载万古霉素介孔羟基磷灰石/壳聚糖可降解复合材料的制备及在骨科应用基础研究
摘要
介孔材料是近年来化学和材料领域研究的热点，以羟基磷灰石（HA）为基质合成介孔材料的研究受到关注。在应用于骨科领域中，介孔HA既具有良好生物活性和骨诱导性，又具有更宽大的孔道空间和吸附性能,可以增加对药物的负载量和对药物释放的控制，同时增大的孔道可以更加利于骨的诱导和骨的长入，为将其用于载药和成骨方面提供了理论依据。单纯的HA植入存在一定的缺陷:力学性能差，脆性大，材料的抗弯强度低，在生理环境中的抗疲劳与破坏强度不高，为一种典型的脆性材料，难以制成与骨组织相匹配的形态，因而无法满足临床上各式各样骨缺损植骨的需要。研究者利用壳聚糖与HA复合来改善材料的相关性能，壳聚糖（CS）具有良好的生物活性和生物相容性，并且本身具有抗菌和提高成骨细胞成活率及活性，促进成骨细胞的分化，还具有促进成骨和基质矿化的作用。CS也具有良好的吸附性，可以作为药物的载体，控制药物的释放。将HA与CS制成复合材料后克服了两种单组分材料各自的不足，集二者的优点于一身，既具有良好的生物活性，较高的机械强度，一定的韧度与一定的溶解速率，又克服了羟基磷灰石的脆性，极低的降解速率和壳聚糖的极低强度和降解过快，是一种不但具有较理想的传导成骨效应，而且具有较好的力学性能的骨替代生物材料。治疗骨髓炎伴骨缺损或骨不连一直是困扰骨科临床工作的难点。本论文旨在将化学领域、生物材料领域、医学领域相互有机交叉，合成一种新的介孔HA/壳聚糖复合生物材料，验证其具有良好的骨诱导性和可降解性，用其载药能够控制药物的释放，既可以填充骨缺损，诱导骨长入，又可以治疗和预防感染，为其下一步在临床应用提供实验基础。
创新点：
⑴将HA和CS这两种材料复合形成复合材料可以充分发挥二者的优点。既具有良好的生物活性，较高的机械强度，一定的韧度与一定的溶解速率，又克服了羟基磷灰石的脆性，极低的降解速率和壳聚糖的极低强度和降解过快。
⑵将HA制成介孔材料，介孔HA既具有HA的良好生物活性和骨诱导性，又比HA具有更宽大的孔道空间和吸附性能,可以增加对药物的负载量和对药物释放的控制，同时增加的孔道可以更加利于骨的诱导和骨长入。
⑶将万古霉素载入介孔HA，形成了局部药物缓释系统（DDS）。介孔HA增加了对万古霉素的负载量，同时对万古霉素的释放起到缓释的作用，在抗感染的同时还能促进骨的修复和长入，比以往采用抗生素骨水泥治疗和预防假体周围感染具有无比的优越性。
The treatment of acute and chronic orthopaedic infection is difficult,time consuming and expensive. Despite the availability of a myriad ofantibiotics and marked advances in surgical treatment, the long-termrecurrence rateremains alarming at a rate of20%to30%. Absence ofantibiotics labelled exclusively for osteomyelitic conditions leave thechoice of therapy to the preferences and experience of the attendingphysicians. Very high concentrations of antibiotics are required at thetarget site but could hardly be attained by conventional routes of drugadministration without provoking serious side effects. Hence,conventional therapy hampers clinicians in striking a balance betweenefficacy and safety in deciding an optimal treatment strategy forosteomyelitic patients. Moreover, patients bear unfulfilled therapeuticoutcomes, development of adverse drug effects and huge healthcare bills. Local drug delivery systems placed in the affected area, release druglocally for prolonged periods and at concentrations generally higher thanthose achieved by conventional drug delivery strategies.So far, a largenumber of systems have been employed as various drug delivery systems,such as biodegradable polymers hydroxyapatite,calcium phosphatecement xerogels, hydrogels, and mesoporous silica. Recently mesoporousmaterials is chemistry and material field research hotspot, and they havegained enhanced interest with particular attention as drug storage andrelease carrier due to their large unique surface area and hole volume,uniform and adjustable pore canal, easily modified surface features forsite specific delivery. Due to these advantages, mesoporous materialsshow a broad application prospect in chemitry, biological medicine,chemical catalysis, nanometer materials and so on.The drug deliverysystems using mesoporous materials as drug carriers have been reported.
Due to the bioactivity, biocompatibility,stability, nontoxic properties,hydroxyapatite with porous surface structure and–OH groups may serveas an ideal candidate drug carrier for the delivery of a variety ofpharmaceutical molecules, and especially mesoporous HA have broader pore canal space and adsorption performance than nano HA. Thus, thedesign and development of mesoporous HA might be able to reach thisgoal. Chitosan as a high molecular bioactive polymer has satisfactorybiocompatibility and biodegradation, and itself can improve survival rateof the osteoblast and promote the osteoblast differentiation and Matrixmineralization.Its surface hydrophilicity is beneficial to celldifferentiation and adhesion.So HA and CS two kinds of materials wereformed composite materials and may fully exrt their advantage. HA wasscattered among chitosan matrix and formed composite obtained goodosteoinduction and osteoconduction. Moreover, biological space ofdissolving chitosan is helpful to tissue reconstruction and then isconductive to drug releasing.
