail slide micromanipulator (Wagner Instruments, Hamden, CT), a platinum wire was slowly lowered to penetrate the surface of the silk droplet without contacting the underlying titanium stud. The wire was centrally lowered onto the titanium stud to ensure even coating deposition. The platinum wire was connected to the negative terminal of the 25 V power supply, while the stud was connected to the positive terminal of the same power supply. The power supply was switched on for 30 s, which resulted in the deposition of a silk gel on the surface of the stud. A second titanium stud was placed onto the newly formed gel, expelling excess solution and resulting in adherence of the two studs.

Coating stainless steel shims and dental implants Flat steel shims or the titanium implants (SLActive 4.1 ømm Straumann Implant, Basel, Switzerland) were directly connected to the positive terminal of the power supply and submerged into 5 mL of 5% or 10% (w/v) silk solution. A negative platinum wire was placed into the solution approximately 10 mm away for the implant and gelation proceeded for 5–120 s to observe the effect of gelation time on coating thickness.

Melted electrogels For melted gel coatings, egels were formed on platinum wires using 5% or 10% silk. The platinum wires were connected to the positive and negative terminal of the 25 V power supply. The power supply was switched on for 30 s. The gel that formed on the positive terminal was transferred to an aluminum plate and placed in a 120 C oven for 2 min. Upon complete melting, the temperature of the oven was adjusted to 80 C to maintain the melted state of the gel.

Adhering titanium studs with melted electrogels and silk solution A syringe was used to dispense either silk solution (20 mL) or the melted egel (20 mL) directly onto the surface of a titanium stud. A second titanium stud was placed onto the liquid, expelling excess solution and resulting in adherence of the two studs.



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Coating shims with melted gel and silk solution To coat flat steel shims, the egels were melted as described above. Fifteen shims were obtained and separated into three groups of five, with each group receiving 1, 5, or 10 dip coats. Each dip coat consisted of submerging 15 mm of the shim into the melted gel solution for 20 s, followed by a 15-s hold period in air. Coating was performed using a Zwick/Roell Z005 (Ulm, Germany) mechanical tester with an environmental heating chamber. The shim to be coated was fixed to the moving crosshead using standard tensile test clamps. The melted gel was placed under the shim in an aluminum dish. The dip coating procedure was performed at 80 C to maintain the melted state of the gel. For dip coating in 5% and 10% w/v silk solutions, the same Zwick mechanical tester and dip protocol setup used for melted gel dipping was employed. The environmental chamber was set to 37 C during the procedure with silk being placed in a petri-dish rather than the aluminum pan.

Measurement of coating thickness A stylus profilometer and pocket thickness gauge (Mitutoyo 7308, Aurora, IL, USA) were used to determine the thickness of the silk coatings deposited onto

(a)

Custom adaptor with fixed 7mm titanium stud

the flat steel shims. Using three films, thickness measurements were obtained at three regions of each film and the average of the readings was taken as the coating thickness.

Mechanical analysis Uniaxial tensile tests were performed on an Instron 3366 universal testing frame (Instron, Norwood, MA, USA) equipped with a 100 N capacity load sensor. Egels were formed directly onto the top surface of a titanium stud fixed to a custom Instron adapter. The adhered stud was allowed to dry for 12 h prior to testing. The custom adaptors were mounted directly onto the Instron (Figure 1), and the adhered stud was held in place via standard Instron pneumatic clamps or vise clamps. For tensile testing, the loading rate was fixed at 0.5 mm min1. Each treatment group was tested for ultimate tensile stress.

Scanning electron microscopy Scanning electron microscopy (SEM) Imaging was used to qualitatively observe the morphology of the titanium implants and egel coatings. Coated and uncoated implants were imaged using a Supra 55VP microscope (Carl Zeiss Inc., Thornwood, NY, USA) at an

(b)

7mm titanium stud adhered using silk electrogel

(c)

(d)

(e)

Figure 1. (a) custom adapter designed for testing the titanium studs adhered using silk electrogels on an Instron uniaxial mechanical tester. Preparation of implant testing material (acrylic and BoneSim) using a (b) dental implant surgical preparation kit. The 4.1 mm diameter insertion sites (c) were drilled into the test material and the hole tapped to accommodate the threaded implant (d). The two halves of the acrylic block (and BoneSim) were held together in a frame (e) or with clamps and separated to remove the implant without damaging the applied coating. Scale bars ¼ 4 mm.




