纳米珍珠层人工骨的制备及实验研究
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摘要
研究背景:
珍珠层(nacre or mother of pearl)是软体动物珍珠贝科或蚌科动物的贝壳内层部分,其主要成分为文石型碳酸钙,并含有少量有机质和微量金属元素。1992年法国科学家Lopez首先在研究中发现珍珠层具有诱导成骨的作用。此后,大量研究均证实这种天然生物材料具有良好的生物相容性,并且具有骨诱导和骨传导作用。学者们认为珍珠层的骨诱导作用来自于其中的有机质成分,其中全部或部分成分可能是具有骨诱导作用的信号分子。于是,国内外学者开展了广泛的、将珍珠层单一材料或以其为原材料制备的复合人工骨植入骨缺损部位的实验研究,这些包括将珍珠层块体植入骨承重部位和将珍珠层颗粒及粉末填充骨缺损的实验,以及将珍珠层复合人工骨植入大段骨缺损的实验,这些实验研究均显示出来源于这种天然材料良好的成骨效果。
理想的骨组织植入修复材料应具有良好的生物相容性,具有足够的强度,能有效充当新骨形成的支架,具骨诱导和骨传导作用进而能有效促进骨形成,并能在体内逐渐发生生物降解,被骨组织替代,以达到完全修复的目的。随着对珍珠层材料研究的深入,学者们发现这种材料虽然具有良好的生物学活性,其降解却足一个相当长期的过程。经研究分析,这主要与珍珠层材料的理化成分及形态大小两种因素有关。一方面,碳酸钙盐在植入区域形成了弱碱性的环境,不利于材料的降解吸收;另一方面,当珍珠层的形态逐渐变小,由块状、颗粒状再到粉末状,其降解吸收则呈现上升的趋势。通过各种研究,人们发现,珍珠层这种天然材料的生物降解主要是建立在非特异性的理化溶解方式上,而活性细胞介导的吸收方式对于这种材料的降解作用有限。
如何改善这种材料的降解性能,使之能够更好地发挥骨修复作用,并被宿主新生骨顺利的长入替代,是当前在这方面研究所面临的最主要问题。引起我们注意的是,目前国内外进行广泛的实验研究所采用的珍珠层粉,均是属于微米级的粉体,由此可知其内部文石晶片和有机质没有发生完全解离,被认为这是影响其降解的主要原因之一。如前所述,由于珍珠层的降解方式主要是建立在非特异性的理化溶解方式上的,可以认为随着珍珠层的粒度进一步下降,将会更有利于其降解。
随着材料学的不断发展,应用纳米技术制备的生物活性材料——纳米人工骨也随之诞生,这种新材料的内部结构与天然骨相似,有助于体内的细胞及大分子对其进行识别,从而使得材料的生物活性、利用度和生物相容性得以提高。经过初步的实验研究发现,纳米材料具有良好的生物相容性及骨引导活性,其生物活性优于微米材料。同时,由于纳米材料中的纳米粒子所具有的量子尺寸效应、小尺寸效应、表面效应和宏观量子隧道效应的特点,其降解性能也因此而得到了提高。
本实验研究是在本课题组前期研究的基础上,将机械研磨法获得的纳米珍珠层粉与消旋聚乳酸,采用与以往相同的制备工艺,即溶剂浇铸、热压铸模、溶质沥滤的工序,复合成多孔结构的纳米级珍珠层粉/消旋聚乳酸复合人工骨,由于机械研磨法制得的纳米珍珠层粉具有保留其原有活性成分的效果,并且其产生的弱碱性的环境与消旋聚乳酸降解后的弱酸性产物发生中和,从理论上分析将有利于这种新材料的降解吸收;将纳米级珍珠层粉/消旋聚乳酸复合人工骨与以往的微米级珍珠层粉/消旋聚乳酸复合人工骨进行比较研究,从材料表征和理化特性、体外细胞学试验以及动物体内植入试验等各个方面进行全面分析,旨在探讨这一新型天然纳米材料纳米珍珠层粉,与人工合成高分子生物材料消旋聚乳酸相结合,所制作的复合人工骨的生物学效应和生物降解能力,为其推广应用提供实验依据。
目的:
1.探讨纳米珍珠层人工骨与微米珍珠层人工骨相比之下的性能变化及其应用的合理性;
2.研究纳米珍珠层人工骨与成骨细胞的生物相容性;
3.观察纳米级珍珠层粉在大鼠骨缺损区的降解吸收情况和以其为原材料制作的复合人工骨在大鼠体内的生物相容性;
4.观察纳米珍珠层人工骨修复兔桡骨缺损的作用。
方法:
1.材料制备:采用机械研磨法获得纳米级珍珠层粉,与消旋聚乳酸复合,按照溶剂浇铸、热压铸模、溶质沥滤的工序,制成中空圆柱体的纳米级珍珠层粉/消旋聚乳酸复合人工骨,同法制作出实心圆柱体的微米级珍珠层粉/消旋聚乳酸复合人工骨。
2.材料表征测定:通过扫描电子显微镜观察纳米级珍珠层粉与微米级珍珠层粉的实际粒径、以及这两种原材料制得的纳米珍珠层复合人工骨和微米珍珠层复合人工骨的孔径,采用比重法测量两种人工骨的孔隙率,并采用MTS858万能力学实验机对这两种人工骨进行生物力学强度的检测,对以上检测结果进行对比分析。
3.体外细胞学实验:纳米珍珠层/消旋聚乳酸复合人工骨与微米珍珠层/消旋聚乳酸复合人工骨分别与传至第3代的成骨细胞共培养,在倒置相差显微镜下观察成骨细胞贴附情况,在扫描电子显微镜下观察成骨细胞在材料上生长情况,用CCK-8法检测成骨细胞与人工骨材料共培养1、3、5、7天后的增殖情况,共培养7天后,BCA法测定细胞蛋白含量,AKP试剂盒测定细胞碱性磷酸酶活性。
4.原材料粉体及人工骨片体内植入实验:将纳米珍珠层粉及其与消旋聚乳酸复合制得的人工骨片,分别植入大鼠股骨骨洞及股部肌囊,同时与微米珍珠层粉及其复合人工骨作对照,并建立空白对照。于术后当天及第2、4、8周分别作X-ray摄片检查,动物处死前予四环素注射作活体荧光标记,处死后行大体、组织学及扫描电镜观察。
5.人工骨植入体内修复大段骨缺损实验:将纳米珍珠层/消旋聚乳酸复合人工骨植入新西兰白兔1.5cm的桡骨缺损区,同时与微米珍珠层/消旋聚乳酸复合人工骨作对照,并设立空白对照。分别于术后当天及4、8、16、24周作X线、骨密度测量,动物处死前予四环素注射作活体荧光标记,然后动物取材作大体及组织学检查。
结果:
1.制得纳米珍珠层/消旋聚乳酸复合人工骨,为3.5mm×3.5mm×15mm的、中央有一直径1mm穿通孔的空心圆柱体;制得微米珍珠层/消旋聚乳酸复合人工骨,为3.5mm×3.5mm×15mm的实心圆柱体。
