Sustained Release Of Rifampicin From Implantable Fibrin Polymer/Bone Allograft Composite Systems

Background and Objective:One-third of the world's population is infected with the Mycobacterium tuberculosis. There were an estimated 13.7 million prevalent cases in 2007, and China accounts for nearly 17 % tuberculosis burden of the world, it's second only to India in the number of TB patients. Since the WHO declared tuberculosis a worldwide emergency in 1993, spinal tuberculosis has resurged after decades of decline in incidence, as a common form of extrapulmonary tuberculosis in china. Even extra-pulmonary tuberculosis is not contagious, public attention are most focused on pulmonary tuberculosis. Unfortunately and obviously osteoarticular tuberculosis has risen parallel to that of pulmonary tuberculosis. It attempted to call much more attention, if considering the large population base and as important cause of disability and mortality.Since rifampicin was introduced as antituberculous agent in 1963, it has been the cornerstone of drug regimens for the treatment of tuberculosis, its use has greatly shortened the duration of chemotherapy for the treatment of TB. However, studies have shown probable reasons lead to poor variable bioavailability and lower plasma concentrations of rifampicin, and intralesional concentration of rifampicin and pyrazinamide in osteoarticular tuberculosis were mostly at subtherapeutic level, it has been associated with clinical failure and drug resistance. Low level resistance to rifampicin implies the antituberculous effect would be optimized with higher concentration rifampicin. In order to maintain effective rifampicin concentration, novel drug delivery systems has been investigated for sustained or controlled release purpose .So aims of this study included: To select an antituberculous drug for sustained release by analyzing drug susceptibility testing of Mycobacterium tuberculosis in spinal tuberculosis, detection of intralesional isoniazid and rifampicin concentration using HPLC method, and the killing effect of higher concentration rifampicin on rifampicin-resistant M. tuberculosis isolated from patients with osteoarticular tuberculosis in vitro.Materials and Methods:(1)The specimens (pus or caseous tissue) were collected from 72 patients with osteoarticular tuberculosis during surgery. After processed in a routine mycobacteriology laboratory procedure, the specimens were inoculated into vials of the BACT/ALERT 3D system. Mycobacterium tuberculosis strains were identified with PNB or TCH culture medium growth trial. The drug susceptibility testing was performed using absolute concentration method,which included 11 first and second line drugs. (2)Two simple, precise and accurate HPLC-based assays for the quantification of rifampicin and isoniazid were established. The intralesional effect of rifampicin and isoniazid of 36 patients with osteoarticular tuberculosis were accessed by concentration:MIC ratio.(3)A set of 18 rifampicin-resistant M. tuberculosis isolates were prospectively collected for further study. Detection of rpoB gene mutations associated with rifampicin resistance in Mycobacterium tuberculosis was performed using non-fluorescent low-density DNA microarrays. Following secondary culture, susceptibility to rifmapincin was tested at increase concentration of rifmapincin by degrees (2,4,8,16,32,64,128,256μg/ml),which is attainable for prolonged periods of time with local chemotherapy.(4)Procurement, processing, sterilization and conservation of the iliac crest allograft scaffold were all performed according to the guidelines of the American Association of Tissue Banks. Its physical characteristics were observed including colour, texture and porosities, the diameter of pores and microstructure were tested with scanning electron microscope.Its porosity and density were tested with liquid displacement technique.(5)Orthogonal experiment design was used in the optimization of a fibrin scaffold for sustained release of rifampicin. The scanning electron microscopy analysis and in vitro study of sustained release of fibrin disc containing rifampicin crystals were carried out to optimize the avaible fibrinogen and thrombin concentration.(6) A new negative pressure impregnation system was designed and manufactured. In the light of previous results, preparation of implantable fibrin polymer/bone allograft composite systems for rifampicin delivery were unerwent using negative pressure impregnation technique using the No.3,5,8 fibrin compositions . Fibrin glue and rifampicin methanol solution were impregnated into the pore of bone allograft under negative pressure respectively. (7) The sustained release of rifampicin from implantable fibrin polymer/bone allograft composite systems was carried out in vitro ,the chemostat filled with goat serum as the release medium was placed in a humidified 37oC /5% CO2 incubator. The HPLC-based assay was used to determined the concentration of rifampincin for optimization the No.3,5 and 8 fibrin compositions .