Name: Multimodal Approach to Assess Bone Regeneration and Scaffold Performance (Video) | JoVE Journal
Uploaded: 2026-02-13T13:45:00.000+00:00
Duration: 6 min 54 s
Description: Explore multimodal imaging techniques for bone regeneration analysis using polarized light, SEM, TEM, and EDS. This protocol enables multiscale assessment of scaffold integration and osteointegration.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was supported by grants to the authors from the following Brazilian agencies: Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPq/Brazil) and Funda&#231;&#227;o Carlos Chagas Filho de Amparo &#224; Pesquisa do Estado do Rio de Janeiro (FAPERJ). We thank LABNANO/CBPF and CENABIO/UFRJ for the electron&#160;microscopy facilities. Language refinement and grammar revision were performed using ChatGPT (OpenAI, GPT-5.1, 2025). All edits were reviewed and verified by the authors.

<ol><li>Reznikov, N., Shahar, R., Weiner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bone+hierarchical+structure+in+three+dimensions%5BTitle%5D)+AND+%22Acta+Biomater%22%5BJournal%5D)">Bone hierarchical structure in three dimensions.</a> Acta Biomater. 10 (9), 3815-3826 (2014).</li>
<li>Buss, D. J., Kröger, R., McKee, M. D., Reznikov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hierarchical+organization+of+bone+in+three+dimensions%3A+A+twist+of+twists%5BTitle%5D)+AND+%22J+Str+Biol%3A+X%22%5BJournal%5D)">Hierarchical organization of bone in three dimensions: A twist of twists.</a> J Str Biol: X. 6, (2022).</li>
<li>Grandfield, K., Gustafsson, S., Palmquist, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Where+bone+meets+implant%3A+The+characterization+of+nano-osseointegration%5BTitle%5D)+AND+%22Nanoscale%22%5BJournal%5D)">Where bone meets implant: The characterization of nano-osseointegration.</a> Nanoscale. 5 (10), 4302-4308 (2013).</li>
<li>Loi, F., Córdova, L. A., Pajarinen, J., Lin, T., Yao, Z., Goodman, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inflammation%2C+fracture+and+bone+repair%5BTitle%5D)+AND+%22Bone%22%5BJournal%5D)">Inflammation, fracture and bone repair.</a> Bone. 86, 119-130 (2016).</li>
<li>Einhorn, T. A., Gerstenfeld, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fracture+healing%3A+mechanisms+and+interventions%5BTitle%5D)+AND+%22Nature+Rev+Rheumatol%22%5BJournal%5D)">Fracture healing: mechanisms and interventions.</a> Nature Rev Rheumatol. 11 (1), 45-54 (2015).</li>
<li>Shah, F. A., Thomsen, P., Palmquist, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Osseointegration+and+current+interpretations+of+the+bone-implant+interface%5BTitle%5D)+AND+%22Acta+Biomater%22%5BJournal%5D)">Osseointegration and current interpretations of the bone-implant interface.</a> Acta Biomater. 84, 1-15 (2019).</li>
<li>Palmquist, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+multiscale+analytical+approach+to+evaluate+osseointegration%5BTitle%5D)+AND+%22J+Mater+Sci+Mater+Med%22%5BJournal%5D)">A multiscale analytical approach to evaluate osseointegration.</a> J Mater Sci Mater Med. 29 (5), 60(2018).</li>
<li>Sim, J. H., Yeo, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Light+Microscopy+Analysis+of+Bone+Response+to+Implant+Surfaces%5BTitle%5D)+AND+%22Microsc+Today%22%5BJournal%5D)">Light Microscopy Analysis of Bone Response to Implant Surfaces.</a> Microsc Today. 24 (4), 28-33 (2016).</li>
<li>Reznikov, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inter-trabecular+angle%3A+A+parameter+of+trabecular+bone+architecture+in+the+human+proximal+femur+that+reveals+underlying+topological+motifs%5BTitle%5D)+AND+%22Acta+Biomater%22%5BJournal%5D)">Inter-trabecular angle: A parameter of trabecular bone architecture in the human proximal femur that reveals underlying topological motifs.</a> Acta Biomater. 44, 65-72 (2016).</li>
<li>Müller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hierarchical+microimaging+of+bone+structure+and+function%5BTitle%5D)+AND+%22Nat+Rev+Rheumatol%22%5BJournal%5D)">Hierarchical microimaging of bone structure and function.</a> Nat Rev Rheumatol. 5 (7), 373-381 (2009).</li>