2.通过电镜观察,纳米级珍珠层粉的平均粒径为50.4±12.4nm,所制得的纳米珍珠层/消旋聚乳酸复合人工骨孔径平均为215.7±77.5μm,普通微米级珍珠层粉的平均粒径为5.0±3.0μm,所制得的微米珍珠层/消旋聚乳酸复合人工骨孔径平均为205.1±72.0μm,结果表明前者为纳米级粉体,后者为微米级粉体,而两种人工骨的孔径无显著差异(P＞0.05);采用比重法测得纳米珍珠层/消旋聚乳酸复合人工骨和微米珍珠层/消旋聚乳酸复合人工骨的孔隙率分别是(65.4±2.9)%和(53.4±2.2)%,前者要高于后者(P＜0.05);生物力学测试结果为纳米珍珠层/消旋聚乳酸复合人工骨与微米珍珠层/消旋聚乳酸复合人工骨在抗压强度和弹性模量方无显著差异(P＞0.05),而前者的抗折弯强度低于后者(p＜0.05)。
3.纳米珍珠层/消旋聚乳酸复合人工骨与微米珍珠层/消旋聚乳酸复合人工骨分别与成骨细胞共培养后,其各时间点CCK-8法检测值与空白对照无显著差异(P＞0.05),成骨细胞均在第5天达到增殖高峰期;培养7天后,实验组细胞蛋白含量高于对照组及空白组(P＜0.05),后两者之间则无显著差异(P＞0.05);碱性磷酸酶活性在三组间均无显著差异(P＞0.05);电镜下可见成骨细胞在两种人工骨上都有良好生长贴附能力。
4.X-ray显示两种粉体在大鼠股骨骨洞植入第2周时都开始出现了降解,第4周时更为明显,纳米珍珠层粉较之微米珍珠层粉降解更快,而空白对照组骨洞阴影仍可见,至8周时,则所有组骨洞均己闭合修复,X-ray下已不可见原钻孔痕迹,恢复正常骨质密度;硬组织磨片四环素荧光双标记结果显示纳米珍珠层粉植入组较其余两组在骨缺损区周围新骨生长速度更快,空白组速度最慢(P＜0.05);电镜观察及常规脱钙切片亦可见到纳米粉体降解较快;由以上两种原材料制得的纳米珍珠层/消旋聚乳酸复合人工骨与微米珍珠层/消旋聚乳酸复合人工骨在大鼠体内均与周围组织结合良好,无明显炎症反应。
5.X-ray显示纳米珍珠层/消旋聚乳酸复合人工骨植入兔桡骨缺损区后其骨愈合速度较对照组微米珍珠层/消旋聚乳酸复合人工骨植入的快,至植入术后24周,实验组骨缺损区接近正常骨密度,对照组骨缺损区密度较低,空白组则呈现骨不连状态;骨密度测量结果显示术后8周、16周、24周实验组的骨密度值高于对照组(P＜0.05),24周实验组的骨密度值与术前所测得的正常值无显著性差异(P＞0.05);动物取材大体所见均显示组织相容性良好,骨组织逐渐由植入材料两端向中央生长;常规切片HE染色及硬组织磨片Stevenel's blue/Van Geison's picro-fuchsin联合染色均可见实验组骨缺损区长势优于对照组,至术后24周,实验组骨髓腔与材料已呈相交通状;硬组织磨片荧光显微镜下观察,两组材料在术后8周处于骨生长最快速时期,16周时速度开始减慢,术后4、8、16周时实验组的新骨生长速度均较对照组的快(p＜0.05)。
结论:
1.纳米珍珠层人工骨的制备原料纳米珍珠层粉采用机械球磨法制作,其粉体粒径达到纳米级颗粒的要求。纳米珍珠层/消旋聚乳酸复合人工骨具有良好的孔径及孔隙率,其生物力学特性达到了人工骨植入的性能指标要求。
2.带有中空孔道的纳米珍珠层/消旋聚乳酸复合人工骨在制备上更为合理,这种结构可以有效避免发生溶质滤出不彻底的问题。中空体的纳米珍珠层/消旋聚乳酸复合人工骨可增加人工骨材料与植入部位内环境的接触面,有利于内部物质的交换,并且在必要时还可以作为促骨生长因子以及药物的承载空间。
3.纳米珍珠层/消旋聚乳酸复合人工骨属于一种无细胞毒性的纳米材料,具有良好的生物相容性,对成骨细胞的生长、增殖与分化功能无不良影响,并且在一定程度上促进了成骨细胞的功能,利于细胞的粘附与生长;同时,这种复合人工骨还具有良好的骨传导功能。
4.纳米珍珠层粉较微米珍珠层粉在体内具有更好的生物降解性能,这种材料的生物降解作用主要是发生在非特异性理化溶解的方式之上的。并且,纳米珍珠层粉的诱导成骨、促进骨组织形成的作用较微米珍珠层粉也明显增强。
5.纳米珍珠层/消旋聚乳酸复合人工骨具有良好的体内生物相容性。纳米珍珠层/消旋聚乳酸复合人工骨的降解性能及成骨活性,较之微米珍珠层/消旋聚乳酸复合人工骨都有了明显的提高,是一种具有良好开发前景的骨组织替代材料。
Background
Nacre(mother of pearl) is the inner layer of the shell of mollusc pearl oyster or bivalve.Nacre is mainly composed of calcium carbonate crystallized in the form of aragonite on an organic matrix scaffold,with microamount metal elements.The osteoinductive function of nacre was first found by French scientist Lopez in 1992. Since then,lots of researches had proved that this kind of biomaterial had satisfactory biocompatibility,osteoinduction and osteoconduction.Scholars considered that the osteoinduction of nacre came from the organic matrix,some of which may be the osteoinductive signaling molecule.Consequently,domestic and foreign scholars carried out extensive experimental studies on implanting nacre or it's composite artificial bone into the bone defect area,such as implanting nacre block in the weight-bearing site,filling nacre particle or nacre powder into bone defect hole,or implanting nacre artificial bone into the diaphyseal bone defect.All this presented satisfactory results.
