| Objective To study the sustained release of isoniazid(INH)and rifampicin(RFP)-containing hydroxyapatite calcium sulfate nano bone in the rabbit spinal tuberculosis model.Hard tissue sections,azure methylene blue staining(AM staining)and transmission electron microscopy(TEM)were used to observe the distribution of the nano bone in the local vertebral body bone of the focus and its correlation with the local anti-tuberculosis treatment effect.Methods 85 New Zealand white rabbits,male and female unlimited,weighing 2.5 ± 0.3KG,were fed with standard cage feed.70 New Zealand white rabbits were randomly selected to construct L4-5 vertebral tuberculosis model with standard strain of Mycobacterium tuberculosis H37 Rv,After 2 weeks,the experimental rabbits with bone destruction,dead bone and cavity formation in diseased vertebrae were selected as the successful model by X-ray and CT examination.30 model rabbits were randomly selected as the experimental group and the control group;10 normal rabbits were randomly selected as the blank control group.After focal debridement,the experimental group(nano drug group)was the model rabbits implanted with isoniazid and rifampin-containing hydroxyapatite calcium sulfate nano bone in the bone defect;the control group(non-nano drug group)and the blank control group were implanted with non-drug contained hydroxyapatite calcium sulfate nano bone.At 24 h,72 h,1 w,2 w,4 w,6 w,8 w,10 w and 12 w after the operation,the experimental rabbits in the nanodrug group and the non-nanodrug group were killed,choose 3 each time,high performance liquid chromatography(HPLC)was used to detect the concentration of drugs in the vertebral body,paravertebral tissue and inferior vena cava blood of the diseased vertebra of rabbits after implantation of nano bone.At the 2w,4w,8w and 12 w after operation,two rabbits in the blank control group were randomly killed at each time point,choose the vertebrae at the edge of the graft,The vertebrae of the three groups of rabbits were sectioned,AM stained and TEM to observe the distribution and morphological characteristics of sustained release nanoparticles in local lesions.Results The experimental rabbits in the nano drug group and the non-nano drug group were successfully constructed with 30 rabbits each group,the experimental rabbits in the blank control group were 10,and the rest experimental rabbits were standby.Two rabbits in the non-nano drug group died in the process of construction of spinal tuberculosis model,one rabbit in the blank control group and one rabbit in the nano drug group died during bone grafting,the reason maybe fat embolism or anaesthetic overdose;one rabbit in non-nano drug group died 9 weeks after bone grafting due to not drinking and eating.all the dead rabbits are replenish in time.Paraplegia occurred in the left lower limb of two rabbits in the non-nano drug group at 4 weeks after operation,a sensation of subcutaneous fluctuation appeared in the lumbar region of one experimental rabbit at 8 weeks after the operation.In the remaining experimental rabbits,the postoperative activity,spirit and feeding were good,and the wounds healed normally.Drug release experiment of nano bone: The drug concentration at each time point of INH in the vertebral bone of model rabbit lesions was 75.66±1.95μg/g、48.46±2.34μg/g、30.69±2.74μg/g、20.34±1.63μg/g、9.36±1.17μg/g、5.19±1.40μg/g、2.73±1.19μg/g,no drug concentration was detected at each time point after the 10 th week.The concentration of paravertebral muscle at each time point was 39.51±2.25μg/g、23.65±1.55μg/g、16.39±2.10μg/g、10.38±1.44μg/g、3.66±0.79μg/g、1.89±0.73μg/g、0.26± 0.10μg/g,similarly,no drug concentration was detected at each time point after the 10 th week.The concentration of INH in inferior vena cava was 0.63±0.05μg/g、0.28±0.03μg/g、0.12±0.01μg/g,no drug concentration was detected after 2 weeks.The concentration of INH in vertebrae and paraspinal muscles was similar at the same time(P>0.05);The INH concentration in the inferior vena cava was significantly lower than that in the former(P < 0.05).The drug concentration at each time point of RFP in the vertebral bone of model rabbit lesions was 10.85±2.45μg/g、22.47±1.94μg/g、38.32±1.73μg/g、24.22±1.45μg/g、17.85±1.50μg/g、9.81±1.30μg/g、6.35±1.30μg/g、5.11±0.53μg/g、1.32±0.33μg/g。The concentration of paravertebral muscle at each time point was 5.39 ±1.50μg/g、20.66 ±1.29μg/g、48.72 ±2.24μg/g、32.27 ±1.63μg/g、15.58 ±1.88μg/g、8.69 ±0.79μg/g、3.43 ± 0.39μg/g,no drug concentration was detected at each time point after the 10 th week.The concentration of INH in inferior vena cava was 0.75±0.09μg/g、1.38±0.23μg/g、0.59±0.04μg/g、0.15±0.03μg/g,no drug concentration was detected after 4 weeks.The concentration of RFP in vertebrae and paraspinal muscles was similar at the same time(P>0.05);The concentration in the inferior vena cava was significantly lower than that in the former(P < 0.05).Animal pictures and AM staining pictures: In the nano drug group,bone damage was serious,there are small vacuol-like changes in bone and more inflammatory cells filled;In the non-nano drug group,dead bone flowed out of the focus area of tuberculosis,the fusion interface of nano bone graft formed a dense sclerotic wall,the normal bone structure in the dense sclerotic wall was changed,and the trabecula was thickened,the gap disappears and forms a dense stratified plate structure,and the Haversian system disappears;In the blank control group,the injury of bone tissue was slight,and a few inflammatory cells were scattered in bone tissue.TEM picture:nano bone implanted into tuberculosis focus 2 weeks: Hydroxyapatite calcium sulfate nano bone particles entered the injured bone cells by endocytosis and were surrounded by lysosomes.In the experimental group,the drug loaded nano bone particles were obviously decomposed at 4 weeks,most of the nano bone particles have been decomposed at 8 weeks,and disappeared completely at 12 weeks,the injured bone cells repaired well and absorbed nano particles.The morphology and structure of bone cells returned to normal.In the control group,the degradation of nanoparticles was slow in 4 weeks and stagnated in 8 weeks,the nucleus of bone cells was pyknosis after 12 weeks,osteocyte apoptosis and nano particles remained in the cytoplasm of bone cells.In the blank control group,no apoptosis was found in the osteoblasts at all time points after implantation of non-drug loaded hydroxyapatite calcium sulfate nano bone,the nano particles were decomposed,and the morphology and structure of bone cells were normal.Conclusion Isoniazid and rifampicin-containing hydroxyapatite-calcium sulfate nano bone can sustained and long-term release anti-tuberculosis drugs in the focus of the rabbit spinal tuberculosis model,drug concentration and duration were higher than those in blood.The biocompatibility of the nanobone was confirmed by AM staining and TEM,The sustained-release nanoparticles pass through the dense sclerotic bone and are absorbed by the cytosol of bone cells,playing the role of treatment and repair at the cell level. |
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