<meta charset="utf8"></head><body>Análise multimodal da formação óssea usando modelos de explante

<ol>
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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<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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		<tr>
			<td>3 mL syringe</td>
			<td>HENKE-JECT</td>
			<td>8300005762</td>
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		<tr>
			<td>37% hydrochloric acid&#160;</td>
			<td>VWR&#160;</td>
			<td>1.00317.1000&#160;</td>
			<td></td>
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		<tr>
			<td>Acetic acid (glacial)&#160;</td>
			<td>Sigma&#160;</td>
			<td>A6283&#160;</td>
			<td></td>
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		<tr>
			<td>Acetone&#160;</td>
			<td>VWR&#160;</td>
			<td>20063-365&#160;</td>
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		<tr>
			<td>Alcian Blue 8GX</td>
			<td>VWR&#160;</td>
			<td>361186</td>
			<td></td>
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		<tr>
			<td>Ammonium hydroxide</td>
			<td>VWR&#160;</td>
			<td>318612</td>
			<td></td>
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		<tr>
			<td>Apatitic tricalcium phosphate&#160;</td>
			<td>Centre for Biomedical and Healthcare Engineering (Mines Saint Etienne, France)</td>
			<td>TV26U&#160;</td>
			<td></td>
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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>
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		<tr>
			<td>BMP2</td>
			<td>Medtronic&#160;</td>
			<td>InductOs 1.5 mg/mL</td>
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		<tr>
			<td>Brillant crocein&#160;</td>
			<td>Aldrich&#160;</td>
			<td>2107507</td>
			<td></td>
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		<tr>
			<td>CTVox&#160;</td>
			<td>Bruker</td>
			<td>-</td>
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		<tr>
			<td>DataViewer&#160;</td>
			<td>Skyscan</td>
			<td>-</td>
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		<tr>
			<td>Diamond blade</td>
			<td>Struers</td>
			<td>MOD13</td>
			<td></td>
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		<tr>
			<td>Diamond saw</td>
			<td>Struers</td>
			<td>Accutom-50</td>
			<td></td>
		</tr>
		<tr>
			<td>DiaPro Mol B3 diamond solution</td>
			<td>Struers</td>
			<td>40600379</td>
			<td></td>
		</tr>
		<tr>
			<td>DiaPro Nap B1 diamond solution</td>
			<td>Struers</td>
			<td>40600373</td>
			<td></td>
		</tr>
		<tr>
			<td>Dibasic sodium phosphate (Na2HPO4)</td>
			<td>Sigma&#160;</td>
			<td>102404598</td>
			<td></td>
		</tr>
		<tr>
			<td>Dibutyl Phtalate</td>
			<td>Chimie-Plus Laboratoires</td>
			<td>28656</td>
			<td></td>
		</tr>
		<tr>
			<td>DragonFly software&#160;</td>
			<td>ORS</td>
			<td>2022.1.0.1231.&#160;</td>
			<td></td>
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		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) high glucose, GlutaMAX(TM), pyruvate</td>
			<td>ThermoFisher Scientific</td>
			<td>31966-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosine Y- Surgipath&#160;</td>
			<td>Sigma&#160;</td>
			<td>1002830105</td>
			<td></td>
		</tr>
		<tr>
			<td>Erythrosin B</td>
			<td>Sigma&#160;</td>
			<td>102141057</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>VWR&#160;</td>
			<td>20820362</td>
			<td></td>
		</tr>
		<tr>
			<td>Eukitt&#160;</td>
			<td>Dutscher</td>
			<td>6.00.01.0003.06.01.01</td>
			<td></td>
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		<tr>
			<td>Falcon 50 mL</td>
			<td>Sarstedt</td>
			<td>62.547.254</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferric chloride hexahydrate (FeCl3, 6H2O)&#160;</td>
			<td>Merck&#160;</td>
			<td>1.03943.0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)&#160;</td>
			<td>Eurobio</td>
			<td>CVFSVF00</td>
			<td></td>
		</tr>
		<tr>
			<td>Fuchsine acid</td>
			<td>Merck&#160;</td>
			<td>1.05231.0025&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Biosera</td>
			<td>MS01NG100J</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Sigma&#160;</td>
			<td>86.118.9&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Isostatic press</td>
			<td>Nova Suisse</td>
			<td>Pmax 1500 bars</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser diffraction granulometry</td>
			<td>&#160;Malvern</td>
			<td>Mastersizer 3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Light green</td>
