We want to thank the technical facilities involved in the collection and processing of specimens, including SC3M (SFR Francois Bonamy (UMS 016), University of Nantes), SFR ICAT (University of Angers), BIO3, HiMolA, and SC4BIO. The Inserm UMR_S 1229 RMeS is supported by grants from the French Government through Inserm, Nantes Universit&#233;, Univ Angers and Oniris VetAgroBio institutions. CL is also grateful to HTL Biotechnology.

Multimodal Analysis of Bone Formation Using Explant Models

<ol>
	<li>Feroz, S., Cathro, P., Ivanovski, S., Muhammad, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+bone+grafts+and+substitutes:+A+review+of+recent+advancements+and+applications.">Biomimetic bone grafts and substitutes: A review of recent advancements and applications.</a> Biomed Eng Adv. 6, 100107 (2023).</li><li>Tsuji, K., 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=BMP2+activity,+although+dispensable+for+bone+formation,+is+required+for+the+initiation+of+fracture+healing.">BMP2 activity, although dispensable for bone formation, is required for the initiation of fracture healing.</a> Nat Genet. 38 (12), 1424-1429 (2006).</li><li>Hadjidakis, D. J., Androulakis, I. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boneremodeling.">Boneremodeling.</a> Ann N Y Acad Sci. 1092, 385-396 (2006).</li><li>Boivin, G., Meunier, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+degree+of+mineralization+of+bone+tissue+measured+by+computerized+quantitative+contact+microradiography.">The degree of mineralization of bone tissue measured by computerized quantitative contact microradiography.</a> Calcified Tiss Int. 70 (6), 503-511 (2002).</li><li>Boivin, G., 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=Influence+of+remodeling+on+the+mineralization+of+bone+tissue.">Influence of remodeling on the mineralization of bone tissue.</a> Osteoporo Int. 20 (6), 1023-1026 (2009).</li><li>Zanghellini, B., 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=Multimodal+analysis+and+comparison+of+stoichiometric+and+structural+characteristics+of+parosteal+and+conventional+osteosarcoma+with+massive+sclerosis+in+human+bone.">Multimodal analysis and comparison of stoichiometric and structural characteristics of parosteal and conventional osteosarcoma with massive sclerosis in human bone.</a> J Str Biol. 216 (3), 108106 (2024).</li><li>Trento, G., 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=formation+around+two+titanium+implant+surfaces+placed+in+bone+defects+with+and+without+a+bone+substitute+material:+A+histological,+histomorphometric,+and+micro-computed+tomography+evaluation.">formation around two titanium implant surfaces placed in bone defects with and without a bone substitute material: A histological, histomorphometric, and micro-computed tomography evaluation.</a> Clin Implant Dent Relat Res. 22 (2), 177-185 (2020).</li><li>Palmquist, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multiscale+analytical+approach+to+evaluate+osseointegration.">A multiscale analytical approach to evaluate osseointegration.</a> J Mater Sci Mater Med. 29 (5), 60 (2018).</li><li>Bud&#225;n, F., 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=Novel+radiomics+evaluation+of+bone+formation+utilizing+multimodal+(SPECT/X-ray+CT)+in+vivo+imaging.">Novel radiomics evaluation of bone formation utilizing multimodal (SPECT/X-ray CT) in vivo imaging.</a> PLoS ONE. 13 (9), e0204423 (2018).