Abstract
Objective
Dental implants provide support for dental crowns and bridges by serving as abutments for the replacement of missing teeth. To prevent bacterial accumulation and growth at the site of implantation, solutions such as systemic antibiotics and localized delivery of bactericidal agents are often employed. The objective of this study was to demonstrate a novel method of controlled localized delivery of antibacterial agents to an implant site using a biodegradable custom fabricated ring.
Methods
The study involved incorporating a model antibacterial agent (metronidazole) into custom designed poly-ε-caprolactone/alginate (PCL/alginate) composite rings to produce the intended controlled release profile. The rings can be designed to fit around the body of any root form dental implants of various diameters, shapes and sizes.
Results
In vitro release studies indicate that pure (100%) alginate rings exhibited an expected burst release of metronidazole in the first few hours, whereas Alginate/PCL composite rings produced a medium burst release followed by a sustained release for a period greater than 4 weeks. By varying the PCL/alginate weight ratios, we have shown that we can control the amount of antibacterial agents released to provide the minimal inhibitory concentration (MIC) needed for adequate protection. The fabricated composite rings have achieved a 50% antibacterial agent release profile over the first 48 h and the remaining amount slowly released over the remainder of the study period. The PCL/alginate agent release characteristic fits the Ritger–Peppas model indicating a diffusion-based mechanism during the 30-day study period.
Significance
The developed system demonstrates a controllable drug release profile and the potential for the ring to inhibit bacterial biofilm growth for the prevention of diseases such as peri-implantitis resulting from bacterial infection at the implant site.
1
Introduction
The use of osseointegrated dental implants for the replacement of lost teeth has become common and well accepted in dentistry. Advances in biomaterials research and titanium implant manufacturing techniques have resulted in a wide selection of implants being used in restorative dentistry. Despite reports of high success rates in dental implants, 5–11% of dental implants fail and require complete removal due to either bacterial infection or improper loading conditions during mastication. Bacteria accumulate on the surface almost immediately after implantation via a series of events that is initiated by the adsorption of the molecules from the saliva to produce a conditioning film called pellicle . This film provides a surface for attachment of the initial colonizers, which are mainly comprised of Gram-positive streptococci (such as Streptococcus oralis , Streptococcus mitis ) and actinomyces species . Within thirty minutes, the first colonies are formed by surface attachment and simultaneous proliferation and co-aggregation . The attached bacteria and their progeny continuously secrete proteins, enzymes , and insoluble extracellular polysaccharides that form a biofilm, which promotes co-existence with secondary colonizers and facilitates survival by resisting antibacterial agents . The continuous development of biofilms can progress into peri-implantitis and often requires the complete removal of the implant .
Several strategies have been developed to prevent implant failure caused by bacterial accumulation on the implant surface. The most common ones include oral ingestion of antibiotics prescribed for a period of time and local injection of antibiotics at the site of implantation during the initial weeks following placement . Advanced strategies include coating the implant surface with anti-adhesive polymers to prevent bacterial accumulation at the implant site. Several antibiotics have been used for clinically such as Chlorhexidine, Tetracyclines and Sanguinarine which are prescribed based on the types of bacterial infection diagnosed . Metronidazole is a first-line of antibiotics for treating anaerobic infections in oral disease, and also has a low minimum inhibitory concentration of 90% (MIC 90 ) against B. fragilis species that are the most frequently encountered anaerobic pathogens clinically . A commercial lipid-like gel based on glyceryl monooleate (GMO) and triglyceride (sesame oil) containing 25% metronidazole (Elyzol; Dumex-Alpharma, Denmark) has been used for over 30 years for periodontal diseases . Some studies report that Elyzol is not effective for periodontal treatment due to poor microbiological inhibition . This is probably due to the fact that the Elyzol gel is susceptible to elimination from the periodontal pocket due to its gel form factor .
Besides systemic administration of antibiotics and/or local delivery of antibacterial agents, matrix-based solutions have also been studied to perform local controlled release of the agents for the treatment of periodontal diseases. Aliphatic polyesters such as poly-ε-caprolactone (PCL) have been widely used for different biomedical application purposes. Due to its biocompatibility and non-toxic degradation within the human body , PCL is one of the most suitable biomaterials used in controlled drug release applications. PCL is a hydrophobic and biodegradable polymer under physiological conditions , and has been researched extensively for the controlled delivery of drugs contained within a matrix. Kyun and co-workers showed that by embedding minocycline in PCL, it is possible to obtain sustained release of the drug within the periodontal pocket for up to seven days and can eliminate bacteria from the periodontal pocket. Manufacturing processes such as electrospinning have produced PCL nanofibers for dental applications such as scaffolds for periodontal regeneration , and electrospun PCL nanofibers with metronidazole for long term sustained drug release . Due to its relatively dense structure, PCL based matrices do not provide the necessary burst release of antibacterial agents needed for the initial 48 h post-implantation.