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operating voltage of 5 KV. No gold sputtering was applied to the samples, and all coated samples were allowed to air dry for 24 h prior to imaging.

Coating adherence Using a Straumann surgical implant kit, acrylic blocks and BoneSimTM organic bone analog (BoneSim, Newaygo, MI, USA) material were prepared to mimic clinical implant sites. The acrylic was cut into 2’’ 2’’ squares and each square cut into equal halves while the entire BoneSim block was cut into two equal halves. A 3.5-mm diameter bit was used to create a centrally aligned 10 mm deep hole (Figure 1(b) to (e)) in the acrylic blocks and five equally space holes in the BoneSim. The holes were tapped using the appropriate bits from the surgical kit. The two halves of the acrylic block were placed in a support frame while the BoneSim was held together using clamps. The coated surgical implants (10% v/w silk, 5 s electrogelation) were placed into the implantation site using dental impact drills and torqued to 35 Ncm2. The two halves of the blocks were then separated and coating adherence assessed using ImageJ image analysis software (NIH, Bethesda, MD, USA) to observe the coated surface area of the implants before and after mock implantation. The process of implantation was performed under both dry and saline soaked conditions.

Statistical analysis Statistical analysis was performed by one-way analysis of variance (ANOVA) and Tukey’s post-hoc test. Differences were considered significant when p 0.05.

Results To evaluate the feasibility of using silk egels and melted gels for implant coatings, each deposition technique

1200 900 600 300

Coating thickness and reproducibility is extremely important for the success of coatings. Egels were formed using 25 V and silk solutions of 5% or 10% w/v. To alter gel thickness, the applied voltage was sustained for 5–120 s, with longer times resulting in greater gel thickness. Thickness of the egel grew linearly with application time (Figure 2(a)). Reproducibly formed coatings with thicknesses of 90–1650 mm were generated depending on silk concentration and time. Similarly, layers produced by dip-coating samples into melted egels demonstrated increased thickness with dip number and silk concentration (Figure 2(b)). Generally, for both the direct electrodeposition and dip coating techniques, 10% w/v samples produced thicker coatings than the corresponding 5% w/v sample. Samples dip coated directly into 5% or 10 % silk solution produced samples that ranged from 3.3 to 56.3 mm. As with the dip coating in melted gels, these coatings grow linearly with dip number. Dip coating in melted gels produced significantly thicker coatings than the corresponding dip number in silk solution for 5% (p 0.05) (Figure 2(c)). For the 10% silk solutions, significantly thicker coatings were obtained for dip numbers greater than one (p 0.05). SEM images in Figure 3 demonstrate the additive deposition of air-dried silk gels onto dental implants. Implants coated for 5 s and 30 s (Figure 3(b) to (c))

(c)

5% 10%

550 Thickness (µm)

1500 Thickness (µm)

(b) 660

5% 10%

Layer thickness

440 330 220 110

0 40 80 Time (sec)

120

50 40 30 20 10 0

0 0

5% 10%

60 Thickness (µm)

(a) 1800

was assessed for reproducibility and thickness. Silk solution concentration and application time were varied to control silk deposition and coating thickness. Coated dental implants were inserted into acrylic and BoneSim sockets mimicking the mechanical properties of bone and delamination was quantified via image analysis. The ultimate mechanical strength of adhered titanium studs was accessed using an Instron mechanical tester.

0

5 Number of Coatings

10

0

5 10 15 Number of Coatings

20

Figure 2. Measured dry thickness of silk coatings obtained from (a) direct electrogelation, (b) dip coating into melted electrogel solution or (c) dip coating in silk solution (n ¼ 4).



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showed thin, uniform silk layers which contoured well to the topography of the implant. Deposition times of 60 s (Figure 3(d)) showed threads that were occluded by the silk coating. Additionally, the 60 s deposition shows that the micro-patterned surface of the implant was fully masked by the thicker, largely smooth silk coating.