Ideal implant restoration material should have satisfactory biocompatibility and high enough strength,serve as the scaffold for the new bone to grow in,have boneinductive and boneconductive functions to prompt the bone formation,and have the ability to biodegrade and be replaced by bone tissue gradually and repair the bone defect completely at last.However,when scholars continue to research,they found that the degradation of this material was a long time period,in spite of its good bioactivity.The degradation was connected with two factors:physicochemical composition and size.On one side,calcium carbonate formulate the faintly alkaline environment at implanting area which was disadvantage for the degradation;on the other side,when the nacre shape turned from block to grain and finally became powder,the degradation of this material presented a ascendant tendency.It was found that this natural material's degradation mainly bases on the physicochemical resolving manner,while the living cell mediated absorbing manner play limited role on the degradation.
How to advance the degradation function and exert its better use for bone repair through new bone growing in and replacing,is the major problem at present.What need to be noticed is that the nacre powder being researched was just belong to micron-scale powder,and its aragonite crystal had not been separated with the internal organic matter.So this maybe a factor affecting its degradation.Since degradation of nacre mainly bases on the physicochemical resolving manner,so it can be considered that as the material's particle size decreases its degradation will increase.
With the development of materialogy,nano-artificial bone,the biomaterial prepared through nanotechnology had emerged.Its structure was similar with natural bone,fit for human body cell and macromolecule to recognize,improving the bioactivity,availability and biocompatibility.Lots of researches showed that nano materials had satisfactory biocompatibility and osteoinduction,which had better bioactivity and biodegradation than non-nano materials.Meanwhile,because of the characteristics of quantum size effect,small size effect,surface effect and macroscopic quantum tunneling effect of nano-particle in this nano-material,its degradation function had been advanced.
Basing on the former research of our study group,this research task is to compose the nano-scale nacre powder made through mechanical grinding with PDLLA(poly-D,L-lactide acid) to prepare porous structure artificial bone in the same tectmology as former,in the process of solvent casting/heat ompressing/particulate leaching.The nano-scale nacre powder reserve the inherent bioactive component.In addition,the faintly alkaline environment brought by nacre powder will be balance out with the weak acid degradation product of PDLLA,then the biodegradation of composite artificial bone will be improved on this theory. Comparing this new nano artificial bone with the former micron nacre/PDLLA composite artificial bone,analyzing on the aspects of material's physicochemical characterization,cytological test in vitro and implanting test in vivo,this research is to explore the biological effect and biodegradation ability of this new composite artificial bone which is composed of the natural nano-material nano-nacre powder and the synthetic macromolecule biomaterial PDLLA.
Objectives:
1.To explore the changes and the applying rationality of the nano-nacre artificial bone developed from the former nacre artificial bone;
2.To research the biocompatibility of nano-nacre artificial bone with osteoblast;
3.To observe the biodegradation of nano-nacre powder in the bone defect area of rats,and the biocompatibility of nano-nacre artificial bone in rat's body;
4.To investigate the function of nano-nacre artificial bone on repairing the radial diaphyseal defects of rabbits.
Methods
1.Prepration of the material:The nano-scale nacre powder made through mechanical grinding was composed with PDLLA(poly-D,L-lactide acid) to prepare the cylinder shape with a center hole artificial bone,while the micron-scale nacre powder was also composed with PDLLA to prepare the cylinder shape artificial bone without hole.Both of them were fabricated in the process of solvent casting/heat compressing/particulate leaching.
2.Test of the characterizations of material:Checked the particle size of nano-nacre powder and micron-nacre powder,and the pore size of two kinds of artificial bone made from those powders under the observation of SEM(scanning electron microscope).Checked the porosity of two kinds of artificial bone through density measurement method.And checked the biomechanical characteristics with the MTS858 universal mechanical testing machine.All the parameters were analyzed for comparing.
3.Cytological experiments:Co-cultured the 3rd generation osteoblast with two kinds of artificial bone.Then observed the growth situation under inverted phase contrast microscope and scanning electron microscope.Checked the proliferation of osteoblast 1,3,5,7 days after being co-cultured with CCK-8 method.Finally,7 days after being co-cultured,the total protein contents were checked with BCA method and the alkaline phosphatase activities were checked with AKP test kit.
