| Research backgroundChronic osteomyelitis is mainly caused by Staphylococcus aureus, in particular, methicillin-resistant S. aureus (MRSA), and it is a common disease in the department of orthopaedic surgery. Despite the progress and development of surgical techniques and antibiotic drugs, it is difficult to treat chronic osteomyelitis for orthopaedic surgeons in clinical work, which brought great psychological and economic burden to patients with. Due to the serious destruction of blood supply in local lesions, by oral or intravenous antibiotics for a long time, blood is difficult to carry antibiotic to the local infection, shorting of effective bactericidal concentration, the infection remaining, the recurrence rate bing higher, and increaseing the side effects of damage the body’s vital organs and the development of drug resistance. The different degree of bone defect often leaves after debridement of chronic osteomyelitis.If the bone defect is big, which will influence the mechanical strength and stability of the backbone, and bone graft is needed to rebiuld defect and promote bone healing. Despite thorough debridement, it still remains a certain number of bacteria. Residual bacteria multiply, when implants are filled, which often leads to implant failure. Therefore, bone filling material as the treatment of chronic osteomyelitis can eradicate local residual bacteria, and can support and reconstruct bone defects, which is a key to cure chronic osteomyelitis. In the 1970s and 1980s, Buchholz, Wahlig, Klemm et al successively reported that treatment of osteomyelitis through the application of gentamicin combined with poly-methylmethacrylate (PMMA) bone cement at the bone infection site resulted in significant improvements. Local application of antibiotics greatly aroused people’s concern. Local drug controlled-release system directly release antibiotics to the lesion site via the sustaining and stable way, to reach high in local drug concentration where does not need blood transportation of antibiotics, which effectively restrains and kills bacteria, and improve osteomyelitis of the cure rate and reduce the recurrence rate. At the same time, the low drug concentration in blood circulation, avoid system drug side effects of vital organs.However, PMMA bone cement does not degrade and thus has many disadvantages:(1) it releases a large amount of heat in the polymerisation process, which leads to some antibiotic failures; (2) it lacks the nano-microporous structure of the natural bone, so antibiotics are not completely released, which partially neutralises the antibiotic effect; (3) it cannot be degraded after implantation in the body, which requires a secondary operation to take out PMMA and reconstruct the bone defects; (4) it can induce secondary infection.The development of tissue bioengineering and forging technologies has resulted in many releasing materials that have good biocompatibility, are biodegradable, and are capable of osteogenesis to replace the PMMA bone cement. One of the most studied materials is hydroxyapatite (HA) and its derivative. Because HA constitutes the main component of bone, it has good biocompatibility and biological activity and is widely used for bone tissue repairing and as a replacement material.In the study, bone-like hydroxyapatite/poly amino acid (BHA/PAA), developed by Chongqing Medical University and Sichuan University National Nano-materials Company, was used as microspheres carrier for carrying antibiotics, combined with polylactic acid-glycolic acid copolymer (PLGA, PLA:PGA50:50) coating rifapintine (RIP) microspheres, which the treatment of chronic osteomyelitis.BHA is carbonated nano-hydroxyapatite (n-HA), and it is more similar to normal bone structure. Its nano-crystal is small, with good biodegradation performance, biocompatibility and induction osteogenesis and its biological activity is stronger. In addition, its nanocrystal is smaller than n-HA, so its apparent area increases, and its adsorption capacity increase for drug and drug microspheres. PAA is similar to collagen molecular structure, with good biocompatibility and biodegradable. BHA’s good biocompatibility, induction osteogenesis and bone conduction activity combines with PAA’s good mechanical performance and processability to create a potential BHA/PAA bone repair material.In the study, BHA/PAA was used as the carrier of RPM. In vitro and in vivo, the drug releasing ability, antibacterial ability cytotoxicity, osteogenesis induction ability and biocompatibility of RPT-loaded BHA/PAA were conducted and assessed, which provides a new approach for the clinical treatment of chronic osteomyelitis.ObjectiveIn vivo, to study drug release, the inhibit bacterial and antibacterial activity, cytotoxicity, and the effect on osteobalst-like MG63 cells proliferation and osteogenesis of BHA/PAA and RMP-BHA/PAA composite bone materials. In vivo, animal models of chronic osteomyelitis were established at rabbit tibia. On this basis of animal models the effect of RMP-BHA/PAA bone filling materials was studied. Through assessment of antibacterial ability, inducing osteogenesis and bone defect recovery, impact of vital organs.Through in vivo and in vitro, we hope to find a new slow-release composite bone material carried antibiotics which has anti-infection ability and is capable of inducing osteogenesis, and further study of chronic osteomyelitis on the