多孔生物陶瓷支架载药控释体系的研究
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摘要
一直以来,临床上棘手于对由创伤、感染和肿瘤切除后所造成的大节段骨缺损进行修复。自体骨虽因其具有优良的骨传导性、骨诱导性、成骨潜能和无免疫原性而被视为骨移植材料的金标准,但自体骨来源数量有限,且取骨手术存在至少10%的并发症,植入体内后,需要较长时间的爬行替代过程。而异体骨和异种骨具有抗原性,尤其在移植较大骨时,常因剧烈的免疫排斥反应导致移植失败,并且还有病原传播危险。近年来,人们开始用组织工程的原理和技术,将具有成骨潜能的细胞诱导分化、增殖并种植于支架材料上,形成工程化人工骨,促进大节段骨缺损的修复。
然而,在体外构建大尺寸组织工程骨会因体外培养的细胞在植入体内后存活率较低、血管化问题和临床时限等问题而受到诸多限制。同时,临床上为更好地促进骨的愈合,还会给患者施以药物治疗。但由于骨组织在生物学上的如密度大、血流量低、渗透性差等特殊性,传统给药方式使药物很难按理想状态到达病变部位,从而疗效低。
羟基磷灰石(HA)的组成和结构与自然骨的极为相似,有良好的生物相容性、骨传导性和骨诱导性,被广泛应用为骨的替代材料。HA能吸附化学药物小分子和蛋白质等生物大分子,所以将其作为药物载体被广为研究。但其作为药物载体存在有如表面易吸附杂质、载药率低和突释问题。
我课题组前期创新地研究并制备了由多孔生物陶瓷球粒堆积的孔隙100%互通、孔隙大小、孔隙率可控且可与生物活性物质均匀混合的大尺寸(Φ1～1.5×3-4cm)多孔支架,以期修复超临界尺寸的骨缺损。本实验旨在在此支架上,负载可促骨生长、促血管化的生物活性物质,使其成为缓释体系,期在修复骨缺损中持续有效地释放活性物质,促进骨生长及愈合。
本实验用溶胶-凝胶法和W/O乳化成球技术制备了HA多孔球形颗粒,并对球粒的孔隙结构进行了表征；考察了丹酚酸B (Sal B)对成骨细胞增殖和分化的影响；将HA球粒作为药物载体,利用其吸附性将Sal B负载其上,同时考察分别用几种不同浓度的PLA和几种不同浓度的壳聚糖对载药球粒进行包裹后药物的释放情况,选择出较佳的包裹物质及其浓度,成功制得壳聚糖包裹的Sal B缓释HA球粒,考察该缓释体系对成骨细胞活性的影响。本实验获得的结论如下：
1.采用溶胶/凝胶法和W/O乳化成球技术制备的HA球粒具有组织生长需要的微观孔结构。
2. Sal B能有效地促进成骨细胞增殖、分化及细胞外基质矿化,提高成骨细胞的总代谢活性和碱性磷酸酶的表达从而促进成骨。当Sal B的质量浓度≤160μg/mL时对成骨细胞的增殖和分化有明显的促进作用,且质量浓度越高效果越明显；但当Sal B的质量浓度高于160μg/mL时,便对成骨细胞产生毒性,导致其凋亡。由此得出,Sal B可作为促骨生长和愈合的生物活性物质应用于骨缺损的修复,且最佳药效浓度为160μg/mL。
3.由于PLA的疏水性,其所形成的膜不能很好的附着在HA球粒表面,易脱落,所以几种不同浓度的PLA对载药球粒的包裹效果都不好。而壳聚糖亲水性和成膜性好,且具有良好的细胞相容性,最终选用2%的壳聚糖溶液包裹制得载Sal B的缓释HA球粒。
4.壳聚糖包裹Sal B缓释HA球粒可在体外长时间内释放具有生物活性的Sal B,维持有效促进成骨细胞增殖的浓度并保持成骨细胞的生物学活性,较长时间内促进成骨细胞分裂和增殖,能满足骨创伤治疗中所需要的持续性促进骨形成的能力,为修复大截段骨缺损提供了新的途径。
In clinic,it has been difficult to repair the large critical size defects arising from trauma,infection,tumor resection.Autologous bone is still the gold standard in bone reconstructive surgery because of its osteoconductivity, osteoinductivity,osteogenic potential and lack of immunogenicity as well.However,this standard auto graft has its own share of problems like inadequate amount,at least10%morbidity and potential complications in bone surgery.And it will take a long time to creep substitution. The bone allograft and xenograft has antigenicity, especially in large bone graft, often fail in the graft due to severe immune rejection, and has danger in the spread of pathogens as well. Therefore, in recent years with the emergence of tissue engineering, people began to use the principles and techniques of tissue engineering to plant the cells which have the osteogenic potential in the scaffold after differentiation, proliferation. And at last the engineered artificial bone has been made and can promote a large segmental bone defect repair. In cultivating tissue engineered bone in vitro, specially in large tissue engineered bone, the currently used bone substitutes still face many unsolved problems including incomplete formation of new bone tissue, failure of neovascularization, and slow growth of the capillary network. Meanwhile, drug is ofen used for better bone healing in clinical.However, due to bone tissue, such as in the biological density, low blood flow, permeability and poor specificity, the traditional methods of drug administration is difficult to arrive at the ideal site of the lesion, and thus its efficacy is low.