</li><li>Hulsart-Billstr&#246;m, G., 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+surprisingly+poor+correlation+between+in+vitro+and+in+vivo+testing+of+biomaterials+for+bone+regeneration:+results+of+a+multicentre+analysis.">A surprisingly poor correlation between in vitro and in vivo testing of biomaterials for bone regeneration: results of a multicentre analysis.</a> Eur Cells Mater. 31, 312-322 (2016).</li><li>Cramer, E. 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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternatives+to+animal+testing:+A+review.">Alternatives to animal testing: A review.</a> Saudi Pharma J. 23 (3), 223-229 (2015).</li><li>Moussi, H., 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=New+formulation+of+injectable+and+degradable+calcium+phosphate/silanized+hyaluronic+acid+composite+foam:+Investigation+in+a+rabbit+model+of+long+bone+defect.">New formulation of injectable and degradable calcium phosphate/silanized hyaluronic acid composite foam: Investigation in a rabbit model of long bone defect.</a> SSRN. , (2024).</li><li>Par&#233;, A., 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=Standardized+and+axially+vascularized+calcium+phosphate-based+implants+for+segmental+mandibular+defects:+A+promising+proof+of+concept.">Standardized and axially vascularized calcium phosphate-based implants for segmental mandibular defects: A promising proof of concept.</a> Acta Biomater. 154, 626-640 (2022).</li><li>Roschger, P., Fratzl, P., Eschberger, J., Klaushofer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+quantitative+backscattered+electron+imaging+for+the+measurement+of+mineral+density+distribution+in+human+bone+biopsies.">Validation of quantitative backscattered electron imaging for the measurement of mineral density distribution in human bone biopsies.</a> Bone. 23 (4), 319-326 (1998).</li><li>Roschger, P., Plenk, H., Klaushofer, K., Eschberger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+scanning+electron+microscopy+approach+to+the+quantification+of+bone+mineral+distribution:+backscattered+electron+image+grey-levels+correlated+to+calcium+K+alpha-line+intensities.">A new scanning electron microscopy approach to the quantification of bone mineral distribution: backscattered electron image grey-levels correlated to calcium K alpha-line intensities.</a> Scanning Micro. 9 (1), 75-86 (1995).</li><li>Porter, A., 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=Quick+and+inexpensive+paraffin-embedding+method+for+dynamic+bone+formation+analyses.">Quick and inexpensive paraffin-embedding method for dynamic bone formation analyses.</a> Sci Rep. 7, 42505 (2017).</li><li>Goldschlager, T., Abdelkader, A., Kerr, J., Boundy, I., Jenkin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Undecalcified+bone+preparation+for+histology,+histomorphometry+and+fluorochrome+analysis.">Undecalcified bone preparation for histology, histomorphometry and fluorochrome analysis.</a> J Vis Exp. (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. B., Rushton, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+surgical+anatomy+of+the+stifle+joint+in+Sheep.">The surgical anatomy of the stifle joint in Sheep.</a> Vet Surg. 27 (6), 596-605 (1998).</li><li>Kleuskens, M. W. A., van Donkelaar, C. C., Kock, L. M., Janssen, R. P. A., Ito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+human+osteochondral+culture+model.">An ex vivo human osteochondral culture model.</a> J Ortho Res. 39 (4), 871-879 (2021).</li></ol>

