This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (NIH) under Award Number R01 AR059088, the Department of Defense (DoD) under Research Award Number W81XWH-12-1-0304, and the Mary and Paul Harrington Distinguished Professorship Endowment.

All animal procedures included in this protocol were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Kansas Medical Center, in compliance with all federal and state laws and regulations.
1. Creation of knee OA in mice
<ol>
	<li>Create a mouse model of knee OA by surgical destabilization of the medial meniscus (DMM) as described by Glasson et al.18 in 22 wild-type BALB/c mice at 10-11 weeks of age. Perform sham surgery as a control procedure on eight mice with the same background and age. 
	NOTE: Both sexes were used for the original project to meet the NIH requirement for consideration of sex as a biological variable, though examination of sex difference is not the scope of this protocol.</li>
	<li>Anesthetize animals by inhalation of Isoflurane. Check the depth of anesthesia by monitoring their respiratory rate/effort and lack of response to toe/tail pinch. Put animals in a supine position.</li>
	<li>Shave the skin in the knee area and clean the skin with Povidone-Iodine + alcohol skin scrub; three alternating cycles.</li>
	<li>Perform the DMM procedure on the right knee under a surgical microscope. Expose the knee joint through a medial parapatellar incision (1.2-1.5 cm in length) and incise the joint capsule. Keep the patella and the patellar tendon intact. After careful exposure of the medial meniscotibial ligament (MML) which anchors the medial meniscus to the tibial plateau, transect it with micro-surgical scissors to destabilize the medial meniscus.</li>
	<li>Perform sham surgery on the right knee as a control procedure, in which the MML was visualized but not transected.</li>
	<li>Close the joint capsule with 8-0 absorbable polyglactin sutures and skin incision with 7-0 non-absorbable sutures for both DMM and sham procedures to assure proper use of the knee once healing has occurred.</li>
	<li>Inject SR Buprenorphine (0.20-0.5 mg/kg) subcutaneously (SC) immediately before the surgical procedure for analgesia, which provides pain relief up to 72 h after a single injection. Monitor operated animals after surgery.</li>
	<li>Euthanize animals using a CO2 chamber at 2, 8, and 16 weeks post-surgery. After unconsciousness, confirm the death of the animals by a physical method (opening the chest cavity). These methods of euthanasia are consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association (AVMA).</li>
	<li>Harvest the knee joints for histological analyses at 2, 8, and 16 weeks after DMM surgery and at 2 and 16 weeks after Sham surgery to obtain mouse knee joints with different degrees of OA severity or SCB thickening.</li>
</ol>
2. Preparation of tissue sections and histologic images
<ol>
	<li>Fix mouse knee joint tissue samples in 2% paraformaldehyde, decalcify them in 25% formic acid, embed in paraffin, and section coronally to examine both the medial and lateral compartments.</li>
	<li>Cut knee specimens from the posterior side of the knee using a microtome and collect tissue sections that are 5-&#181;m-thick at 70-80 &#181;m intervals to obtain about 40 tissue slides across the entire knee joint. A micrometer-assisted estimation suggests that slide numbers 1-6 are from the far-posterior, 11-18 from mid-posterior, 23-30 from mid-anterior, and 35-40 from the far-anterior portion of the knee joint. Discard or collect intervening sections for additional stains.</li>
	<li>Perform Safranin-O and fast green stains according to the manufacturer&#39;s instructions to specifically identify cartilage cells and matrices on every five slides. Perform Hematoxylin-Eosin staining according to the manufacturer&#39;s instructions to examine the knee joints at the cellular and tissue levels as described previously19,20,21,22.</li>
	<li>Acquire histologic images with a microscope equipped with a digital camera. General histopathologic analysis and histologic OA grading were conducted as described previously15,19,20,21,22.</li>
</ol>
3. Quantitative measurement of osteoarthritic subchondral bone with ImageJ software
<ol>
	<li>Download the ImageJ software and open histologic images of interest.
	<ol>
		<li>Download the ImageJ bundled with Java 1.8.0_172 from&#160;https://imagej.nih.gov/ij/.</li>
		<li>Open the ImageJ program. Click the File tab on the Ribbon and click the Open option to open the histologic image.</li>
