Preparation And Study Of The Nano-Nacre Artificial Bone

BackgroundNacre(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.Methods1.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.Results1.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).Conclusions1.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.