Abstract
The aim of this study was to formulate Pentoxyfylline drug (PTX) as a local bioadhesive Carbopol (Cbp) based gels for the aid of bone induction around an endosseus oral implant. The local delivery of the drug will probably avoid most of the problems associated with its systemic use including; disturbances in gastrointestinal tract and the central nervous system. Two concentrations of 1% and 3% Cbp containing 1% PTX were prepared. The gels were investigated for their physicochemical properties. Cbp based gels were found to be translucent with good homogeneity, uniform distribution of the drug and absence of any lumps. The pH of the gels was within neutrality, 7.1, which is considered to be acceptable to avoid the risk of any possible irritation in the oral cavity. The Cbp gels exhibited satisfactory bioadhesive properties and a pseudo-plastic rheological behavior. Cumulative drug released from the gels showed a controlled-release for more than 24 hours with the order of 3% >1% and the drug was released by diffusion mechanism from both gels. Statistical analysis revealed non-significant difference in drug content, rheological property and release rate of the stored gels for six months compared to the fresh ones. In vivo experimental results in rabbits have shown significant difference in bone depth induction of 3% and 1% Cbp gels with the formation of strong organized bone over the control group. Local administration of Pentoxifylline could be regarded as a valid approach in the management of osseointegration.
Introduction
Implant success is primarily dependent upon or achieved by osseointegration which is defined as the direct contact between the implant surfaces and ordered, living bone (Brånemark et al., Citation2001). Various approaches have been attempted to enhance the level of osseointegration via increasing Bone-to Implant Contact (BIC). It is generally observed that the degree of BIC varies depending on implant macro/micro structures, surface characteristics, different healing periods, and the presence or absence of loading (Cochran, Citation1999).
Despite the ongoing improvement in implant characteristics, bone intrinsic potential for regeneration may be stimulated with adjuvant therapies to standard surgical procedures, as it is important to achieve the best possible implant osseointegration and to ensure long-term implant stability. Attempts were performed to achieve faster osseointegration with the use of pharmaceutical compounds that increase bone formation around an endosseus implant. Most bone-modulating drugs currently used, such as biphosphonates, calcitonin, estrogen, selective estrogen receptor modulators and vitamin D analogues, inhibit bone resorption instead of primarily stimulating new bone formation (Garret et al., Citation2001). While bone-forming agents that are capable of rebuilding bone; anabolic agents and recombinant bone morphogenetic protein-2(BMP-2) were reported (Saito et al., Citation2001). Kinoshita and coworkers (Kinoshita et al., Citation2000) reported that daily injections of pentoxifylline (PTX) stimulated bone formation and increased systemic bone mass in mice.
PTX is a xanthine derivative that inhibits phosphodiesterases (PDEs), resulting in the elevation of intracellular cyclic adenosine monophosphate (cAMP) (Tsutsumimoto et al., Citation1999). It has been widely used as an agent for the treatment of cerebrovascular disorders or occlusive arterial diseases. PTX has recently been found to prevent or attenuate the action of tumor necrosis factor and has been used as a treatment for acute septic shock or chronic cachexia. Additional reports have documented that PTX modulates the release of other cytokines, such as interleukin-1 (IL-1) and IL-6 (Tsutsumimoto et al., Citation2002).
Systemic administration of PTX can induce adverse reactions such as disturbances in gastrointestinal tract and the central nervous system including nausea, vomiting, diarrhea, headache, dizziness and tremors (Tsutsumimoto et al., Citation1999). Therefore local drug delivery avoids most of these problems, by limiting the drug to the target site (site specific approach) with little or no systemic uptake. In addition, the local concentration achieved can be much higher than is possible via systemic route.
