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. 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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. 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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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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 resu...

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.

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			<td>0.20 filters</td>
			<td>VWR&#160;</td>
			<td>28145-501</td>
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			<td>18 G needle (1,2x40 mm)</td>
			<td>Sterican</td>
			<td>4665120</td>
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			<td>3 mL syringe</td>
			<td>HENKE-JECT</td>
			<td>8300005762</td>
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			<td>37% hydrochloric acid&#160;</td>
			<td>VWR&#160;</td>
			<td>1.00317.1000&#160;</td>
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			<td>Acetic acid (glacial)&#160;</td>
			<td>Sigma&#160;</td>
			<td>A6283&#160;</td>
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			<td>Acetone&#160;</td>
			<td>VWR&#160;</td>
			<td>20063-365&#160;</td>
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			<td>Alcian Blue 8GX</td>
			<td>VWR&#160;</td>
			<td>361186</td>
			<td></td>
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			<td>Ammonium hydroxide</td>
			<td>VWR&#160;</td>
			<td>318612</td>
			<td></td>
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			<td>Apatitic tricalcium phosphate&#160;</td>
			<td>Centre for Biomedical and Healthcare Engineering (Mines Saint Etienne, France)</td>
			<td>TV26U&#160;</td>
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		<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>
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			<td>Brillant crocein&#160;</td>
			<td>Aldrich&#160;</td>
			<td>2107507</td>
			<td></td>
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			<td>CTVox&#160;</td>
			<td>Bruker</td>
			<td>-</td>
			<td></td>
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			<td>DataViewer&#160;</td>
			<td>Skyscan</td>
			<td>-</td>
			<td></td>
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			<td>Diamond blade</td>
			<td>Struers</td>
			<td>MOD13</td>
			<td></td>
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			<td>Diamond saw</td>
			<td>Struers</td>
			<td>Accutom-50</td>
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			<td>DiaPro Mol B3 diamond solution</td>
			<td>Struers</td>
			<td>40600379</td>
			<td></td>
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			<td>DiaPro Nap B1 diamond solution</td>
			<td>Struers</td>
			<td>40600373</td>
			<td></td>
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			<td>Dibasic sodium phosphate (Na2HPO4)</td>
			<td>Sigma&#160;</td>
			<td>102404598</td>
			<td></td>
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			<td>Dibutyl Phtalate</td>
			<td>Chimie-Plus Laboratoires</td>
			<td>28656</td>
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			<td>DragonFly software&#160;</td>
			<td>ORS</td>
			<td>2022.1.0.1231.&#160;</td>
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			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) high glucose, GlutaMAX(TM), pyruvate</td>
			<td>ThermoFisher Scientific</td>
			<td>31966-021</td>
			<td></td>
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			<td>Eosine Y- Surgipath&#160;</td>
			<td>Sigma&#160;</td>
			<td>1002830105</td>
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			<td>Erythrosin B</td>
			<td>Sigma&#160;</td>
			<td>102141057</td>
			<td></td>
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			<td>Ethanol absolute</td>
			<td>VWR&#160;</td>
			<td>20820362</td>
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			<td>Eukitt&#160;</td>
			<td>Dutscher</td>
			<td>6.00.01.0003.06.01.01</td>
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			<td>Falcon 50 mL</td>
			<td>Sarstedt</td>
			<td>62.547.254</td>
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			<td>Ferric chloride hexahydrate (FeCl3, 6H2O)&#160;</td>
			<td>Merck&#160;</td>
			<td>1.03943.0250</td>
			<td></td>
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		<tr>
			<td>Fetal Bovine Serum (FBS)&#160;</td>
			<td>Eurobio</td>
			<td>CVFSVF00</td>
			<td></td>
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		<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>
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		<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>
			<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&#160;</td>
			<td>28975.325</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylidine Ponceau</td>
			<td>Aldrich&#160;</td>
			<td>19.976-1&#160;</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...