Implant coating adherence and mechanical strength Using the SEM images and thickness results as a guide, 5 s egel coatings were applied to implants and allowed to dry prior to insertion into the acrylic and BoneSim sockets (Figure 4(a)). Acrylic block and BoneSim were selected as the socket materials as they most closely resemble the mechanical properties of actual bone

(Figure 4(b)).33,34 Figure 4(a) shows an implant at various stages of the coating procedure from left to right: uncoated implant, the as-formed gel coating, and dry gel. Drying produced a uniformly thick, uncompromised silk coating that conformed well to the threads and maintained the micro-patterns of the sintered surface. Table 1 summarizes the results of the implant insertion tests for implants coated via direct electrodeposition. Image analysis demonstrated that for both materials tested, about 90% of the silk coating remained adhered to the implant after insertion. The acrylic block showed the lowest coating adherence with 87.5 9.6% of the silk remaining after complete insertion. The implants placed in the dry BoneSim

(a)

0 seconds

(b)

5 seconds

(c)

30 seconds

(d)

60 seconds

Figure 3. Representative SEMs of dental implants showing coated threads after no exposure to silk (a) (0 s) or direct electrogelation for (b) 5, (c) 30, or (d) 60 s and 24 h of drying at room temperature. Scale bars ¼ 1 mm.

(a)

Implant coating

(b)

Implant insertion

Figure 4. Representative images of 4.1 mm dental implants at various stages of coating (a) from left to right: uncoated, as-formed electrogel and dry electrogel. Dental implants inserted into test materials (b), acrylic (left) and BoneSimÕ (right), after electrogel coating was allowed to dry. Scale bars ¼ 4 mm.



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Table 1. Silk coating adherence. Implant substrate

Coating remaining after insertion, mean (SD)

Acrylic block Dry BoneSimÕ Wet BoneSimÕ

87.5 (9.6)% 94.8 (3.6)% 89.3 (3.1)%

Percentage of the silk electrogel (10% w/v) coating remaining on the titanium implant after insertion into the test material in both wet and dry conditions (n ¼ 5).

groups using an unpaired t-test revealed that the mean adhesive strength of the egel coating was significantly higher than that of the melted gels (p 0.023). The adhesive strength of titanium studs adhered using silk solution was found to be 0.096 0.034 MPa, significantly lower than both the egel and melted gel coatings (p 0.01). These adhesion strengths are within the range of 0.07–4.83 MPa35–38 obtained with biomaterials such collagen, chitosan, and hydroxyapatite.

Discussion 0.5

** Silk solution 0.4

Melted Electrogel

Adhesive Strength (MPa)

Electrogel 0.3

* 0.2

* ** 0.1

0 Coating Technique

Figure 5. Mean measured adhesive strength of coatings obtained from direct electrogelation (30 s of deposition), dip coating into melted electrogel solution or dip coating into 5% silk solution. Vertical lines represent standard deviations (n ¼ 4). Line indicates significance at p 0.05, and * indicates significance at p 0.01.

retained 94.8 3.6% of the silk coating. In order to more closely mimic a clinical setting, insertion into BoneSim was also performed in a saline bath, and this treatment was referred to as ‘‘wet BoneSim.’’ Under these conditions, 89.3 3.1% of the silk remained adhered to the implants after insertion, which was statistically identical to the dry tests. The mean adhesive strength of coatings produced via dip coating in 5% (w/v) silk solution, melted egel deposition and direct electrodeposition, 30-s depositions on to titanium studs are shown in Figure 5. The mean adhesive strength of the melted egel and the electrodeposition coatings was 0.23 0.03 MPa and 0.37 0.09 MPa, respectively. Comparison of the