<li>Martinez-Zelaya, V. R., Archilha, N. L., Calasans-Maia, M., Farina, M., Rossi, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trabecular+architecture+during+the+healing+process+of+a+tibial+diaphysis+defect%5BTitle%5D)+AND+%22Acta+Biomater%22%5BJournal%5D)">Trabecular architecture during the healing process of a tibial diaphysis defect.</a> Acta Biomater. 120, 181-193 (2021).</li>
<li>Buss, D. J., Reznikov, N., McKee, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crossfibrillar+mineral+tessellation+in+normal+and+Hyp+mouse+bone+as+revealed+by+3D+FIB-SEM+microscopy%5BTitle%5D)+AND+%22J+Str+Biol%22%5BJournal%5D)">Crossfibrillar mineral tessellation in normal and Hyp mouse bone as revealed by 3D FIB-SEM microscopy.</a> J Str Biol. 212 (2), 107603(2020).</li>
<li>Binkley, D. M., Deering, J., Yuan, H., Gourrier, A., Grandfield, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ellipsoidal+mesoscale+mineralization+pattern+in+human+cortical+bone+revealed+in+3D+by+plasma+focused+ion+beam+serial+sectioning%5BTitle%5D)+AND+%22J+Str+Biol%22%5BJournal%5D)">Ellipsoidal mesoscale mineralization pattern in human cortical bone revealed in 3D by plasma focused ion beam serial sectioning.</a> J Str Biol. 212 (2), 107615(2020).</li>
<li>Reznikov, N., Almany-Magal, R., Shahar, R., Weiner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+imaging+of+collagen+fibril+organization+in+rat+circumferential+lamellar+bone+using+a+dual+beam+electron+microscope+reveals+ordered+and+disordered+sub-lamellar+structures%5BTitle%5D)+AND+%22Bone%22%5BJournal%5D)">Three-dimensional imaging of collagen fibril organization in rat circumferential lamellar bone using a dual beam electron microscope reveals ordered and disordered sub-lamellar structures.</a> Bone. 52 (2), 676-683 (2013).</li>
<li>Reznikov, N., Bilton, M., Lari, L., Stevens, M. M., Kröger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fractal-like+hierarchical+organization+of+bone+begins+at+the+nanoscale%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Fractal-like hierarchical organization of bone begins at the nanoscale.</a> Science. 360 (6388), 2189(2018).</li>
<li>Grandfield, K., Vuong, V., Schwarcz, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrastructure+of+Bone%3A+Hierarchical+Features+from+Nanometer+to+Micrometer+Scale+Revealed+in+Focused+Ion+Beam+Sections+in+the+TEM%5BTitle%5D)+AND+%22Calcified+Tissue+Int%22%5BJournal%5D)">Ultrastructure of Bone: Hierarchical Features from Nanometer to Micrometer Scale Revealed in Focused Ion Beam Sections in the TEM.</a> Calcified Tissue Int. 103 (6), 606-616 (2018).</li>
<li>Micheletti, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((mineral+organization+at+the+mesoscale%3A+A+review+of+mineral+ellipsoids+in+bone+and+at+bone+interfaces%5BTitle%5D)+AND+%22Acta+Biomater%22%5BJournal%5D)">mineral organization at the mesoscale: A review of mineral ellipsoids in bone and at bone interfaces.</a> Acta Biomater. 142, 1-13 (2022).</li>
<li>Micheletti, C., Shah, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bone+hierarchical+organization+through+the+lens+of+materials+science%3A+Present+opportunities+and+future+challenges%5BTitle%5D)+AND+%22Bone+Rep%22%5BJournal%5D)">Bone hierarchical organization through the lens of materials science: Present opportunities and future challenges.</a> Bone Rep. 22, (2024).</li>
<li>Shah, F. A., Ruscsák, K., Palmquist, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((50+years+of+scanning+electron+microscopy+of+bone-a+comprehensive+overview+of+the+important+discoveries+made+and+insights+gained+into+bone+material+properties+in+health%2C+disease%2C+and+taphonomy%5BTitle%5D)+AND+%22Bone+Res%22%5BJournal%5D)">50 years of scanning electron microscopy of bone-a comprehensive overview of the important discoveries made and insights gained into bone material properties in health, disease, and taphonomy.</a> Bone Res. 7 (1), (2019).</li>