Hydroxyapatite (HA) has very similar composition and structure to natural bone and with good biocompatibility, bone osteoconductivity and osteoinductivity.It has been widely used as bone substitute materials. HA can absorb chemical molecules and biological macromolecules such as proteins, so as a drug carrier has been widely studied. But it has problems with adsorption of impurities, low drug loading and burst release.
A novel scaffold with large size and controlled porous structure has been designed and prepared for the aim of massive tissue engineered bone in previous experiment of our group. It was composed of HA spherules and porous HA tube.HA spherules can easily compound with biological active substance before being filled into the HA tubes. This study was designed to load the biological active substances promoting bone growth, neovasculariztion on the scaffold and get a new drug delivery system.This system can release the biological active substances in a sustained and effective manner in order to promote bone growth and healing in the repair of bone defects.
HA spherules were prepared by the method of sol-gel and water/oil emulsification technology and the porous structures were characterized. Sal B,the major water-soluble components of the traditional Chinese medicine radix salviae miltiorrhizae, was investigated on osteoblast proliferation and differentiation and was loaded on the HA spherules.Then the drug-loaded spherules were wrapped with several different concentrations of PLA and several different concentrations of chitosan. The case of drug release and the best package substance with the right concentration were investigated. Finally,the influence of Sal B-loaded HA sustained-release spherules on the activity of osteoblasts cultured in vitro was studied.The main conclusions were obtained as follows:
1. The HA spherules,prepared by the method of sol-gel and water/oil emulsification technology,possessed the porous structures can meet the organization growth.
2. Sal B has been proven to be effective in promoting osteoblast proliferation and differentiation. SalB has the potential toameliorate bone healing by stimulating both the total metabolic activity and ALP activity of osteoblastic cells. It was suggested that Sal B at a mass concentration=160μg/mL significantly contributed to osteoblast proliferation. Moreover, the higher mass concentration indicated more obvious proliferation. However, Sal B at a mass concentration>160μg/mL had toxic effect on ostaoblasts. It showed that Sal B can promote bone growth and healing as the biological active substances used in the repair of bone defects, and the best effective concentration was160μg/mL.
3. The results showed that PLA can form film but can not be attached to surface of HA sperules due to its hydrophobicity.So it was easy to come off and wrappd poor.However,it were explored that chitosan has good hydrophilicity,good film-formed and good cell compatibility,which was used to wrap on the surface of the HA spherules to get the Sal B-loaded HA sustained release spherules and2%concentration percentage was choosen in this study.
