<meta charset="utf8"></head><body>ניתוח מולטימודאלי של היווצרות עצם באמצעות מודלים explant

<ol>
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E. A., Ito, K., Hofmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+bone+models+and+their+potential+in+preclinical+evaluation.">Ex vivo bone models and their potential in preclinical evaluation.</a> Curr Osteoporo Rep. 19 (1), 75-87 (2021).</li><li>Chan, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+trabecular+bone+explant+model+of+osteocyte-osteoblast+co-culture+for+bone+mechanobiology.">A trabecular bone explant model of osteocyte-osteoblast co-culture for bone mechanobiology.</a> Cell Mol Bioeng. 2 (3), 405-415 (2009).</li><li>Doke, S. K., Dhawale, S. 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(35), e1707 (2010).</li><li>Kazanci, M., Roschger, P., Paschalis, E. P., Klaushofer, K., Fratzl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+osteonal+tissues+by+Raman+spectral+mapping:+Orientation-composition.">Bone osteonal tissues by Raman spectral mapping: Orientation-composition.</a> J Str Biol. 156 (3), 489-496 (2006).</li><li>Unal, M., Ahmed, R., Mahadevan-Jansen, A., Nyman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compositional+assessment+of+bone+by+Raman+spectroscopy.">Compositional assessment of bone by Raman spectroscopy.</a> Analyst. 146 (24), 7464-7490 (2021).</li><li>Oliver, W. C., Pharr, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+technique+for+determining+hardness+and+elastic+modulus+using+load+and+displacement+sensing+indentation+experiments.">An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments.</a> J Mater Res. 7 (6), 1564-1583 (1992).</li><li>Proffen, B. L., McElfresh, M., Fleming, B. C., Murray, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+anatomical+study+of+the+human+knee+and+six+animal+species.">A comparative anatomical study of the human knee and six animal species.</a> Knee. 19 (4), 493-499 (2012).</li><li>Allen, M. J., Houlton, J. E. F., Adams, S. 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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.20 filters</td>
			<td>VWR&#160;</td>
			<td>28145-501</td>
			<td></td>
		</tr>
		<tr>
			<td>18 G needle (1,2x40 mm)</td>
			<td>Sterican</td>
			<td>4665120</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL syringe</td>
			<td>HENKE-JECT</td>
			<td>8300005762</td>
			<td></td>
		</tr>
		<tr>
			<td>37% hydrochloric acid&#160;</td>
			<td>VWR&#160;</td>
			<td>1.00317.1000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid (glacial)&#160;</td>
			<td>Sigma&#160;</td>
			<td>A6283&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone&#160;</td>
			<td>VWR&#160;</td>
			<td>20063-365&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue 8GX</td>
			<td>VWR&#160;</td>
			<td>361186</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide</td>
			<td>VWR&#160;</td>
			<td>318612</td>
			<td></td>
		</tr>
		<tr>
			<td>Apatitic tricalcium phosphate&#160;</td>
			<td>Centre for Biomedical and Healthcare Engineering (Mines Saint Etienne, France)</td>
			<td>TV26U&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Azophloxine</td>
			<td>Sigma&#160;</td>
			<td>210633</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzoyl peroxide</td>
			<td>Sigma&#160;</td>
			<td>8.01641.0250</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP2</td>
			<td>Medtronic&#160;</td>
			<td>InductOs 1.5 mg/mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Brillant crocein&#160;</td>
			<td>Aldrich&#160;</td>
			<td>2107507</td>
			<td></td>
		</tr>
		<tr>
			<td>CTVox&#160;</td>
			<td>Bruker</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>DataViewer&#160;</td>
			<td>Skyscan</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond blade</td>
			<td>Struers</td>
			<td>MOD13</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond saw</td>
			<td>Struers</td>
			<td>Accutom-50</td>
			<td></td>
		</tr>
		<tr>
			<td>DiaPro Mol B3 diamond solution</td>
			<td>Struers</td>
			<td>40600379</td>
			<td></td>
		</tr>
		<tr>
			<td>DiaPro Nap B1 diamond solution</td>
			<td>Struers</td>
			<td>40600373</td>
			<td></td>
		</tr>
		<tr>
			<td>Dibasic sodium phosphate (Na2HPO4)</td>
			<td>Sigma&#160;</td>
			<td>102404598</td>
			<td></td>
		</tr>
		<tr>
			<td>Dibutyl Phtalate</td>
			<td>Chimie-Plus Laboratoires</td>
			<td>28656</td>
			<td></td>
		</tr>
		<tr>
			<td>DragonFly software&#160;</td>
			<td>ORS</td>
			<td>2022.1.0.1231.&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) high glucose, GlutaMAX(TM), pyruvate</td>
			<td>ThermoFisher Scientific</td>