		<li>Find the file directory address, select the picture file, and click Open.</li>
	</ol>
	</li>
	<li>Calibrate ImageJ with the micrometer on the histologic images.
	<ol>
		<li>Use the straight-line tool to sketch one unit of length on the micrometer and click Analyze &#62; (then) Set scale. Set the Known distance and Pixel aspect ratio to 1 and click OK. ImageJ can convert the pixel length to the unit length on micrometer.</li>
		<li>Set the measured factor to area. Click Analyze &#62; Set Measurement and check Area and Limit to threshold box under new window. This step sets ImageJ to measure the parameter &#34;Area&#34; within selected &#34;Threshold&#34;.</li>
	</ol>
	</li>
	<li>Measure total subchondral bone (SCB) area of interest.
	<ol>
		<li>Define the SCB region of interest (ROI) as shown in the orange boxes of Figure 1A, which covers the SCB cortical plate and a portion of the underlying trabecular bone adjacent to the cortical plate in the medial femoral condyle (MFC) and medial tibial plateau (MTP) with specific dimensions for each ROI. Osteoarthritic SCB thickening usually occurs in these areas. Define the SCB ROI with the same shape and dimension in each MFC or MTP for all examined joints to ensure that the same size of the specific ROI was measured for all animals.</li>
		<li>Sketch the outline of the total SCB area of interest by using the Polygon selection tool under the main window of ImageJ. 
		NOTE: The selection tools give the system a threshold to limit the measured area.</li>
		<li>Measure the total SCB area: After the threshold is selected, click Analyze &#62; Measure. A &#34;Results&#34; window with area measurement will open.</li>
	</ol>
	</li>
	<li>Measure the bone substance area containing solid bone without bone marrow.
	<ol>
		<li>Click Edit &#62; Clear Outside to exclude the area outside the total SCB area. 
		NOTE: Only the total SCB area is visible after clicking Clear Outside option. The picture outside the total SCB area will turn black. This step allows observers to focus on the bone substance area within the area of interest.</li>
		<li>Click Image &#62; Adjust &#62; Color threshold to open the &#34;Threshold Color&#34; window. Click Original at the bottom of the &#34;Threshold Color&#34; window to restore the picture to the original status. Use selection tools in step 3.3.2 to draw a small box in the bone substance region. Click the Sample option at the bottom of the &#34;Threshold Color&#34; window to define the bone substance area. 
		NOTE: The &#34;Sample&#34; option in the &#34;Threshold Color&#34; window allows ImageJ to select all the same pixels on the total SCB area as the bone substance sample area. The selected bone substance area will turn red.</li>
		<li>Click Select at the bottom of the threshold color balance window to create an area measurement threshold. Click Analyze &#62; Measure at ImageJ main menu, and the bone substance area measurement result will show on the &#34;Results&#34; window.</li>
		<li>Save the data of the total SCB area and bone substance area.</li>
	</ol>
	</li>
	<li>Calculate the ratio of bone substance area (mm2) to total SCB area (mm2) of interest which represents the bone substance thickness (mm2/1.0 mm2) within the total SCB area.</li>
	<li>Measure the SCB thickness of histologic sections/images (as described in steps 3.1-3.5) of far-posterior, mid-posterior, mid-anterior, and far-anterior areas (as described in step 2.2) of DMM-induced OA to assess area-specific SCB thickness of 6 knee joints (Figure 1B). 
	&#8203;NOTE: This can validate the reliability of this quantitative measurement protocol because it is known that osteoarthritic SCB changes co-localize with cartilage lesions and that osteoarthritic cartilage damage with SCB thickening is more severe in the weight-bearing areas (mid-portion) of rodent knee joints14,15. Therefore, it is appropriate to use mid-sections for quantitative measurement of osteoarthritic SCB thickening.</li>
</ol>
4. Statistics
<ol>
	<li>Perform statistical analyses using data from quantitative measurement and visual grading of SCB thickness. Determine the inter- and intra-observer variability and reproducibility by Pearson&#39;s correlation coefficient analyses.</li>
	<li>Determine the significance of differences between study groups using Student&#39;s t-tests or one-way ANOVA, followed by a post-hoc test (Tukey) using spreadsheet software. Consider a p-value of less than 0.05 to be statistically significant.</li>
</ol>