4.Implantation of the nacre powder and artificial bone slice:The nano-nacre powder and the artificial bone slice made from this powder and PDLLA (poly-D,L-lactide acid) were implanted into the femoral holes and the femoral muscle sacs of rats respectively.Normal micron-nacre powder and the artificial bones made from this powder and PDLLA were implanted as control group, and blank group was set up without implanting anything.X-ray photography were performed immediately and at 2,4 and 8 weeks after operation.The animals were injected with tetracycline for fluorescent mark before being killed.Then, the gross specimens,histological and SEM observations were performed.
5.Implantation of the artificial bone for repairing segmental bone defect:Created 1.5cm radial defects on both sides of New Zealand white rabbits.Then implanted the nano-nacre artificial bone into the bone defect of left side,and implanted the micron-nacre artificial bone into the other side as control group.Meanwhile, blank group was set up without implanting anything.X-ray photography and bone mineral density checking were performed immediately and at 4,8,16 and 24 weeks after operation.The animals were injected with tetracycline for fluorescent mark before being killed.Then,the gross specimens and histological observations were performed.
Results
1.Nano-nacre artificial bone(NNAB) was prepared,3.5mm×3.5mm×15mm,in cylinder shape with a center hole;While micron-nacre artificial bone(MNAB) was prepared,3.5mm×3.5mm×15mm,in cylinder shape without hole.
2.It was checked with the SEM that the average scale of nano-nacre powder was 50.4±12.4nm and the average pore size of it's correlative artificial bone was 215.7±77.5μm,whereas the micron-scale nacre powder was 5.0±3.0μm and the correlative artificial bone had the 205.1±72.0μm average pore size,which denoted that the former powder belong to nano-scale powder while the later just micron-scale,and the difference of pore size between two kinds of artificial bones was not significant(P＞0.05).The porosities of nano-nacre artificial bone and the control group were(65.4±2.9)%and(53.4±2.2)%respectively,the former was higher than the later(P＜0.05).The biomechanical tests showed that two kinds of artificial bones had not significant difference on compressive strength and Young's modulus(P＞0.05),while the flexural strength of nano-nacre artificial bone was less than the control group(P＜0.05).
3.The results of CCK-8 showed that the difference were not significant in each group,the proliferation of osteoblast reached the peak at the 5th day;7 days after being co-cultured,the total protein content of study group was higher than control group and blank group(P＜0.05),while the difference between control group and blank group was not significant(P＞0.05);The difference of alkaline phosphatase activities among three groups was not significant(P＞0.05);The SEM view showed that osteoblast attached and grew well in two kinds of artificial bone.
4.X-ray photography showed that two kinds of powder started to degrade in 2 weeks;this phenomenon became more appear in 4 weeks,nano-nacre powder degraded faster than micron-nacre powder,while the hole shadow was easy to be found;in 8 weeks,all the femoral holes recovered and returned to normal bone mineral density in all groups.Analysis of tetracycline fluorescent double marks in the hard tissue grinding slices indicated that new bone grew fastest around the bone defect area in study group,while most slowly in blank group(P＜0.05); SEM(scanning electron microscope) observation showed that nano-nacre powder degraded more quickly.The same result can be found through the demineralized sections morphometric analysis,and both of the composite artificial bones made from those two kinds of nacre powder had the good connection with the adjacent tissue in rats body without apparent inflammatory response.
5.X-ray photography showed that rabbit's bone defects healed faster in study group since NNAB implanted than in control group since MNAB implanted.At 24 weeks after operation,bone density in radial defects had nearly accessed to the normal area,while lower in control group,and turned up nonunion in blank group;The checking of BMD(bone mineral density) showed that results in study group were higher than those in control group at 8,16 and 24 week(P＜0.05), and the difference between the BMD values in study group at 24 week and those in blank group was not significant(P＞0.05).The gross specimens showed satisfactory histocompatibility both in study group and in control group,with bone tissue growing from two sides into the center of implanted materials; Normal slices in HE stain and hard tissue grinding slices in Stevenel's blue/Van Geison's picro-fuchsin stain showed that the bone growth tendency was better in study group than that in control group,and the medullary cavity had been penetrated to the implanted materials in study group at 24 week;Analysis of tetracycline fluorescent double marks in the hard tissue grinding slices indicated that new bone in both groups grew fastest 8 weeks after surgery,while slow down at 16 week.The growth speeds of new bone were faster in study group than those in control group at 4,8 and 16 week(P＜0.05).
Conclusions
1.Nano-nacre powder,raw material of NNAB,has the nano-scale particle size. NNAB has satisfactory pore size and porosity.Its biomechanical characters meet with the characteristic requisition of implanting artificial bone.
2.The preparation of this NNAB with a center hole is more reasonable.With this structure the problem that the particulate is difficult to be leached out thoroughly can be resolved.The center hole of NNAB can increase the contact surface with the internal environment of implanting area,be beneficial for the internal material to exchange,or be utilized as the container of bone growth factor or medicine if need.
3.NNAB belong to a kind of nano-material without cytotoxicity,has satisfactory biocompatibility.It has not harmful effects on the growth,proliferation and differentiation of osteoblast.It can somewhat promote the function of cell and benefit the adhesion and growth of cell.It has satisfactory bone conductive function and meet with the basic requisition of implanting material of artificial bone.
4.The degradation of nano-nacre powder is better.It's degradation mainly bases on the physicochemical resolving manner.Moreover,the functions of osteoinduction and prompting bone formulation of nano-nacre powder have enlarged.
5.NNAB possesses satisfactory biocompatibility.It's degradation and ossification abilities are better than those of MNAB.All those indicat that NNAB is a promising bone substitute material.