Gel based topical formulations seems to be one of the most suitable vehicles for local drug delivery to the oral cavity tissues. These delivery systems have several advantages such as the ease of administration, non greasy, patient compliance and better drug release and diffusion (Danester & Evone, Citation2008). Moreover gels can be easily prepared, they process a higher biocompatibility, can be eliminated rapidly through the normal catabolic pathway and decrease the risk of irrigative or allergic host reactions at the application site (Reddy et al., Citation2005). To increase an adherence between the bases and oral tissues, polymers with bioadhesive properties were selected as gelling agents (Ghandi & Robinson, Citation1994, Songkro et al., Citation2009). Among these bioadhesive polymers is Carbopol (Cbp) 934; it is a polyacrylic acid polymer, cross linked with allyl sucrose. This acidic carboxylic group partially dissociates in aqueous solution, producing a flexible coil structure. It has been reported that Cbp 934P is a bioadhesive polymer and has been investigated extensively by the pharmaceutical industry because of its high viscosity at low concentration as well as its low toxicity (Sweetman, Citation2011).
Therefore, the aim of this work was in vitro and in vivo evaluation of bioadhesive Cbp gel of pentoxyfilline for bone induction.
Materials and methods
Materials
PTX BP93/USPXXIII and Cbp 934, of Goodrich Chemical Co., were supplied by Pharaonia Pharmaceuticals, Alexandria, Egypt. Ketamine Hydrochloride were purchased from Eipico, Egypt; Xylazine, M.H.; ADWIA, Egypt; Mepecaine, Alexandria Co. for Pharmaceuticals, Egypt; Pentobarbitone; Diazepam, Roche, France. Other chemicals were of pharmaceutical grade.
Preparation of gels
Cbp gels (1 and 3% w/w) were prepared using the previously reported method by Bonacucina et al. (Citation2004). An appropriate amount of Cbp powder was slowly added into water under constant stirring using a magnetic stirrer. The mixture had been kept at room temperature for 24 hours to get rid of the entrapped air bubbles and about 2–3 drops of triethanolamine was added, and well mixed until the gel was formed.
For drug loaded gels, an adequate weighed quantity of PTX (1%) was sprinkled gently onto the prepared gel using a magnetic stirrer until a homogenous clear gel was obtained. The gels were kept at room temperature for 24 hours before being used in different evaluation studies.
In vitro characterizations
Drug content evaluation
Drug content was determined by dissolving accurately weighed quantity of gels in Sorenson phosphate buffer (pH 6.6). After appropriate dilution, absorbance was recorded by using UV spectrophotometer (Ultraviolet-Spectrophotometer, Pharmacia double beam, England) at 273 nm. Concentrations used in calibration curve was from 5 µg/ml to 25 µg/ml
Rheological study
The viscosities were determined using Brookfield viscometer (Model DV-II+ Pro, Brookfield, Middleboro, MA) at 25 °C. A sample of the gel (1% and 3%) was placed in a suitable beaker and the instrument continuously sheared the material at various rates, using spindle number 6. Measurement was done over a range of speed of 3, 4, 5, 6, 10, 12, 20, 30, 50, and 60 rpm with 60 seconds between two successive speeds and then in a descending order. All measurements were made in triplicate.
Differential scanning calorimetric study
Samples (3–4 mg) were placed in aluminum pan and heated at a rate of 10 °C/min to temperature of 300 °C. The instrument (Perkin Elmer, Germany) was calibrated with indium and dry nitrogen was used as a carrier gas with a flow rate of 25 ml/min. These studies were performed for the drug, polymer, drug–polymer physical mixture in a ratio of 1:1 and drug-loaded gel.
Spreadability study
Spreadability of the gels was done by two methods
Diameter method
According to Varshosaz et al. (Varshosaz et al., Citation2002), two smooth polished glass blocks were selected; one block was fixed on a table and the level was adjusted. The upper block was passed down through a pulley by a thread, the end of which was tied to a pan. 150 mg was weighed from the selected formulations of the freshly prepared gel. Each separately was placed on the center of the fixed block and above which the second block was placed and pressed with some weight (200 g). The diameter formed of the gel between the blocks was measured after five minutes application of force to indicate the spreadability of the gels.
Another method of spreadability
Excess of gel sample was applied in between two glass slides and was compressed to uniform thickness by placing 300 gm weight for five minutes (Shankar et al., Citation2009). Weight (100 gm) was added to the pan and the time required separating the two slides, i.e. the time in which the upper glass slide moves over the lower plate was taken as measure of spreadability (S).