			<td>31966-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosine Y- Surgipath&#160;</td>
			<td>Sigma&#160;</td>
			<td>1002830105</td>
			<td></td>
		</tr>
		<tr>
			<td>Erythrosin B</td>
			<td>Sigma&#160;</td>
			<td>102141057</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>VWR&#160;</td>
			<td>20820362</td>
			<td></td>
		</tr>
		<tr>
			<td>Eukitt&#160;</td>
			<td>Dutscher</td>
			<td>6.00.01.0003.06.01.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL</td>
			<td>Sarstedt</td>
			<td>62.547.254</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferric chloride hexahydrate (FeCl3, 6H2O)&#160;</td>
			<td>Merck&#160;</td>
			<td>1.03943.0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)&#160;</td>
			<td>Eurobio</td>
			<td>CVFSVF00</td>
			<td></td>
		</tr>
		<tr>
			<td>Fuchsine acid</td>
			<td>Merck&#160;</td>
			<td>1.05231.0025&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Biosera</td>
			<td>MS01NG100J</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Sigma&#160;</td>
			<td>86.118.9&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Isostatic press</td>
			<td>Nova Suisse</td>
			<td>Pmax 1500 bars</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser diffraction granulometry</td>
			<td>&#160;Malvern</td>
			<td>Mastersizer 3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Light green</td>
			<td>Prolabo&#160;</td>
			<td>28947135</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium carbonate</td>
			<td>Sigma&#160;</td>
			<td>A13149&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>MD-Mol polishing cloth</td>
			<td>Struers</td>
			<td>40500077</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylcyclohexane</td>
			<td>VWR&#160;</td>
			<td>8.06147.1000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylcyclohexane&#160;</td>
			<td>VWR&#160;</td>
			<td>8.06147.1000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylcyclohexane&#160;</td>
			<td>VWR&#160;</td>
			<td>8.06147.1000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylmethacrylate</td>
			<td>Sigma&#160;</td>
			<td>8.00590.2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-CT, micro-scanner&#160;</td>
			<td>Bruker</td>
			<td>Skyscan 1272</td>
			<td></td>
		</tr>
		<tr>
			<td>Monobasic sodium phosphate (NAH2PO4)</td>
			<td>Sigma&#160;</td>
			<td>71496</td>
			<td></td>
		</tr>
		<tr>
			<td>Mortar</td>
			<td>Fritsch&#160;</td>
			<td>Pulverisette 6</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N, Dimethylanilin</td>
			<td>Sigma&#160;</td>
			<td>803060</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoindentation station</td>
			<td>Anton Paar</td>
			<td>NHT2</td>
			<td></td>
		</tr>
		<tr>
			<td>ND-Nap polishing cloth</td>
			<td>Struers</td>
			<td>40500080</td>
			<td></td>
		</tr>
		<tr>
			<td>OATS Osteochondral Autograft Transfer System Set, 4,75 mm</td>
			<td>Arthrex</td>
			<td>AR-1981-04S</td>
			<td></td>
		</tr>
		<tr>
			<td>OATS Osteochondral Autograft Transfer System Set, 8 mm</td>
			<td>Arthrex</td>
			<td>AR-1981-08S</td>
			<td></td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>Ral</td>
			<td>M15</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma&#160;</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Peel-a-way disposable embbedding moulds</td>
			<td>Polysciences, Inc</td>
			<td>18646C-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (P/S)</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphomolybdic acid&#160;</td>
			<td>Sigma&#160;</td>
			<td>221856-100 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphotungstic acid</td>
			<td>Aldrich&#160;</td>
			<td>12863-5&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Polishing machine</td>
			<td>Sturers</td>
			<td>Dap V</td>
			<td></td>
		</tr>
		<tr>
			<td>Poupinel</td>
			<td>MEMMERT</td>
			<td>TV26U&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Raman microspectrometer</td>
			<td>Renishaw</td>
			<td>InVia Qontor</td>
			<td></td>
		</tr>
		<tr>
			<td>Safran du G&#226;tinais&#160;</td>
			<td>Labonord&#160;</td>
			<td>11507737</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>Carl Zeiss</td>
			<td>Evo LS 10</td>
			<td></td>
		</tr>
		<tr>
			<td>SEM</td>
			<td>Zeiss</td>
			<td>Carl Zeiss Evo LS10</td>
			<td></td>
		</tr>
		<tr>
			<td>SiC foils/Grinding papers</td>
			<td>Struers</td>
			<td>40400008 (#320), 40400011 (#1000), 40400122 (#2000), 40400182 (#4000)</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver paint</td>
			<td>Electron microscopy sciences</td>
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comprehensive characterization of tissue mineralization in an ex vivo model