The present study demonstrated that silk coatings can be deposited onto titanium substrates using the electrogelation approach, resulting in sufficient adhesion and mechanical strength to withstand implantation stresses. Both techniques, direct electrodeposition and melted gel deposition, take advantage of the mechanical properties of silk fibroin, as well as the unique electrodeposition capabilities to form conformal surface, biocompatible coatings. An advantage of the direct silk deposition system over existing techniques is the control and reproducibility of the layers. Other coating systems are often limited by the challenge of generating chemically homogeneous, uniformly thick coatings.38 Hydroxyapatite coatings require vacuum plasma spray application of a bond coat and secondary titanium structural coat before the final plasma spraying of hydroxyapatite can be performed.35 Collagen coatings involve the use of acetic acid for solubilization and chloroform for solution sterilization, prior to manual pipetting onto the titanium substrates.38 To form chitosan coatings, toxic reagents such as 3-isocyanatopropyltriethoxysilan and glutaraldehyde are used for silanization and fixation to the titanium substrate.37 These techniques involve various harsh or toxic processing steps that make coating deposition challenging and can limit clinical applicability. Figure 2 shows that the thickness of the layers with both egel coating techniques grew linearly with application time and dip number. For the direct egel deposition technique, coating thickness ranged from 55 to 1118 mm for the 5% w/v silk and 65 to 1654 mm for the 10% w/v coatings. The dip coated treatments ranged from 37 to 180 mm and 35 to 450 mm for the 5% and 10% w/v silk melted gels, respectively. However, these dip coatings showed higher variation and were technically more difficult to produce with good reproducibility than the direct egel deposition. Dip coating directly into silk solution produced very reproducible coatings ranging from 3 to 56 mm in thickness. The deposition rate for these dip coated samples was slower than both other coating strategies. Twenty dip coats in 10% silk solution were required to produce



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a coating thicknesses equivalent to one dip in the 5% melted gel. The deposited gels had higher concentrations of protein compared to the stock solution, and this concentration difference could partly explain the thicker coatings with fewer dips into the melted solution. All the thickness values obtained using the various silk coating strategies were in line with other coating materials such as hydroxyapatite, zirconium oxide, collagen, and poly(lactic-co-glycolic acid) which range from 1 to 150 mm.38–43 Delamination of coatings is a major problem with current coating systems, in particular for coatings produced via plasma spray.44 Coating delamination can result in the release of particles and ultimately leads to clinical failure.44 Implant coating studies suggested that 25- to 50-mm-thick coatings performed well in fatigue testing, while coatings of 75 mm or thicker showed delamination.39,40 The silk coatings produced and tested for delamination were approximately 171 mm thick, more than the optimal range (Figure 2(a))39,45; however, minimal delamination was observed. To reduce the thickness of the egel coatings, thickness applied voltage and deposition times can be tuned and brought to the optimal 25–50 mm thickness range. Ultimately, these coatings will be placed in vivo and thus would require sterilization. The silk coating can be sterilized either post-deposition using autoclaving, ethanol exposure, and ethylene oxide or UV exposure. These processes can alter the coating, thus the most appropriate method would have to be determined to minimize delamination and related changes. The silk coating can also be formed directly using pre-sterilized silk solutions via filtration, allowing the formation of delamination-resistant coatings without the need of further processing. Delamination studies suggest that the adhesion of the silk to the underlying titanium surface exceeds the strength required to withstand implantation. SEM imaging supported this observation with little occlusion of the implant threads for samples that received 30 s (Figure 3(a) to (c)) or less of silk deposition. Increasing deposition to 60 s resulted in masking of implant threads and would make delamination of the coating more likely (Figure 3(d)).39 Moreover, these thicker coatings masked the underlying sintered surface which has been shown to improve osseointegration.19 An optimal coating would be able to resist coating delamination and maintain the micro-patterned surface morphology of the implant. Coating delamination is not dictated by coating thickness alone, but a number of variables including the adhesion strength between the implant and the coating material. The adhesive strength of other coatings derived from biological sources, such as collagen, chitosan, and hydroxyapatite, ranged from 0.07 to

4.83 MPa.35–38 Although the adhesion strength obtained for the silk coatings, 0.23 and 0.38 MPa for melted and direct deposition, respectively (Figure 5), fell within the range of competing biological coating systems. The difference in adhesion between the electrodeposited and dip coated techniques is likely due to the differences in coating assembly. The dip coatings are composed of loosely associated, randomly assembled silk layers with no distinct organization. The lack of tight molecular packing results in the lower adhesive strength for this coating technique. During electrodeposition, there was alignment of the charged silk molecules assembled onto the charged substrate.32 This allows for tighter packing of the proteins with end-toend stacking of the negative and positive silk fibroin regions ultimately resulting in increased adhesion to the metal surface. As with any coating, the outcome of the implant in the case of coating delamination can be affected by the bone-to-implant contact area. Because the socket size created of the silk coated implants was the same as those created for uncoated implants, if delamination were to occur, bone-implant contact would still be maximized, improving opportunities for success. It is important to note that highest adhesion values obtained for many of the other biological coatings required co-deposition or chemical bonding.37,38 This highlights another key advantage of the silk coatings, their inherent ease of application. Most other coating systems require harsh, expensive, or otherwise difficult processing conditions in order to deposit and maintain mechanical robustness.46 The silk coatings are aqueous based, biocompatible, controllably degradable, and coating deposition requires no chemical modifications.22–25 This allows for simplified implant functionalization and the incorporation of therapeutics and their release over controllable time frames.32,47,48