The authors have no conflicts of interest to disclose.

Repair of bone defects is a major challenge in regenerative medicine to restore mobility, reduce pain, and improve the quality of life of affected individuals. The use of explant models offers a number of advantages compared to in vivo studies for the investigation of bone defect repair. In addition to ethical considerations, this model allows for the rigorous control of experimental conditions and the reduction of biological variability, thereby facilitating the generation of more accurate and reproducible results. Furthermore, the mechanical and biological properties of sheep bone are closely aligned with those of human bone in comparison to other animals23,24. This makes the sheep explant model a particularly relevant choice for preclinical studies and a representative model of human clinical conditions. 
Along with the development of imaging and material sciences methodology, the explant model described here represents a good model to investigate the effects of regenerative medicine on skeletal tissues in a controlled environment reducing experimental bias. The dimensions and morphology of the explant are optimal for detailed analysis of neo-osteogenesis and material properties, enabling comprehensive assessment and characterization25. These findings emphasize the model's potential for clinical translation, offering a robust framework for evaluating bone graft substitutes and enhancing the likelihood of successful clinical outcomes in bone regeneration therapies. Furthermore, the model facilitates precise evaluation of osteointegration and biomaterial performance, which are critical factors in developing effective bone repair strategies.
To ensure the success of the study, it is critical to follow the protocol's key steps meticulously. One of the most important aspects is ensuring the sample is fully dehydrated during the embedding process. Any remaining moisture can hinder proper resin embedding, leading to a blurred and non-transparent resin, which could compromise the clarity and accuracy of the results and could also result in holes during histology sectioning. This careful attention to detail is essential for producing reliable and reproducible findings. In order to ensure accurate conversion of grey levels into a calcium content map, it is essential to employ calibration using a Faraday cup and C, Al, and Si standards for SEM analysis. It is crucial to ensure consistency in the use of the same SEM for all samples, as differences in backscattered electron detectors can lead to variability in the results. In Raman spectroscopy, calibration of the wavenumber and thorough baseline correction are essential to exclude fluorescence background and differentiate between peaks from the embedding resin and the sample. For nanoindentation, achieving a perfectly smooth surface is critical, given the displacement limits of indenters, and calibration with a known standard, such as fused silica, is necessary to ensure reliable measurements.
The modifications and troubleshooting of these techniques frequently address the anisotropic nature of bone material, which has the potential to influence measurement consistency. To illustrate, in Raman spectroscopy, the polarization of the incident laser can have a considerable impact on the recorded intensity. The use of a 20x objective with a low numerical aperture can assist in the mitigation of this issue. It is also of paramount importance to ensure consistent calibration and measurement conditions across all samples in order to maintain data integrity. The inherent anisotropy of bone represents a limitation of these methodologies, as it can affect the uniformity of measurements, particularly in techniques such as Raman spectroscopy. Furthermore, the necessity for precise calibration and environmental control may limit the general applicability if these aspects are not meticulously managed.
The potential applicability of these approaches extends significantly to the evaluation of novel biomaterials and therapeutic strategies for bone repair. The model's capacity to closely mimic human bone physiology provides a robust platform for preclinical testing, enabling a thorough assessment of the safety and efficacy of new treatments in a controlled setting. This model's utility is particularly relevant for accelerating the translational pathway from laboratory research to clinical practice, facilitating the early identification of promising candidates for bone regeneration therapies. By providing a reliable and reproducible method for evaluating bone repair materials, this technique supports the advancement of regenerative medicine and the development of clinically viable solutions.

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.