<li>Georgiadis, M., Müller, R., Schneider, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Techniques+to+assess+bone+ultrastructure+organization%3A+Orientation+and+arrangement+of+mineralized+collagen+fibrils%5BTitle%5D)+AND+%22J+Royal+Soc+Interface%22%5BJournal%5D)">Techniques to assess bone ultrastructure organization: Orientation and arrangement of mineralized collagen fibrils.</a> J Royal Soc Interface. 13 (119), (2016).</li>
<li>Martinez-Zelaya, V. R., Archilha, N. L., Calasans-Maia, M., Farina, M., Rossi, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trabecular+architecture+during+the+healing+process+of+a+tibial+diaphysis+defect%5BTitle%5D)+AND+%22Acta+Biomater%22%5BJournal%5D)">Trabecular architecture during the healing process of a tibial diaphysis defect.</a> Acta Biomater. 120, 181-193 (2021).</li>
<li>Keklikoglou, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Micro-computed+tomography+for+natural+history+specimens%3A+a+handbook+of+best+practice+protocols%5BTitle%5D)+AND+%22Eur+J+Taxonomy%22%5BJournal%5D)">Micro-computed tomography for natural history specimens: a handbook of best practice protocols.</a> Eur J Taxonomy. (522), (2019).</li>
<li>Peddie, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Volume+electron+microscopy%5BTitle%5D)+AND+%22Nat+Rev+Meth+Primers%22%5BJournal%5D)">Volume electron microscopy.</a> Nat Rev Meth Primers. 2 (1), 51(2022).</li>
<li>Costa De-Moraes, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histomorphometric+and+microtomographic+evaluation+of+hydroxyapatite+coated+implants+and+L-PRF+in+over+drilled+bone+sites+in+sheep%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Histomorphometric and microtomographic evaluation of hydroxyapatite coated implants and L-PRF in over drilled bone sites in sheep.</a> Sci Rep. 15 (1), 14761(2025).</li>
<li>Liu, Y., Xie, D., Zhou, R., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((3D+X-ray+micro-computed+tomography+imaging+for+the+microarchitecture+evaluation+of+porous+metallic+implants+and+scaffolds%5BTitle%5D)+AND+%22Micron%22%5BJournal%5D)">3D X-ray micro-computed tomography imaging for the microarchitecture evaluation of porous metallic implants and scaffolds.</a> Micron. 142, (2021).</li>
<li>Wee, H., Khajuria, D. K., Kamal, F., Lewis, G. S., Elbarbary, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessment+of+Bone+Fracture+Healing+Using+Micro-Computed+Tomography%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Assessment of Bone Fracture Healing Using Micro-Computed Tomography.</a> J Vis Exp. (190), e64262(2022).</li>
<li>Ramezanzadehkoldeh, M., Skallerud, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroCT-based+finite+element+models+as+a+tool+for+virtual+testing+of+cortical+bone%5BTitle%5D)+AND+%22Med+Eng+Physics%22%5BJournal%5D)">MicroCT-based finite element models as a tool for virtual testing of cortical bone.</a> Med Eng Physics. 46, 12-20 (2017).</li>
<li>Shefelbine, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prediction+of+fracture+callus+mechanical+properties+using+micro-CT+images+and+voxel-based+finite+element+analysis%5BTitle%5D)+AND+%22Bone%22%5BJournal%5D)">Prediction of fracture callus mechanical properties using micro-CT images and voxel-based finite element analysis.</a> Bone. 36 (3), 480-488 (2005).</li>
<li>O'Neill, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Micro-computed+tomography+assessment+of+the+progression+of+fracture+healing+in+mice%5BTitle%5D)+AND+%22Bone%22%5BJournal%5D)">Micro-computed tomography assessment of the progression of fracture healing in mice.</a> Bone. 50 (6), 1357-1367 (2012).</li>
<li>Morgan, E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Micro-computed+tomography+assessment+of+fracture+healing%3A+Relationships+among+callus+structure%2C+composition%2C+and+mechanical%5BTitle%5D)+AND+%22Bone%22%5BJournal%5D)">Micro-computed tomography assessment of fracture healing: Relationships among callus structure, composition, and mechanical.</a> Bone. 44 (2), 335-344 (2009).</li>