珍珠层(nacre or mother of pearl)是软体动物珍珠贝科或蚌科动物的贝壳内层部分,其主要成分为文石型碳酸钙,并含有少量有机质和微量金属元素。1992年法国科学家Lopez首先在研究中发现珍珠层具有诱导成骨的作用。此后,大量研究均证实这种天然生物材料具有良好的生物相容性,并且具有骨诱导和骨传导作用。学者们认为珍珠层的骨诱导作用来自于其中的有机质成分,其中全部或部分成分可能是具有骨诱导作用的信号分子。于是,国内外学者开展了广泛的、将珍珠层单一材料或以其为原材料制备的复合人工骨植入骨缺损部位的实验研究,这些包括将珍珠层块体植入骨承重部位和将珍珠层颗粒及粉末填充骨缺损的实验,以及将珍珠层复合人工骨植入大段骨缺损的实验,这些实验研究均显示出来源于这种天然材料良好的成骨效果。
理想的骨组织植入修复材料应具有良好的生物相容性,具有足够的强度,能有效充当新骨形成的支架,具骨诱导和骨传导作用进而能有效促进骨形成,并能在体内逐渐发生生物降解,被骨组织替代,以达到完全修复的目的。随着对珍珠层材料研究的深入,学者们发现这种材料虽然具有良好的生物学活性,其降解却足一个相当长期的过程。经研究分析,这主要与珍珠层材料的理化成分及形态大小两种因素有关。一方面,碳酸钙盐在植入区域形成了弱碱性的环境,不利于材料的降解吸收;另一方面,当珍珠层的形态逐渐变小,由块状、颗粒状再到粉末状,其降解吸收则呈现上升的趋势。通过各种研究,人们发现,珍珠层这种天然材料的生物降解主要是建立在非特异性的理化溶解方式上,而活性细胞介导的吸收方式对于这种材料的降解作用有限。
如何改善这种材料的降解性能,使之能够更好地发挥骨修复作用,并被宿主新生骨顺利的长入替代,是当前在这方面研究所面临的最主要问题。引起我们注意的是,目前国内外进行广泛的实验研究所采用的珍珠层粉,均是属于微米级的粉体,由此可知其内部文石晶片和有机质没有发生完全解离,被认为这是影响其降解的主要原因之一。如前所述,由于珍珠层的降解方式主要是建立在非特异性的理化溶解方式上的,可以认为随着珍珠层的粒度进一步下降,将会更有利于其降解。
随着材料学的不断发展,应用纳米技术制备的生物活性材料——纳米人工骨也随之诞生,这种新材料的内部结构与天然骨相似,有助于体内的细胞及大分子对其进行识别,从而使得材料的生物活性、利用度和生物相容性得以提高。经过初步的实验研究发现,纳米材料具有良好的生物相容性及骨引导活性,其生物活性优于微米材料。同时,由于纳米材料中的纳米粒子所具有的量子尺寸效应、小尺寸效应、表面效应和宏观量子隧道效应的特点,其降解性能也因此而得到了提高。
本实验研究是在本课题组前期研究的基础上,将机械研磨法获得的纳米珍珠层粉与消旋聚乳酸,采用与以往相同的制备工艺,即溶剂浇铸、热压铸模、溶质沥滤的工序,复合成多孔结构的纳米级珍珠层粉/消旋聚乳酸复合人工骨,由于机械研磨法制得的纳米珍珠层粉具有保留其原有活性成分的效果,并且其产生的弱碱性的环境与消旋聚乳酸降解后的弱酸性产物发生中和,从理论上分析将有利于这种新材料的降解吸收;将纳米级珍珠层粉/消旋聚乳酸复合人工骨与以往的微米级珍珠层粉/消旋聚乳酸复合人工骨进行比较研究,从材料表征和理化特性、体外细胞学试验以及动物体内植入试验等各个方面进行全面分析,旨在探讨这一新型天然纳米材料纳米珍珠层粉,与人工合成高分子生物材料消旋聚乳酸相结合,所制作的复合人工骨的生物学效应和生物降解能力,为其推广应用提供实验依据。
目的:
1.探讨纳米珍珠层人工骨与微米珍珠层人工骨相比之下的性能变化及其应用的合理性;
2.研究纳米珍珠层人工骨与成骨细胞的生物相容性;
3.观察纳米级珍珠层粉在大鼠骨缺损区的降解吸收情况和以其为原材料制作的复合人工骨在大鼠体内的生物相容性;
4.观察纳米珍珠层人工骨修复兔桡骨缺损的作用。
方法:
1.材料制备:采用机械研磨法获得纳米级珍珠层粉,与消旋聚乳酸复合,按照溶剂浇铸、热压铸模、溶质沥滤的工序,制成中空圆柱体的纳米级珍珠层粉/消旋聚乳酸复合人工骨,同法制作出实心圆柱体的微米级珍珠层粉/消旋聚乳酸复合人工骨。
2.材料表征测定:通过扫描电子显微镜观察纳米级珍珠层粉与微米级珍珠层粉的实际粒径、以及这两种原材料制得的纳米珍珠层复合人工骨和微米珍珠层复合人工骨的孔径,采用比重法测量两种人工骨的孔隙率,并采用MTS858万能力学实验机对这两种人工骨进行生物力学强度的检测,对以上检测结果进行对比分析。
3.体外细胞学实验:纳米珍珠层/消旋聚乳酸复合人工骨与微米珍珠层/消旋聚乳酸复合人工骨分别与传至第3代的成骨细胞共培养,在倒置相差显微镜下观察成骨细胞贴附情况,在扫描电子显微镜下观察成骨细胞在材料上生长情况,用CCK-8法检测成骨细胞与人工骨材料共培养1、3、5、7天后的增殖情况,共培养7天后,BCA法测定细胞蛋白含量,AKP试剂盒测定细胞碱性磷酸酶活性。
4.原材料粉体及人工骨片体内植入实验:将纳米珍珠层粉及其与消旋聚乳酸复合制得的人工骨片,分别植入大鼠股骨骨洞及股部肌囊,同时与微米珍珠层粉及其复合人工骨作对照,并建立空白对照。于术后当天及第2、4、8周分别作X-ray摄片检查,动物处死前予四环素注射作活体荧光标记,处死后行大体、组织学及扫描电镜观察。
5.人工骨植入体内修复大段骨缺损实验:将纳米珍珠层/消旋聚乳酸复合人工骨植入新西兰白兔1.5cm的桡骨缺损区,同时与微米珍珠层/消旋聚乳酸复合人工骨作对照,并设立空白对照。分别于术后当天及4、8、16、24周作X线、骨密度测量,动物处死前予四环素注射作活体荧光标记,然后动物取材作大体及组织学检查。
结果:
1.制得纳米珍珠层/消旋聚乳酸复合人工骨,为3.5mm×3.5mm×15mm的、中央有一直径1mm穿通孔的空心圆柱体;制得微米珍珠层/消旋聚乳酸复合人工骨,为3.5mm×3.5mm×15mm的实心圆柱体。
2.通过电镜观察,纳米级珍珠层粉的平均粒径为50.4±12.4nm,所制得的纳米珍珠层/消旋聚乳酸复合人工骨孔径平均为215.7±77.5μm,普通微米级珍珠层粉的平均粒径为5.0±3.0μm,所制得的微米珍珠层/消旋聚乳酸复合人工骨孔径平均为205.1±72.0μm,结果表明前者为纳米级粉体,后者为微米级粉体,而两种人工骨的孔径无显著差异(P＞0.05);采用比重法测得纳米珍珠层/消旋聚乳酸复合人工骨和微米珍珠层/消旋聚乳酸复合人工骨的孔隙率分别是(65.4±2.9)%和(53.4±2.2)%,前者要高于后者(P＜0.05);生物力学测试结果为纳米珍珠层/消旋聚乳酸复合人工骨与微米珍珠层/消旋聚乳酸复合人工骨在抗压强度和弹性模量方无显著差异(P＞0.05),而前者的抗折弯强度低于后者(p＜0.05)。
