人类牙骨质的纳米结构表征
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摘要
明确表征人类牙骨质中的微观结构、特别是纳米结构有助于仿生学研究，对于牙损伤和牙周疾病的治疗，尤其是种植体表面微形貌的仿生设计具有重要意义。本论文利用扫描电子显微镜、光学显微镜表征了健康的人类牙齿中无细胞牙骨质的微米结构和纳米结构特点。首先比较多种不同制样方法制备的牙根牙骨质样品的微观结构形貌，找到较为完美保持牙骨质微观结构、特别是纳米结构的样品制备方法。进一步利用扫描电子显微镜研究发现：无细胞牙骨质主要由无机的纳米粒子和有机的纳米纤维组成，这些纳米粒子表现出沿着纳米纤维线性排列生长的趋势。基于毛细管微模塑技术（MIMIC），应用光固化树脂复制了无细胞牙骨质中有机组分的分布及其在存在形态，利用盐酸溶液刻蚀除去无机纳米粒子之后，在聚合物中留下了许多相互独立的类似纺锤形的纳米孔状结构。这些纳米孔结构一方面说明牙骨质中的无机纳米粒子被有机组分分割包围、彼此不相接触，另一方面表明这些纳米粒子不是球形的，而是椭球形或者纳米片状的。通过本论文的研究，我们获得了牙骨质表面形貌的更直观及科学的认识，为人工牙根仿生学研究提供更客观的理论依据，对口腔组织工程学研究具有重要的意义。
Cementum is a complex composite hard tissue that joins root dentin toalveolar bone by the way of a periodontal ligament (PDL). Differs from enameland dentin, composition of cementum is similar to that of bone. However,unlike the bone, cementum does not undergo continuous remodeling, andcontinues to grow in thickness throughout life. As scientists believe thatresearch on human cementum would provide essential information to engineermaterials for tooth replacement, more and more interests have been attracted inthis field during the past ten years. Many excellent works have been reportedon determining the composition, structures, types and distribution of cementumin human teeth. Cho et al found that cementum is formed as primary acellularcementum and secondary cellular cementum. Acellular cementum, which isfound on the cervical third and middle portions of the root, plays the mostimportant role in fixing the fibers of the PDL.
As the development of bionics, it has been found that the specialfunctionalities of some organisms are more likely related to their unique micro-or nanostructures rather than their intrinsic property of materials. Therefore,determining the microstructure of cementum might help us design new kinds ofreplacement materials for teeth injury and disease treatment, which mighteventually allow restoration of functions for patients with periodontal diseases.Using different characterization techniques including scanning electronicmicroscopy (SEM)，transmission electron microscopy (TEM), atomic forcemicroscopy (AFM), X-ray diffraction (XRD)、 Fourier transform infraredspectroscopy (FTIR) and light microscopy with polarization techniques,structure of cementum has been widely studied. For example, Strocchi et al have reported the orientation of collagen fiber bundles in cementum matrix.Based on electron microscopy characterization of the tissue, several researcherssuggested that the Sharpey’s fibers embedding into the cementum on the rootsurface at right angle, are more calcified in center than that of the peripheralportion. A limitation of the published works regarding cementum microstr-ucture is the sample preparation process including sectioning followed bychemical treatment of the sectioned surfaces, especially the chemical treatmentincluding tissue fixation and demineralization, in addition to different types ofenzymatic treatments. These treatments would cause artifacts in the structure,especially the nanoscopic microstructures. Therefore, most works reported inthe literature are focused on the histological study of cementum structures overmicrometer scale, studies on the nanostructures of cementum are seldomknown.