<li>Reznikov, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inter-trabecular+angle%3A+A+parameter+of+trabecular+bone+architecture+in+the+human+proximal+femur+that+reveals+underlying+topological+motifs%5BTitle%5D)+AND+%22Acta+Biomater%22%5BJournal%5D)">Inter-trabecular angle: A parameter of trabecular bone architecture in the human proximal femur that reveals underlying topological motifs.</a> Acta Biomater. 44, 65-72 (2016).</li>
<li>Zaborowska, M., Shah, F. A., Trobos, M., Palmquist, A. Retrieval, sample processing, and analyses of bone-anchored implants. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aZaborowska%2C+M+%22Bone+Repair+Biomater+Regenerat+Clin+Appl+Second+Edition%22">Bone Repair Biomater Regenerat Clin Appl Second Edition</a>. , 447-466 (2018).</li>
<li>Thorfve, A., Palmquist, A., Grandfield, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+analytical+techniques+for+evaluation+of+osseointegrated+titanium+implants%5BTitle%5D)+AND+%22Mater+Sci+Technol%22%5BJournal%5D)">Three-dimensional analytical techniques for evaluation of osseointegrated titanium implants.</a> Mater Sci Technol. 31 (2), 174-179 (2015).</li>
<li>Mastrogiacomo, M., Campi, G., Cancedda, R., Cedola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synchrotron+radiation+techniques+boost+the+research+in+bone+tissue+engineering%5BTitle%5D)+AND+%22Acta+Biomater%22%5BJournal%5D)">Synchrotron radiation techniques boost the research in bone tissue engineering.</a> Acta Biomater. 89, 33-46 (2019).</li>
<li>Raguin, E., Rechav, K., Shahar, R., Weiner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Focused+ion+beam-SEM+3D+analysis+of+mineralized+osteonal+bone%3A+lamellae+and+cement+sheath+structures%5BTitle%5D)+AND+%22Acta+Biomater%22%5BJournal%5D)">Focused ion beam-SEM 3D analysis of mineralized osteonal bone: lamellae and cement sheath structures.</a> Acta Biomater. 121, 497-513 (2021).</li>
<li>McMahon, G., Malis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultramicrotomy+of+nanocrystalline+materials%5BTitle%5D)+AND+%22Microscopy+Res+Tech%22%5BJournal%5D)">Ultramicrotomy of nanocrystalline materials.</a> Microscopy Res Tech. 31 (4), 267-274 (1995).</li>
<li>Bassim, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Minimizing+damage+during+FIB+sample+preparation+of+soft+materials%5BTitle%5D)+AND+%22J+Microsc%22%5BJournal%5D)">Minimizing damage during FIB sample preparation of soft materials.</a> J Microsc. 245 (3), 288-301 (2012).</li>
<li>Witzke, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+Sample+Preparation+Using+Focused+Ion+Beam+and+Ultramicrotomy+of+Human+Dental+Enamel+and+Dentine+for+Multimicroscopic+Imaging+at+Micro-+and+Nanoscale%5BTitle%5D)+AND+%22Materials%22%5BJournal%5D)">Comparative Sample Preparation Using Focused Ion Beam and Ultramicrotomy of Human Dental Enamel and Dentine for Multimicroscopic Imaging at Micro- and Nanoscale.</a> Materials. 15 (9), 3084(2022).</li>
<li>Carter, J. L. W., Karakulak Uz, T., Ibrahim, B., Pigott, J. S., Gordon, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comparison+of+energy+dispersive+spectroscopy+in+transmission+scanning+electron+microscopy+with+scanning+transmission+electron+microscopy%5BTitle%5D)+AND+%22Ultramicroscopy%22%5BJournal%5D)">A comparison of energy dispersive spectroscopy in transmission scanning electron microscopy with scanning transmission electron microscopy.</a> Ultramicroscopy. 270, 114106(2025).</li>
<li>Grandfield, K., McNally, E. A., Palmquist, A., Botton, G. A., Thomsen, P., Engqvist, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+biointerfaces+in+three+dimensions%3A+electron+tomography+of+the+bone-hydroxyapatite+interface%5BTitle%5D)+AND+%22J+Royal+Soc+Interface%22%5BJournal%5D)">Visualizing biointerfaces in three dimensions: electron tomography of the bone-hydroxyapatite interface.</a> J Royal Soc Interface. 7 (51), 1497-1501 (2010).</li>
<li>Grandfield, K., Palmquist, A., Engqvist, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+structure+of+laser-modified+Ti6Al4V+and+bone+interface+revealed+with+STEM+tomography%5BTitle%5D)+AND+%22Ultramicroscopy%22%5BJournal%5D)">Three-dimensional structure of laser-modified Ti6Al4V and bone interface revealed with STEM tomography.</a> Ultramicroscopy. 127, 48-52 (2013).</li>
</ol>