<table><tbody><tr><td>0.20 filters</td><td>VWR&amp;nbsp;</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&amp;nbsp;</td><td>VWR&amp;nbsp;</td><td>1.00317.1000&amp;nbsp;</td><td></td></tr><tr><td>Acetic acid (glacial)&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>A6283&amp;nbsp;</td><td></td></tr><tr><td>Acetone&amp;nbsp;</td><td>VWR&amp;nbsp;</td><td>20063-365&amp;nbsp;</td><td></td></tr><tr><td>Alcian Blue 8GX</td><td>VWR&amp;nbsp;</td><td>361186</td><td></td></tr><tr><td>Ammonium hydroxide</td><td>VWR&amp;nbsp;</td><td>318612</td><td></td></tr><tr><td>Apatitic tricalcium phosphate&amp;nbsp;</td><td>Centre for Biomedical and Healthcare Engineering (Mines Saint Etienne, France)</td><td>TV26U&amp;nbsp;</td><td></td></tr><tr><td>Azophloxine</td><td>Sigma&amp;nbsp;</td><td>210633</td><td></td></tr><tr><td>Benzoyl peroxide</td><td>Sigma&amp;nbsp;</td><td>8.01641.0250</td><td></td></tr><tr><td>BMP2</td><td>Medtronic&amp;nbsp;</td><td>InductOs 1.5 mg/mL</td><td></td></tr><tr><td>Brillant crocein&amp;nbsp;</td><td>Aldrich&amp;nbsp;</td><td>2107507</td><td></td></tr><tr><td>CTVox&amp;nbsp;</td><td>Bruker</td><td>-</td><td></td></tr><tr><td>DataViewer&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>ORS</td><td>2022.1.0.1231.&amp;nbsp;</td><td></td></tr><tr><td>Dulbecco&#039;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&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>1002830105</td><td></td></tr><tr><td>Erythrosin B</td><td>Sigma&amp;nbsp;</td><td>102141057</td><td></td></tr><tr><td>Ethanol absolute</td><td>VWR&amp;nbsp;</td><td>20820362</td><td></td></tr><tr><td>Eukitt&amp;nbsp;</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, 6H&lt;sub&gt;2&lt;/sub&gt;O)&amp;nbsp;</td><td>Merck&amp;nbsp;</td><td>1.03943.0250</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)&amp;nbsp;</td><td>Eurobio</td><td>CVFSVF00</td><td></td></tr><tr><td>Fuchsine acid</td><td>Merck&amp;nbsp;</td><td>1.05231.0025&amp;nbsp;</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Biosera</td><td>MS01NG100J</td><td></td></tr><tr><td>Hematoxylin</td><td>Sigma&amp;nbsp;</td><td>86.118.9&amp;nbsp;</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>&amp;nbsp;Malvern</td><td>Mastersizer 3000</td><td></td></tr><tr><td>Light green</td><td>Prolabo&amp;nbsp;</td><td>28947135</td><td></td></tr><tr><td>Lithium carbonate</td><td>Sigma&amp;nbsp;</td><td>A13149&amp;nbsp;</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&amp;nbsp;</td><td>8.06147.1000&amp;nbsp;</td><td></td></tr><tr><td>Methylcyclohexane&amp;nbsp;</td><td>VWR&amp;nbsp;</td><td>8.06147.1000&amp;nbsp;</td><td></td></tr><tr><td>Methylcyclohexane&amp;nbsp;</td><td>VWR&amp;nbsp;</td><td>8.06147.1000&amp;nbsp;</td><td></td></tr><tr><td>Methylmethacrylate</td><td>Sigma&amp;nbsp;</td><td>8.00590.2500</td><td></td></tr><tr><td>Micro-CT, micro-scanner&amp;nbsp;</td><td>Bruker</td><td>Skyscan 1272</td><td></td></tr><tr><td>Monobasic sodium phosphate (NAH2PO4)</td><td>Sigma&amp;nbsp;</td><td>71496</td><td></td></tr><tr><td>Mortar</td><td>Fritsch&amp;nbsp;</td><td>Pulverisette 6</td><td></td></tr><tr><td>N,N, Dimethylanilin</td><td>Sigma&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>221856-100 g</td><td></td></tr><tr><td>Phosphotungstic acid</td><td>Aldrich&amp;nbsp;</td><td>12863-5&amp;nbsp;</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&amp;nbsp;</td><td></td></tr><tr><td>Raman microspectrometer</td><td>Renishaw</td><td>InVia Qontor</td><td></td></tr><tr><td>Safran du G&amp;acirc;tinais&amp;nbsp;</td><td>Labonord&amp;nbsp;</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><td>12686-15</td><td></td></tr><tr><td>Standard stub with Faraday cup, carbon, aluminium and silicon standards</td><td>Micro-Analysis Consultants Ltd</td><td>8602</td><td></td></tr><tr><td>T25 flask</td><td>Corning</td><td>430639</td><td></td></tr><tr><td>Xylene</td><td>VWR&amp;nbsp;</td><td>28975.325</td><td></td></tr><tr><td>Xylidine Ponceau</td><td>Aldrich&amp;nbsp;</td><td>19.976-1&amp;nbsp;</td><td></td></tr></tbody></table>

comprehensive characterization of tissue mineralization in an ex vivo model

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.