Herein, we characterized the nanostructures in acellular cementum ofhealth human teeth by SEM. Twelve teeth including6maxillary first premolars,2mandibular first premolars and4mandibular second premolars, wereobtained from11to16year old human subjects requiring operation oforthodontic extraction following a protocol approved by the UCSF Committeeon Human Research. Informed consents were obtained from all the patients. Allteeth used in this study were periodontally healthy, and preserved in watercontaining thymol after extraction．
In the second chapter, we studied the effects of sample preparationtechniques on the morphology of nanostructures in sectioned teeth specimens.In order to investigate the effects of sectioning technique on determination ofthe nanostructures in tooth specimens, we compared the morphology ofsamples sectioned by freeze fracturing and diamond wafering blade cuttingmethods. From the SEM images showing the morphology of dentin andcementum in specimens fabricated by the freeze fracturing method, we can see the microstructures of dentinal tubule clearly in dentin, and the sub-micrometerscale texture structures in cementum were preserved very well. However, in thesamples sectioned by diamond wafering blade, both the micrometer scalestructures of dentinal tubule and sub-micrometer scale structures in cementumwere destroyed severely. Thus we know that the freeze fracturing method caneffectively preserve the microstructures of tooth specimen in sectioning process.Therefore, we supposed that the freeze fracturing method is an ideal techniquefor sectioning the teeth specimens.
We have also studied the effects of different after-treatment of the toothroot specimens on the determination of the microstructures. The freezefractured specimens were divided into three groups. Group (A) was washedwith distilled water and ethanol in sequence without any after-treatment. Group(B) was immersed into0.25wt%trypsin solution at room temperature for48h,and then washed with distilled water and ethanol in sequence. These twogroups of specimens were dried in oven of60oC for72h before mounting onSEM stubs. In order to burn off the organic component, specimens of Group (C)were heated from room temperature to600oC (10oC/min) in an oven, andannealed at600oC for6h, then mounted on the SEM stubs directly aftercooling to room temperature. The microstructures in these different sampleswere characterized and compared by optical microsopy.
In the third chapter, nanostructures in acellular extrinsic fiber cementumhave been determined. A comparative study on the nanoscopic morphology ofacellular cementum was carried out on root samples prepared by differentmethods. By comparing the microscopic morphology of three groups ofspecimens prepared by different after-treatments, we have found that acellularextrinsic fiber cementum is mainly composed of two kinds of nanostructures:inorganic nanoparticles and organic nanofibers (As it is well known that themain composite of these inorganic nanoparticles is hydroxyapatite). And the characterized results showed that the size of inorganic nanoparticles rangesfrom20to70nm, the organic nanofibers are with diameter of less than80nm.Burning off the organic component of tooth specimens, we could see that theburnt cementum was composed of nanoparticles. Some of these nanoparticleswere larger than100nm, which are obviously larger than the un-burnt ones.This suggests that the burning treatment would cause fusion of inorganicnanoparticles in cementum.
The morphology difference between acellular extrinsic fiber cementumand dentin in burnt has also been characterized. We have found that some ofthe inorganic nanoparticles in acellular cementum fused into inorganicnanofibers during the process of annealing. This result suggests that theinorganic nanoparticles in acellular cementum are linearly arranged along theorganic fibers, which is different from the disordered nanoparticles in dentin.
By MIMIC method we have prepared polymer multi-porous structuresusing the teeth specimens as templates.By immersing dried tooth specimens inmonomers of UV curable resin and polymerizing under UV light, we haveembedded the specimens in polymer resin. The inorganic component of teeth ismainly hydroxylapatite, which is soluble in hydrochloric acid. By immersingthe polymer embedded with tooth specimens in hydrochloric acid (10wt%) fora period, the inorganic component was etched out, resulting in multiporouspolymer structures templated from human teeth. SEM images of nanostructureson the surface formed during the etching treatment, where is originally theinterface between the polymer resin and tooth cementum. The nanofiber-likestructures shown by the images should be copy of organic components (mainlySharpey’s fiber) in acellular cementum, which are almost perpendicular to thesurface of tooth roots. From the enlarged SEM images we can see that amongthe fiber-like nanostructures there are a lot of nano-pores. The size of themrange from20to70nm, which is similar to the size of inorganic nanoparticles characterized in chapter2. We believe that these nano-pores were formed byusing inorganic nanoparticles of cementum as templates. Another interestingphenomenon is that most of these nano-pores are of spindle shape rather thanround. This suggests that the inorganic nanoparticles in cementum might beoval or even nanosheet in shape. As these nano-pores are isolated from eachother, we speculate that the inorganic nanoparticles of acellular cementumdistribute isolatedly in the organic components.
