| BackgroundAlthough in recent years the incidence of tuberculosis has declined worldwide,extrapulmonary tuberculosis incidence rate rises gradually.Bone and joint tuberculosis is the most important type of extrapulmonary tuberculosis,which accounted for about 10%-11%of it.Currently,the repair of bone defects and maintaining effective concentration of anti-TB drugs at concentrations lesions remain the two major themes of bone and joint tuberculosis treatment.Because of special hardened wall structure,the traditional oral anti-TB drugs is difficult to penetrate the lesion and maintain an effective drug concentration.A large number of long-term oral anti-TB drugs easily lead to serious systemic side effects,it has also become a major problem faced by traditional therapies required.As a new mode of administration,sustain-release drug delivery system can maintained long-term and stable drug release,which have broad application prospects in the field of treatment of bone and joint tuberculosis as well as other chronic infectious diseases.In recent years,extensive and in-depth studies of polylactic acid(PLA)and with glycolic acid copolymer(PLGA)at home and abroad have prove that they can effectively extend the duration of action of drugs,improve orientation and reduce the toxic side effects,which may be an ideal drug delivery system.Bone and joint tuberculosis patients with surgical treatment often leave residual cavity defect after debridement,implantation of autologous bone or bone substitute materials is helpful for rebuilding the local mechanical structure and promoting new bone formation.Although autogenous bone is most commonly used in clinical as graft materials,but the damage caused by secondary surgery,intractable pain,the inability of pre-combination with anti-tuberculosis and other reasons have hindered the autologous bone from becoming the best implant materials for the treatment of bone and joint tuberculosis.While conventional calcium phosphate materials can be completely degraded in the body and can be pre-combined with long-term anti-TB drugs,but the lack of bone conductibility limits its wide use especially in the area with poor blood supply.Studies have shown that absorbable materials have the advantage of gradual degradation compared with conventional materials,which can not only supply a certain degree of mechanical strength of the defect area earlier,but also ensure it will not disturb the growth of new tissue later.Genex(?)is a kind of Biphasic calcium composite materials with a negative surface charge which is a new-type osteogenic implantation materials consisting of a porous ?-tricalcium phosphate phase and a calcium sulfate phase produced by Bioson Corporation.Compared with the traditional calcium phosphate materials,the unique negative charge on its surface can have an directional effect of dissimilar charge adsorption.This property is conducive to the accumulation and adsorption of various molecules,bone morphogenetic protein-related factors such as bone morphogenetic protein and other substancesin in plasma and body fluid,which is benefit for inducting bone formation.Because of its non-toxic,easy shaping,ability of inducing bone formation in vivo as well as the advantages of completely degradation,Genex(?)has been widely used in clinical bone repair surgery.Taking its capacity of pre-combining with anti-TB drug delivery system into account,Genex(?)can become an ideal implant materialsfor the treatment of bone and joint tuberculosis.Purpose1.To fabricate an implantable composite materials with sustained-release effect,certain degradation with time to adapt osteogenesis,biocompatible and excellent performance of osteogenesis.2.To determine the optimum composition ratio of composite materials through the detection of the maximum compressive strength and compressive force.To observe the physical properties of composite materials by scanning electron microscopy.3.To verify the sustained release properties,biocompatibility and osteogenic properties of the composite materials through in vitro and in vivo experiments.Materials and Methods1.The RIF/PLGA microspheres were prepared by a modified oil-in-water single-emulsion solvent evaporation process,the anti-TB composite support materials was fabricated by the combination of RIF/PLGA microspheres and Biphasic calcium composite materials.2.To manufacture the composite support materials with the blending ratio of 0%,2.5%,5%,7.5%and 10%and then shaped it into a cylinder with diameter of 3 mm and height of 6 mm.After the composite materials is solidified,measuring the maximum compressive strength and compressive force by an electronic universal testing machine to determine the optimum mixing ratio of composite materials;To observing the physical properties,particle size and distribution through scanning electron microscopy.3.To calculate encapsulation efficiency and drug loading of microspheres by UV spectrophotometer through direct determination.Putting pure rifampicin,microspheres and composite scaffolds into dialysis bags and placed on a horizontal shaking bed.Measured the concentration of drug of supernatant at 1,3,7,14,21 and 28 days to evaluate the drug release properties of microspheres and composite materials in vitro.4.Verifying the drug release properties of composite materials in vivo through implanting composite materials in muscle pouches of adult male SD rat.Animals were sacrificed at 1,3,7,14,21 and 28 days post-operation,concentration of drugs in muscle and plasma were measured for evaluating the performance of sustain-release.5.36 adult male SD rats were randomly divided into positive control group,experimental group and negative control group.Rifampicin and composite scaffolds were implanted into the quadriceps muscle pouch of the positive control group and experimental group respectively while the negative control group implanted nothing.Animals were sacrificed after 7 and 28 days,the ratio of liver weight and body weight among 3 groups were calculated,which was used for evaluating the biocompatibility of anti-tuberculosis scaffold together with liver pathological section.Made a circular bone defect with a diameter of 5mm at each side of skull,blank scaffolds,composite