osteochondral bone explant preparation for comprehensive assessment of the quality of regenerated bone tissues

Multimodal Analysis of Bone Formation Using Explant Models

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.

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

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

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.

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

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

osteochondral-bone-explant-preparation-for-comprehensive-assessment

Research

JoVE Journal

Bioengineering

49 Views. To begin, dehydrate the osteochondral bone explant collected from sheep in a 40 milliliter glass vial containing 25 milliliters of dehydration solution. Place the vials on a rotating wheel for one hour at room temperature. After the last wash, replace the dehydration solution with 25 milliliters of xylene.Place the glass vials on the rotating wheel for one hour at room temperature. Next, add 10%benzoyl peroxide, 10%dibutyl phthalate, and 450 microliters of N, N-Dimethylaniline diluted ...