Mesoporous hydroxyapatite/chitosan composite have beensuccessfully prepared and synthesized using a freeze-drying method, andthen characterized by Scanning Electron Microscope, X-ray diffractionand IR. Results showed that mesoporous HA/CS presented higher surfacearea and porosity. The effect of mesoporous HA/CS on early adhesion,proliferation and differentiation of osteoblast cells in vitro was measured.. MTT cytotoxicity test and cell adhesion test show that the materials havegood biocompatibility and make cell viability and proliferation. In vitrotesting using osteoblast-like cells showed that on the surfaces of cells areable to adhere, proliferate, and migrate through the pores. These cellsmaintained similar expression levels of osteoblastic-associated markersnamely collagen type I (COL-1), alkaline phosphatase(ALP). Histologicanalysis and radiological analysis in vivo also prove that HA/CS canrepair bone defect as a new kind of bone grafting material. Thisexperiment In vivo evaluated local tissue vancomycin concentrations byHigh Performance Liquid Chromatography Tandem mass spectrometry(HPLC-TS) and antibacterial activity against methicillin-resistantStaphylococcus aureus (MRSA) using a muscular rabbit model.Moreover, the number of viable MRSA on HA/CS was significantly morethan seen on mesoporous HA/CS. We can conclude that the mesoporousHA/CS loaded with vancomycin represents a promising candidate forbone regeneration as a drug delivery. This experiment evaluated localtissue vancomycin concentrations and antibacterial activity againstmethicillin-resistant Staphylococcus aureus (MRSA) using a muscular rabbit model. Mesoporous HA/CS loaded with vancomycin samples wereimplanted into the muscular pockets of the rabbit extremity. The resultsrevealed that local tissue vancomycin concentrations of mesoporousHA/CS was higher than it of HA/CS at different time(3d,7d,14d,28d)anddifferent distance site(1cm,2cm). Antibacterial assays indicate thatmesoporous HA/CS loaded with vancomycin have the effect of on growthof organisms and keep the diameter of inhibition zone until it disappearedfor20d. Moreover, the number of viable MRSA on HA/CS wassignificantly more than seen on mesoporous HA/CS. These resultsdemonstrated that mesoporous HA/CS could enhance the drug loadingamount and releasing amount in vivo and offer good ability to killMRSA.
In the present investigation, a novel and rational approach was madeto combine antibiotic drugs with HA/CS scaffold matrix, which candeliver the drug locally and in a sustained fashion. Nano-HA scaffoldshave been studied in detail to cure osteomyelitis in animal model.Mesoporous HA/CS presented a higher surface area and porosity whencompared with conventional micro-structured HA. BMP-2, Collagen-1 gene expression of osteoblastic associated markers also presented a highexpression. MTT assay and Cell adhesion assay showed that Mesoporoushydroxyapatite/chitosan provided a more adequate environment for celladhesion and migration and possessed good biocompatibility, improvingcell response. Because the VCM molecules can be adsorbed onto outersurface and inner area of mesoporous materials, it is suitable forcontrolled release as a drug carrier. This study provides novel andimportant information on the in vivo and in vitro effects of HA/CS loadedwith VCM, and suggests that this mesoporous materials releases VCMfor a long time after implantation and disinfects against MRSA, andsatisfies the demand as a drug delivery system.
创新点：
⑴将HA和CS这两种材料复合形成复合材料可以充分发挥二者的优点。既具有良好的生物活性，较高的机械强度，一定的韧度与一定的溶解速率，又克服了羟基磷灰石的脆性，极低的降解速率和壳聚糖的极低强度和降解过快。
⑵将HA制成介孔材料，介孔HA既具有HA的良好生物活性和骨诱导性，又比HA具有更宽大的孔道空间和吸附性能,可以增加对药物的负载量和对药物释放的控制，同时增加的孔道可以更加利于骨的诱导和骨长入。
⑶将万古霉素载入介孔HA，形成了局部药物缓释系统（DDS）。介孔HA增加了对万古霉素的负载量，同时对万古霉素的释放起到缓释的作用，在抗感染的同时还能促进骨的修复和长入，比以往采用抗生素骨水泥治疗和预防假体周围感染具有无比的优越性。
The treatment of acute and chronic orthopaedic infection is difficult,time consuming and expensive. Despite the availability of a myriad ofantibiotics and marked advances in surgical treatment, the long-termrecurrence rateremains alarming at a rate of20%to30%. Absence ofantibiotics labelled exclusively for osteomyelitic conditions leave thechoice of therapy to the preferences and experience of the attendingphysicians. Very high concentrations of antibiotics are required at thetarget site but could hardly be attained by conventional routes of drugadministration without provoking serious side effects. Hence,conventional therapy hampers clinicians in striking a balance betweenefficacy and safety in deciding an optimal treatment strategy forosteomyelitic patients. Moreover, patients bear unfulfilled therapeuticoutcomes, development of adverse drug effects and huge healthcare bills. Local drug delivery systems placed in the affected area, release druglocally for prolonged periods and at concentrations generally higher thanthose achieved by conventional drug delivery strategies.So far, a largenumber of systems have been employed as various drug delivery systems,such as biodegradable polymers hydroxyapatite,calcium phosphatecement xerogels, hydrogels, and mesoporous silica. Recently mesoporousmaterials is chemistry and material field research hotspot, and they havegained enhanced interest with particular attention as drug storage andrelease carrier due to their large unique surface area and hole volume,uniform and adjustable pore canal, easily modified surface features forsite specific delivery. Due to these advantages, mesoporous materialsshow a broad application prospect in chemitry, biological medicine,chemical catalysis, nanometer materials and so on.The drug deliverysystems using mesoporous materials as drug carriers have been reported.