scaffold materials and the negative control treatment were perform respectively.X-ray detection,micro-CT scanning and pathological examination of the skull were applied after 6 weeks to evaluate the osteogenic properties of composite scaffold materials.ResultThe compressive strength and the maximum compression force of pure Genex(?)were(11.50±2.88)Mpa and(0.08±0.02)kN respectively.The compressive strength and the maximum compression force would not change statistically until the mixing ratio was more than 7.5%.When the mixing ratio was up to 10%,the compressive strength and the maximum compression force of composite scaffold material were(6.67±0.82)Mpa and(0.05±0.01)kN respectively,which were decreased significantly compared to that of pure biphase bone cement(P<0.05).Taking compressive strength and the maximum compression force into account,therefore to determine the optimum mixing ratio of microspheres:composite scaffolds materials=7.5:100(W:W).Scanning electron microscope showed that microspheres has an uniformly smooth surface spherical with an average diameter of about 20 ?m.The microspheres were uniformly distributed,no significant overlap was observed.Interlocking micro-structure and internal materials uniformly distributed pores of blank scaffold were visible through scan electron microscope.The only difference between blank biphasic bone cement and composite scaffolds was the microspheres combined with composite scaffolds materials.After the adoption of the direct method was repeated 3 times,the average encapsulation efficiency and drug loading rates of microspheres were(56.05±5.33)%and(29.80±2.88)%.In vitro-release assay,measured by UV spectrophotometry,cumulative drug release rate of pure rifampicin is reached(99.23±0.24)%on the third day,while the microspheres group and composite materials group were(34.59±2.17)%and(34.59±2.17)%severally.The cumulative drug release rate of the microspheres group and composite materials group at the first 14 days was(61.21 ±2.26)%and(53.55±2.56)%severally,which reached(84.27±2.65)%and(69.88±3.03)%respectively after 28 days.3.Local muscle drug concentration,measured by high performance liquid chromatography,after 1 day,3 days,7 days,14 days,21 days and 28days were(54.83±1.11)?g/g,(73.94±6.46)?g/g,(30.94±1.50)?g/g,(18.45±0.71)?g/g,(17.87±1.24)?g/g and(14.96±0.33)?g/g respectively;systemic blood concentrations were(5.49±0.37)?g/g,(4.75±0.23)?g/g,(3.35±0.55)?g/g,(1.26±0.09)?g/g,(1.04±0.05)?g/g and(0.90±0.03)?g/g respectively.4.The ratio of liver weight and body weight of positive control group,experimental group and negative control group in 7 days were(3.985±0.26)%,(3.985±0.26)%and(3.02±0.13)%severally,while that reached(2.92±0.27)%,(2.92±0.27)%and(2.95±0.11)%severally at 28days.The ratio of liver weight and body weight both at 7days and 28days post-operation were significantly higher than those in the experimental group and negative control group(P<0.05),nothing significantly difference(P>0.05)were observed between the experimental and control groups.The result of liver pathological section was similar to that of hepatic index:for the experimental group and negative control group hepatic lobule is clearly visible,liver cells arranged in neat,no periportal bile duct hyperplasia,and no inflammatory cell infiltration.Liver pathological sections of positive control group shows a large number of inflammatory edema of liver cells and mild periportal bile duct hyperplasia,the situation was slightly better although liver damage was still very obvious compare to the other two groups.The X-ray test results 1 day post-operation showed that the density of the composite scaffold group and pure biphasic bone cement group were significantly higher than the normal bone tissue at the beginning,the density of the defect area was close to the normal density of bone tissue six weeks after the implantation.The density of negative control group of the defect area did not change significantly after 6 weeks,which was still significantly lower than the surrounding bone tissue.Micro-CT three-dimensional imaging showed that hardly any regenerated new bone except in the margin on the the defect region of negative control group at 6 weeks after surgery.As for the other 2 groups,a lot of new bone formation could be seen and the defect area was almost filled with regenerated new bone and residual biodegradable materials.Micro-CT analysis showed that bone volume and bone volume fraction of the negative control group was significantly lower than that of the other 2 groups 6 weeks post-operation(P<0.05),nothing significant difference has been found between the experimental and control groups(P>0.05).Pathological section of the negative control group showed that only a small amount of new bone surrounded the defects area after six weeks,no new bone appears on the center of skull defect and the defect area was linked by a layer of fibrous connective tissue.Histology performance between the experimental and control groups were similar:a lot of new bone formation were visible in the area surrounding the defect,abundant capillary and materials with complete degradation could in the regenerate new bone.Obvious subperiosteal osteogenesis were shown in the center of defect region.Conclusion1.Successfully fabricated a new kind of composite anti-TB scaffold materials consist of calcium sulfate/?-tricalcium phosphate composite bone cement with negatively charged and RIF/PLGA microspheres.2.Successfully demonstrated the long-term release property of microspheres and composite materials as well as the biocompatibility of composite materials through in vitro and in vivo experiment.3.After six weeks with the experimental observations,we successfully demonstrated that combining with sustained release microspheres hadn't affected the osteogenic property of calcium sulfate/?-tricalcium phosphate composite bone cement with negatively charged,which still exhibits excellent osteogenesis performance.4.We have build a new type of composite anti-TB scaffold materials with multiple satisfactory properties after verifying,which might supply an effective alternative for bone and joint TB patients. |
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