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The authors declare no competing conflicts of interest.

Measuring SCB thickening is an important component of histologic assessments of OA severity. Most existing OA grading systems focus mainly on cartilage changes15,16,17. A widely used murine osteoarthritic SCB thickness grading method that categorizes SCB thickening as mild, moderate, and severe is largely subjective, and its reliability has not been fully validated15. The present study has developed and v...

Osteoarthritis (OA), characterized radiographically by joint space narrowing due to the loss of articular cartilage, osteophytes, and subchondral bone (SCB) sclerosis, is the most common form of arthritis1,2. Although the role of peri-articular bone in the etiology of OA is not fully understood, osteophyte formation and SCB sclerosis are generally thought to be the results of the disease process rather than causative factors, but changes in peri-articular bone architecture/shape and biology may contribute to the development and progression of OA3,4. The development of an accurate and easily executed OA grading system, including SCB measurement, is critical for comparative studies among research laboratories and in evaluating the efficacy of therapeutic agents designed to prevent or attenuate OA progression.
SCB is built with a thin dome-like bone plate and an underlying layer of trabecular bone. The SCB plate is the cortical lamella, lying parallel to and immediately under the calcified cartilage. Small branches of arterial and venous vessels, as well as nerves, penetrate through the channels in the SCB plate, communicating between the calcified cartilage and the trabecular bone. The subchondral trabecular bone contains blood vessels, sensory nerves, bone marrow and is more porous and metabolically active than the SCB plate. Therefore, SCB exerts shock-absorbing and supportive functions and is also important for cartilage nutrient supply and metabolism in normal joints5,6,7,8.
SCB thickening (in histology) and sclerosis (in radiography) are the major hallmarks of OA and key research areas of OA pathophysiology. Measuring SCB thickening is an important component of histologic assessments of OA severity. Previously reported digital microradiography for measuring rodent SCB mineral density9 as well as micro-computed tomography (micro-CT) based quantitative SCB measurement in rodent models of OA10,11,12,13 have improved our understanding of SCB structure and the role of SCB changes in OA pathophysiology. SCB area and thickness has also been quantified with histological slides using a sophisticated computer system with specific and expensive bone histomorphometry software14. Nevertheless, visual estimate-based semi-quantitative OA grading systems, including SCB thickening grading, are more widely used than micro-CT at the present time because the grading systems are easy to use, particularly for screening numerous histologic images. However, most existing OA grading systems focus mainly on cartilage changes15,16,17. A widely used osteoarthritic SCB thickness grading method that categorizes SCB thickening as mild, moderate, and severe is largely subjective, and its reliability has not been fully validated15. A reliable and easily executed step-by-step osteoarthritic SCB thickness measurement protocol is either not fully developed or un-standardized.
This study aimed to develop a reproducible, sensitive, and easily executed protocol to quantitatively measure the SCB thickness in a mouse model of OA. Our rigorous measurement tests and statistical analysis demonstrated that this ImageJ software-assisted quantitative measurement protocol could quantify the SCB thickness in both normal and osteoarthritic knee joints. The newly developed protocol is reproducible and more sensitive to mild SCB changes than the widely used visual grading systems. It can be used for detecting early osteoarthritic SCB changes and for assessing in vivo efficacy of OA treatments in concert with OA cartilage grading.

<table><tbody><tr><td>Safranin-O</td><td>Sigma-Aldrich</td><td>S8884</td><td></td></tr><tr><td>Fast green</td><td>Sigma-Aldrich</td><td>F7252</td><td></td></tr><tr><td>Hematoxylin</td><td>Sigma-Aldrich</td><td>GHS216</td><td></td></tr><tr><td>Eosin</td><td>Sigma-Aldrich</td><td>E4382</td><td></td></tr><tr><td>illustrator</td><td>Adobe</td><td>Not applicable</td><td></td></tr></tbody></table>

software-assisted quantitative measurement of osteoarthritic subchondral bone thickness

Subchondral bone thickening and sclerosis are the major hallmarks of osteoarthritis (OA), both in animal models and in humans. Currently, the severity of the histologic subchondral bone thickening is mostly determined by visual estimation based semi-quantitative grading systems. This article presents a reproducible and easily executed protocol to quantitatively measure subchondral bone thickness in a mouse model of knee OA induced by destabilization of the medial meniscus (DMM). This protocol utilized ImageJ software to quantify subchondral bone thickness on histologic images after defining a region of interest in the medial femoral condyle and the medical tibial plateau where subchondral bone thickening usually occurs in DMM-induced knee OA. Histologic images from knee joints with a sham procedure were used as controls. Statistical analysis indicated that the newly developed quantitative subchondral bone measurement system was highly reproducible with low intra- and inter-observer variabilities. The results suggest that the new protocol is more sensitive to subtle or mild subchondral bone thickening than the widely used visual grading systems. This protocol is suitable for detecting both early and progressing osteoarthritic subchondral bone changes and for assessing in vivo efficacy of OA treatments in concert with OA cartilage grading.