Another example of a biodegradable hydrophilic matrix material is alginate, a naturally occurring polysaccharide produced from the structural component of brown algae and bacteria . In addition to its non-toxicity and biodegradability, alginate forms hydrogels under mild conditions when immersed in a divalent ion solution . Consequently, alginate has been used extensively in pharmaceutical industries as a dental impression material , wound dressing material between bone and periosteum in the jaws , and in oral drug delivery . Creating a composite of two polymeric biomaterials is one approach to developing new biomaterials exhibiting combinations of properties that cannot be obtained by the individual polymers themselves. Sodium alginate has significant burst release property in the first couple of hours while polymers such as PCL and/or PLGA can delay drug diffusion to slow the release rate at which drug molecules are exposed in an aqueous environment . While several laboratory solutions have been proposed, clinically relevant solutions demand ease of use by the dentist and any proposed solution should not add to the complexity of the placement procedure, particularly paying attention to prevent scratching the surface of the metal implant. Ideally, the solution must be integrated into the implant with minimal post-implantation visits made by the patient while protecting them from further infection during the healing process.
In this study, we have developed a ring like implantable device encapsulated with a candidate antibacterial agent whose release can be customized for the lifetime of the implant. This device is made from a composite of biocompatible and biodegradable polymers (calcium-alginate hydrogels and poly-ε-caprolactone) encapsulated with metronidazole for the localized delivery of the agent at the implant site. This application ensures high concentration of drug around the implant, and reduces side effects associated with a systemic drug application. A possible placement of the annular ring would be around the body of a root form dental implant adjacent to the bone crest as shown in Fig. 1 . We have devised a fabrication process to produce customized blends of the two polymers (Alginate and PCL) to form custom shaped rings of virtually any dimension, size and shape to fit various dental implants. Molds of the desired shape were prepared into which discrete regions of poly-ε-caprolactone/alginate was solvent casted and encapsulated with known quantities of metronidazole (MZ). Our studies show that by tuning the relative weight percentages of PCL to alginate, we can customize the release profiles of MZ from the composite annular ring. Finally, the mechanical properties (both Young’s Modulus and stiffness) of the composite ring have also been investigated.
2
Materials and methods
2.1
Materials
Metronidazole (MZ), calcium chloride (CaCl 2 ), sodium hydroxide (NaOH) 1.0 N solution, hydrogen chloride (HCl) acid 0.1 N solution and sodium alginate were obtained from (Sigma-Aldrich, MO, USA). Potassium phosphate monobasic (KH 2 PO 4 ), potassium chloride (KCl) (Sigma–Aldrich, MO, USA), sodium hydrogen phosphate (Na 2 HPO 4 ) (Sigma–Aldrich, WI, USA) and sodium chloride (Calbiochem, CA, USA) were used to prepare phosphate buffered saline (PBS) solution. The silicone elastomer base and curing agent (Dow Corning, MI, USA) were used to prepare the PDMS (poly-dimethyl siloxane) mold. Poly-ε-caprolactone (MW 70,000) (Scientific Polymer Products Inc., New York, USA) was used to produce the PCL rings and Dichloromethane (Alfa Aesar, MA, USA) as the solvent.
2.2
Methods
2.2.1
Fabrication of PDMS molds
To fabricate alginate rings with the desired dimensions, we initially developed flexible poly-dimethyl-siloxane (PDMS) templates. These templates were made by a polymerization technique using steel washers of desired dimensions 3 mm inner diameter, 7 mm outer diameter and 1.5 mm thickness. The steel washers were placed on the bottom of a plastic petri dish and a thoroughly mixed 10:1 uncured silicone elastomer base and curing agent was then poured over the washers and degassed for 30 min using a vacuum. The degassed PDMS was then cured in an oven at 70 °C overnight. The cured PDMS was then peeled off the petri dish surface and the developed templates were then plasma treated using a plasma cleaner (PDC 32G, Harrick Plasma, New York, USA) at pressurized oxygen of 400millitorrs for two minutes to improve the wettability of the surface. The process is shown in Fig. 2 .