4.The chitosan-coated Sal B-loaded HA sustained-release spherules prepared in this study can release Sal-B in vitro for a long period, effectively promoted osteoblast proliferation, maintained biological activity of osteoblasts, contributed to osteoblast differentiation and proliferation, increased utilization rate of drug in local region, decreased adverse reaction, and persistently contributed to bone formation. It is helpful for construction and repair of bioceramic scaffolds and is of high clinical value in treatment of large segmental bone defects.
然而,在体外构建大尺寸组织工程骨会因体外培养的细胞在植入体内后存活率较低、血管化问题和临床时限等问题而受到诸多限制。同时,临床上为更好地促进骨的愈合,还会给患者施以药物治疗。但由于骨组织在生物学上的如密度大、血流量低、渗透性差等特殊性,传统给药方式使药物很难按理想状态到达病变部位,从而疗效低。
羟基磷灰石(HA)的组成和结构与自然骨的极为相似,有良好的生物相容性、骨传导性和骨诱导性,被广泛应用为骨的替代材料。HA能吸附化学药物小分子和蛋白质等生物大分子,所以将其作为药物载体被广为研究。但其作为药物载体存在有如表面易吸附杂质、载药率低和突释问题。
我课题组前期创新地研究并制备了由多孔生物陶瓷球粒堆积的孔隙100%互通、孔隙大小、孔隙率可控且可与生物活性物质均匀混合的大尺寸(Φ1～1.5×3-4cm)多孔支架,以期修复超临界尺寸的骨缺损。本实验旨在在此支架上,负载可促骨生长、促血管化的生物活性物质,使其成为缓释体系,期在修复骨缺损中持续有效地释放活性物质,促进骨生长及愈合。
本实验用溶胶-凝胶法和W/O乳化成球技术制备了HA多孔球形颗粒,并对球粒的孔隙结构进行了表征；考察了丹酚酸B (Sal B)对成骨细胞增殖和分化的影响；将HA球粒作为药物载体,利用其吸附性将Sal B负载其上,同时考察分别用几种不同浓度的PLA和几种不同浓度的壳聚糖对载药球粒进行包裹后药物的释放情况,选择出较佳的包裹物质及其浓度,成功制得壳聚糖包裹的Sal B缓释HA球粒,考察该缓释体系对成骨细胞活性的影响。本实验获得的结论如下：
1.采用溶胶/凝胶法和W/O乳化成球技术制备的HA球粒具有组织生长需要的微观孔结构。
2. Sal B能有效地促进成骨细胞增殖、分化及细胞外基质矿化,提高成骨细胞的总代谢活性和碱性磷酸酶的表达从而促进成骨。当Sal B的质量浓度≤160μg/mL时对成骨细胞的增殖和分化有明显的促进作用,且质量浓度越高效果越明显；但当Sal B的质量浓度高于160μg/mL时,便对成骨细胞产生毒性,导致其凋亡。由此得出,Sal B可作为促骨生长和愈合的生物活性物质应用于骨缺损的修复,且最佳药效浓度为160μg/mL。
3.由于PLA的疏水性,其所形成的膜不能很好的附着在HA球粒表面,易脱落,所以几种不同浓度的PLA对载药球粒的包裹效果都不好。而壳聚糖亲水性和成膜性好,且具有良好的细胞相容性,最终选用2%的壳聚糖溶液包裹制得载Sal B的缓释HA球粒。
4.壳聚糖包裹Sal B缓释HA球粒可在体外长时间内释放具有生物活性的Sal B,维持有效促进成骨细胞增殖的浓度并保持成骨细胞的生物学活性,较长时间内促进成骨细胞分裂和增殖,能满足骨创伤治疗中所需要的持续性促进骨形成的能力,为修复大截段骨缺损提供了新的途径。
In clinic,it has been difficult to repair the large critical size defects arising from trauma,infection,tumor resection.Autologous bone is still the gold standard in bone reconstructive surgery because of its osteoconductivity, osteoinductivity,osteogenic potential and lack of immunogenicity as well.However,this standard auto graft has its own share of problems like inadequate amount,at least10%morbidity and potential complications in bone surgery.And it will take a long time to creep substitution. The bone allograft and xenograft has antigenicity, especially in large bone graft, often fail in the graft due to severe immune rejection, and has danger in the spread of pathogens as well. Therefore, in recent years with the emergence of tissue engineering, people began to use the principles and techniques of tissue engineering to plant the cells which have the osteogenic potential in the scaffold after differentiation, proliferation. And at last the engineered artificial bone has been made and can promote a large segmental bone defect repair. In cultivating tissue engineered bone in vitro, specially in large tissue engineered bone, the currently used bone substitutes still face many unsolved problems including incomplete formation of new bone tissue, failure of neovascularization, and slow growth of the capillary network. Meanwhile, drug is ofen used for better bone healing in clinical.However, due to bone tissue, such as in the biological density, low blood flow, permeability and poor specificity, the traditional methods of drug administration is difficult to arrive at the ideal site of the lesion, and thus its efficacy is low.