A micro-CT image of the explant is shown in Figure 2. Using Manual segmentation cannot optimally separate bone from cement, present in the central canal, using global thresholding. To improve the recognition of trabecular bone and cement, we propose to use deep learning. Deep learning is powerful for recognizing biomaterial characteristics and helps to improve the separation between bone and cement, enabling a better assessment of cement-bone interactions. This is of the utmost importance in order to accurately quantify the amount of bone formed at the interface with cement and not to incorporate false pixels in this calculation.
However, simply quantifying bone at the interface is not enough to characterize newly formed bone. A more in-depth study of bone matrix quality and cellular composition is required to better understand the strength of the new bone and the cellular mechanisms that led to the formation of this new matrix. The bone matrix can be characterized using two complementary approaches: qBEI and Raman microspectroscopy. An example of a qBEI image is presented in Figure 3. With qBEI, each grayscale-encoded pixel is transformed into a calcium concentration. This is a prerequisite for assessing the distribution of calcium content in trabecular bone and for understanding (i) the degree of mineralization of newly-formed bone at the interface with cement and (ii) whether the presence of cement disrupts trabecular bone mineralization. The advantage of qBEI is its ability to acquire a high-resolution image in a very short time, unlike Raman microspectroscopy which requires a longer acquisition time. qBEI parameters such as Camean, Capeak and Cawidth can then be derived from the calcium distribution and used to compare the response between two cements or biomaterials.
On the other hand, qBEI does not provide any information on the quality of the organic phase of the bone matrix at the interface with the biomaterial. Additional information regarding the material properties of the organic phase is derived from Raman microspectroscopy. Raman microspectroscopy is performed on the same specimen as used for qBEI after a quick surface grinding and polishing in order to remove the conductive carbon layer. In Raman microspectroscopy, spectra are acquired at each pixel location. Examples of raw unprocessed and processed Raman spectra are provided in Figure 4. Each spectrum contains information on the composition of the pixel analyzed and includes peaks related to the embedding resin, in this case, pMMA, the mineral phase, peaks v1PO4 and v1CO3, and the organic phase, peaks Pro, Hyp, CML, Amide III, PG, CH2, Pen. and Amide I. In the example in Figure 4, the untreated spectrum shows the contribution of the coating resin at ~812 cm-1 and a non-zero curved baseline due to fluorescence, although a near-infrared red laser is used. Spectrum processing is a prerequisite for ensuring accurate calculation of the various Raman parameters, in particular the elimination of background fluorescence. When acquiring data, the user should ensure both accurate positioning and avoidance of detector saturation to confirm the presence of the v1PO4 peak located ~960 cm-1. Following post-processing, peak intensity ratios are computed according to the location of the characteristic vibrational mode.
However, it is questionable whether changes in material composition led to changes in biomechanical response. To gather information on the biomechanics of newly formed bone, we systematically carry out nanoindentation investigations on the same sample used for Raman microspectroscopy. Tissue mechanical information is obtained from nanoindentation curves represented by load versus depth curves (Figure 5). It is worth noting that the depth does not go back to zero at the end of the test, representing the plastic deformation and the permanent damage created to the material. The indentation creep rate is derived from the depth versus time curve.
Finally, histological staining is used to assess the tissue response to the biomaterial at the cellular level (Figure 6). The histological stains proposed allow us to determine the presence of osteoid tissue, but also the identification of cells (macrophages, multinucleated giant cells, osteoclasts, osteoblasts) and the possible presence of a fibrous capsule at the interface with the biomaterial. This is very important for understanding the biocompatibility of a biomaterial, but also the persistence of new bone when the biomaterial is degraded.
Overall, the multi-method approach enables an in-depth study of bone formation, the quality of newly formed bone, and cellular composition. When carried out in the proposed sequential order, all investigations can be performed on the same sample, reducing cost and processing time.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67235/67235fig01.jpg" /> 
Figure 1: Graphical abstract. A summary of the protocol steps is provided here. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67235/67235fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67235/67235fig02.jpg" /> 
Figure 2: Micro-CT and deep learning analyses. (A) 3D reconstruction of micro-CT images. (B) Bone and cement segmentation: comparison between manual segmentation and deep learning. Scale bar = 5 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67235/67235fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67235/67235fig03.jpg" /> 
Figure 3: Quantitative backscattered electron imaging for determining calcium content. (A) The backscattered image composed of grey levels obtained in the scanning electron microscopy (SEM) is converted by the image analysis software into (B) a calcium map showing the hot spots of the bone sample. (C) The distribution of the calcium content versus bone area is plotted, and the three major qBEI parameters, Camean, Capeak, and Cawidth, are computed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67235/67235fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67235/67235fig04.jpg" /> 
Figure 4: Example of Raman spectra. (A) Raw unprocessed Raman spectrum obtained in the spectral range 800 - 1800 cm-1. The fluorescence background is clearly visible as the spectra do not start from 0. The resin contribution (pMMA) is clearly visible at ~812 cm-1. (B) Processed spectrum. The position of the different vibrational modes of interest is indicated on the spectrum. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67235/67235fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67235/67235fig05.jpg" /> 
Figure 5: Investigation of tissue mechanical properties by nanoindentation. (A) The nanoindentation device is composed of an optical part used to define the location of the region of interest at the surface of the specimen block, and then the block is translated under the indentation part. (B) Load versus time and depth versus time are plotted during indentation test and are used to generate the (C) load versus depth curve. In this example the chosen depth was fixed at 400 nm. The maximum load (Lm), the depth at the end of loading (hl), the depth after the pause period (hm), the slope of the unloading segment (S) and the area under the loading-unloading phase of the test (Wplast) are derived from the load versus depth curve and are used to compute the different nanoindentation parameters. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67235/67235fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67235/67235fig06.jpg" /> 
Figure 6: Histological analysis of osteochondral explants treated with cement after 28 days of culture. (A) Movat staining demonstrates the tissue structure, including bone and the cement filling the defect. (B) HES staining illustrates the tissue integration and cellular response around the cement. Scale bar: 2.5 mm. (C) Magnification of the highlighted area in (B), which details the interface between the cement and the surrounding tissue, where cells can be observed invading the cement. Scale bar: 500 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67235/67235fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Advanced Techniques for Characterizing Tissue Mineralization in Bone Regeneration Research

