Study Of Biomimetic Mineralization And Regulation

Bone,enamel,dentin and cementum are unique biominerals in craniofacial tissues,which have the remarkable mechanical properties of toughness,strength,stiffness and resistance to wearing due to their specific structures.The hydroxyapatite(HA)is the main component in these hard tissues in which HA crystals are highly oriented into complex architecture and hierarchical structures.These unique properties of hard tissues became acctractive for not only biologists to study the mechanism of biomineralization,but also a lot of scientists to synthesize new biomaterials or new functional materials with biomimetical techniques.In vivo,the biomineralization is mediated by specialized cells,such as osteoblasts and odontoblasts synthesize the extracellular matrix in which the collagen is the predominant matrix protein,and the ameloblasts synthesize different proteins to form enamel extracellular matrix in which the amelogenin is the abundant component.These matrix macromolecules not only involve in regulating the content of mineral ions and sequester these ions into the mineralizing area,but also directly induce the nucleation,orientiation,and growth of mineral particles with their highly ordered structures formed by self-assembling.Most of biomeralization is formed from a transient mineral phase,such as amorphous calcium phosphate(ACP)or octacalcium phosphate (OCP).Control over biomineral properties can be accomplished by regulation of particle size,shape,crystal orientation,and polymorphic structure.However,it is still unclear that how the mineral ions initially to be recruitted to form the primary crystals,how the other physical and chemical factors affect the biomineralization,and the how the collagen works for the process of the biomineralization.In our study,we designed a set of models to set up a system to mimic the process of mineralization in vitro.In this system,the condition of mineralization could be controled to study the whole process of mineralization in detail and the effect of single factors like fluoride, pH value,temperature,reaction time and collagen matrix on biomineralization.Then we investigated the function roles of fluoride ions and collagen in apatite formation,and the effects of pH value,temperature and time on biominerlization.In our first experiment,we design and made a set of standard models.With this model,the cation selective membrane could be fixed and the membrane is able to efficiently control of the flux of calcium ion and the formation of calcium phosphates precipitates,thus we can observe the initiation of mineralization. After 1 day reaction,the results show the amophous calcium phosphate firstly deposited on the cation seletive membrane and formed the nanoscale particles,then these mineral particles merged together to form into short strains.After 3 days reaction,there are new crystals forming firstly in the area where the amophous calcium phosphate had formed previously,and these newly formed crystals appeared in different shapes,such as rods,and nano threads and ribbons.These different crystals indicates different calcium phosphate were formed in the same reaction.In the following work,we test the different physical and chemical factors for biomineralization with this mondel system.It shows fluoride ions have substantial effect on apatite formation even in a very small concentration,and ppm level of fluoride can induce to form prism crystals.The results inditates fluoride can accelerates the transition of OCP to HA,and improves the crystallinity of apatite and induces formation of F-containing hydroxyapatite Ca₅(PO₄)₃OH_1-XF_X.The pH value is also a sensitive factor for mineralization.In our study,the weak acid condition shows induce the crystal grow into prism shape while the slight basic condition will induce the formation of sheets of calcium phosphate.When we used the neutral pH value,5ppm fluoride condition,the better shape of calcium phsphate crystals were sythesized after 21 days reaction.These crystals equally growed into small prisms with 3um height,and their shape and arrangement are very similar as natural enamel crystals.Analysis by energy dispersive x-ray diffraction shows most of these precipitate are OCP,including a small amount of HA.In our third experiment,we add collagen gel into the reaction space to observe the effect of organic matix on biominerali -zation.The results show there are small strands of calcium phosphate crystals forming on the cation selective membrane,and most of these product are HA by means of EDX analysis.In addtion,the amount of HA in the precipitates increased as the concentration of collagen increased.However,there is no difference between 20%and 30%collagen,which indicates 20% collagen is maximum concentraion suitable for our experiment.The interesting experiment is we synthesized dentine-like crystals in the condition of 10%gelatine,pH value 6.8,and 5ppm fluoride.These crystals appeared small and thin ribbons,which were arranged into a net of calcium precipatates along the orientation of collagen fibers.Some of them are like peritubular dentin,and some of them looks like intertubular dentin.Most of these precipates are OCP by means of EDX analysis.Finally,we try different conditions and synthesized the HA crystals in the condition of 7.2 pH value,5ppm fluoride and 20%gelatin.The HA crystals appeared typical hexagonal prism or hexagonal cone with around 100nm diameter and 2um height,growing along c-axis direction.The neighboring crystal connected or mergered into strands of crystals or longer crystals,which similar as dentin crystals.Analysis by energy dispersive x-ray diffraction shows most of these precipitate are HA.Our study set up a model system for biomineralization which can be easily and accurately controlled to simulate the process of biomeralization.With this model system,we study the effect of physical and chemical factors on biomeralization,and also got better condition for the biomeralization study in vitro.The significant part of our study is when the organic matrix was added into this biomineralization system,we found the collagen could induce the nucleation,growth,orientation and arragement of HA crystals.Finally,we synthesized the dentin-like and enamel-like calcium phosphate crystals.Not like the cation-selective membrane system reported in previous studys,in which the cation-selective membrane and the dialysis membrane are simply be fixed by rubber rings,and the reaction space can't be controlled.We set up a set of stardard model in which the membranes could be fixed and not broken in this model,and thus the experiment could be repeated accuratelly. In addition,the reaction space could be quantitively created to study the effect of the matrix protein on the biomineralization. Significantly,we synthesized the dentin-like minerals after we tested the different concentration of fluoride,pH value,temperature, collagen matrix and reaction period.These quite large mount of crystals covered the whole cation-selective membrane,about 1mm height,which has perfect prism structure,quality as nature hydroxyapatite.All these results give us good indications to study dentin regenesis and biomimicly synthesize new functional organic-inoraganic components as well as uncover the mechanism of biomineraliza ton in the future.