The spreadability of the prepared gels was calculated by using the following formula:
where S is the spreadability (g cm/sec), M represents the weight tied to the upper slide (g), L represents the length moved by the glass slide (cm) and T stands for the time taken to separate the slides completely from each other (sec).
Bioadhesion study
The previous spreadability test was repeated but by applying gradual increase of weights until detachment occurred to indicate the detachment force (bioadhesive force) in terms of gm (Sudhakar et al., Citation2006).
In vitro release study
An accurately weighed amount of gel formulation equivalent to 1.5 mg of PTX was placed in a stainless steel cup (10 mm diameter and 5 mm depth) and covered with polyester gauze acting as a mechanical barrier to prevent gel escape without interfering with drug release (Ismail et al., Citation2000) and was fixed to the cup through a specially designed stainless steel ring. The cup was placed at the bottom of 10 ml beaker containing 2 ml of release medium (Sorenson phosphate buffer pH 6.6) (Sridevi et al., Citation1995). The beaker was placed in a thermostatically controlled water bath without shaking adjusted to 37 ± 0.5 °C which is the simulating temperature of the buccal cavity. At pre-determined time intervals, total volume was withdrawn and replaced by equal, pre-heated fresh medium at 37 ± 0.5 °C. Samples were analyzed spectrophotometrically for PTX at 273 nm. All experiments were done in triplicates.
Kinetic analysis of release data
To determine the mechanism of drug release from different gel formulations, The in vitro release data were analyzed by the following commonly used exponential equation of Korsmeyer et al. (Citation1983):
The Korsmeyer et al equation in logarithmic form is
Where Mt/M∞: the fraction of released drug at time t; K: release constant and depends on structural and geometric characteristics of the drug/polymer system and n: release exponent indicative of the release mechanism.
Dissolution efficiency calculations
As a parameter for the comparison of the release profiles, dissolution efficiency (DE) of profiles was calculated from the area under the curve at time t (measured using the trapezoidal rule) and expressed as a percentage of the area of the rectangle described by 100% dissolution in the same time (Khan, Citation1975).
Effect of ageing
The effect of ageing was studied out on the prepared gel formulations, characterized by optimal properties. The gel was stored in well closed aluminum tubes and kept at ambient conditions for six months. The changes in physical appearance, drug content, rheological and release characteristics of the gels were determined after 3 and 6 months.
In vivo experimental protocol
The study was approved by Pharos Institutional animal ethics committee. 18 white adult male New Zealand rabbits aged 9–12 months weighing (3–3.5 kg) were used in the entire study. The animals were kept in individual cages with free access to food and water. They were randomly assigned to three treatment groups (6 animals/group). Group A acted as a control, Group B received topical PTX gel 1% and Group C received topical PTX gel 3% (PTX was applied locally in the right osteotomy site before implant insertion for each rabbit tibia and left side implants were considered as control). A total of Titanium of diameter 3.3 mm and 8 mm length implants were inserted in the tibia of each rabbit.
Under aseptic conditions the surgical procedure was carried out under general anesthesia produced by an intramuscular injection of Xylazine 5 mg/kg body weight and ketamine hydrochloride 30 mg/kg body weight. Local anesthesia with 1 ml of 5% Xylocaine was administrated to the tibial metaphysis where the implants were to be inserted.
Assessment of peri-implant bone density
Intra-oral peri-apical radiographic films (size 2) were utilized to obtain radiographs immediately following implant placement (baseline) and immediately after sacrifice. Radiographic exposure was carried out using ANY-RAY portable x-ray machine (Vatech Company Limited, Korea.). All films were processed under standard conditions by automatic processor (HOPE Dental-max Hoop Co, USA) and scanned using Az-tek III scanner (Irvine, CA). The bone density was measured by Image J software program (public domain, NIH).
Histological examination of the tibia
At the end of the predetermined experimental periods (8 weeks) all rabbits were sacrificed by an over dose of a pentobarbitone 60 mg/ml/kg body weight into an ear vein. Following sacrifice the tibiae were dissected free and fixed in 10% buffered formic acid. Further after, the samples were fixated in 70% ethanol following dehydration in a graded series of ethanol and finally embedded in polmethyl methacrylate resin. After polymerization the specimens were sectioned in the sagital plan and through the long axis of the implants, and then were further ground to about 20 µm thickness. Analysis was performed using scanning electron microscopy (SEM Joel (JSM-T20), scanning microscopy, Japan).
