First evidence of octacalcium phosphate@osteocalcin nanocomplex as skeletal bone component directing collagen triple–helix nanofibril mineralization

Tibia trabeculae and vertebrae of rats as well as human femur were investigated by high-resolution TEM at the atomic scale in order to reveal snapshots of the morphogenetic processes of local bone ultrastructure formation. By taking into account reflections of hydroxyapatite for Fourier filtering the appearance of individual alpha–chains within the triple–helix clearly shows that bone bears the feature of an intergrowth composite structure extending from the atomic to the nanoscale, thus representing a molecular composite of collagen and apatite. Careful Fourier analysis reveals that the non–collagenous protein osteocalcin is present directly combined with octacalcium phosphate. Besides single spherical specimen of about 2 nm in diameter, osteocalcin is spread between and over collagen fibrils and is often observed as pearl necklace strings. In high-resolution TEM, the three binding sites of the γ-carboxylated glutamic acid groups of the mineralized osteocalcin were successfully imaged, which provide the chemical binding to octacalcium phosphate. Osteocalcin is attached to the collagen structure and interacts with the Ca–sites on the (100) dominated hydroxyapatite platelets with Ca-Ca distances of about 9.5 Å. Thus, osteocalcin takes on the functions of Ca–ion transport and suppression of hydroxyapatite expansion.


FIB preparation:
Imaging of collagen fibrils and discussion of preparation artefacts.

Beam damage:
TEM experiments and discussion.

Figure S9
Beam damage experiments (sample for FIB cut): Time dependent high-resolution at dose rate and magnification as used for the actual experiment.
12. Figure S10 Beam damage experiments (sample for FIB cut): Time dependent high-resolution at high dose rate and high magnification. 13. Figure S11 Beam damage experiments (FIB cut of biomimetic sample): Imaging triple-helical fibrils at high-resolution in fluorapatite -gelatine composites.

FIB preparation: Imaging of collagen fibrils and discussion of preparation artefacts
The combination of focused ion beam (FIB) TEM sample preparation and transmission electron microscopy offers several advantages for submicron and nanostructural investigations. The FIB technique involves specimen thinning using a beam of ions of sufficient energy to remove material. There are several advantages of this technique compared to the usually used microtomy, one benefit is eliminating the need for time-consuming, multi-stage chemical fixation and embedding procedure. Additionally, the mechanical sectioning with a diamond knife potentially can introduce mechanical damages of such brittle objects. As disadvantage it should be mentioned that artifacts are created by the high energy gallium beam such as destruction of the inorganic crystal structure (glass formation) and damaging of the organic components. where the morphology and surface between bone and bone ceramics was characterized. 9 Mostly, FIB was used to generate polished surfaces in order to carry out EDX investigations on the bone/implant or the cell/bone-ceramic interface. 10 of human forearm bone-anchored amputation prosthesis. 13 This group mostly characterized the interface morphology of implants and bone by using EFTEM and high-resolution TEM [14][15][16][17][18][19][20][21] extended to electron tomography on bone-titanium oxide interface in humans at the nanometer scale. 19,22 In the above mentioned cases, it was proved by TEM imaging that the collagen fibril is not affected and that the 67 nm striation is preserved by the FIB milling procedure. 8,17 Besides gallium ion milling also argon ion beam polishing is used which is a fast preparation technique for evaluating interfaces of osseo-integrated implants. 23 In the course of our former extensive investigations we could show that in case of the beam sensitive organic/inorganic composites prepared by FIB milling encompassing human and artificial otoconia, bovine teeth, oxalate dihydrate polyacrylic acid composites, desmosponges, layered organic solar cells, nanocolloidal mesocrystals and gelatin-fluorapatite composites the sample structure is preserved. 5,23-32 For example, high-resolution of the apatite (002) lattice of 3.44 Å combined with the imaging of individual collagen triple-helices proved to be possible in FIB cut samples of fluorapatite-gelatine composites. 5,31 FIB cut samples prepared in cross section can be helpful to image repair layers covering pain sensitive nerve channels of bovine dentin at high-resolution. The dentin repair was achieved by applying specific toothpaste containing a composite of nanosized hydroxyapatite and gelatine. 32 In our study we used FIB cuts of rat tibia (see Figure 1,2,4,5,S6,S9,S10 and SEM images S1-S4).
In order to rule out artefacts caused by the FIB cut preparation we also investigated conventional diamond thin cuts of human femur ( Figure 3,8,9 and Figure S7) and rat vertebra ( Figure 6 and Figure S7).