In summary, through a comparative study of tooth root samples preparedby different methods, we have found that the acellular cementum is mainlycomposed of inorganic nanoparticles of20-70nm and organic nanofibers withdiameter less than80nm. Based on the micro-molding in capillaries (MIMIC)strategy, we have prepared polymer multi-porous structures by using theinorganic components of tooth as templates. It has been found that thenano-pores are similar in size with the nanoparticles of cementum. Moreover,most of these nano-pores are isolated, not connect to each other, which suggeststhat the inorganic nanoparticles are separated by the organic components anddistribute isolatedly in cementum. By comparing the nanoscopic morphology ofcementum with dentin, we have found that the inorganic nanoparticles in theacellular cementum are arranged linearly along the nanofibers, while theinorganic nanoparticles in dentin are disordered. We believe that the presentwork would provide references in biomimetic preparation of tissue engineeringscaffolds for tooth treatment, and give us inspiration to design new types oftooth implant with better biological interface.
Cementum is a complex composite hard tissue that joins root dentin toalveolar bone by the way of a periodontal ligament (PDL). Differs from enameland dentin, composition of cementum is similar to that of bone. However,unlike the bone, cementum does not undergo continuous remodeling, andcontinues to grow in thickness throughout life. As scientists believe thatresearch on human cementum would provide essential information to engineermaterials for tooth replacement, more and more interests have been attracted inthis field during the past ten years. Many excellent works have been reportedon determining the composition, structures, types and distribution of cementumin human teeth. Cho et al found that cementum is formed as primary acellularcementum and secondary cellular cementum. Acellular cementum, which isfound on the cervical third and middle portions of the root, plays the mostimportant role in fixing the fibers of the PDL.
As the development of bionics, it has been found that the specialfunctionalities of some organisms are more likely related to their unique micro-or nanostructures rather than their intrinsic property of materials. Therefore,determining the microstructure of cementum might help us design new kinds ofreplacement materials for teeth injury and disease treatment, which mighteventually allow restoration of functions for patients with periodontal diseases.Using different characterization techniques including scanning electronicmicroscopy (SEM)，transmission electron microscopy (TEM), atomic forcemicroscopy (AFM), X-ray diffraction (XRD)、 Fourier transform infraredspectroscopy (FTIR) and light microscopy with polarization techniques,structure of cementum has been widely studied. For example, Strocchi et al have reported the orientation of collagen fiber bundles in cementum matrix.Based on electron microscopy characterization of the tissue, several researcherssuggested that the Sharpey’s fibers embedding into the cementum on the rootsurface at right angle, are more calcified in center than that of the peripheralportion. A limitation of the published works regarding cementum microstr-ucture is the sample preparation process including sectioning followed bychemical treatment of the sectioned surfaces, especially the chemical treatmentincluding tissue fixation and demineralization, in addition to different types ofenzymatic treatments. These treatments would cause artifacts in the structure,especially the nanoscopic microstructures. Therefore, most works reported inthe literature are focused on the histological study of cementum structures overmicrometer scale, studies on the nanostructures of cementum are seldomknown.
Herein, we characterized the nanostructures in acellular cementum ofhealth human teeth by SEM. Twelve teeth including6maxillary first premolars,2mandibular first premolars and4mandibular second premolars, wereobtained from11to16year old human subjects requiring operation oforthodontic extraction following a protocol approved by the UCSF Committeeon Human Research. Informed consents were obtained from all the patients. Allteeth used in this study were periodontally healthy, and preserved in watercontaining thymol after extraction．
In the second chapter, we studied the effects of sample preparationtechniques on the morphology of nanostructures in sectioned teeth specimens.In order to investigate the effects of sectioning technique on determination ofthe nanostructures in tooth specimens, we compared the morphology ofsamples sectioned by freeze fracturing and diamond wafering blade cuttingmethods. From the SEM images showing the morphology of dentin andcementum in specimens fabricated by the freeze fracturing method, we can see the microstructures of dentinal tubule clearly in dentin, and the sub-micrometerscale texture structures in cementum were preserved very well. However, in thesamples sectioned by diamond wafering blade, both the micrometer scalestructures of dentinal tubule and sub-micrometer scale structures in cementumwere destroyed severely. Thus we know that the freeze fracturing method caneffectively preserve the microstructures of tooth specimen in sectioning process.Therefore, we supposed that the freeze fracturing method is an ideal techniquefor sectioning the teeth specimens.