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.

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

613 Views.  Nantes Université, Univ Angers, Oniris, CHU Nantes, INSERM, Regenerative Medicine and Skeleton, RMeS, UMR 1229. 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.

Video: Author Spotlight: Advanced Techniques for Characterizing Tissue Mineralization in Bone Regeneration Research

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Medicine

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. These cell lines, after treatment with ascorbic acid (AA) and &#946;-glycerophosphate (&#946;-GP), undergo complete osteogenic transdifferentiation from proliferation to mineralization and produce matrix vesicles (MVs) that trigger apatite nucleation in the extracellular matrix (ECM).
Based on Alizarin Red-S (AR-S) staining and analysis of the composition of minerals in cell lysates using ultraviolet (UV) light or in vesicles using TEM imaging followed by EDX quantitation and ion mapping, we can infer that osteosarcoma Saos-2 and osteoblastic hFOB 1.19 cells reveal distinct mineralization profiles. Saos-2 cells mineralize more efficiently than hFOB 1.19 cells and produce larger mineral deposits that are not visible under UV light but are similar to hydroxyapatite (HA) in that they have more Ca and F substitutions.
The results obtained using these techniques allow us to conclude that the process of mineralization differs depending on the cell type. We propose that, at the cellular level, the origin and properties of vesicles predetermine the type of minerals.

We present a protocol to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. Their mineralization profiles were analyzed by Alizarin Red-S (AR-S) staining, ultraviolet (UV) light visualization, transmission electron microscopy (TEM) imaging and energy dispersive X-ray microanalysis (EDX).

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in ...

Chemistry

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence of calcification inhibitors, that concomitantly lead to a loss of vessel elasticity and function. Vascular calcification is an important contributor to morbidity and mortality in many pathologies, including chronic kidney disease, diabetes mellitus, and atherosclerosis. Current research models to study vascular calcification are limited and are only viable at the late stages of calcification development in vivo. In vitro tools for studying vascular calcification use end-point measurements, increasing the demands on biological material and risking the introduction of variability to research studies. We demonstrate the application of a novel fluorescently labeled probe that binds to in vitro calcification development on human vascular smooth muscle cells and determines the real-time development of in vitro calcification. In this protocol, we describe the application of our newly developed calcification assay, a novel tool in disease modeling that has potential translational applications. We envisage this assay to be relevant in a broader spectrum of mineral deposition research, including applications in bone, cartilage, or dental research.

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Cardiovascular disease is the leading cause of death worldwide. Vascular calcification contributes substantially to the burden of cardiovascular morbidity and mortality. This protocol describes a simple method to quantify vascular smooth muscle cell-mediated calcium precipitation in vitro by fluorescent imaging.

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence ...

Biochemistry

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.

In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections of mineralized tissues.

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious ...