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Different imaging techniques have been employed to investigate bone architecture and assess the effectiveness of biomaterials in inducing osteointegration7,21,22,23,24. In this protocol, we established a workflow that incorporates X-ray microtomography, optical microscopy, and electron microscopy to analyze the mineralized bone tissue formed in a rat tibia def...

Bone is a complex, hierarchically organized tissue1,2. Understanding its structural organization is essential for addressing fundamental questions related to how cells contribute to tissue architecture and how structural changes impact mechanical function3. In this context, bone remodeling after injury remains a central topic of investigation. Fracture healing is a complex multistage process, beginning with an initial inflammatory response, followed by angiogenesis and the differentiation of progenitor cells into chondrocytes and osteoblasts4,5. Much has been debated regarding the role of biomaterials to stimulate osteointegration, particularly about biocompatibility and their ability to prevent fibrous encapsulation6.
A commonly adopted method to assess biomaterial-stimulated osteointegration is optical microscopy, which enables both quantitative and qualitative histological analyses. This approach has been shown to be a reliable and effective way to evaluate the success of bone implants7,8. Nevertheless, several issues related to bone hierarchical organization cannot be explored by using a single imaging approach3, particularly the histological one, which is limited at least in part by the magnification restriction of the method.
Advances in three-dimensional imaging have proven essential for understanding bone hierarchical organization. Techniques such as microcomputed tomography (microCT; either synchrotron radiation or conventional laboratory-based X-ray sources)9,10,11, dual-beam scanning electron microscopy (FIB-SEM) associated with slice-and-view tomography12,13,14 and transmission electron tomography15,16 provided new insights into bone architecture at multiple scales.
Based on different 3D imaging approaches, a model of bone hierarchical organization has been proposed, which initially identified nine distinct levels of bone structural organization1. This scheme has recently been revised and expanded to encompass approximately 12 hierarchical levels, serving as a valuable reference for exploring the relationship between structure and function in bone tissue2,17. Importantly, this proposed hierarchical model provides a foundation for evaluating the success of bone repair following injury. Evaluating whether the regenerated bone tissue reproduces the native hierarchical structure is a key standard in assessing the quality of bone healing, particularly when biomaterials are used within the damaged site.
Although the use of multiple imaging techniques to investigate bone organization has been discussed6,7,18,19,20, little has been explored regarding a workflow that integrates different imaging modalities to assess bone mineral organization across multiple spatial scales. As mentioned, bone is a complex composite tissue, consisting of an organic phase (cells, collagen, and macromolecules) and a mineralized matrix. From a sample preparation perspective, this complexity presents technical challenges regarding solvent permeation through the sample, sectioning of a hard specimen, and the integration of different imaging strategies on&#160;the same sample.
The key innovation in this work is the establishment of a reproducible, correlative, and multiscale imaging workflow using three-dimensional imaging and complementary techniques. As illustrated in Figure 1A, the workflow is designed so that the same sample can be sequentially examined using different imaging modalities. Because some of these techniques are destructive or require sectioning, the workflow follows an order that begins with non-destructive methods and progresses to approaches that involve cutting or material removal.
This sample preparation and analysis workflow is broadly applicable to hard or mineralized specimens, including cortical and trabecular bone, pre-implantation scaffolds, and the evaluation of the bone/biomaterials interface after implantation. By combining imaging modalities that span different fields of view and resolution ranges (Figure 1B), the workflow enables the analysis of samples from the centimeter scale down to the micrometer and nanometer scales. While the sequence is optimized to support correlative analysis of a single specimen, each technique can also be applied independently according to the experimental goal. This approach enables a detailed assessment of biomaterials&#39; osteointegration and the evaluation of the regenerated bone growth in the healing area.

