Serial Experimental Studies On CH/PEG-SA/MMC Drug Delivery System

Despite the advances in surgical techniques, epidural scarfing tissue afterlaminectomy and windowing cause to be a significant reason for failed back surgerysyndrome(FBSS). FBSS is a group of disorders with persistent or recurrent symptomsfollowing spinal surgery, with the hallmarks of back pain, sciatica, and functionalimpairment. FBSS is characterized by severe chronic and disabling pain thatgenerally is resistant to physiotherapy and pharrmacologic treatment. There are manycauses of FBSS; among of the most common is epidural fibrosis. There are numerousreports suggesting that fibrosis and adhesions can cause compression or tethering ofnerve root, which may cause recurrent radicular pain and physical., impairment. Thescar formation and adhesion is increasing with time course and size of laminaopening; The scar adhesion has minimal dura compression effec; The scar adhesionmay induce neovascularization and axon swelling in cauda equina area; Thepathological effects are time, volume and distance dependent; The scar adhesion caninduce dura mater thickening and can really induce some pathological change to duramater and nerve root in cauda equina area. It seriously influences the the long-termeffect of surgical operation of spine,and brings difficulties to re-orthopaedic surgery.This increases hospital stay and suffering of the patients. Therefore, how availablyprevent from epidural sarring adhesion after postoperation is the urgently solving topic referring to the realm of spinal surgery. In recent years, the domestic andinternational scholars have carried on a great deal of research and made certainprogress on the found of primary experiments and clinical practices.So we developeda sort of new type drug delivery system(DDS),cooperating with South ChinaAgricultural University. Serial experimental studies gave some remarkable results asfollows: While possesseing excellent biocompatility, the sort of new type DDS canslowly deliver the Mitomycin C(MMC) at the implant site.It can effectively inhibitthe hyperplasy of epidural scarring tissue after spinal postoperation.This experiment divides into three parts.Section one: Development of chitosan/polyethylene glycols succinate film drugdelivery system and the biocompatibility to fibroblast.Objective: To develop the chitosan (CH)/polyethylene glycols succinate acid(PEG-SA) film and study the biocompatibility to fibroblast.Methods: The experiment was accomplished in the Central Laboratory, ZhujiangHospital, Southern Medical University from May to December in 2006. Experimentalmaterials: CH/PEG-SA or CH/PEG was prepared by casting the mixed solution ofdialysed CH and PEG-SA or PEG in a freeze dryer. Fibroblasts of SD rats newly bornfrom two to five days were seeded on the CH/PEG-SA, CH/PEG and CH filmsrespectively. Experiment evaluation:â‘ MTT assay was used to determine theabsorbance (A) value of fibroblasts seeding on different films after 1/2,1,3, 5 daysculture. Then relative adhesion rate at 1/2 day was calculated according to A_490nm ofdifferent films/A_490nm of culture plate×100%.â‘¡Surface topography were analyzed byinverted phase contrast microscope. The experimental data is analyzed with One-WayANOVA and LSD on relative adhesion rate and Repeated-Measures ANOVA onproliferation of fibroblasts by the SPSS13.0.Results:â‘ Fibroblasts adhered well on the CH/PEG-SA, but poorly on CH/PEG, CH and even gradually died. MTT results showed that the A value of the CH/PEG-SAgroup was about (0.074±0.009) at the first 12 hours after seeding and reached(0.141±0.031) at the 5th day, which was 6.17 and 6.13 times higher than those of theCH group (P=0.000).â‘¡Growth characteristics of fibroblasts: The fibroblastproliferated best on CH/PEG-SA, with the highest activity, strongest proliferation andquickest velocity, and then was those on CH/PEG. There was significant differencebetween the two (P=0.046). However, the fibroblasts proliferated less on the surfaceCH, with the significant difference in comparison with other groups (P=0.000).