Reproducibility comparison between visual estimate grading and ImageJ-assisted quantitative measurement: 
SCB thickness in 48 regions of interest (ROI) (24 MFC and 24 MTP), defined from a mid-section of each knee from 24 knees/animals was scored by three independent individuals using the existing 0-3 visual scoring scheme as described in the literature15,23, where 0 = normal (no SCB thickening), 1 = mild, 2 = moderate, and 3 = severe SCB thi...

Watch this Scientific Journal Video about Software-Assisted Quantitative Measurement of Osteoarthritic Subchondral Bone Thickness at JoVE.com

Software-Assisted Quantitative Measurement of Osteoarthritic Subchondral Bone Thickness

This methodology article presents a software-assisted quantitative measurement protocol to quantify histologic subchondral bone thickness in murine osteoarthritic knee joints and normal knee joints as controls. This protocol is highly sensitive to subtle thickening and is suitable for detecting early osteoarthritic subchondral bone changes.

Subchondral bone thickening and sclerosis are the major hallmarks of osteoarthritis (OA), both in animal models and in humans. Currently, the severity of ...

software-assisted-quantitative-measurement-osteoarthritic-subchondral

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Medicine

2.8K Views.  University of Kansas Medical Center. Subchondrale botverdikking, of sclerose, is een van de kenmerken van artrose, zowel in diermodellen als bij mensen. Momenteel wordt de ernst van histologische subchondrale botverdikking meestal bepaald door visuele schatting op basis van semi-kwantitatieve beoordelingssystemen. Deze studie heeft tot doel een reproduceerbaar en gemakkelijk uit te voeren protocol te ontwikkelen om de subchondrale botdikte kwantitatief te meten in een muismodel van knie-artrose geïnduceerd door destabilisatie v...

Video: Software-Assisted Quantitative Measurement of Osteoarthritic Subchondral Bone Thickness

Quantitative Autonomic Testing

Disorders associated with dysfunction of autonomic nervous system are quite common yet frequently unrecognized. Quantitative autonomic testing can be invaluable tool for evaluation of these disorders, both in clinic and research. There are number of autonomic tests, however, only few were validated clinically or are quantitative. Here, fully quantitative and clinically validated protocol for testing of autonomic functions is presented. As a bare minimum the clinical autonomic laboratory should have a tilt table, ECG monitor, continuous noninvasive blood pressure monitor, respiratory monitor and a mean for evaluation of sudomotor domain. The software for recording and evaluation of autonomic tests is critical for correct evaluation of data. The presented protocol evaluates 3 major autonomic domains: cardiovagal, adrenergic and sudomotor. The tests include deep breathing, Valsalva maneuver, head-up tilt, and quantitative sudomotor axon test (QSART). The severity and distribution of dysautonomia is quantitated using Composite Autonomic Severity Scores (CASS). Detailed protocol is provided highlighting essential aspects of testing with emphasis on proper data acquisition, obtaining the relevant parameters and unbiased evaluation of autonomic signals. The normative data and CASS algorithm for interpretation of results are provided as well.

Standardized, comprehensive and fully quantitative testing of autonomic functions is described. The autonomic tests consist of evaluation of all three major autonomic domains including cardiovagal, adrenergic and sudomotor. The severity and distribution of dysautonomia is quantitated using Composite Autonomic Severity Scores.

Disorders associated with dysfunction of autonomic nervous system are quite common yet frequently unrecognized. Quantitative autonomic testing can be ...

Measuring Local Tissue Strains in Tendons via Open-Source Digital Image Correlation

There is considerable scientific interest in understanding the strains that tendon cells experience in situ and how these strains influence tissue remodeling. Based on this interest, several analytical techniques have been developed to measure local tissue strains within tendon explants during loading. However, in several cases, the accuracy and sensitivity of these techniques have not been reported, and none of the algorithms are publicly available. This has made it difficult for the more widespread measurement of local tissue strains in tendon explants. Therefore, the objective of this paper was to create a validated analysis tool for measuring local tissue strains in tendon explants that is readily available and easy to use. Specifically, a publicly available augmented-Lagrangian digital image correlation (ALDIC) algorithm was adapted for measuring 2D strains by tracking the displacements of cell nuclei within mouse Achilles tendons under uniaxial tension. Additionally, the accuracy of the calculated strains was validated by analyzing digitally transformed images, as well as by comparing the strains with values determined from an independent technique (i.e., photobleached lines). Finally, a technique was incorporated into the algorithm to reconstruct the reference image using the calculated displacement field, which can be used to assess the accuracy of the algorithm in the absence of known strain values or a secondary measurement technique. The algorithm is capable of measuring strains up to 0.1 with an accuracy of 0.00015. The technique for comparing a reconstructed reference image with the actual reference image successfully identified samples that had erroneous data and indicated that, in samples with good data, approximately 85% of the displacement field was accurate. Finally, the strains measured in mouse Achilles tendons were consistent with the prior literature. Therefore, this algorithm is a highly useful and adaptable tool for accurately measuring local tissue strains in tendons.