Conclusions Two distinct methods of forming silk-based gel coatings on implant systems of various geometries (studs, shims, and screws) were described and the mechanical properties characterized. Silk egels and melted gels formed reproducible conformal coatings capable of withstanding insertion into mock bone sockets. The electrogelation coating process exploits the unique mechanical and physical properties of silk proteins in tunable modes to modulate adherence, coating thickness and offers a relatively simple and flexible system with which to functionalize dental implants. The all aqueous process, the avoidance of chemical crosslinking, and the ability to sequester and deliver labile therapeutics from the silk suggest this approach offers a novel strategy for functionalized implant coatings.



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Acknowledgements We thank Dr. Ethan Golden for his help with preparation of the implant sockets and Scott MacCorkle for help with fabrication of the Instron adaptors.

Declaration of conflicting interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding This work was funded by the NIH grant numbers EB002520 and AR061988.

References 1. Leblebicioglu B, Rawal S and Mariotti A. A review of the functional and esthetic requirements for dental implants. J Am Dent Assoc 2007; 138: 321–329. 2. Donos N, Mardas N and Chadha V. Clinical outcomes of implants following lateral bone augmentation: systematic assessment of available options (barrier membranes, bone grafts, split osteotomy). J Clin Periodontol 2008; 35: 173–202. 3. Clementini M, Morlupi A, Canullo L, et al. Success rate of dental implants inserted in horizontal and vertical guided bone regenerated areas: a systematic review. Int J Oral Maxillofac Surg 2012; 41: 847–852. 4. Juodzbalys G, Raustia AM and Kubilius R. A 5-year follow-up study on one-stage implants inserted concomitantly with localized alveolar ridge augmentation. J Oral Rehabil 2007; 34: 781–789. 5. Johnson GK and Guthmiller JM. The impact of cigarette smoking on periodontal disease and treatment. Periodontol 2000 2007; 44: 178–194. 6. Granstro¨m G. Osseointegration in irradiated cancer patients: an analysis with respect to implant failures. J Oral Maxillofac Surg 2005; 63: 579–585. 7. Michaeli E, Weinberg I and Nahlieli O. Dental implants in the diabetic patient: systemic and rehabilitative considerations. Quintessence Int 2009; 40: 639–645. 8. Mellado-Valero A, Ferrer-Garcia JC, Calvo-Catala J, et al. Implant treatment in patients with osteoporosis. Med Oral Patol Oral Cir Bucal 2010; 15: e52–e57. 9. Persson LG, Ericsson I, Berglundh T, et al. Osseintegration following treatment of peri-implantitis and replacement of implant components. J Clin Periodontol 2001; 28: 258–263. 10. Anil S, Anand PS, Alghamdi H, et al. Dental implant surface enhancement and osseointegration. In: Turkyilmaz PI (ed.) Implant dentistry – a rapidly evolving practice. InTech: Rijeka, Croatia, 2011. 11. Oshida Y, Tuna EB, Akto¨ren O, et al. Dental implant systems. Int J Mol Sci 2010; 11: 1580–1678. 12. Allegrini Jr S, Rumpel E, Kauschke E, et al. Hydroxyapatite grafting promotes new bone formation and osseointegration of smooth titanium implants. Ann Anat 2006; 188: 143–151.