scientific research provides theoretical basis and new method.Methods:this experiment is made up of four parts1. The determination of composite artificial bone material of BHA/PAA loaded with RPM in vitro drug slow-release, the biocompatibility and antibacterial abilityIn vitro, the bacteriostatic and antibacterial ability of saphylococcus aureus and E.coli was detected via bacteriostatic ring test and scanning electron microscope (SME) for the composite artificial bone material of BHA/PAA loaded with RPM load; cell biocompatibility and safety of the drug-loaded composite bone was determined by MTT method and acute hemolysis test.2. The determination of composite artificial bone material of BHA/PAA loaded with RPM in vitro osteogenesis abilityBHA/PAA as comparison, by inverted microscope, electron microscope scanning, MTT proliferation experiment, determination of calcium and alkaline phosphatase (ALP), alizarin red staining method, it was detected for osteobalst-like MG63 cell on morphology, adhesion, proliferation, osteogenesis and physical and chemical properties, and it was assessed that the composite artificial bone material of BHA/PAA loaded with RPM fluenced on osteobalst-like MG63 cell growth and osteogenesis ability.3. The establishment and assessment of experimental animal model of chronic osteomyelitisRabbit right proximal tibia intramedullary was injected 5% morrhuate sodium and Staphylococcus aureus suspension, at the same time, and half animal medullary cavity filled in the foreign body cotton ball to induce and establish chronic osteomyelitis. The relationship between the severity of chronic osteomyelitis-induced and the foreign body cotton ball was evaluated, to establish animal model of chronic osteomyelitis provides an ideal suitable method.4. The curative effect of chronic osteomyelitis of rabbit tibia and the toxicity studies of important organs for composite artificial bone material of BHA/PAA loaded with RPMThe animals of chronic osteomyelitis which were successfully established, after through thorough debridement, were implanted with RPM-loaded RMP/PAA composite material of artificial bone, without RPM-loaded BHA/PAA materials and blank control group. Via the gross observation, imaging and histologic and bacteriologic method, antibacterial effect of materials was detected; Through imaging, histology, scanning electron microscopy (SEM), calcein fluorescent marker, the ability of materials osteogenesis; Through the histological examination heart, liver, lung, and kidney structure change, it was evaluated that the composite artificial bone material had or had not toxic side effects to the important organs of animals.Results1. The determination of composite artificial bone material of BHA/PAA loaded with RPM in vitro drug slow-release, the biocompatibility and antibacterial abilityIn vitro BHA of RPM/PAA composite artificial bone material for saphylococcus aureus and escherichia coli had obvious bacteriostatic and antibacterial effect, good slow release effect. Especially, for saphylococcus aureus, its antibacterial ability was stronger and longer; In vitro, the composite artificial bone had not toxic side effects to L929 cells and red blood cell, and had good biocompatibility.2. The determination of composite artificial bone material of BHA/PAA loaded with RPM in vitro osteogenesis abilityOsteobalst-like MG63 cell was cultivated with BHA/PAA composite artificial bone material with or without RPM. In the inverted phase contrast microscope, there were a lot of MG63 cell around the material adhesion, growth, and the cell count increasing with the extension of culture time; After 7 days, by scanning electron microscope, osteobalst-like MG63 cell adhesion, growth was in good condition on material, and a lot of calcium nodule formed on material; MTT cell proliferation experiment results showed that the cell count of each group increased with the extension of incubation time, through each of material groups compassion with control group,MTT value was difference(P<0.05);MG63 cell was cultivated in different materials and in blank control group, and the determination results of ALP activity and cell calcium concentration indicated:both increased with the extension of incubation time, the results of statistical analysis was difference (P<0.05) in each of material groups compassion with control group.3. The establishment and assessment of experimental animal model of chronic osteomyelitisGroup A, the experimental group,injection of 0.2 ml 3 × 10 CFU/ml bacteria suspension;group B, the control group, injection of 0.2 ml 3×108 CFU/ml bacteria suspension, and fill in the diameter of 0.4 cm eyewinker tampons;Group C, blank group,0.2 ml saline injection, only. At 4 weeks after modeling, imaging examination, in addition to the blank group rabbit proximal tibia structure form normal, the other two groups at different levels had shown that the trabecular bone was thinned and sparse, and bone density reduced and bone dissolved, with dead bone formation.Through HE stainning organization pathology inspection, it was obviously visible the inflammatory cell infiltration, bone marrow cavity cell necrosis, interstitial hemorrhage from the periosteum to the bone marrow cavity at different levels.By gross observation and radiological detection standards for saphylococcus aureus osteomyelitis severity classification of animal models: the blank group belongs