			<td>Prolabo&#160;</td>
			<td>28947135</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium carbonate</td>
			<td>Sigma&#160;</td>
			<td>A13149&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>MD-Mol polishing cloth</td>
			<td>Struers</td>
			<td>40500077</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylcyclohexane</td>
			<td>VWR&#160;</td>
			<td>8.06147.1000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylcyclohexane&#160;</td>
			<td>VWR&#160;</td>
			<td>8.06147.1000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylcyclohexane&#160;</td>
			<td>VWR&#160;</td>
			<td>8.06147.1000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylmethacrylate</td>
			<td>Sigma&#160;</td>
			<td>8.00590.2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-CT, micro-scanner&#160;</td>
			<td>Bruker</td>
			<td>Skyscan 1272</td>
			<td></td>
		</tr>
		<tr>
			<td>Monobasic sodium phosphate (NAH2PO4)</td>
			<td>Sigma&#160;</td>
			<td>71496</td>
			<td></td>
		</tr>
		<tr>
			<td>Mortar</td>
			<td>Fritsch&#160;</td>
			<td>Pulverisette 6</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N, Dimethylanilin</td>
			<td>Sigma&#160;</td>
			<td>803060</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoindentation station</td>
			<td>Anton Paar</td>
			<td>NHT2</td>
			<td></td>
		</tr>
		<tr>
			<td>ND-Nap polishing cloth</td>
			<td>Struers</td>
			<td>40500080</td>
			<td></td>
		</tr>
		<tr>
			<td>OATS Osteochondral Autograft Transfer System Set, 4,75 mm</td>
			<td>Arthrex</td>
			<td>AR-1981-04S</td>
			<td></td>
		</tr>
		<tr>
			<td>OATS Osteochondral Autograft Transfer System Set, 8 mm</td>
			<td>Arthrex</td>
			<td>AR-1981-08S</td>
			<td></td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>Ral</td>
			<td>M15</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma&#160;</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Peel-a-way disposable embbedding moulds</td>
			<td>Polysciences, Inc</td>
			<td>18646C-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (P/S)</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphomolybdic acid&#160;</td>
			<td>Sigma&#160;</td>
			<td>221856-100 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphotungstic acid</td>
			<td>Aldrich&#160;</td>
			<td>12863-5&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Polishing machine</td>
			<td>Sturers</td>
			<td>Dap V</td>
			<td></td>
		</tr>
		<tr>
			<td>Poupinel</td>
			<td>MEMMERT</td>
			<td>TV26U&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Raman microspectrometer</td>
			<td>Renishaw</td>
			<td>InVia Qontor</td>
			<td></td>
		</tr>
		<tr>
			<td>Safran du G&#226;tinais&#160;</td>
			<td>Labonord&#160;</td>
			<td>11507737</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>Carl Zeiss</td>
			<td>Evo LS 10</td>
			<td></td>
		</tr>
		<tr>
			<td>SEM</td>
			<td>Zeiss</td>
			<td>Carl Zeiss Evo LS10</td>
			<td></td>
		</tr>
		<tr>
			<td>SiC foils/Grinding papers</td>
			<td>Struers</td>
			<td>40400008 (#320), 40400011 (#1000), 40400122 (#2000), 40400182 (#4000)</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver paint</td>
			<td>Electron microscopy sciences</td>
			<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>
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comprehensive characterization of tissue mineralization in an ex vivo model

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

<meta charset="utf8"></head><body>Autor em destaque: Técnicas avançadas para caracterizar a mineralização de tecidos na pesquisa de regeneração óssea

Caracterização abrangente da mineralização tecidual em um modelo ex vivo

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

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

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

Research

JoVE Journal

Bioengineering

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

366 visualizações.  Nantes Université, Univ Angers, Oniris, CHU Nantes, INSERM, Regenerative Medicine and Skeleton, RMeS, UMR 1229. Nossa pesquisa se concentra na compreensão e melhoria da regeneração óssea. Aqui, pretendemos caracterizar extensivamente a mineralização tecidual usando abordagens técnicas variadas e complementares para facilitar a identificação precoce de candidatos promissores para terapias de regeneração óssea. Para avaliar a qualidade e a estrutura do tecido recém-mineralizado, usamos abordagens complementares, incluindo imagens 3D de alta resolução com m...