13. Ogawa T and Nishimura I. Genes differentially expressed in titanium implant healing. J Dent Res 2006; 85: 566–570. 14. Fujibayashi S, Neo M, Kim H-M, et al. Osteoinduction of porous bioactive titanium metal. Biomaterials 2004; 25: 443–450. 15. Wang J, de Boer J and de Groot K. Preparation and characterization of electrodeposited calcium phosphate/ chitosan coating on Ti6Al4V plates. J Dent Res 2004; 83: 296–301. 16. Bra˚nemark P-I, Breine U, Adell R, et al. Intra-osseous anchorage of dental prostheses: I. Experimental studies. Scand J Plast Reconstr Surg Hand Surg 1969; 3: 81–100. 17. Branemark P-I, Zarb GA, Albrektsson T, et al. Tissueintegrated prostheses. Osseointegration in clinical dentistry. Plast Reconstr Surg 1986; 77: 496–497. 18. Meraw SJ and Reeve CM. Qualitative analysis of peripheral peri-implant bone and influence of alendronate sodium on early bone regeneration. J Periodontol 1999; 70: 1228–1233. 19. Lavenus S, Louarn G and Layrolle P. Nanotechnology and dental implants. Int J Biomater 2010; 2010: 9. 20. Alt V, Bitschnau A, O¨sterling J, et al. The effects of combined gentamicin–hydroxyapatite coating for cementless joint prostheses on the reduction of infection rates in a rabbit infection prophylaxis model. Biomaterials 2006; 27: 4627–4634. 21. Junker R, Dimakis A, Thoneick M, et al. Effects of implant surface coatings and composition on bone integration: a systematic review. Clin Oral Implants Res 2009; 20: 185–206. 22. Jin HJ, Karageorgiou V, Kim UJ, et al. Water-stable silk films with reduced b-sheet content. Adv Funct Mater 2005; 15: 1241–1247. 23. Hu X, Shmelev K, Sun L, et al. Regulation of silk material structure by temperature-controlled water vapor annealing. Biomacromolecules 2011; 12: 1686–1696. 24. Altman GH, Diaz F, Jakuba C, et al. Silk-based biomaterials. Biomaterials 2003; 24: 401–416. 25. Zhang J, Pritchard E, Hu X, et al. Stabilization of vaccines and antibiotics in silk and eliminating the cold chain. Proc Nat Acad Sci U S A 2012; 109: 11981–11986. 26. Tien LW, Wu F, Tang-Schomer MD, et al. Silk as a multifunctional biomaterial substrate for reduced glial scarring around brain-penetrating electrodes. Adv Funct Mater 2013; 23: 3185–3193. 27. Elia R, Newhide DR, Pedevillano PD, et al. Silk–hyaluronan-based composite hydrogels: a novel, securable vehicle for drug delivery. J Biomater Appl 2011; 27: 749–762. 28. Wang X, Kluge JA, Leisk GG, et al. Sonication-induced gelation of silk fibroin for cell encapsulation. Biomaterials 2008; 29: 1054–1064. 29. Guziewicz N, Best A, Perez-Ramirez B, et al. Lyophilized silk fibroin hydrogels for the sustained local delivery of therapeutic monoclonal antibodies. Biomaterials 2011; 32: 2642–2650. 30. Kojic N, Panzer MJ, Leisk GG, et al. Ion electrodiffusion governs silk electrogelation. Soft Matter 2012; 8: 2897–2905. 31. Lin Y, Xia X, Shang K, et al. Tuning chemical and physical cross-links in silk electrogels for morphological



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34.

35.

36.

37.

38.

39.

9

analysis and mechanical reinforcement. Biomacromolecules 2013; 14: 2629–2635. Yucel T, Kojic N, Leisk GG, et al. Non-equilibrium silk fibroin adhesives. J Struct Biol 2010; 170: 406–412. Migliorati M, Benedicenti S, Signori A, et al. Thread shape factor: evaluation of three different orthodontic miniscrews stability. Eur J Orthod 2012; 35: 401–405. Mansour A, Ercoli C, Graser G, et al. Comparative evaluation of casting retention using the ITI solid abutment with six cements. Clin Oral Implants Res 2002; 13: 343–348. Aebli N, Stich H, Schawalder P, et al. Effects of bone morphogenetic protein-2 and hyaluronic acid on the osseointegration of hydroxyapatite-coated implants: an experimental study in sheep. J Biomed Mater Res 2005; 73A: 295–302. Zhang Z, Jiang T, Ma K, et al. Low temperature electrophoretic deposition of porous chitosan/silk fibroin composite coating for titanium biofunctionalization. J Mater Chem 2011; 21: 7705–7713. Yuan Y, Chesnutt BM, Wright L, et al. Mechanical property, degradation rate, and bone cell growth of chitosan coated titanium influenced by degree of deacetylation of chitosan. J Biomed Mater Res B Appl Biomater 2008; 86B: 245–252. Wang RCC, Hsieh M-C, Yang S-P, et al. Characteristics and cyto-compatibility of Collagen/Ca–P coatings on Ti6Al4V substrate. Surf Coating Technol 2011; 205: 4683–4689. Lynn AK and DuQuesnay DL. Hydroxyapatite-coated Ti–6Al–4V: part 2: the effects of post-deposition heat treatment at low temperatures. Biomaterials 2002; 23: 1947–1953.

