Author: Hanna Cho

Publisher:

Published: 2017

Total Pages:

ISBN-13:

DISCLOSURES: Jinha Kwon (N), Ran Zhuang (N), Do-Gyoon Kim (N), Hanna Cho (N) INTRODUCTION: Bone is a highly-heterogeneous composite material consisting of soft organic constituents (i.e., mostly type I collagen) and hard inorganic mineral (i.e., crystalline carbonated apatite). The collagen molecules are secreted by osteoblasts (i.e., bone forming cells) to build a structural matrix, which is strengthened by the subsequent mineral deposition. Thus, the mineralization during the bone formation and remodeling process is a key factor of modulating bone stiffness by controlling the structural and compositional heterogeneity of bone, often referred as bone quality. To clarify its underlying mechanism, a tool to characterize the bone quality at the same length scale as collagen fibrils and carbonated apatite (i.e., nanoscale level) is essential. In previous studies, Scanning Electron Microscope (SEM) and Transmission Electron Microscopy (TEM) have been widely used to observe the microstructure of bone matrix in the nanometer scale2,3. However, these techniques can only measure morphological information of the sample and require an electrical coating on the sample. To measure nanomechanical properties, nanoindentation has been widely used but its microscale tip fails to separate the regions of collagen and mineral. To overcome these limitations, we applied an advanced Atomic Force Microscopy (AFM) technique, called bi-modal AFM4, which can simultaneously map the nanoscale morphology and nanomechanical properties by utilizing two mode frequencies. Using the bi-modal AFM, we successfully characterized the chronical change of bone quality in a dental implant sample with 4 weeks of healing period, in which age of the bone tissue can be easily identified by the location from the metal implant. METHODS: Following IACUC approval. an adult male beagle dog (10-15 kg) received a dental implant at the second premolar in its mandible. At the 4-weeks of post-implantation healing period, the animal was euthanized to dissect the bone implant construct. The specimen was fixed in a formalin solution for 7 days, and embedded in methyl metharylate resin, and cut to expose bone and implant interface. Finally, the section was polished with 1 u00b5m diamond paste and prepared on a glass slide. Upon completion of sample preparation, the sample was characterized by a commercial AFM system (MFP-3D infinity, Asylum Researchu00ae) using a commercial AFM cantilever (AC160TS-R3, spring constant 26 N/m, OLYMPUSu00ae). To perform the bi-modal AFM, two flexural resonant modes of the AFM cantilever (instead of one resonant mode as in the typical tapping mode operation) were excited and the resulting responses in these two frequencies were monitored by a laser detector system. The first resonant mode is used to get the topographic information of the sample, while a higher resonant mode is used to discriminate different mechanical properties and, thereby, to visualize relative material compositions.RESULTS SECTION: Figure 1 shows the optical microscopic image of the metal and bone matrix at the bone implant interface. Because the interfacial bone matrix undergoes active modeling and remodeling after implantation, the relatively newer bone matrix likely exists at the location closer to the metal implant. Thus, the red and blue box in Figure 1b represent a newer and older bone region, respectively, where the advanced bi-modal AFM was performed. The AFM results are shown in Figure 2: a-c in the old bone region and d-f in the newer bone region. While Figures 2a-b and d-e show the topography map in a 20x20 u00b5m2 and 4x4 u00b5m2 area, respectively, Figures 2c and 2f show its stiffness map in the 4x4 u00b5m2 area. The lower resolution morphology maps shown in Figures 2a and 2d cannot distinguish the difference between these two regions. The higher-resolution morphology maps in Figures 2b and 2e shows somewhat better discrimination in the morphological information, but it is not easy to interpret how different they are. The results get fully comprehensible in the high-resolution stiffness maps in Figures 2e and 2f, in which the brighter color represents higher stiffness. In the stiffness maps, the triangular shapes with higher stiffness are clearly interpreted as minerals, while the lower stiffness particles are collagen. These results explicitly characterize the bone quality by identifying the heterogeneity of bone. Moreover, it is evident that the collagen fibrils get highly aligned along the crystalline structure of minerals along with the progress of the bone healing and remodeling process. DISCUSSION: The morphology and stiffness maps of bone matrix in a newly formed and pre-existing regions in a bone implant system were successfully obtained through an advanced bi-modal AFM technique. The current findings show the structural difference of bone matrix depending on the tissue age, in which the arrangement of collagen fibril is ordered as the remodeling proceeds. In addition, the stiffness maps obtained by the bi-modal AFM techniques help to understand its mechanical structure in nanometer scale. The alignment of the old bone matrix was clearly shown through the stiffness map, although it was hard to observe the alignment through morphological information only. In the future study, we will perform a careful calibration on the stiffness mapping to quantify Youngu2019s modulus and measure variable regions and samples to investigate the mechanism of bone mineralization process. SIGNIFICANCE/CLINICAL RELEVANCE: This is the first study to characterize bone quality depending on tissue age in nanometer scale through an advanced bi-modal AFM technique, which help to obtain a better understanding of bone healing process.REFERENCES: [1] Martin, R. B et al., Skeletal tissue mechanics 2015, [2] Natalie R. et al., Acta Biomaterialia 2014; 3815u20133826, [3] Natalie R. et al., Bone 2013; 93u2013104, [4] Garcia, R.et al., European Polymer Journal 49, 2013;1897u20131906.