3.纳米珍珠层/消旋聚乳酸复合人工骨与微米珍珠层/消旋聚乳酸复合人工骨分别与成骨细胞共培养后,其各时间点CCK-8法检测值与空白对照无显著差异(P＞0.05),成骨细胞均在第5天达到增殖高峰期;培养7天后,实验组细胞蛋白含量高于对照组及空白组(P＜0.05),后两者之间则无显著差异(P＞0.05);碱性磷酸酶活性在三组间均无显著差异(P＞0.05);电镜下可见成骨细胞在两种人工骨上都有良好生长贴附能力。
4.X-ray显示两种粉体在大鼠股骨骨洞植入第2周时都开始出现了降解,第4周时更为明显,纳米珍珠层粉较之微米珍珠层粉降解更快,而空白对照组骨洞阴影仍可见,至8周时,则所有组骨洞均己闭合修复,X-ray下已不可见原钻孔痕迹,恢复正常骨质密度;硬组织磨片四环素荧光双标记结果显示纳米珍珠层粉植入组较其余两组在骨缺损区周围新骨生长速度更快,空白组速度最慢(P＜0.05);电镜观察及常规脱钙切片亦可见到纳米粉体降解较快;由以上两种原材料制得的纳米珍珠层/消旋聚乳酸复合人工骨与微米珍珠层/消旋聚乳酸复合人工骨在大鼠体内均与周围组织结合良好,无明显炎症反应。
5.X-ray显示纳米珍珠层/消旋聚乳酸复合人工骨植入兔桡骨缺损区后其骨愈合速度较对照组微米珍珠层/消旋聚乳酸复合人工骨植入的快,至植入术后24周,实验组骨缺损区接近正常骨密度,对照组骨缺损区密度较低,空白组则呈现骨不连状态;骨密度测量结果显示术后8周、16周、24周实验组的骨密度值高于对照组(P＜0.05),24周实验组的骨密度值与术前所测得的正常值无显著性差异(P＞0.05);动物取材大体所见均显示组织相容性良好,骨组织逐渐由植入材料两端向中央生长;常规切片HE染色及硬组织磨片Stevenel's blue/Van Geison's picro-fuchsin联合染色均可见实验组骨缺损区长势优于对照组,至术后24周,实验组骨髓腔与材料已呈相交通状;硬组织磨片荧光显微镜下观察,两组材料在术后8周处于骨生长最快速时期,16周时速度开始减慢,术后4、8、16周时实验组的新骨生长速度均较对照组的快(p＜0.05)。
结论:
1.纳米珍珠层人工骨的制备原料纳米珍珠层粉采用机械球磨法制作,其粉体粒径达到纳米级颗粒的要求。纳米珍珠层/消旋聚乳酸复合人工骨具有良好的孔径及孔隙率,其生物力学特性达到了人工骨植入的性能指标要求。
2.带有中空孔道的纳米珍珠层/消旋聚乳酸复合人工骨在制备上更为合理,这种结构可以有效避免发生溶质滤出不彻底的问题。中空体的纳米珍珠层/消旋聚乳酸复合人工骨可增加人工骨材料与植入部位内环境的接触面,有利于内部物质的交换,并且在必要时还可以作为促骨生长因子以及药物的承载空间。
3.纳米珍珠层/消旋聚乳酸复合人工骨属于一种无细胞毒性的纳米材料,具有良好的生物相容性,对成骨细胞的生长、增殖与分化功能无不良影响,并且在一定程度上促进了成骨细胞的功能,利于细胞的粘附与生长;同时,这种复合人工骨还具有良好的骨传导功能。
4.纳米珍珠层粉较微米珍珠层粉在体内具有更好的生物降解性能,这种材料的生物降解作用主要是发生在非特异性理化溶解的方式之上的。并且,纳米珍珠层粉的诱导成骨、促进骨组织形成的作用较微米珍珠层粉也明显增强。
5.纳米珍珠层/消旋聚乳酸复合人工骨具有良好的体内生物相容性。纳米珍珠层/消旋聚乳酸复合人工骨的降解性能及成骨活性,较之微米珍珠层/消旋聚乳酸复合人工骨都有了明显的提高,是一种具有良好开发前景的骨组织替代材料。
Background
Nacre(mother of pearl) is the inner layer of the shell of mollusc pearl oyster or bivalve.Nacre is mainly composed of calcium carbonate crystallized in the form of aragonite on an organic matrix scaffold,with microamount metal elements.The osteoinductive function of nacre was first found by French scientist Lopez in 1992. Since then,lots of researches had proved that this kind of biomaterial had satisfactory biocompatibility,osteoinduction and osteoconduction.Scholars considered that the osteoinduction of nacre came from the organic matrix,some of which may be the osteoinductive signaling molecule.Consequently,domestic and foreign scholars carried out extensive experimental studies on implanting nacre or it's composite artificial bone into the bone defect area,such as implanting nacre block in the weight-bearing site,filling nacre particle or nacre powder into bone defect hole,or implanting nacre artificial bone into the diaphyseal bone defect.All this presented satisfactory results.