(8) The toxic effects of rifmapicin on the proliferation and osteogenic differentiation of MSCs were assessed. by MTT-measurements, extracellular matrix calcication , immunohistochemistry staining, real-time RT-PCR, and western blot. (9) For in vivo study, the No.5 implantable fibrin polymer/bone allograft composite systems was implanted in the goat's os ilium and lumbar interbody. The local rifampicin concentration was determined by an assay of effusion drained from implanted sites.Results:(1) Thirty one of the total fifty specimens were culture positive(43.1%). Twenty eight specimens (90.32%) were identified as M. tuberculosis infection and three (9.68%) as M. bovis. The drug susceptibility testing outcomes showed a resistance level of 11.27% for isoniazid, 2.78% for rifampicin and ethambutol, 5.56% for streptomycin, 2.78% for dipasic and 12.68% for levofloxacin.(2)The mean intralesional rifampicin concentration of 23 of 36 patients was 0.78±0.45ug/ml, which was mostly at subtherapeutic level,6 of which was 4.60±0.76 ug/ml(exceeding 10 fold MIC),the other seven were undetectable. The mean intralesional isoniazid concentration of 34 of 36 patients was 0.543±0.132(0.332-0.704) ug/ml, which was achieving 10 fold MIC.(3)Mutations in the RRDR of the rpoB gene were identified in 17 (94.4%) of the 18 rifampin resistant isolates. 1 Rifs strain exhibited no mutations in this region. The most prevalent mutation sites were in codons 531 (55.56%), 526 (11.11 %), 516 (16.67 %) and 513 (11.11%).High level rifampin resistant strains (250μg/ ml rifampin resistant) had higher mutation frequency at 531-Ser than low level rifampin resistant strains (50μg/ ml rifampin resistant) ( P < 0.05) .Isolates with mutations of rpoB were highly resistant to rifampicin,11 of which with MICs exceeding 256 μg/ml(not determined).The MICs for the remaining 7 resistant isolates were between 32 and 256μg/ml. Especially for low level rifampicin resistant strains, M. tuberculosis growth was inhibited under certain higher concentration. (4) The freeze-dried iliac crest bone allograft was ivory white, clean and porous, presenting sandwich structure with tricortical and cancellous bone. The rifampicin crystal was cemented in the pore by fibrin. Its mean density ,porosity and pore diameter was 0.39±0.03 g/ml, 61.23±9.12% , 293±23.2 um,respectively. (5) The No.3,5,8 fibrin discs showed satisfying microstructure and sustained release property: the rifampicin crystals were surrounded into the three-dimensional fibrin network structures. The release curves were relatively smooth.They could maintain microbial killing effect of rifampicin for at least 49 days. (6) The three group implantable fibrin polymer/bone allograft composite systems for rifampicin were cardinal red, the pore of bone allogrft were filled with fibrin and rifampicin. The rifampicin crystals were surrounded into the three-dimensional fibrin network structures.(7)All the NO.3,5,8 implantable systems for rifampicin could maintain microbial killing effect of rifampicin for at least 42 days,49 day and 49 days,respectively. But release curve of NO.5 was smoother, sudden release could be observed in NO.8.All had effective antituberculous effect in vitro.(8)The rifampicin obviously inhibited MSCs proliferation at concentrations of 32ug/ml; In vitro osteogenesis, rifampicin decreased the ALP,COL 1A1 and OPNexpression in a dose-dependent manner, obviously when the concentration of rifampicin exceeding 16ug/ml. (9) In vivo sustained release in osteogenic microenvironment,the concentration of rifampicin showed time-based decrease, but 13.55ug/ml could be achived in 42 days; Bone repair process was not affected in long term followup.Conclusion :(1)The procedures were accurate and low-cost for first and second line antituberculous drugs susceptibility testing,and could be recommended for refining the optimal individual chemotherapy regimen in spinal tuberculosis. The drug resistance epidemiology of osteoarticular tuberculosis was parallel to that of pulmonary tuberculosis, rifampicin resistance was low.(2) The intralesional rifampicin concentration was mostly at subtherapeutic level; (3) At a high concentration, rifampicin inhibits proliferation and differentiation of human bone marrow mesenchymal stem cells and could compromise the bone-healing process in vitro. (4) The implantable systems with NO. 5 fibrin composition was optimal for rifampicin release and maintain microbial killing effect. (5)The implantable fibrin polymer/bone allograft composite systems for rifampicin could maintain microbial killing effect for at least 42 days in goat model, and result in solid lumbar interbody bone union.