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

Research

JoVE Journal

Bioengineering

591 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

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury leading to permanent pathological damage. Existing conventional assays allow the analysis of leukocyte adhesion molecules only after the extraction of leukocytes from the blood. This requires the blood to undergo several steps before peripheral blood leukocytes (PBLs) can be ready for analysis, which in turn can stimulate PBLs influencing the research findings. The autoperfused micro flow chamber assay, however, allows scientists to study early leukocytes functional dysregulation using the systemic flow of a live mouse while having the freedom of manipulating a coated chamber. Through a disease model, the functional expression of leukocyte adhesion molecules can be assessed and quantified in a micro-glass chamber coated with immobilized endothelial adhesion molecules ex vivo. In this model, the blood flows between the right common carotid artery and left external jugular vein of a live mouse under anesthesia, allowing the interaction of native PBLs in the chamber. Real-time experimental analysis is achieved with the assistance of an intravital microscope as well as a Harvard Apparatus pressure device. The application of a flow regulator at the input point of the glass chamber allows comparable physiological flow conditions amongst the experiments. Leukocyte rolling velocity is the main outcome and is measured using the National Institutes of Health open-access software ImageJ. In summary, the autoperfused micro flow chamber assay provides an optimal physiological environment to study leukocytes endothelial interaction and allows researchers to draw accurate conclusions when studying inflammation.

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Here, we present a protocol that allows the investigator to assess leukocyte recruitment dynamics ex vivo by connecting a chamber coated with endothelial-derived adhesion molecules to the circulatory system of a mouse. This method offers significant advantages since it allows for leukocyte assessment under relative biological conditions.

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen&#39;s ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.&#160; The detailed setup methodology of this new model is explained.

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to characterize in animal models due to physiological differences between animals and humans. Tissue engineering offers opportunities to provide in vitro models of functional human NMJs that can be used to diagnose and investigate NMJ pathologies and test potential therapeutics. By incorporating optogenetic proteins into induced pluripotent stem cells (iPSCs), we generated neurons that can be stimulated with specific wavelengths of light. If the NMJ is healthy and functional, a neurochemical signal from the motoneuron results in muscle contraction. Through the integration of optogenetics and microfabrication with tissue engineering, we established an unbiased and automated methodology for characterizing NMJ function using video analysis. A standardized protocol was developed for NMJ formation, optical stimulation with simultaneous video recording, and video analysis of tissue contractility. Stimulation of optogenetic motoneurons by light to induce skeletal muscle contractions recapitulates human NMJ physiology and allows for repeated functional measurements of NMJ over time and in response to various inputs. We demonstrate this platform&#39;s ability to show functional improvements in neuromuscular connectivity over time and characterize the damaging effects of patient MG antibodies or neurotoxins on NMJ function.

We describe a reproducible, automated, and unbiased imaging system for characterizing neuromuscular junction function using human engineered skeletal muscle tissue and optogenetic motoneurons. This system allows for the functional quantification of neuromuscular connectivity over time and detects diminished neuromuscular function caused by neurotoxins and myasthenia gravis patient serum.

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to ...

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical communities. The resolution of results obtained from current methods of measuring certain biomarkers can take several days or weeks to obtain, as they can be limited in resolution both spatially and temporally (e.g., fluid compartment microdialysis of interstitial fluid analyzed by enzyme-linked immunosorbent assay [ELISA], high-performance liquid chromatography [HPLC], or mass spectrometry); thus, their guidance of timely diagnosis and treatment is disrupted. In the present study, a unique technique for detecting and measuring peptide transmitters in vivo through the use of a capacitive immunoprobe biosensor (CI probe) is reported. The fabrication protocol and in vitro characterization of these probes are described. Measurements of sympathetic stimulation-evoked neuropeptide Y (NPY) release in vivo are provided. NPY release is correlated to the sympathetic release of norepinephrine for reference. The data demonstrate an approach for the fast and localized measurement of neuropeptides in vivo. Future applications include intraoperative real-time assessment of disease progression and minimally invasive catheter-based deployment of these probes.

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

Established immunochemical methods to measure peptide transmitters in vivo rely on microdialysis or bulk fluid draw to obtain the sample for offline analysis. However, these suffer from spatiotemporal limitations. The present protocol describes the fabrication and application of a capacitive immunoprobe biosensor that overcomes the limitations of the existing techniques.

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical ...

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

We describe the surgical technique and decellularization process for composite rat hindlimbs. Decellularization is conducted using low-concentration sodium dodecyl sulfate through an ex vivo machine perfusion system.

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...