Ideal implant restoration material should have satisfactory biocompatibility and high enough strength,serve as the scaffold for the new bone to grow in,have boneinductive and boneconductive functions to prompt the bone formation,and have the ability to biodegrade and be replaced by bone tissue gradually and repair the bone defect completely at last.However,when scholars continue to research,they found that the degradation of this material was a long time period,in spite of its good bioactivity.The degradation was connected with two factors:physicochemical composition and size.On one side,calcium carbonate formulate the faintly alkaline environment at implanting area which was disadvantage for the degradation;on the other side,when the nacre shape turned from block to grain and finally became powder,the degradation of this material presented a ascendant tendency.It was found that this natural material's degradation mainly bases on the physicochemical resolving manner,while the living cell mediated absorbing manner play limited role on the degradation.
How to advance the degradation function and exert its better use for bone repair through new bone growing in and replacing,is the major problem at present.What need to be noticed is that the nacre powder being researched was just belong to micron-scale powder,and its aragonite crystal had not been separated with the internal organic matter.So this maybe a factor affecting its degradation.Since degradation of nacre mainly bases on the physicochemical resolving manner,so it can be considered that as the material's particle size decreases its degradation will increase.
With the development of materialogy,nano-artificial bone,the biomaterial prepared through nanotechnology had emerged.Its structure was similar with natural bone,fit for human body cell and macromolecule to recognize,improving the bioactivity,availability and biocompatibility.Lots of researches showed that nano materials had satisfactory biocompatibility and osteoinduction,which had better bioactivity and biodegradation than non-nano materials.Meanwhile,because of the characteristics of quantum size effect,small size effect,surface effect and macroscopic quantum tunneling effect of nano-particle in this nano-material,its degradation function had been advanced.
Basing on the former research of our study group,this research task is to compose the nano-scale nacre powder made through mechanical grinding with PDLLA(poly-D,L-lactide acid) to prepare porous structure artificial bone in the same tectmology as former,in the process of solvent casting/heat ompressing/particulate leaching.The nano-scale nacre powder reserve the inherent bioactive component.In addition,the faintly alkaline environment brought by nacre powder will be balance out with the weak acid degradation product of PDLLA,then the biodegradation of composite artificial bone will be improved on this theory. Comparing this new nano artificial bone with the former micron nacre/PDLLA composite artificial bone,analyzing on the aspects of material's physicochemical characterization,cytological test in vitro and implanting test in vivo,this research is to explore the biological effect and biodegradation ability of this new composite artificial bone which is composed of the natural nano-material nano-nacre powder and the synthetic macromolecule biomaterial PDLLA.
Objectives:
1.To explore the changes and the applying rationality of the nano-nacre artificial bone developed from the former nacre artificial bone;
2.To research the biocompatibility of nano-nacre artificial bone with osteoblast;
3.To observe the biodegradation of nano-nacre powder in the bone defect area of rats,and the biocompatibility of nano-nacre artificial bone in rat's body;
4.To investigate the function of nano-nacre artificial bone on repairing the radial diaphyseal defects of rabbits.
Methods
1.Prepration of the material:The nano-scale nacre powder made through mechanical grinding was composed with PDLLA(poly-D,L-lactide acid) to prepare the cylinder shape with a center hole artificial bone,while the micron-scale nacre powder was also composed with PDLLA to prepare the cylinder shape artificial bone without hole.Both of them were fabricated in the process of solvent casting/heat compressing/particulate leaching.
2.Test of the characterizations of material:Checked the particle size of nano-nacre powder and micron-nacre powder,and the pore size of two kinds of artificial bone made from those powders under the observation of SEM(scanning electron microscope).Checked the porosity of two kinds of artificial bone through density measurement method.And checked the biomechanical characteristics with the MTS858 universal mechanical testing machine.All the parameters were analyzed for comparing.
3.Cytological experiments:Co-cultured the 3rd generation osteoblast with two kinds of artificial bone.Then observed the growth situation under inverted phase contrast microscope and scanning electron microscope.Checked the proliferation of osteoblast 1,3,5,7 days after being co-cultured with CCK-8 method.Finally,7 days after being co-cultured,the total protein contents were checked with BCA method and the alkaline phosphatase activities were checked with AKP test kit.