Beam damage in TEM: Experiments and discussion
In order to test and to determine the stability of the FIB cut sample, we performed time- whereas the FFT seems still not to be affected, keeping the resolution of 1.48 Å. Therefore, for this area we exclude that under the actual recording conditions the crystal is already affected. In order to prove that the observed structures are not simply beam damage artifacts, we recorded high-resolution images with high electron doses. In this way, we take into account damage that is caused by the additional time needed for focusing the sample and for recording the images at high magnification. As we have already shown in numerous publications the triple-helical structure is well preserved in the apatite structure in FIB cuts 5,31 as well in classical diamond ultramicrotomy. 33,34 In Figure S11a,b high-resolution is shown from a FIB cut of a fluorapatitegelatine composite, where a bundle of triple-helices is revealed. Generally, formation of bubbles is observed during decomposition of bulk apatite samples due to electron irradiation with a high dose in the TEM 35 and also formation of CaO. 36,37 In our case, formation of bubbles and CaO was not observed. Application of higher electron dose even may lead to increased crystallinity in bone as reported by Hong et al. 38 or transition of hexagonal to monoclinic apatite. 39 In case of OCP the beam damage should be a higher concern than in apatite because of the presence of hydrated layers. Nevertheless, as shown by e.g. Suvorova and Buffat using a field emission microscope at 300 kV and magnification as high as 510.000x to 640.000x it is possible to image OCP in high-resolution. 40 Xin et al. also imaged successfully OCP in high-resolution at 200 kV with field emission and tested the stability of OCP by different recording times. 41 Also, our group investigated thin, biomimetically grown OCP platelets in high-resolution which were synthesized to be used as dentine repair material. 32  When one performs electron microscopy on organic and biological materials, beam damage will play a decisive role. [42][43][44][45][46][47][48] Therefore, the attainable resolution is not given by the point resolution of the microscope but by the critical electron dose on the sample. Various undesirable phenomena will occur, such as loss of high-resolution structural information as well as longrange order accompanied by chemical decomposition, mass loss, and decrease of mechanical stability. Finally, with the higher brightness of the field emission guns (FEG) and higher acceleration voltage, the probability of knock-on damage will also increase for organic and biological samples. Therefore, it is important to estimate roughly the influence of the electron beam damage on unstained biological samples. In general, proteins are extremely beam sensitive, and some are even destroyed by a dose of ~1 e Å -2 . 42 Former measurements by electron diffraction on glucose embedded bacteriorhodopsin 47 show that the finer structural detail of 7 Å vanishes at a dose of 1.4 e Å -2 , and that the 3 Å reflection already reaches the critical dose below 1 e Å -2 at room temperature. In a very early paper by Glaeser et al. the beam damage of glucose embedded catalase crystals is described. 48 They found that the logarithm of critical electron dose is linear with the achievable resolution. In summary, for biological samples, the main restriction of resolution is given by the beam sensitivity of the protein and thus only a low dose of about 1-10 e Å -2 can be applied. For example, if a dose of 10 e Å -2 is exerted, one should be able to measure ~3 nm as smallest object detail. Cryo TEM studies are achieving reproducible 3D resolutions in the order of 6 to 10 Å as shown on an archeon S-layer displayed structural details of ~10 Å at -120 °C with spot scan illumination using a total dose of about 1 e Å -2 . 49 Kimura et al. imaged the surface of bacteriorhodopsin with a resolution of ~4 Å at 4.2 K, using low dose and an acceleration voltage of 400 kV. 50 The total dose amounted to about less than 10 e Å -2 .
Thus, by using a cooling holder, with low dose and by embedding the sample into a polymer matrix combined with higher acceleration voltage, one is be able to reach at best a resolution of 4 Å. Another possibility is to use reduced acceleration voltage of 80 kV since the knock-on damage threshold of carbon atoms lies above this range. 51 Nowadays promising steps are taken in the direction of even applying 20 kV by using chromatic aberration correction for atomic resolution combined with spherical aberration-corrector. 52 In our actual experiments, beam damage artefacts should be minimized, since we used the low-dose technique, meaning focusing on a vicinal area and using low illumination. Also, we applied lower magnification of about 320.000x than usual (500.000-800.000x). In addition, collagen is a highly interconnected biopolymer and thus more stable than the above discussed proteins. For example, in our former holography experiments we used 24 e Å -2 at 200 kV for imaging the 67 nm striation pattern without occurrence of beam damage of a non-stained collagen sample. 53 In another work, we were able to record HR-TEM images of silicification of collagen on H. sieboldii glass sponges. 54 Finally, we experienced since over a decade of TEM and FIB cut preparation on apatite-gelatine composites that the apatite serves as a protection matrix and stabiliser for the buried and integrated protein inside. 5      The dose rate amounts about 900 e/Å 2 and achieved resolution is 1.72 Å as read out from the FFT. There is no indication of occurrence of nanobubbles or for CaO formation. (c) Highresolution of slightly shifted area shown in (a). Defocus and astigmatism were adjusted with respect to (b). The arrow marks the same crystal in (b) and (c). Again, even at the accumulated dose of ~178.500 e/Å 2 the neighbor area is still well preserved.  molecule is observed where the individual alpha-chains tend to a parallel arrangement, which may causes less mechanical stress with respect to the apatite lattice. 31