Due to the bioactivity, biocompatibility,stability, nontoxic properties,hydroxyapatite with porous surface structure and–OH groups may serveas an ideal candidate drug carrier for the delivery of a variety ofpharmaceutical molecules, and especially mesoporous HA have broader pore canal space and adsorption performance than nano HA. Thus, thedesign and development of mesoporous HA might be able to reach thisgoal. Chitosan as a high molecular bioactive polymer has satisfactorybiocompatibility and biodegradation, and itself can improve survival rateof the osteoblast and promote the osteoblast differentiation and Matrixmineralization.Its surface hydrophilicity is beneficial to celldifferentiation and adhesion.So HA and CS two kinds of materials wereformed composite materials and may fully exrt their advantage. HA wasscattered among chitosan matrix and formed composite obtained goodosteoinduction and osteoconduction. Moreover, biological space ofdissolving chitosan is helpful to tissue reconstruction and then isconductive to drug releasing.
Mesoporous hydroxyapatite/chitosan composite have beensuccessfully prepared and synthesized using a freeze-drying method, andthen characterized by Scanning Electron Microscope, X-ray diffractionand IR. Results showed that mesoporous HA/CS presented higher surfacearea and porosity. The effect of mesoporous HA/CS on early adhesion,proliferation and differentiation of osteoblast cells in vitro was measured.. MTT cytotoxicity test and cell adhesion test show that the materials havegood biocompatibility and make cell viability and proliferation. In vitrotesting using osteoblast-like cells showed that on the surfaces of cells areable to adhere, proliferate, and migrate through the pores. These cellsmaintained similar expression levels of osteoblastic-associated markersnamely collagen type I (COL-1), alkaline phosphatase(ALP). Histologicanalysis and radiological analysis in vivo also prove that HA/CS canrepair bone defect as a new kind of bone grafting material. Thisexperiment In vivo evaluated local tissue vancomycin concentrations byHigh Performance Liquid Chromatography Tandem mass spectrometry(HPLC-TS) and antibacterial activity against methicillin-resistantStaphylococcus aureus (MRSA) using a muscular rabbit model.Moreover, the number of viable MRSA on HA/CS was significantly morethan seen on mesoporous HA/CS. We can conclude that the mesoporousHA/CS loaded with vancomycin represents a promising candidate forbone regeneration as a drug delivery. This experiment evaluated localtissue vancomycin concentrations and antibacterial activity againstmethicillin-resistant Staphylococcus aureus (MRSA) using a muscular rabbit model. Mesoporous HA/CS loaded with vancomycin samples wereimplanted into the muscular pockets of the rabbit extremity. The resultsrevealed that local tissue vancomycin concentrations of mesoporousHA/CS was higher than it of HA/CS at different time(3d,7d,14d,28d)anddifferent distance site(1cm,2cm). Antibacterial assays indicate thatmesoporous HA/CS loaded with vancomycin have the effect of on growthof organisms and keep the diameter of inhibition zone until it disappearedfor20d. Moreover, the number of viable MRSA on HA/CS wassignificantly more than seen on mesoporous HA/CS. These resultsdemonstrated that mesoporous HA/CS could enhance the drug loadingamount and releasing amount in vivo and offer good ability to killMRSA.
In the present investigation, a novel and rational approach was madeto combine antibiotic drugs with HA/CS scaffold matrix, which candeliver the drug locally and in a sustained fashion. Nano-HA scaffoldshave been studied in detail to cure osteomyelitis in animal model.Mesoporous HA/CS presented a higher surface area and porosity whencompared with conventional micro-structured HA. BMP-2, Collagen-1 gene expression of osteoblastic associated markers also presented a highexpression. MTT assay and Cell adhesion assay showed that Mesoporoushydroxyapatite/chitosan provided a more adequate environment for celladhesion and migration and possessed good biocompatibility, improvingcell response. Because the VCM molecules can be adsorbed onto outersurface and inner area of mesoporous materials, it is suitable forcontrolled release as a drug carrier. This study provides novel andimportant information on the in vivo and in vitro effects of HA/CS loadedwith VCM, and suggests that this mesoporous materials releases VCMfor a long time after implantation and disinfects against MRSA, andsatisfies the demand as a drug delivery system.
引文
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