Bone defects, whether caused by trauma, tumor resection, congenital anomalies, or infection, represent a major challenge for regenerative medicine. These alterations compromise the structural integrity of the skeletal system, leading to discomfort, functional impairment, and a reduction in patients&#39; quality of life.
To overcome these challenges, innovative bone repair strategies have emerged, with a focus on enhancing osteogenesis and bone tissue regeneration. These approaches include the use of implantable, injectable, or 3D-printable bone substitutes, which can be of natural origin (e.g., bio-sourced macromolecules, animal-derived hydroxyapatite) or synthetic (e.g., bioglasses, calcium phosphates)1. To enhance their low inherent ability to guide and stimulate bone regeneration, bone substitutes can be loaded with osteoinductive factors, such as bone morphogenetic proteins (BMPs), to promote osteogenic differentiation of progenitor cells and enhance bone formation2.
Bone formation is based on the initial formation of a collagen matrix, which is then mineralized by hydroxyapatite crystals, thereby reinforcing the bone structure3. This process confers specific stiffness and strength to the bone. The quality of the mineralized tissue is intricately governed by its microstructural attributes and degree of mineralization4. This quality plays a pivotal role in bone healing and the functionality of the regenerated bone5. However, characterizing bone mineralization remains a challenging task due to the inherent variability across multivariate studies6,7,8.
In addition, initial evaluations of the biocompatibility, cytocompatibility, and differentiation potential of bone graft substitutes are typically conducted in vitro. However, methodological disparities impede the seamless comparison of outcomes. Furthermore, these in vitro studies do not fully capture the multicellular interactions and complex dialogue between cell populations, including bone marrow cells, which are essential for regulating the bone regeneration process9. This lack of accurate representation of the bone microenvironment may compromise the accuracy of subsequent preclinical studies10.
Although in vivo assessments provide a more accurate representation of physiological contexts, they are constrained by ethical, logistical, and financial considerations. Consequently, ex vivo evaluations play a pivotal role as an interface between in vitro and in vivo studies, serving as a necessary intermediate step before moving on to experiments on living subjects11,12,13.
In this context, the implementation of comprehensive characterization methodologies is needed to appraise the quality of regenerated bone tissue and to ensure the relevance of the strategy before moving on to a preclinical model. Consequently, we propose a protocol based on the analysis of an explant model using sheep knee joint tissue. This innovative methodology involves implanting BMP2-loaded cement into the explants and conducting a detailed analysis of tissue mineralization after 28 days of culture.
The technical approaches employed in this study are diverse and complementary, collectively providing a comprehensive approach to evaluating the quality of regenerated bone tissue (Figure 1). High-resolution micro-CT imaging enables detailed 3D visualization of the bone structure, providing valuable insights into the mineral density, morphology, and integrity of the newly formed tissue. This technique is crucial for assessing the efficacy of bone regeneration and monitoring the progression of mineralization over time. Nanoindentation is a precise approach for determining the mechanical properties of the tissue, such as its hardness and strength. By measuring the response of the material to a force applied on a nanometric scale, this method enables the assessment of the robustness and quality of the mineralized tissue. Histological examinations using common staining such as hematoxylin/eosin/saffron (HES), Goldner&#39;s trichrome, and Movat&#39;s pentachrome provide invaluable insights into tissue structure and composition. These stainings&#39; allow differentiation of the various tissue components, including cells, extracellular matrix, and mineral deposits, thereby enabling a comprehensive qualitative assessment of the bone regeneration process. Backscatter scanning electron microscopy (SEM) mapping offers a high-resolution visualization of the surface of the samples, allowing detailed analysis of the degree of mineralization of the bone matrix, as well as the interfaces between the implanted material and the host tissue. Finally, Raman spectroscopy provides information regarding the molecular composition of the tissue, particularly through the identification of specific components such as proteins, lipids, and minerals. This approach enables the characterization of the mineralized matrix and the detection of growth factors such as BMP2, thereby providing crucial information on the persistence of pro-osteogenic stimuli in the regeneration medium.
Using a multi-disciplinary approach, integrating various analytical techniques, our study aims to provide a thorough and comprehensive assessment of the quality of regenerated bone tissue, thus providing a solid basis for the evaluation of bone graft substitutes and their potential clinical application.