2.2.2
Preparation of drug loaded alginate ring
5.4% (w/v) metronidazole (MZ) was added to 1, 2 and 4% (w/v) alginate solution dissolved in pure water. The suspensions were mixed thoroughly for 45 min to enhance dissolution of the alginate powder. After dissolution, 95 μl of the colloidal solution was loaded into each treated PDMS mold and then immersed in a compartment of a 12 well culture plate containing 2 mL CaCl 2 solution to induce gelation. The set up was left at room temperature to be cured for a period of minimum 2 h. The calcium-alginate hydrogel rings were removed from the mold, rinsed in 1 mL distilled water to remove any excess Ca 2+ ions and then air dried for 24 h ( Fig. 2 ).
2.2.3
Preparation of drug loaded PCL rings
5.4% (w/v) metronidazole (MZ) and 10% PCL (w/v) were made in dichloromethane solution. The resulting clear solution was loaded into each PDMS mold and then solvent dried under a chemical hood. After solvent evaporation, a layer by layer amount of the PCL/MZ mixture solution was deposited within the PDMS mold and then continuously dried until a solvent casted PCL ring was formed within the mold dimensions. The molds with PCL rings were then transferred into a vacuum chamber for 15 min to remove any remaining dichloromethane solvent solution from the drug loaded ring.
2.2.4
Preparation of drug loaded rings made from PCL/alginate composite
Metronidazole loaded composite rings were prepared from known weight ratios of PCL polymer and alginate hydrogel using a two-step processing method. The metronidazole (81.25 mg, 5.4%, w/w) was mixed with a volume of dichloromethane (15 ml) in glass bottom beakers at room temperature until it was completely dissolved, giving a clear solution; PCL (1.5 g, 10%w/w) was then added to the solution. Three drug loadings were used: 50:1, 10:1 and 5:1 (Total amount of MZ in PCL: Total amount of MZ in alginate). The solution of alginate-MZ mixture was prepared as described before and placed on the PDMS mold and crosslinked with 4% CaCl 2 solution. The crosslinked alginate rings were dried out for two minutes at 80 °C and then for 1 h at room temperature. In the next step, the dissolved PCL solution was solution casted on to the desired PDMS mold containing the alginate ring and dried under a chemical hood at room temperature. The PDMS mold was not covered, so as to enable a relatively fast evaporation rate of 70 μl/20 min per casting. This process was then repeated in a layer-by-layer deposition pattern until a solid PCL component filled up within the mold and surrounded the alginate rings. Following this step, the PCL/alginate rings were dried for 15 min in a vacuum chamber to remove any air bubbles and residual solvent. After sufficient casting and drying, the PCL/alginate ring was peeled away from the mold and placed in PBS solution for further experimentation. The process of fabrication is shown in Fig. 2 .
2.2.5
Mechanical testing
A compression test was performed in a material testing system (MTS, Model 100R, TestResources, Minnesota, USA) with a 50 N load cell (Interface Force Inc., Arizona. USA) to measure the force–displacement relation of the PCL, PCL/alginate composite and Alginate rings at a displacement rate of 0.005 mm/s. The elastic modulus was obtained based on the stress–strain relation using a simple linear elastic model. The stiffness of the specimen under compression was calculated using the applied compressive force divided by the specimen displacement.
2.2.6
Standard curve generation
An initial stock concentration of 0.25 mg/mL of MZ in 10 mL of pH 7.4 PBS was prepared. The solution was mixed thoroughly using a vortex and allowed to stand at 37 ± 0.5° C for 30 min to achieve complete dissolution. When no visible particles were observed, aliquots of 100, 200, 400, 600, 800 and 1000 μl were taken from the stock solution and diluted. A wavescan was performed using a UV–vis spectrophotometer (Beckman Coulter UV 200) to determine the maximum wavelength ( λ max ) of MZ. The absorbance of the various concentrations was then measured at the λ max to construct a standard curve. The λ max for MZ is 319 nm.
2.2.7
In vitro release studies: pure alginate rings, pure PCL rings and PCL/alginate rings
To investigate the amount of drug released from pure alginate rings, 10 mL of dissolution medium (0.1 M phosphate buffered saline; pH 6.8) was placed in each well of a 6 well culture plate equilibrated to 37 °C. An alginate ring sample was then placed in each well of the plate and stored at 37 ± 0.5° C. At regular time intervals (every hour for the first 8 h and then every 12 h afterwards), an aliquot of 30 μl was collected for analysis. The collected sample was replaced by the same amount (30 μl) of fresh PBS solution of the same pH to maintain infinite sink condition throughout the study. The samples were diluted and assayed at 319 nm. To investigate the drug release from PCL and PCL/alginate rings, samples were immersed in 1 ml PBS under room temperature. 100 μl PBS were collected from different time points and samples were placed into fresh 1 ml PBS for the next time study. The metronidazole content in each sample was determined with a spectrophotometer at 319 nm. A calibration curve was prepared for each set of measurements, with a correlation coefficient >0.99. Six samples were examined for each ring type.