Measuring Local Tissue Strains in Tendons via Open-Source Digital Image Correlation

This paper describes an open-source digital image correlation algorithm for measuring local 2D tissue strains within tendon explants. The accuracy of the technique has been validated using multiple techniques, and it is available for public use.

There is considerable scientific interest in understanding the strains that tendon cells experience in situ and how these strains influence tissue ...

Engineering

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...

Bioengineering

Addressing Practical Issues in Atomic Force Microscopy-Based Micro-Indentation on Human Articular Cartilage Explants

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the biological field. However, as with any other microscopic approach, methodological challenges can arise. In particular, the characteristics of the sample, sample preparation, type of instrument, and indentation probe can lead to unwanted artifacts. In this protocol, we exemplify these emerging issues on healthy as well as osteoarthritic articular cartilage explants. To this end, we first show via a step-by-step approach how to generate, grade, and visually classify ex vivo articular cartilage discs according to different stages of degeneration by means of large 2D mosaic fluorescence imaging of the whole tissue explants. The major strength of the ex vivo model is that it comprises aged, native, human cartilage that allows the investigation of osteoarthritis-related changes from early onset to progression. In addition, common pitfalls in tissue preparation, as well as the actual AFM procedure together with the subsequent data analysis, are also presented. We show how basic but crucial steps such as sample preparation and processing, topographic sample characteristics caused by advanced degeneration, and sample-tip interaction can impact data acquisition. We also subject to scrutiny the most common problems in AFM and describe, where possible, how to overcome them. Knowledge of these limitations is of the utmost importance for correct data acquisition, interpretation, and, ultimately, the embedding of findings into a broad scientific context.

We present a step-by-step approach to identify and address the most common problems associated with atomic force microscopy micro-indentations. We exemplify the emerging problems on native human articular cartilage explants characterized by various degrees of osteoarthritis-driven degeneration.

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the ...

A Probing Device for Quantitatively Measuring the Mechanical Properties of Soft Tissues during Arthroscopy

Probing in arthroscopic surgery is performed by pulling or pushing the soft tissue, which provides feedback for understanding the condition of the soft tissue. However, the output is only qualitative based on the &#8220;surgeon&#8217;s feeling&#8221;. Herein is described a probing device developed to address this issue by measuring the resistance of soft tissues quantitatively with a tri-axial force sensor. Under both conditions (i.e., pull- and push-probing certain tissues mimicking the acetabular labrum and cartilage), this probing device is found to be useful for measuring some mechanical properties in joints during arthroscopy.

Probing during arthroscopy surgery is normally done to assess the condition of the soft tissue, but this approach has always been subjective and qualitative. This report describes a probing device that can measure the resistance of the soft tissue quantitatively with a tri-axial force sensor during arthroscopy.

Probing in arthroscopic surgery is performed by pulling or pushing the soft tissue, which provides feedback for understanding the condition of the soft ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

Cortical Bone Assessment Using the Ultrasonic BDAT Device

Cortical Bone Assessment Using Ultrasonic Guided Waves: A Reproducibility Study in a Healthy Population