<meta charset="utf8"></head><body>מחבר זרקור: טכניקות מתקדמות לאפיון מינרליזציה של רקמות במחקר התחדשות עצם

אפיון מקיף של מינרליזציה של רקמות במודל Ex Vivo

Our research focuses on understanding and ameliorating bone regeneration. Here we aim to extensively characterize tissue mineralization using various and complementary technical approaches to facilitate the early identification of promising candidates for bone regeneration therapies. To evaluate the quality and structure of newly mineralized tissue, we use complementary approaches, including high resolution 3D imaging with micro-CT associated with deep learning analysis.Nano indentation, histological examinations, back scattering scanning electron microscope mapping, and Raman spectroscopy. Repair of bone defects is a major challenge in regenerative medicine. We developed explant models that facilitate the evaluation of bone regeneration, and the monitoring of mineralization over time.

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous ...

comprehensive-characterization-tissue-mineralization-an-ex-vivo

Research

JoVE Journal

Bioengineering

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

393 Views.  Nantes Université, Univ Angers, Oniris, CHU Nantes, INSERM, Regenerative Medicine and Skeleton, RMeS, UMR 1229. המחקר שלנו מתמקד בהבנה ובשיפור התחדשות העצם. כאן אנו שואפים לאפיין באופן נרחב מינרליזציה של רקמות באמצעות גישות טכניות שונות ומשלימות כדי להקל על זיהוי מוקדם של מועמדים מבטיחים לטיפולי התחדשות עצם. כדי להעריך את האיכות והמבנה של רקמה חדשה שעברה מינרליזצ...

Video: מחבר זרקור: טכניקות מתקדמות לאפיון מינרליזציה של רקמות במחקר התחדשות עצם

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous modalities that are currently available for analysis. Here, we propose a workflow for a comprehensive evaluation of new bone formation using a relevant large animal osseous ex vivo explant. A bone defect (diameter = 3.75 mm; depth = 5.0 mm) is created in an explanted sheep femoral head and injected with a macroporous bone substitute loaded with a pro-osteogenic growth factor (bone morphogenetic protein 2 - BMP2). Subsequently, the explant is maintained in culture for a 28-day period, allowing cellular colonization and subsequent bone formation. To evaluate the quality and structure of newly mineralized tissue, the following successive methods are set up: (i) Characterization and high-resolution 3D images of the entire explant using micro-CT, followed by deep learning image analyses to enhance the discrimination of mineralized tissues; (ii) Nano-indentation to determine the mechanical properties of the newly formed tissue; (iii) Histological examinations, such as Hematoxylin/Eosin/Saffron (HES), Goldner's trichrome, and Movat's pentachrome to provide a qualitative assessment of mineralized tissue, particularly with regard to the visualization of the osteoid barrier and the presence of bone cells; (iv) Back-scattering scanning electron microscopy (SEM) mapping with internal reference to quantify the degree of mineralization and provide detailed insights into surface morphology, mineral composition, and bone-biomaterial interface; (v) Raman spectroscopy to characterize the molecular composition of the mineralized matrix and to provide insights into the persistence of BMP2 within the cement through the detection of peptide bonds. This multimodal analysis will provide an effective assessment of newly formed bone and comprehensive qualitative and quantitative insights into mineralized tissues. Through the standardization of these protocols, we aim to facilitate interstudy comparisons and improve the validity and reliability of research findings.

The proposed protocol entails a global approach to assess bone formation in the context of bone regeneration using multimodal analyses. It aims to provide qualitative and quantitative information on new bone formation, enhancing the rigor and validity of basic and pre-clinical investigations.

