Introduction: Adhesions following surgery represent a significant problem often resulting in pain, disability, and additional surgeries. Compounds are available for the prevention of postoperative adhesions, but effectiveness is difficult to assess; current models of adhesion comparison are limited to qualitative methods with much potential bias.
Objectives: The objectives of the research performed were to create a quantitative model of adhesion strength assessment, to successfully encapsulate ketorolac tromethamine (KT) into poly(lactic-co-glycolic acid) microspheres, and to characterize of the microspheres for use in preventing adhesion formation.
Quantitative Model
Methods: The primary focus of this research was the creation of an adhesion complex that was suitable to quantitative testing using the Material Testing System (MTS™ System Corp, Eden Prairie, MN) machine platform. Following a midline infraumbilical laparotomy, bowel packing and retraction, and adequate exposure of the uterine horns and adjacent pelvic sidewall, a salpingostomy is made using electrocautery 1cm caudal the uterus-fallopian tube junction. A 7cm 8fr. latex urinary catheter, reinforced with a coaxial internal semi-rigid 5fr. polypropylene catheter, is inserted until it lies entirely within the lumen of the uterus. A 10cm segment of 6.35mm ID latex rubber drain tubing is secured to the dorsal aspect of the broad ligament medial the uterine horn; this is placed to prevent sidewall-broad ligament adhesion avoiding interference with the sidewall-uterus adhesion. The uterus and latex rubber drain are attached to the sidewall of the pelvis. The peritoneum lateral to the attached uterus is coagulated along the full length of the catheter insert at a setting of 6/10 (17W output) using a shielded electrocautery tip; cauterized area corresponds to the uterine horn lie and is limited to the peritoneum only. This injury is mirrored on the cannulated uterus to desiccate the superficial layer, and repeated on the contralateral side. Upon completion of the injury, antibiotic rinse is administered, excess fluid is removed via suction, packing and retractors are removed and abdomen is closed.
Following a 2-week survival, a midline laparotomy is again performed; the surgeon visual assesses the pelvic uterine horn adhesion. Euthanasia is achieved and the entire complex of cannulated uterine horn, sutures, adherent pelvic sidewall and muscle is removed en bloc. The sample is marked cranial and caudally for reference; muscle, broad ligament, and latex rubber drain are cautiously dissected away. The remaining uterine horn and pelvic sidewall is cut at 1.5cm and 4.5cm from the cranial suture. The catheters are replaced by a 0.64cm OD by 8cm stainless steel rod. The length of uterus being tested is measured and recorded. Rubber O-rings, 0.95cm OD and 1.28cm OD, are placed to prevent lateral movement and stabilize the specimen during testing. The peritoneal sidewall is tightly secured within a jig clamp and the complex is loaded on the MTS machine platform. The hydraulically controlled ram moves the stainless steel rod a total of 40mm at 1.6mm/sec; force and displacement measurements are recorded during the pull. Histological assessment adjacent to the testing site is completed.
To establish the statistical significance of this study SigmaStat® (Systat Software, Inc., Ashburn, VA) was employed. Pearson and Spearman analyses were performed to identify correlations among variables, and an analysis of variance (ANOVA), blocking on the animal was performed using a post hoc Tukey standardized range test to evaluate the main effects. Also, each grading method was tested with paired t-test to establish statistical differences while blocking on the animal. Significance was accept at p<0.05.
Ketorlac Tromethamine Encapsulated Microspheres
Methods: Ketorolac tromethamine was utilized as a water soluble adjuvant. In order to encapsulate it within PLGA microspheres, a standard water-in-oil-in-water emulsion technique was utilized. Two stabilizers PVA and PVP were examined at concentrations of 1%, 2%, and 5%. Theoretical loadings of 0.1 and 0.2 were studied; all six solutions were used to produce microspheres at both theoretical loadings. The samples were analyzed for drug loading efficiency, yield, microsphere surface morphology determination, particle size analysis, release profiles, and differential scanning calorimetry studies. To establish statistical significance in this section, SigmaStat® (Systat Software, Inc., Ashburn, VA) was employed. Whenever possible, Student's t-tests were used; when normality or variance did not allow for this method, Mann-Whitney Rank Sum Tests were utilized. Analysis of variance (ANOVA) was employed when more than two variables were compared. Significance was accepted at p<0.05.
Results and Discussion: The MTS measures force by displacement which can be quantitatively analyzed and interpreted, while the histology provided a comprehensive description, including vascularity, density, collagen content, and organization of the adhesion being quantified. Blocking on the swine, the cranial and caudal histology scores were shown to have a positive correlation (p < 0.002, r2 = 0.46) and were not significantly different from one another. Furthermore, the visual score showed no correlative relationship with either histological score or with any MTS force parameters. This model is unique and advantageous in its ability to quantitatively assess the strength of the adhesion complex, minimizing the potential for bias. The significant advantage of this method is that the adhesions are created in a manner appropriate for quantitative assessment using the MTS system. The ultimate utility of this technique lies not with the testing of adhesions themselves, but in testing and comparing of adhesion prevention techniques.
The encapsulation studies showed that ketorolac tromethamine was successful encapsulated in all six solutions. The data collected regarding to the criteria of yield, and drug loading efficiency failed to produce a ‘most efficient’ solution for encapsulation ability. The microspheres showed a smooth surface morphology and appeared to consist of three distinct size ranges. This observation was corroborated by particle size analysis. In the release studies, it was observed that there were discrepancies between the different solutions although the yield and encapsulation efficiencies were similar. There are several possible explanations for the variable release rates. When examined in conjunction with the size analysis data it would seem that the most plausible is that the initial burst and transition regions are directly affected by the particle sizes. Differential scanning calorimetry verified encapsulation and showed a relationship between encapsulated ketorolac tromethamine and Tg.
The results presented and discussed in this thesis represent the initial steps toward the creation of a pharmaceutical adjuvant for the prevention of pelvic adhesions. The analysis shows that KT can be successfully encapsulated into PLGA. While the release time seen above was longer than the originally sought release of 14 days, the desired initial burst effect was large and this could be potentially advantageous.
Future work: The next important step to take in developing KT microspheres for pharmaceutical drug delivery is to determine the effect of sterilization on the microsphere structure, size, and duration of drug release under in vitro conditions. Once microsphere sterilization has been characterized and if there are no significant issues, in vitro and in vivo testing can be pursued.
In order to determine the optimum single size or combination of sizes, it is necessary to determine an optimum therapeutic concentration for adhesion growth prevention. To achieve this, a co-culture of mouse fibroblast cells and macrophage cells would be useful. From this in-vitro study, an optimum, minimum, and toxicity level for KT can be determined. Provided this information, the efficacy, size, and release rate of the produced microspheres can be reevaluated and a single or combination of sizes can be isolated via filtration.
Without in-vitro studies, it is impossible to move forward. From the studies, the synthesis technique can be refined to produce microspheres with the most desirable properties following sterilization. Once this is completed, animal studies and eventual implementation as a pharmaceutical adjuvant for the prevention of pelvic adhesions can commence.