to the level 0 grade, control group 2-3 grade, the experimental group 3-4 grade.4. The curative effect of chronic osteomyelitis of rabbit tibia and the toxicity studies of important organs for composite artificial bone material of BHA/PAA loaded with RPM4.1 The curative effect of chronic osteomyelitis of rabbit tibia with composite artificial bone material of RFM-loaded BHA/PAAantibacterial ability:after debridement surgery and treatment for 12 weeks, by the gross observation, the proximal tibial form in group A restored to normal, no bone damage, osteonecrosis and purulent secretion; The proximal tibia shape in B, C, the two groups deformated and broadened, and bone destruction was more serious than before treatment, to see a large number of purulent secretion. To pick out lesions bone tissue after debridement and postoperative treatment for 4 weeks, through bone tissue bacterial culture, the bacteria count per gram bone specimens, statistical analysis results showed that the bacteria count in group A was significantly less than group B, C (P<0.01),while the bacteria count in group B,C was no significant difference (P> 0.05).Debridement and postoperative treatment for 12 weeks, HE staining histopathological examination showed that no obvious inflammation and inflammatoy cells were not seen in group A, while inflammatoy cells were obviously in group B,C.Osteogenesis ability:debridement and postoperative treatment for 12 weeks, via the examination of gross observation, histopathology, immunohistochemistry, scanning electron microscopy (SEM) and fluorescent tags, it was evaluated for the effect of RPM-loaded or no RPM-loaded BHA/PAA composite artificial bone inducing osteogenesis. Via Gross observation, the proximal tibia shape in group A restored to normal structure;The material was completely covered by the new fofmation bone,was not seen and connected closely with the normal bone, and bone defect already restored. The proximal tibia shape in groups B, C deformed and broadened, and bone destructed, with different degree of hyperplasia, sclerosis, osteonecrosis in bone defect edge; the material in Group B was visible and did not connect closely with the normal bone, without new bone formation aroud the material. Thtough HE staining pathology examination, it was obviously visible cartilage cells, cartilage matrix and new bone trabecular formation in group A, with the most material degradation and absorption; It have not new bone formation in groups B and C, while the material was obviously visible in group B. Via fluorescent markers, the green fluorescence was obviously visible in the materials and around materials in group A; While the green fluorescent was small, scattered and fuzzy. The immune histological examination of I collagen antibody marking showed that a large of collagen fiber formed in group A, like woven bone, surrounding materials, which showed that osteogenetic activity was obvious, without collagen fiber or a small amount of collagen fiber formation in group B and C. Via scanning electron microscopy (SEM), a large of the trabecular bone crystals formed and presented the radial growth within the material, and the material degradation and absorption was obvious in group A. The trabecular bone formation was not obvious, only a small amount scattered within the material in Group B, and the material degradation and absorption was obvious.4.2 The studies of toxic side effect of composite artificial bone material of B HA/PA A loaded with RPMThe result of blood biochemical tests showed:the level of ALT and AST, Cr, BUN was no significant difference in experimental group and blank control group, via comparison between each other at each time point within the same level(P>0.05). Via HE staining pathology examination, the results of heart, liver, lung and kidney structure was normal and no difference in the experimental group, control group.Conclusion1. In vitro, the composite artificial bone material of RPM-loaded BHA/PAA had obvious bacteriostatic and antibacterial effect to Saphylococcus aureus and E.coli, especially for Saphylococcus aureus. Its antibacterial ability increased with the increasing of RIP dose. In vitro, via the toxicity experiment, the composite artificial bone material had not toxicity, did not produce mechanical damage to L929 cells and red blood cells, and good biocompatibility.2. In vitro, the composite artificial bone material of RPM-loaded BHA/PAA did not inhibit the growth of osteobalst-like MG63 cell,and promoted MG63 cell adhesion, growth, proliferation, maturation and the formation of calcium nodules; it did not influenced on the calcium content and alkaline phosphatase activity of osteobalst-like MG63 cell, and promoted their secretion, with good biocompatibility MG63 cells.3. The composite artificial bone material of RPM-loaded BHA/PAA was a good antibacterial and osteogenesis induced bone slow-release materials; in a short period, it was not obvious side effects of the important organs of rabbit; in chronic osteomyelitis after thorough debridement, it could be filled in and supported bone defect, ultimately curing osteomyelitis and restoration bone defect. The drug slow-release material is expected to be in the scientific research and clinical work to become an ideal replacement material of bone filling of used for the treatment of chronic osteomyelitis. |
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