<table><tbody><tr><td>Acetone&amp;nbsp;</td><td>Merck</td><td>67-64-1</td><td></td></tr><tr><td>Aluminium cylinder</td><td>home-made</td><td>n/a</td><td></td></tr><tr><td>Avizo version 2022.1</td><td>ThermoFisher</td><td>n/a</td><td>software for image filtering, segmentation and 3D visualization</td></tr><tr><td>Cacodylic Acid, Sodium Salt, trihydrate</td><td>Ted Pella</td><td>18851</td><td>powder for Cacodylate Buffer. Prepare a stock solution of 0.2 M</td></tr><tr><td>Diamond Polishing Compound 3 &amp;micro;m size</td><td>Ted Pella</td><td>895-8</td><td></td></tr><tr><td>Diamond Polishing Compound 6 &amp;micro;m size</td><td>Ted Pella</td><td>895-9</td><td></td></tr><tr><td>Diamond Wheel x.006</td><td>South Bay Technology</td><td>DWH 3063</td><td></td></tr><tr><td>Diamond Wheel x.012</td><td>South Bay Technology</td><td>DWH 4122</td><td></td></tr><tr><td>Felt polishing cloth</td><td>Ted Pella</td><td>816-20</td><td></td></tr><tr><td>Flat Embedding Capsules</td><td>EMS</td><td>70021</td><td></td></tr><tr><td>Glass cover slip</td><td>Knittel</td><td>n/a</td><td></td></tr><tr><td>Glass slide</td><td>Knittel</td><td>n/a</td><td></td></tr><tr><td>Glutaraldehyde, 25% EM grade</td><td>Ted Pella</td><td>18426/18427</td><td></td></tr><tr><td>Gold sputter K550X</td><td>EMITECH</td><td>n/a</td><td></td></tr><tr><td>ImageJ</td><td>National Institutes of Health (NIH)</td><td></td><td>software for image aligment, filtering, segmentation and 3D visualization</td></tr><tr><td>IMOD version 5.1</td><td>University of Colorado</td><td></td><td>software for image aligment, segmentation and 3D visualization</td></tr><tr><td>Lapping e Polishing Machine</td><td>South Bay Technology</td><td>model 910</td><td></td></tr><tr><td>Low-Speed Diamond Wheel Saw&amp;nbsp;</td><td>South Bay Technology</td><td>model 650</td><td></td></tr><tr><td>Low Viscosity Embedding Kit</td><td>EMS</td><td>14300</td><td>kit for SPURR resin. Prepare according to the Data sheet specifications&amp;nbsp;</td></tr><tr><td>Micro-CT Skyscan 1273&amp;nbsp;</td><td>Bruker</td><td>n/a</td><td></td></tr><tr><td>Microscope Jeol 2100F 200kV&amp;nbsp;</td><td>Jeol</td><td>n/a</td><td></td></tr><tr><td>Microscope Olympus BX51</td><td>Olympus</td><td>n/a</td><td></td></tr><tr><td>Microscope Tescan Lyra3</td><td>Tescan</td><td>n/a</td><td></td></tr><tr><td>Microscope Zeiss Axioplan</td><td>Zeiss</td><td>n/a</td><td>equipped with a first order lambda plate</td></tr><tr><td>Paraformaldehyde, EM Grade</td><td>Sigma</td><td>P6148</td><td>powder for formaldehyde solution. Prepare a stock solution of 16%</td></tr><tr><td>Plastic slide</td><td>Exakt</td><td>41500</td><td></td></tr><tr><td>QuickStick 135 Mounting Wax</td><td>South Bay Technology</td><td>n/a</td><td></td></tr><tr><td>Sandpapers&amp;nbsp; grit sizes 1200</td><td>n/a</td><td>n/a</td><td></td></tr><tr><td>Sandpapers&amp;nbsp; grit sizes 2500</td><td>n/a</td><td>n/a</td><td></td></tr><tr><td>Sandpapers&amp;nbsp; grit sizes 4100</td><td>n/a</td><td>n/a</td><td></td></tr><tr><td>Super Glue</td><td>Loctite</td><td>n/a</td><td></td></tr><tr><td>Water Soluble Coolant&amp;nbsp;</td><td>South Bay Technology</td><td>n/a</td><td></td></tr><tr><td>Whatmann grade 1 filter paper</td><td>sigma</td><td>WHA1001150</td><td></td></tr></tbody></table>