Hydroxyapatite (HA) has very similar composition and structure to natural bone and with good biocompatibility, bone osteoconductivity and osteoinductivity.It has been widely used as bone substitute materials. HA can absorb chemical molecules and biological macromolecules such as proteins, so as a drug carrier has been widely studied. But it has problems with adsorption of impurities, low drug loading and burst release.
A novel scaffold with large size and controlled porous structure has been designed and prepared for the aim of massive tissue engineered bone in previous experiment of our group. It was composed of HA spherules and porous HA tube.HA spherules can easily compound with biological active substance before being filled into the HA tubes. This study was designed to load the biological active substances promoting bone growth, neovasculariztion on the scaffold and get a new drug delivery system.This system can release the biological active substances in a sustained and effective manner in order to promote bone growth and healing in the repair of bone defects.
HA spherules were prepared by the method of sol-gel and water/oil emulsification technology and the porous structures were characterized. Sal B,the major water-soluble components of the traditional Chinese medicine radix salviae miltiorrhizae, was investigated on osteoblast proliferation and differentiation and was loaded on the HA spherules.Then the drug-loaded spherules were wrapped with several different concentrations of PLA and several different concentrations of chitosan. The case of drug release and the best package substance with the right concentration were investigated. Finally,the influence of Sal B-loaded HA sustained-release spherules on the activity of osteoblasts cultured in vitro was studied.The main conclusions were obtained as follows:
1. The HA spherules,prepared by the method of sol-gel and water/oil emulsification technology,possessed the porous structures can meet the organization growth.
2. Sal B has been proven to be effective in promoting osteoblast proliferation and differentiation. SalB has the potential toameliorate bone healing by stimulating both the total metabolic activity and ALP activity of osteoblastic cells. It was suggested that Sal B at a mass concentration=160μg/mL significantly contributed to osteoblast proliferation. Moreover, the higher mass concentration indicated more obvious proliferation. However, Sal B at a mass concentration>160μg/mL had toxic effect on ostaoblasts. It showed that Sal B can promote bone growth and healing as the biological active substances used in the repair of bone defects, and the best effective concentration was160μg/mL.
3. The results showed that PLA can form film but can not be attached to surface of HA sperules due to its hydrophobicity.So it was easy to come off and wrappd poor.However,it were explored that chitosan has good hydrophilicity,good film-formed and good cell compatibility,which was used to wrap on the surface of the HA spherules to get the Sal B-loaded HA sustained release spherules and2%concentration percentage was choosen in this study.
4.The chitosan-coated Sal B-loaded HA sustained-release spherules prepared in this study can release Sal-B in vitro for a long period, effectively promoted osteoblast proliferation, maintained biological activity of osteoblasts, contributed to osteoblast differentiation and proliferation, increased utilization rate of drug in local region, decreased adverse reaction, and persistently contributed to bone formation. It is helpful for construction and repair of bioceramic scaffolds and is of high clinical value in treatment of large segmental bone defects.
引文
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