40. Sollazzo V, Pezzetti F, Scarano A, et al. Zirconium oxide coating improves implant osseointegration in vivo. Dent Mater 2008; 24: 357–361. 41. Tredwin CJ, Georgiou G, Kim H-W, et al. Hydroxyapatite, fluor-hydroxyapatite and fluorapatite produced via the sol–gel method: bonding to titanium and scanning electron microscopy. Dent Mater 2013; 29: 521–529. 42. Ostrowski N, Lee B, Roy A, et al. Biodegradable poly(lactide-co-glycolide) coatings on magnesium alloys for orthopedic applications. J Mater Sci Mater Med 2013; 24: 85–96. 43. Zhao X, Hu T, Li H, et al. Electrochemically assisted co-deposition of calcium phosphate/collagen coatings on carbon/carbon composites. Appl Surf Sci 2011; 257: 3612–3619. 44. Le Gue´hennec L, Soueidan A, Layrolle P, et al. Surface treatments of titanium dental implants for rapid osseointegration. Dent Mater 2007; 23: 844–854. 45. Wang BC, Lee TM, Chang E, et al. The shear strength and the failure mode of plasma-sprayed hydroxyapatite coating to bone: the effect of coating thickness. J Biomed Mater Res 1993; 27: 1315–1327. 46. Dorozhkin SV. Calcium orthophosphate coatings, films and layers. Prog Biomater 2012; 1: 1–40. 47. Pritchard EM and Kaplan DL. Silk fibroin biomaterials for controlled release drug delivery. Expert Opin Drug Deliv 2011; 8: 797–811. 48. Numata K and Kaplan DL. Silk-based delivery systems of bioactive molecules. Adv Drug Deliv Rev 2010; 62: 1497–1508.


Antifouling coatings for dental implants: Polyethylene glycol-like coatings on titanium by plasma polymerization.

Electrodeposited silk coatings for bone implants.

Evaluation of thin amorphous calcium phosphate coatings on titanium dental implants deposited using magnetron sputtering.

Tuning the surface microstructure of titanate coatings on titanium implants for enhancing bioactivity of implants.

Investigating the structure and biocompatibility of niobium and titanium oxides as coatings for orthopedic metallic implants.

Surface Modifications and Their Effects on Titanium Dental Implants.

Dental follicle stem cells in bone regeneration on titanium implants.

Fibronectin-Grafted Titanium Dental Implants: An In Vivo Study.

Comparative study of hydroxyapatite and titanium dental implants in dogs.

[Improving the biocompatibility of silicone implants using spider silk coatings: immunohistochemical analysis of capsule formation].

Magnesium-containing mixed coatings on zirconia for dental implants: mechanical characterization and in vitro behavior.

In vitro color evaluation of esthetic coatings for metallic dental implants and implant prosthetic appliances.

Peptide aptamers: Novel coatings for orthopaedic implants.

Enhanced osteogenesis on titanium implants by UVB photofunctionalization of hydrothermally grown TiO₂ coatings.

Corrosion behavior and mechanical properties of bioactive sol-gel coatings on titanium implants.

Morphology, composition, and bioactivity of strontium-doped brushite coatings deposited on titanium implants via electrochemical deposition.

nanostructured titanium with balanced actions to bacterial and mammalian cells for dental implants.

Titanium nitride coatings in clinical dentistry.

Bioactive coatings for orthopaedic implants-recent trends in development of implant coatings.

Ultraviolet photofunctionalization of titanium implants.

Chitosan-Recombinamer Layer-by-Layer Coatings for Multifunctional Implants.

Graphene-reinforced calcium silicate coatings for load-bearing implants.

Direct metal laser sintering titanium dental implants: a review of the current literature.

Predicting Peri-implant Stresses Around Titanium and Zirconium Dental Implants-A Finite Element Analysis.