multimodal approach to assess bone regeneration and scaffold performance

Multimodal Approach to Assess Bone Regeneration and Scaffold Performance

The clinical success of an implant largely depends on its ability to promote bone repair and facilitate osteointegration. While significant advancements have been made in designing new scaffolds, a thorough evaluation of implant success at various bone length scales has not been sufficiently investigated. Bone is a hierarchically organized tissue, with an intricate structure that ranges from the organization of collagen fibrils and their mineralization at the nanometer and micrometer scales to the macroscopic arrangement of compact and cancellous bone. This hierarchical organization is crucial for ensuring bone function and mechanical performance. In this context, a comprehensive assessment of the effects of biomaterials on bone repair at multiple length scales is essential for evaluating the effectiveness and safety of implanted materials. However, preparing samples for integrated multimodal analyses poses a technical challenge. In this work, we present a protocol designed to evaluate bone regeneration across various hierarchical levels and analyze the interface between newly formed bone and the implanted scaffold. This approach provides a robust method for assessing the effectiveness of scaffolds in inducing bone repair. The combination of selected 3D and 2D imaging techniques is crucial for a proper evaluation of the complex mineralized tissue that forms during the bone healing process

The initial analysis of resin-embedded samples is performed using microcomputed tomography (microCT). This non-destructive technique facilitates 3D visualization of the entire defect area, enabling both quantitative and qualitative analyses of the sample. Contrast in microCT imaging derives mostly from the density differences of the materials present in the sample. Figure 1 illustrates the microCT analysis of a non-critical defect in a rat tibia that was implanted with the CHA37 biomaterial....

We propose a protocol for assessing bone regeneration at various hierarchical levels. This multimodal approach allows for structural analysis across different length scales, effectively addressing challenges in sample preparation. It also provides a reliable workflow for evaluating the effectiveness of biomaterials in promoting bone regeneration.

The clinical success of an implant largely depends on its ability to promote bone repair and facilitate osteointegration. While significant advancements ...