Statistical analysis of data
Data analysis was carried out with the software package Microsoft Excel version 2007 and DD Solver, add-in program for MS Excel for modeling and comparison of drug release profiles. Results were expressed as a mean ± standard deviation. Statistically significant difference was determined using the student t-test and ANOVA one way with p < 0.05 as a minimal level of significance (Graph Pad Prism, San Diego, CA).
Results and Discussion
Cbp is a hydrophilic polyacrylic acid polymer and its carboxyl groups become highly ionized after neutralization, forming a gel due to the electrostatic repulsion among charged polymer chains interconnected by cross links (Shin et al., Citation2005).
Gels prepared with Cbp were found to be translucent, showed good homogeneity with absence of any lumps. The pH of the gels was 7.1, which was considered to be acceptable to avoid the risk of any irritation in the oral cavity. Content uniformity studies showed that the drug was distributed uniformly in all formulations (). The amount of gelling agent was selected on the basis of optimum quantity required for gel preparation, which has been reported in various literatures (El-Dakrouri et al., Citation2010; Sareen et al., Citation2011).
Table 1. Characteristics of the formulated Cbp-based gel of PTX for bone induction.
Rheological Study
Viscosity is an important parameter for characterizing the gels as it affects the spreadability, extrudability and release of drug. Rheological study of the formulations indicated that gels exhibited pseudo-plastic rheology, as evinced by shear thinning and a decrease in viscosity with increased angular velocity (). This shear thinning behavior is a desirable property for topical preparations as they should be thin during application and thick otherwise.
Figure 1. Rheograms of the formulated Cbp-based gels: (a) 1% Cbp, (b) 3% Cbp.
The viscosity of Cbp gel significantly increased as a function of carbopol concentration in the gel (p < 0.05). This may be due to the increase in formation of three dimensional cross linking structure of the gel (Shankar et al., Citation2009).
Islam et al. (Citation2004) studied the rheological behavior of topical Cbp gels in a pH range between 5.0 and 8.0 and reported that gels were showing the behavior of elastic systems. The elasticity of these networks can be explained by that triethylamine as a swelling agent enable closely packing and tight binding between the long chains and side chains and by the solvent–solvent and solvent–polymer interactions.
The formulations showed no thixotropy behavior because the up curve and down curve were nearly superimposed indicating a rapid recovery of their structure as the shear stress is decreased. The statistical analysis supported this assumption and indicated that there was no significant difference between the up and down curves for gel formulations formed since p value was higher than 0.05. This result was in agreement with those reported by Danester & Evone (Citation2008).
Differential scanning calorimetric study
Differential scanning calorimetric (DSC) has been widely used as a rapid thermal method for examining drug–excipient compatibility (Wissing et al., Citation2000). As shown in , the thermogram of PTX shows the characteristic endothermic peak at 107.54 °C corresponding to drug melting. The thermogram of drug loaded gel showed that the endothermic peak of the drug remained almost unchanged indicating no structural changes occurred for PTX in the Cbp based gel. This finding was in agreement with the work of Das & Palei (Citation2011) for refecoxib loaded Cbp gel.
Figure 2. DSC thermogram of the Cbp polymer, pure drug (PTX), physical mixture of PTX: Cbp (1:1) and PTX Cbp-based gel.
Spreadability
The spreadability plays an important role in patient compliance and helps in uniform application of gel to the affected area. A good gel takes less time to spread and will have high spreadability (Shankar et al., Citation2009).The spreading of the gel could be measured by the diameter formed when the gel was compressed between two blocks of glass plates. shows that increasing the polymer concentration in the gels causes a reduction of spreadability (p < 0.05). The diameter was found to be 7.2 and 5.2 cm for 1% and 3% Cbp respectively which could be indicative of good spreadability.