2.2.8
Imaging
To image the distribution of the alginate hydrogel within the rings, 4% (w/v) alginate was stained with 1% trypan blue (Invitrogen) and casted with PCL as described above for imaging.
2.2.9
Drug release model
The Fickian and non-Fickian solute release behavior from swelling controlled drug–polymer systems were determined using several established models: Ritger–Peppas model, Baker and Lonsdale model, Hixon and Crowell model, Higuchi model, and first order kinetics model. The equations were implemented in Sigma-Plot 10.0 as shown below:
Ritger–Peppas model
Baker and Lonsdale model
Hixon and Crowell model
Higuchi model
First order kinetics model
where F is the percentage of drug released at time t , k is the rate coefficient.
2.2.10
Statistical analysis
All experiments were carried out in triplicate and reported in mean ± SD of n = 6. Comparisons were performed using a one-way ANOVA (analysis of variance) and then post hoc tests were applied. The significance of results were considered at p < 0.05.
2
Materials and methods
2.1
Materials
Metronidazole (MZ), calcium chloride (CaCl 2 ), sodium hydroxide (NaOH) 1.0 N solution, hydrogen chloride (HCl) acid 0.1 N solution and sodium alginate were obtained from (Sigma-Aldrich, MO, USA). Potassium phosphate monobasic (KH 2 PO 4 ), potassium chloride (KCl) (Sigma–Aldrich, MO, USA), sodium hydrogen phosphate (Na 2 HPO 4 ) (Sigma–Aldrich, WI, USA) and sodium chloride (Calbiochem, CA, USA) were used to prepare phosphate buffered saline (PBS) solution. The silicone elastomer base and curing agent (Dow Corning, MI, USA) were used to prepare the PDMS (poly-dimethyl siloxane) mold. Poly-ε-caprolactone (MW 70,000) (Scientific Polymer Products Inc., New York, USA) was used to produce the PCL rings and Dichloromethane (Alfa Aesar, MA, USA) as the solvent.
2.2
Methods
2.2.1
Fabrication of PDMS molds
To fabricate alginate rings with the desired dimensions, we initially developed flexible poly-dimethyl-siloxane (PDMS) templates. These templates were made by a polymerization technique using steel washers of desired dimensions 3 mm inner diameter, 7 mm outer diameter and 1.5 mm thickness. The steel washers were placed on the bottom of a plastic petri dish and a thoroughly mixed 10:1 uncured silicone elastomer base and curing agent was then poured over the washers and degassed for 30 min using a vacuum. The degassed PDMS was then cured in an oven at 70 °C overnight. The cured PDMS was then peeled off the petri dish surface and the developed templates were then plasma treated using a plasma cleaner (PDC 32G, Harrick Plasma, New York, USA) at pressurized oxygen of 400millitorrs for two minutes to improve the wettability of the surface. The process is shown in Fig. 2 .
2.2.2
Preparation of drug loaded alginate ring
5.4% (w/v) metronidazole (MZ) was added to 1, 2 and 4% (w/v) alginate solution dissolved in pure water. The suspensions were mixed thoroughly for 45 min to enhance dissolution of the alginate powder. After dissolution, 95 μl of the colloidal solution was loaded into each treated PDMS mold and then immersed in a compartment of a 12 well culture plate containing 2 mL CaCl 2 solution to induce gelation. The set up was left at room temperature to be cured for a period of minimum 2 h. The calcium-alginate hydrogel rings were removed from the mold, rinsed in 1 mL distilled water to remove any excess Ca 2+ ions and then air dried for 24 h ( Fig. 2 ).
2.2.3
Preparation of drug loaded PCL rings
5.4% (w/v) metronidazole (MZ) and 10% PCL (w/v) were made in dichloromethane solution. The resulting clear solution was loaded into each PDMS mold and then solvent dried under a chemical hood. After solvent evaporation, a layer by layer amount of the PCL/MZ mixture solution was deposited within the PDMS mold and then continuously dried until a solvent casted PCL ring was formed within the mold dimensions. The molds with PCL rings were then transferred into a vacuum chamber for 15 min to remove any remaining dichloromethane solvent solution from the drug loaded ring.