4.Implantation of the nacre powder and artificial bone slice:The nano-nacre powder and the artificial bone slice made from this powder and PDLLA (poly-D,L-lactide acid) were implanted into the femoral holes and the femoral muscle sacs of rats respectively.Normal micron-nacre powder and the artificial bones made from this powder and PDLLA were implanted as control group, and blank group was set up without implanting anything.X-ray photography were performed immediately and at 2,4 and 8 weeks after operation.The animals were injected with tetracycline for fluorescent mark before being killed.Then, the gross specimens,histological and SEM observations were performed.
5.Implantation of the artificial bone for repairing segmental bone defect:Created 1.5cm radial defects on both sides of New Zealand white rabbits.Then implanted the nano-nacre artificial bone into the bone defect of left side,and implanted the micron-nacre artificial bone into the other side as control group.Meanwhile, blank group was set up without implanting anything.X-ray photography and bone mineral density checking were performed immediately and at 4,8,16 and 24 weeks after operation.The animals were injected with tetracycline for fluorescent mark before being killed.Then,the gross specimens and histological observations were performed.
Results
1.Nano-nacre artificial bone(NNAB) was prepared,3.5mm×3.5mm×15mm,in cylinder shape with a center hole;While micron-nacre artificial bone(MNAB) was prepared,3.5mm×3.5mm×15mm,in cylinder shape without hole.
2.It was checked with the SEM that the average scale of nano-nacre powder was 50.4±12.4nm and the average pore size of it's correlative artificial bone was 215.7±77.5μm,whereas the micron-scale nacre powder was 5.0±3.0μm and the correlative artificial bone had the 205.1±72.0μm average pore size,which denoted that the former powder belong to nano-scale powder while the later just micron-scale,and the difference of pore size between two kinds of artificial bones was not significant(P＞0.05).The porosities of nano-nacre artificial bone and the control group were(65.4±2.9)%and(53.4±2.2)%respectively,the former was higher than the later(P＜0.05).The biomechanical tests showed that two kinds of artificial bones had not significant difference on compressive strength and Young's modulus(P＞0.05),while the flexural strength of nano-nacre artificial bone was less than the control group(P＜0.05).
3.The results of CCK-8 showed that the difference were not significant in each group,the proliferation of osteoblast reached the peak at the 5th day;7 days after being co-cultured,the total protein content of study group was higher than control group and blank group(P＜0.05),while the difference between control group and blank group was not significant(P＞0.05);The difference of alkaline phosphatase activities among three groups was not significant(P＞0.05);The SEM view showed that osteoblast attached and grew well in two kinds of artificial bone.
4.X-ray photography showed that two kinds of powder started to degrade in 2 weeks;this phenomenon became more appear in 4 weeks,nano-nacre powder degraded faster than micron-nacre powder,while the hole shadow was easy to be found;in 8 weeks,all the femoral holes recovered and returned to normal bone mineral density in all groups.Analysis of tetracycline fluorescent double marks in the hard tissue grinding slices indicated that new bone grew fastest around the bone defect area in study group,while most slowly in blank group(P＜0.05); SEM(scanning electron microscope) observation showed that nano-nacre powder degraded more quickly.The same result can be found through the demineralized sections morphometric analysis,and both of the composite artificial bones made from those two kinds of nacre powder had the good connection with the adjacent tissue in rats body without apparent inflammatory response.
5.X-ray photography showed that rabbit's bone defects healed faster in study group since NNAB implanted than in control group since MNAB implanted.At 24 weeks after operation,bone density in radial defects had nearly accessed to the normal area,while lower in control group,and turned up nonunion in blank group;The checking of BMD(bone mineral density) showed that results in study group were higher than those in control group at 8,16 and 24 week(P＜0.05), and the difference between the BMD values in study group at 24 week and those in blank group was not significant(P＞0.05).The gross specimens showed satisfactory histocompatibility both in study group and in control group,with bone tissue growing from two sides into the center of implanted materials; Normal slices in HE stain and hard tissue grinding slices in Stevenel's blue/Van Geison's picro-fuchsin stain showed that the bone growth tendency was better in study group than that in control group,and the medullary cavity had been penetrated to the implanted materials in study group at 24 week;Analysis of tetracycline fluorescent double marks in the hard tissue grinding slices indicated that new bone in both groups grew fastest 8 weeks after surgery,while slow down at 16 week.The growth speeds of new bone were faster in study group than those in control group at 4,8 and 16 week(P＜0.05).
Conclusions
1.Nano-nacre powder,raw material of NNAB,has the nano-scale particle size. NNAB has satisfactory pore size and porosity.Its biomechanical characters meet with the characteristic requisition of implanting artificial bone.
2.The preparation of this NNAB with a center hole is more reasonable.With this structure the problem that the particulate is difficult to be leached out thoroughly can be resolved.The center hole of NNAB can increase the contact surface with the internal environment of implanting area,be beneficial for the internal material to exchange,or be utilized as the container of bone growth factor or medicine if need.
3.NNAB belong to a kind of nano-material without cytotoxicity,has satisfactory biocompatibility.It has not harmful effects on the growth,proliferation and differentiation of osteoblast.It can somewhat promote the function of cell and benefit the adhesion and growth of cell.It has satisfactory bone conductive function and meet with the basic requisition of implanting material of artificial bone.
4.The degradation of nano-nacre powder is better.It's degradation mainly bases on the physicochemical resolving manner.Moreover,the functions of osteoinduction and prompting bone formulation of nano-nacre powder have enlarged.
5.NNAB possesses satisfactory biocompatibility.It's degradation and ossification abilities are better than those of MNAB.All those indicat that NNAB is a promising bone substitute material.
引文
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