Bioadhesion
The basic parameters for designing bioadhesive gels are ease of removal of the gel from the primary package, ease of application of the product to the desired region and retention at the application site without disintegration (Gülin et al., Citation2012). The adhesive characteristic is an important parameter in the design of an oral gel, since a desirable gel contact and retention at the mucosal surface will ensure better clinical efficacy. It is worth mentioning that factors such as the molecular weight of polymer, the type and degree of cross-linking agent, molecular architecture and the polymer amount in the gel influenced the mucoadhesive performance (Anlar et al., Citation1993).
As shown in , the increase of Cbp concentration in the gels showed significant increase in bioadhesive strength (p < 0.05). Cbp, due to its chemical nature, high-molecular-weight polymer that readily swells in water; the swelling exposes a large adhesive surface for maximum contact with the mucin (the glycoprotein predominant in the mucous layer) and thus provides excellent mucoadhesiveness (Bansal et al., Citation2009). The pKa of Cbp polymers is 6.0 ± 0.5, above that point, the carboxylic acid groups are ionized greatly, thus reducing hydrogen bonding. Under more alkaline conditions, the Cbp gels are very highly swollen, and the chains are stiffened by electrostatic repulsion of the anionic charges along backbone (Shin et al., Citation2000).
A direct relationship between the viscosity and bioadhesive strength was shown in , as the viscosity of Cbp gel increased, the bioadhesive strength increased. It has been reported by Tur & Ch'ng (Citation1998) that the relationship between the viscosity of Cbp gel systems and bioadhesion may facilitate optimization of mucoadhesive performance, leading to the development of more effective bioadhesive dosage forms. To prepare the bioadhesive systems, one must control the degree of ionization of the polymer by manipulating the pH of the media or, alternatively, choose a bioadhesive with a pKa that can provide a suitable extent of ionization. This phenomenon is essential in the design of bioadhesive dosage forms since external pressure cannot be applied to them in the mucosal membranes.
In vitro drug release
In vitro release profiles give important information on the efficiency of a delivery system proposed for controlled release of drugs. Cbp based gels of PTX were prepared and evaluated with a view to obtain a controlled release of the drug at the site of action in order to decrease the side effects and to increase its clinical efficacy. The results revealed that the release rate of PTX was controlled for a period of time with both formulations. depicts the PTX released from gel formulae after 24 hours was 83.29% ± 1.9 and 66.67% ± 3.5 from 1% and 3% gel respectively. The release of PTX from gels significantly decreased (p < 0.05) due to the increase in the polymer concentration in gel formulations. It has been reported that Cbp at pH > 4.5 swells and after full hydration does not dissolve but osmotic pressure from inside breaks up the structure, mainly by sloughing off eroded particles of the gel and thus the drug is dissolved readily (Efentakis et al., Citation2007).
Figure 3. Release profile of PTX from the formulated Cbp-based gels in Sorenson phosphate buffer pH 6.6 at 37 °C. (Each point represent the mean ± SD, n = 3).
The choice of an appropriate in vitro model has to take into account the need to resemble the in vivo behavior as strictly as possible, therefore, Sorenson phosphate buffer pH 6.6 was used to simulate the gingival fluid environment (Deasy et al., Citation1989). It has been reported that PTX solubility in water is 77 mg/ml (Bohm et al., Citation2003) and sink condition was maintained by removing the whole volume as a sample and replacing it by fresh medium (Ismail, Citation2006).
Viscosity of the vehicle is an important physical property of topical formulations that affects release of drug since it may reduce drug diffusion rate due to formation of rigid structure of the vehicle (Pose-Vilarnovo et al., Citation2004). Hereby, a reverse relationship was also observed between viscosity of the formulations and rate of PTX release.
Kinetic analysis
The kinetic analysis of the release profile was carried out according to the Korsmeyer et al. equation:
where Mt is the cumulative amount of drug released at time “t”; M∞ is the total amount of drug-incorporated; K is the proportionality constant the value of which depends on the structural and geometrical properties of the matrix; and “n” is the release exponent, its value depends on the mechanism of drug release.