We have also studied the effects of different after-treatment of the toothroot specimens on the determination of the microstructures. The freezefractured specimens were divided into three groups. Group (A) was washedwith distilled water and ethanol in sequence without any after-treatment. Group(B) was immersed into0.25wt%trypsin solution at room temperature for48h,and then washed with distilled water and ethanol in sequence. These twogroups of specimens were dried in oven of60oC for72h before mounting onSEM stubs. In order to burn off the organic component, specimens of Group (C)were heated from room temperature to600oC (10oC/min) in an oven, andannealed at600oC for6h, then mounted on the SEM stubs directly aftercooling to room temperature. The microstructures in these different sampleswere characterized and compared by optical microsopy.
In the third chapter, nanostructures in acellular extrinsic fiber cementumhave been determined. A comparative study on the nanoscopic morphology ofacellular cementum was carried out on root samples prepared by differentmethods. By comparing the microscopic morphology of three groups ofspecimens prepared by different after-treatments, we have found that acellularextrinsic fiber cementum is mainly composed of two kinds of nanostructures:inorganic nanoparticles and organic nanofibers (As it is well known that themain composite of these inorganic nanoparticles is hydroxyapatite). And the characterized results showed that the size of inorganic nanoparticles rangesfrom20to70nm, the organic nanofibers are with diameter of less than80nm.Burning off the organic component of tooth specimens, we could see that theburnt cementum was composed of nanoparticles. Some of these nanoparticleswere larger than100nm, which are obviously larger than the un-burnt ones.This suggests that the burning treatment would cause fusion of inorganicnanoparticles in cementum.
The morphology difference between acellular extrinsic fiber cementumand dentin in burnt has also been characterized. We have found that some ofthe inorganic nanoparticles in acellular cementum fused into inorganicnanofibers during the process of annealing. This result suggests that theinorganic nanoparticles in acellular cementum are linearly arranged along theorganic fibers, which is different from the disordered nanoparticles in dentin.
By MIMIC method we have prepared polymer multi-porous structuresusing the teeth specimens as templates.By immersing dried tooth specimens inmonomers of UV curable resin and polymerizing under UV light, we haveembedded the specimens in polymer resin. The inorganic component of teeth ismainly hydroxylapatite, which is soluble in hydrochloric acid. By immersingthe polymer embedded with tooth specimens in hydrochloric acid (10wt%) fora period, the inorganic component was etched out, resulting in multiporouspolymer structures templated from human teeth. SEM images of nanostructureson the surface formed during the etching treatment, where is originally theinterface between the polymer resin and tooth cementum. The nanofiber-likestructures shown by the images should be copy of organic components (mainlySharpey’s fiber) in acellular cementum, which are almost perpendicular to thesurface of tooth roots. From the enlarged SEM images we can see that amongthe fiber-like nanostructures there are a lot of nano-pores. The size of themrange from20to70nm, which is similar to the size of inorganic nanoparticles characterized in chapter2. We believe that these nano-pores were formed byusing inorganic nanoparticles of cementum as templates. Another interestingphenomenon is that most of these nano-pores are of spindle shape rather thanround. This suggests that the inorganic nanoparticles in cementum might beoval or even nanosheet in shape. As these nano-pores are isolated from eachother, we speculate that the inorganic nanoparticles of acellular cementumdistribute isolatedly in the organic components.
In summary, through a comparative study of tooth root samples preparedby different methods, we have found that the acellular cementum is mainlycomposed of inorganic nanoparticles of20-70nm and organic nanofibers withdiameter less than80nm. Based on the micro-molding in capillaries (MIMIC)strategy, we have prepared polymer multi-porous structures by using theinorganic components of tooth as templates. It has been found that thenano-pores are similar in size with the nanoparticles of cementum. Moreover,most of these nano-pores are isolated, not connect to each other, which suggeststhat the inorganic nanoparticles are separated by the organic components anddistribute isolatedly in cementum. By comparing the nanoscopic morphology ofcementum with dentin, we have found that the inorganic nanoparticles in theacellular cementum are arranged linearly along the nanofibers, while theinorganic nanoparticles in dentin are disordered. We believe that the presentwork would provide references in biomimetic preparation of tissue engineeringscaffolds for tooth treatment, and give us inspiration to design new types oftooth implant with better biological interface.
引文
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