Biology

HE Staining and Micro-CT to Evaluate Murine Trabecular Bone Microarchitecture

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton is crucial for gaining insight into the pathophysiology of osteoporosis and improving its treatment. However, handling bone samples can be complex due to their hard and dense properties. Secondly, specialized software makes image processing and analysis difficult. In this protocol, we present a cost-effective and easy-to-use solution for trabecular bone microstructure analysis. Detailed steps and precautions are provided. Micro-CT is a non-destructive three-dimensional (3D) imaging technique that provides high-resolution images of trabecular bone structure. It allows for the objective and quantitative evaluation of bone quality, which is why it is widely regarded as the gold standard method for bone quality assessment. However, histomorphometry remains indispensable as it offers crucial cellular-level parameters, bridging the gap between two-dimensional (2D) and 3D assessments of bone specimens. As for the histologic techniques, we chose to decalcify the bone tissue and then perform traditional paraffin embedding. In summary, combining these two methods can provide more comprehensive and accurate information on bone microstructure.

Author Spotlight: An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model 

This protocol presents an economical and efficient method for quantitative evaluation of bone microarchitecture in a mouse model of osteoporosis by combining Hematoxylin-Eosin (HE) staining and micro-computed tomography (Micro-CT) techniques.

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton ...

A Human Bone Marrow 3D Model to Investigate the Dynamics and Interactions Between Resident Cells in Physiological or Tumoral Contexts

The medullary niche is a complex ecosystem that is essential to maintain homeostasis for resident cells. Indeed, the bone marrow, which includes a complex extracellular matrix and various cell types, such as mesenchymal stem cells, osteoblasts, and endothelial cells, is deeply involved in hematopoietic stem cell regulation through direct cell-cell interactions, as well as cytokine production. To closely mimic this in vivo structure and conduct experiments reflecting the responses of the human bone marrow, several 3D models have been created based on biomaterials, relying primarily on primary stromal cells. Here, a protocol is described to obtain a minimal and standardized system that is easy to set up and provides features of bone marrow-like structure, which combines different cell populations including endothelial cells, and reflects the heterogeneity of in vivo bone marrow tissue. This 3D bone marrow-like structure-assembled using calcium phosphate-based particles and human cell lines, representative of the bone marrow microenvironment-allows the monitoring of a wide variety of biological processes by combining or replacing different primary cell populations within the system. The final 3D structures can then either be harvested for image analysis after fixation, paraffin-embedding, and histological/immunohistochemical staining for cell localization within the system, or dissociated to collect each cellular component for molecular or functional characterization.

Here, we describe an easy-to-implement, standardized, microphysiological system that reflects the complexity of the human bone marrow's in vivo structure, providing a pertinent model to finely study a broad range of normal and pathological events.

The medullary niche is a complex ecosystem that is essential to maintain homeostasis for resident cells. Indeed, the bone marrow, which includes a complex ...

Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models

Cardiovascular disease is the leading cause of death in the world, and vascular calcification is the most significant predictor of cardiovascular events; however, there are currently no treatment or therapeutic options for vascular calcification. Calcification begins within specialized extracellular vesicles (EVs), which serve as nucleating foci by aggregating calcium and phosphate ions. This protocol describes methods for obtaining and assessing calcification in murine aortas and analyzing the associated extracted EVs. First, gross dissection of the mouse is performed to collect any relevant organs, such as the kidneys, liver, and lungs. Then, the murine aorta is isolated and excised from the aortic root to the femoral artery. Two to three aortas are then pooled and incubated in a digestive solution before undergoing ultracentrifugation to isolate the EVs of interest. Next, the mineralization potential of the EVs is determined through incubation in a high-phosphate solution and measuring the light absorbance at a wavelength of 340 nm. Finally, collagen hydrogels are used to observe the calcified mineral formation and maturation produced by the EVs in vitro.

Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models

This paper presents the methodology for obtaining and assessing vascular calcification by isolating murine aortas followed by extracting calcified extracellular vesicles to observe the mineralization potential.

Cardiovascular disease is the leading cause of death in the world, and vascular calcification is the most significant predictor of cardiovascular events; ...

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

Summary

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Chapters in this video

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Models of Bone Metastasis

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

A Simple Critical-sized Femoral Defect Model in Mice

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

A Human Bone Marrow 3D Model to Investigate the Dynamics and Interactions Between Resident Cells in Physiological or Tumoral Contexts

Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models