2.2.4
Preparation of drug loaded rings made from PCL/alginate composite
Metronidazole loaded composite rings were prepared from known weight ratios of PCL polymer and alginate hydrogel using a two-step processing method. The metronidazole (81.25 mg, 5.4%, w/w) was mixed with a volume of dichloromethane (15 ml) in glass bottom beakers at room temperature until it was completely dissolved, giving a clear solution; PCL (1.5 g, 10%w/w) was then added to the solution. Three drug loadings were used: 50:1, 10:1 and 5:1 (Total amount of MZ in PCL: Total amount of MZ in alginate). The solution of alginate-MZ mixture was prepared as described before and placed on the PDMS mold and crosslinked with 4% CaCl 2 solution. The crosslinked alginate rings were dried out for two minutes at 80 °C and then for 1 h at room temperature. In the next step, the dissolved PCL solution was solution casted on to the desired PDMS mold containing the alginate ring and dried under a chemical hood at room temperature. The PDMS mold was not covered, so as to enable a relatively fast evaporation rate of 70 μl/20 min per casting. This process was then repeated in a layer-by-layer deposition pattern until a solid PCL component filled up within the mold and surrounded the alginate rings. Following this step, the PCL/alginate rings were dried for 15 min in a vacuum chamber to remove any air bubbles and residual solvent. After sufficient casting and drying, the PCL/alginate ring was peeled away from the mold and placed in PBS solution for further experimentation. The process of fabrication is shown in Fig. 2 .
2.2.5
Mechanical testing
A compression test was performed in a material testing system (MTS, Model 100R, TestResources, Minnesota, USA) with a 50 N load cell (Interface Force Inc., Arizona. USA) to measure the force–displacement relation of the PCL, PCL/alginate composite and Alginate rings at a displacement rate of 0.005 mm/s. The elastic modulus was obtained based on the stress–strain relation using a simple linear elastic model. The stiffness of the specimen under compression was calculated using the applied compressive force divided by the specimen displacement.
2.2.6
Standard curve generation
An initial stock concentration of 0.25 mg/mL of MZ in 10 mL of pH 7.4 PBS was prepared. The solution was mixed thoroughly using a vortex and allowed to stand at 37 ± 0.5° C for 30 min to achieve complete dissolution. When no visible particles were observed, aliquots of 100, 200, 400, 600, 800 and 1000 μl were taken from the stock solution and diluted. A wavescan was performed using a UV–vis spectrophotometer (Beckman Coulter UV 200) to determine the maximum wavelength ( λ max ) of MZ. The absorbance of the various concentrations was then measured at the λ max to construct a standard curve. The λ max for MZ is 319 nm.
2.2.7
In vitro release studies: pure alginate rings, pure PCL rings and PCL/alginate rings
To investigate the amount of drug released from pure alginate rings, 10 mL of dissolution medium (0.1 M phosphate buffered saline; pH 6.8) was placed in each well of a 6 well culture plate equilibrated to 37 °C. An alginate ring sample was then placed in each well of the plate and stored at 37 ± 0.5° C. At regular time intervals (every hour for the first 8 h and then every 12 h afterwards), an aliquot of 30 μl was collected for analysis. The collected sample was replaced by the same amount (30 μl) of fresh PBS solution of the same pH to maintain infinite sink condition throughout the study. The samples were diluted and assayed at 319 nm. To investigate the drug release from PCL and PCL/alginate rings, samples were immersed in 1 ml PBS under room temperature. 100 μl PBS were collected from different time points and samples were placed into fresh 1 ml PBS for the next time study. The metronidazole content in each sample was determined with a spectrophotometer at 319 nm. A calibration curve was prepared for each set of measurements, with a correlation coefficient >0.99. Six samples were examined for each ring type.
2.2.8
Imaging
To image the distribution of the alginate hydrogel within the rings, 4% (w/v) alginate was stained with 1% trypan blue (Invitrogen) and casted with PCL as described above for imaging.
2.2.9
Drug release model
The Fickian and non-Fickian solute release behavior from swelling controlled drug–polymer systems were determined using several established models: Ritger–Peppas model, Baker and Lonsdale model, Hixon and Crowell model, Higuchi model, and first order kinetics model. The equations were implemented in Sigma-Plot 10.0 as shown below:
Ritger–Peppas model
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