Analysis of Osteochondral Explants Using Micro-CT and Deep Learning Segmentation Techniques

To begin, use an osteochondral autograph transfer system to create a 4.75 millimeter diameter defect in the lateral condyle of a euthanized sheep. Using an orthopedic hammer, create a defect 10 millimeters in depth, and retrieve the osteochondral explant from the condyle. Prepare the calcium phosphate cement, supplemented with 40 micrograms per milliliter of BMP 2, and load the cement paste into a three milliliter syringe.Then inject the cement into the explant defect with an 18 gauge needle. Turn on the micro tomography x-ray machine, and place the explant tube on the sample holder. Set the resolution to 10.7 micrometers, and exposure time to 1, 200 milliseconds, with a one millimeter aluminum filter at 80 kilovolts and 125 microamperes.Average three images for each 0.45 degree rotation increment to enhance the signal to noise ratio. To conduct image segmentation, use the integrated segmentation wizard to train a deep learning model for distinguishing between bone and cement. Select a representative zone containing bone, cement, and background from the reconstructed micro-computed tomography images, and segment this first frame.Now, in the model tab, generate a deep learning model and select the 3D net routine. Then right click on the generated model, and set the experimental parameters as the depth of five, patch size 32 by 32, algorithm, stride ratio of 0.25. And 10 x data.Use the segmented frame to train the deep learning model by clicking the train button. Once the training is complete, define a second frame and automatically segment it using the predict function. Then click on export to publish the trained model, and apply it to the entire micro-computed tomography dataset by selecting segment, exported model, segment, full dataset.

ניתוח צמחים אוסטאוכונדרליים באמצעות מיקרו-CT וטכניקות סגמנטציה של למידה עמוקה

Osteochondral Bone Explant Preparation for Comprehensive Assessment of the Quality of Regenerated Bone Tissues

To begin, dehydrate the osteochondral bone explant collected from sheep in a 40 milliliter glass vial containing 25 milliliters of dehydration solution. Place the vials on a rotating wheel for one hour at room temperature. After the last wash, replace the dehydration solution with 25 milliliters of xylene.Place the glass vials on the rotating wheel for one hour at room temperature. Next, add 10%benzoyl peroxide, 10%dibutyl phthalate, and 450 microliters of N, N-Dimethylaniline diluted 1 to 20 in propanol, and place the glass vial at minus 20 degrees Celsius overnight. Now, place the explant into a medium-sized embedding mold in a plastic box.Pour the methylmethacrylate benzoyl peroxide dibutyl phthalate N, N-aniline solution into the mold. Ventilate the mold with nitrogen flow for five minutes. Close the box hermetically and place it at four degrees Celsius for 48 hours for MMA polymerization and hardening.After two days, remove the resin containing the explant from the mold. Cut the polymethylmethacrylate block containing the explant using a diamond saw along its long axis to generate a 1.5 millimeter thick section at 3000 RPM with a speed of three millimeters per minute. Grind the section with silicon carbide paper with ascending numbers.Carbon coat the thick section with a 10 nanometer carbon film. Mount the section on an aluminum stub. Then, create a silver paint bridge between the top of the section and the stub to allow for electron charging evacuation to the ground.Place the stub on the scanning electron microscopy, or SEM, stage. Close the chamber and initiate a vacuum. Turn on the electron beam and adjust the SEM settings to operate in back-scattered electron mode.Set the gray level standards to 25 for carbon, 225 for aluminum, and 253 for silicon. After calibrating the SEM with the standards, acquire images of the specimen in back-scattered electron mode. Use the back-scattered electron image of the specimen to convert the gray levels into calcium content.Then, plot the calcium distribution content of the image. Place the section on the stage of the Raman microspectrometer. Calibrate wave number for accuracy, and align the laser prior to measurement.To collect the Raman spectra, use a 785 nanometer laser at 30 milliwatts. Set the time of integration to 20 seconds, repeat three times, and use a spectral range of 350 to 1, 800 per centimeter with a grading of 1, 200 lines per millimeter. Once the nanoindentation system is calibrated, place the sample in the optical system and identify the location for the nanoindentation.Then, move the sample under the indentation device. Set the indentation depth to 900 nanometers with a loading-unloading speed of 40 millimeters per minute and a 15 second pause between loading and unloading. Set the cosine coefficient for bone tissue to 0.3 and start the nanoindentation.