Fragility fractures are still a worldwide health burden in the context of population aging. In particular, the global number of hip fractures is expected to double between 2020 and 2050. Therefore, it is essential to detect patients at risk of fragility fracture at the population scale. The current golden standard is dual X-ray absorptiometry (DXA), providing the areal bone mineral density (aBMD). Ultrasonic devices, usually more portable and cheaper than X-ray devices, represent interesting DXA alternatives as screening tools. However, operator dependency is usually recognized as their main drawback. In this study, the measurement protocol of the bi-directional axial transmission (BDAT) ultrasonic device is presented in detail. The dedicated ultrasonic probe is placed at the one-third distal radius of the non-dominant forearm using conventional coupling gel. The guided interface provides in quasi-real time (about 2 Hz) four parameters of interest: velocities of the first arriving signal (vFAS) and the A0 mode (vA0),&#160;cortical thickness (Ct.Th) and porosity (Ct.Po), as well as four quality parameters. The operator moves the probe slowly at the measurement site, carefully observing the feedback provided by the interface until finding a stable position and starting a series of 10 acquisitions. When at least four consistent series are obtained, the measurement ends, and an automatic report is generated. The measurement usually takes about 5 min to complete. To determine the robustness of this protocol, a reproducibility study was conducted among 3 operators (one expert and two novices) and 14 healthy participants (6 women, 8 men, 21-53 years old). The intraclass correlation coefficients (ICC) were found good for vA0 (0.76), Ct.Po (0.80) or excellent for Ct.Th (0.87) and vFAS (0.91). The standard deviations were found to be less than 10% of the total ranges in clinical practice.

Here, we present the measurement protocol of the bi-directional axial transmission (BDAT) ultrasonic device in detail and test it in a reproducibility study, considering 14 healthy participants and 3 operators. Reliability, measured with the intraclass correlation coefficients (ICC), was good to excellent for four parameters of interest.

Fragility fractures are still a worldwide health burden in the context of population aging. In particular, the global number of hip fractures is expected ...

Destabilization of the Medial Meniscus and Cartilage Scratch Murine Model of Accelerated Osteoarthritis

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are used to mimic many aspects of the disease. The present protocol describes the destabilization and cartilage scratch model (DCS) of post-traumatic osteoarthritis. Based on the widely used destabilization of the medial meniscus (DMM) model, DCS introduces three scratches on the cartilage surface. The current article highlights the steps to destabilize the knee by transecting the medial meniscotibial ligament followed by three intentional superficial scratches on the articular cartilage. The possible analysis methods by dynamic weight-bearing, microcomputed tomography, and histology are also demonstrated. While the DCS model is not recommended for studies that focus on the effect of osteoarthritis on the cartilage, it enables the study of osteoarthritis development in a shorter time window, with special focus on (1) osteophyte formation, (2) osteoarthritic and injury pain, and (3) the effect of cartilage damage in the whole joint.

The present protocol describes the controlled microblade scratches on the surface of the articular cartilage after destabilizing the mouse knee by cutting the medial miniscotibial ligament. This animal model presents an accelerated form of osteoarthritis (OA) suitable for studying osteophyte formation, osteosclerosis, and early-stage pain.

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are ...

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

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

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

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

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

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

Identification of Bone Erosions in HR-pQCT Images in Rheumatoid Arthritis

Erosion Identification in Metacarpophalangeal Joints in Rheumatoid Arthritis using High-Resolution Peripheral Quantitative Computed Tomography

Bone erosions are a pathological feature of several forms of inflammatory arthritis including rheumatoid arthritis (RA). The increased presence and size of erosions are associated with poor outcomes, joint function, and disease progression. High-resolution peripheral quantitative computed tomography (HR-pQCT) provides unparalleled in vivo visualization of bone erosions. However, at this resolution, discontinuities in the cortical shell (cortical breaks) that are associated with normal physiological processes and pathology are also visible. The Study grouP for xtrEme Computed Tomography in Rheumatoid Arthritis previously used a consensus process to develop a definition of pathological erosion in HR-pQCT: a cortical break detected in at least two consecutive slices, in at least two perpendicular planes, non-linear in shape, with underlying trabecular bone loss. However, despite the availability of a consensus definition, erosion identification is a demanding task with challenges in inter-rater variability. The purpose of this work is to introduce a training tool to provide users with guidance on identifying pathological cortical breaks on HR-pQCT images for erosion analysis. The protocol presented here uses a custom-built module (Bone Analysis Module (BAM) - Training), implemented as an extension to an open-source image processing software (3D Slicer). Using this module, users can practice identifying erosions and compare their results to erosions annotated by expert rheumatologists.

Author Spotlight: Enhancing Rheumatoid Arthritis Research Through HR-pQCT Imaging Analysis 

Bone erosions are an important pathological feature of rheumatoid arthritis. The purpose of this work is to introduce a training tool to provide users with guidance on identifying pathological cortical breaks on high resolution peripheral quantitative computed tomography images for erosion analysis.

Bone erosions are a pathological feature of several forms of inflammatory arthritis including rheumatoid arthritis (RA). The increased presence and size ...

Software-Assisted Quantitative Measurement of Osteoarthritic Subchondral Bone Thickness

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