Our research evaluates how different biomaterials influen-cee-ate on bone architecture during the regeneration process. Bone regeneration requires a multiscale evaluation, as it is a hierarchically organized the tissue. This protocol enables analysis of the sample at the different scales.To begin, secure the resin-embedded tibia containing the implanted biomaterial onto the specimen holder and attach it to the instrument arm. Orient the sample according to the desired sectioning direction. Obtain a low-speed diamond saw disc to section the bone into approximately 0.7-millimeter slices.Fill the liquid reservoir until the fluid level completely covers the bottom edge of the blade. Ensure the sample is stable and horizontally aligned with the disc surface. Adjust the specimen position using the positioning knobs until the target cut region is aligned with the blade.Begin cutting the sample using a higher speed setting and apply low pressure until an indentation forms. Then reduce the speed to complete the sectioning smoothly and minimize vibration. Stop the saw immediately after the cut is complete.Then examine the cut sample using a stereo microscope for mounting. For mounting, use a homemade assembly consisting of an inner solid stainless steel cylinder and an outer hollow cylinder that provides mechanical support during polishing. Next, place the inner cylinder on a hot plate and heat it to approximately 120 to 140 degrees Celsius.Choose a suitable wax and apply a small amount of wax onto the surface of the heated cylinder and allow it to melt completely. Then place the sample with the surface of interest facing down onto the wax-coated area and gently press the sample to ensure full contact with the melted wax. Then remove the aluminum cylinder from the hot plate and allow the wax to solidify at room temperature.For coarse and intermediate polishing, select sandpaper with grit sizes of 1, 200, 2, 500, and 4, 100 to reduce the sample thickness to approximately 0.15 millimeters. Start with 1, 200-grit sandpaper, followed by 2, 500-grit while applying light pressure. Periodically monitor the sample thickness during polishing using reflection microscopy.Once the target thickness is reached, polish both sides of the sample using 4, 100-grit sandpaper. Continuously clean the sample with distilled water during each polishing step. For final polishing, choose a diamond paste with six or three-micrometer particle size, based on the surface roughness of the sample.Apply a small amount of diamond paste onto a felt polishing cloth, mounted on a polishing disc, and polish the sample. To detach the sample from the cylinder, warm the wax on a hot plate. After removing the unit from the hot plate, take the sample out from the cylinder.Then gently polish both sides of the sample using minimal pressure and dry the polished surface with filter paper. Prior to microscopy analysis, mount the sample between a glass slide and coverslip using immersion oil as the mounting medium. Examine collagen fiber orientation using polarized light microscopy, starting with low magnification between 5x and 10x.Finally, prepare the sample for scanning electron microscopy to further analyze the bone-biomaterial interface. After polishing and initial optical microscopy examination, intrinsic sample features allowed the region identified under light microscopy to be matched to the corresponding location in the micro-computed tomography dataset. Polarized light microscopy differentiated preexisting bone, shown in blue, from newly formed bone during healing, depicted in orange.Higher magnification polarized light microscopy revealed an interface where bone grew in close opposition to preexisting cortical bone with Type I collagen fibers oriented perpendicularly in a thin region. Scanning electron microscopy imaging enabled precise localization of areas of interest. A volume of the selected region was further examined using slice-and-view tomography.The face of the trench displayed bone morphology on a growing trabecula. Transmission electron microscopy enables nano scale examination of the bone-biomaterial interface, which can be combined with selected area electron diffraction. Selected area electron diffraction patterns indicated that both the biomaterial region and the bone region were composed of hydroxyapatite crystals.Energy-dispersive X-ray spectroscopy mapping of the imaged interface region showed phosphorus and calcium. This multimodal workflow provides complementary insights that cannot be achieved by a single imaging technique. This workflow requires significant time, technical expertise, and access to specialized instrumentation.It can investigate functional correlations, long-term bone remodeling, and biomaterial design.

multimodal-approach-to-assess-bone-regeneration-scaffold

Research

JoVE Journal

Biology

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Use of Human Perivascular Stem Cells for Bone Regeneration

A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels.

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

Biological Compatibility Profile on Biomaterials for Bone Regeneration

In this study, we detail methods of decellularization, physical characterization, imaging, and in vivo implantation of plant-based biomaterials, as well as methods for cell seeding and differentiation in the scaffolds. The described methods allow the evaluation of plant-based biomaterials for bone tissue engineering applications.

Decellularized Apple-Derived Scaffolds for Bone Tissue Engineering In Vitro and In Vivo

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 murine bone marrow injury model is a useful tool for studying postnatal osteogenesis through intramembranous ossification. This simplified surgical protocol describes the bone marrow ablation procedure for downstream assessment of new bone formation and cellular responses following injury.

Improved Methodology for Studying Postnatal Osteogenesis via Intramembranous Ossification in a Murine Bone Marrow Injury Model

Here, we present an integrated protocol for generating controlled osteochondral lesions in sheep knees to establish an experimental model suitable for bone and cartilage tissue regeneration studies. The protocol outlines a method for inducing a full-thickness osteochondral defect in the femoral condyle, utilizing techniques that minimize animal welfare impacts and variability in results.

Establishment and Evaluation of a Sheep Model of Full-thickness Osteochondral Defect

This study established a rat femoral half-segmental defect model to evaluate the mechanical and osteogenic performance of bone substitute materials under load-bearing conditions without the use of internal or external fixation.

Half-segmental Diaphyseal Bone Defect Model in Rats for Evaluating Bone Substitute Performance in Load-bearing Regions

Explore multimodal imaging techniques for bone regeneration analysis using polarized light, SEM, TEM, and EDS. This protocol enables multiscale assessment of scaffold integration and osteointegration.

Multimodal Approach to Assess Bone Regeneration and Scaffold Performance

Explore More Videos