â‘¢Surface topography of fibroblasts seeding for 1, 3, 5 days: The transparent and roundcells seeding on CH were free, folded and unadhered, presenting shrinkage and lowproliferation; The fibroblasts were proliferated much better on the CH/PEG-SA,CH/PEG film than that on the surface CH film. The confluent cells were shaped infusiform and arranged tightly in fasciculation or finger print, with the narrow intervaland gathering growth.Conclusion: Incorporating SA in CH/PEG network can improve the mechanicalproperties and flexibility of the films. The fibroblasts are adhered and proliferatedbetter on the CH/PEG-SA film than that on the surface CH film, indicating the goodbiocompatibility of CH/PEG-SA.Section two: Development of chitosan/polyethylene glycols succinateMitomycin C film drug delivery system.and releasing character in vitro.Objective: To develop the chitosan/polyethylene glycols succinate acid/Mitomycin C(CH/PEG- SA/MMC) film drug delivery system and its releasing effect in vitro.Methods: Mitomycin C loading in composite films are determined using aUV-visible spectrophotometer.Freeze-dried films(90mg) were dispersed in 1ml PBSbuffer(pH 7.4). calculated the concentrations of Mitomycin C releasing in vitro referto the standard curve of realationship between the concentrations of Mitomycin C and the value of UV-visible spectrophotometer. We try to reveal the relationship betweenthe films strucure and the drug releasing. The experimental data is analyzed withLinear Regression and Curve Fit by the SPSS13.0.Results: The equation of Linear Regression: y=0.593x³-2.563x²+25.944x-0.236, R Square =1.000. The film has a well drug delivery capability, the samplesweighing 20mg each are soaked into the liqiud of PBS, the titer of Mitomycin Creleasing after the 12 days is 14.9616μg/mL at its peak, afterward again in the 18thday and 32th day appear to twice release high peak, distinguish to 14.4824μg/mL and11.4092μg/mL, then maintain a slow releasing in the following days. After 60 days,the titer is 0.1793μg/mL which is higher than Mitomycin Cs ID50 (10.4713μg/L)to fibroblast.Conclusion: The film can keep drug release very slowly for a long time invitro,which is relevant to its structure. The rate of drug diffusion was substantiallyhigher than that of polymer degradation, so the release profile was more dependent ondrug diffuson rather than polymer degradation. The film has formed a DDS unit in thespecial structure so that the initial burst was low that can be neglected, and then drugsdiffused slowly through the interspaces of polymer matrices.Section three: Therapeutic effect of the CH/PEG-SA/MMC film drug deliverysystem on epidural scarfing tissue after laminectomy of SD rat.Objective: To investigate the therapeutic effect of the CH/PEG-SA/MMC filmDDS on epidural cicatricial tissue.Methods: Thirty SD white rats were selected,and randomly assigned to 6 groupswith 5 rats in each group. A rat model of lumbar laminectomy was used.The animalsall were followed byimplant of 20 mg of the CH film in groupâ… , 20mg of CH/PEGfilm in groupâ…¡, 20mg of CH/PEG-SA film in groupâ…¢, 0.05mg/mL of the MMC5min in groupâ…£, 20mg of 20mg CH/PEG-SA/MMC film in group V,and those in groupâ…¥were left without anything grafting.Specimens were harvestd 4 weeks afterthe above procedures and were then subjected to immunohistochemical andhistological examinations to compare their therapeutic effect on epidural cicatricialtissue. The experimental data is analyzed with Kruskal Wallis Test on the wholeobservation, and one-way ANOVA and LSD on Hyp by the SPSS13.0.Results: There is significant difference among the groups. (P=0.000)â‘ In groupâ…£,â…¤,the content of hydroxyproline (Hyp) were smaller than other 3 group, but therewas no significant difference between them (P=0.654),with no visible histologiealmanifestation of cicatricial tissue;â‘¡In groupⅠ～Ⅴ, there was more significantdifference than groupâ…¥on the content of Hyp (P=0.001,0.017,0.000,0.000,0.000).And the the histological manifestation were more serious in groupâ…¥.â‘¢There is no significant difference in groupâ… ,â…¡andâ…¢(P=0.177,0.880,0.136)Conclusion:â‘ The CH/PEG-SA/MMC film DDS could be implanted as primarygraft into the remaining vertebral plate defect after lumbar laminectomy to effectivelytreat epidural cicatricial tissue,serving as an ideal treatment option for epiduralcicatricial accretio and reaching the attenuation-synergetic effect.â‘¡Conventionaland simple local MMC following lumbar laminectomy was effective in treatingepidural cicatricial tissue,but which can't insure a long-term to effectively be curedand its fiber alignment is irregular.â‘¢Pure partial application CH film only has the afunction of mechanical barrier and limitedly separate the incursion of the cicatricialtissue, but can't obstruct fibroblast become fiber cell to band together to gather,proliferate, synthesize, secrete the collagen fibers toward the wound in theinflammation process and become scar organization.In summary, the CH/PEG-SA/MMC film DDS are simple to get.The DDS have afine releasing character in vivo and the plastic plants have good plasticity in thevertebral cavity, the DDS may have a widespread application prospect in treating scarring tissue adhesion.