The n exponent of the Korsmeyer et al equation was 0.4 and 0.5 for 1% and 3% gel formulations; respectively (). This indicated that drug diffusion through the gel was the rate limiting step for the release of drug from these gels. Diffusion is related to transport of drug from the gel matrix into the surrounding in vitro medium and it depends on the drug concentration. As gradient varies, the drug is released and the distance for diffusion increases. This could explain why the drug diffuses at a comparatively slower rate as the distance for diffusion increases (Bansal et al., Citation2009). Statistical analysis showed that Cbp concentration did not show any significant effect on the n exponent value (p > 0.05).
Table 2. Comparing the DE%, T50%, release rate constant (k) and exponent n of different Cbp-based gel formulations of Pentoxyfilline in Sorenson phosphate buffer pH 6.6.
% Dissolution efficiency after six hour and T50% were also used to compare the drug release characteristics of the two gel formulations and presented in .
Effect of ageing
As shown in and from the results of statistical analysis using student-t test at p > 0.05, a non-significant difference was obtained in the drug content, release behavior and the rheological behavior upon storage of the formulated gels at ambient temperature for 6 months. However further stability studies have to be carried out for extended period of time by considering relative humidity.
Figure 4. Effect of ageing on drug content of the Cbp-based gels at ambient temperature. (Each point represent the mean ± SD, n = 3).
Figure 5. Effect of ageing on the release profiles of PTX from: (a) 1% Cbp (b) 3% Cbp gels in Sorenson phosphate buffer pH 6.6 at 37 °C. (Each point represent the mean ± SD, n = 3).
Figure 6. Effect of ageing on rheological property of the Cbp-based gels: (a) 1% and (b) 3% at ambient temperature.
In vivo study
presents the results of the % increase in bone induction of all studied groups. The study revealed that the % increase of bone induction after 2 months was significantly enhanced (p < 0.05) in both groups who applied Cbp gels, where 55.394% increase was observed for the group applied 3% gel and 19.507 % for 1% compared to only 1.73% for the control group.
Figure 7. Percentage increase in bone density after local application of PTX Cbp based gels after two months in rabbits.
Moreover, SEM revealed the achievement of surface topography and close bone implant contact in all studied groups by the end of the study period. In the PTX 3% group a well organized bone was even detected in the threads and irregularities along the implant ().
Figure 8. (a) SEM of group C (3% gel) at the end of study period showing intimate bone implant contact with no gap (500×). (b) SEM of Control group A showing a gap extending along the bone implant interface. (500×).
This result was confirmed by histological examination for the dissected tibiae and demonstrated evidence of new bone formation next to the implant. The newly formed bone assumed morphology complementary to threads and has a tendency to migrate to the space formed between the implant and the bone. In the 3% group an intimate contact between the implant surface and the bone was observed and the new bone showed numerous Haversian system and interstitial bone, cellular proliferation and differentiation revealed a strong and more organized bone (). On the contrary photomicrograph of the ground section of the control group demonstrates formation of osteoms and formation of immature bone ().
Figure 9. Photomicrograph (100×) ground section of: (a) 3% gel group showing well organized mature bone with no gap and (b) control group showing immature bone.
Conclusion
Local delivery of PTX drug as Cbp based gels in concentration of 1 and 3% have shown significant results in controlled drug release for 24 hours, good bioadhesion properties with good spreadability. In vivo experimental results in rabbits have shown significant difference in bone depth induction of 3% Cbp based gels and formation of strong and more organized bone over the control group.
Therefore it could be concluded that local administration of PTX could be regarded as a valid approach in the management of osseointegration, by limiting the drug to the target site with little or no systemic uptake and hence avoiding most of the problems associated with systemic therapy like disturbances in gastrointestinal tract and the central nervous system.
Acknowledgements
The authors would like to acknowledge and thanks Hala H.Yassin, Associate Professor of Periodontology, Head of Oral Medicine & Periodontology Department, Faculty of Dentistry, MSA University-Cairo, Egypt and Mahmoud G. Salloum Lecturer in Prosthodentic Department, Faculty of Dentistry, Pharos University in Alexandria for their participation in the in vivo experimental part of this work.
Declaration of interest
The authors report no conflict of interest related to the data submitted in this manuscript.
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