Name: tissue collection and rna extraction from the human osteoarthritic knee joint
Uploaded: 2021-07-22T14:00:00.000+00:00
Duration: 6 min 6 s
Description: Watch this Scientific Journal Video about Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint at JoVE.com

The authors thank the study participants who made this research possible and dedicate this report to new scientists in the osteoarthritis field.

This study protocol was approved and followed institutional guidelines set by the Henry Ford Health System Institutional Review Board (IRB #13995).
1. Patient selection
<ol>
	<li>Identify the patients from among those scheduled to undergo TKA with an orthopedic surgeon.</li>
	<li>Select the patients based on the eligibility criteria defined by the study protocol. Examples of inclusion criteria include being 18 years of age or older and having a confirmed diagnosis of knee osteoarthritis. Examples of exclusion criteria include undergoing partial knee replacement or having a confirmed diagnosis of rheumatoid arthritis.</li>
	<li>Contact the patients to obtain informed consent prior to the surgery.</li>
</ol>
2. Sample processing (for RNA)
NOTE: Perform all tissue processing in a class II biosafety cabinet and follow sterile techniques. Always wear appropriate PPE (nitrile gloves, lab coat, safety goggles) when processing human samples. Several bone fragments are produced during TKA, a large amount of bone/cartilage will potentially be available for dissection. Due to disease progression, articular cartilage degeneration may be more severe on some bone portions, which can be factored into experimental design. Only tissues that mandate electrocautery for resection have thermal edge damage, and a concerted surgical effort is made to procure most tissues with a scalpel to minimize damage. Resected tissues must be kept hydrated at all times with sterile PBS.
<ol>
	<li>Disinfect all work surfaces and equipment with 70% ethanol, RNase decontaminant, DEPC-treated water, and again with 70% ethanol. Wipe away residual liquid with clean, lint-free tissues. Forceps, bone cutters, and scalpels are either autoclaved or soaked in 70% ethanol for at least 10 min prior to use. Keep equipment submerged in 70% ethanol when not in immediate use.</li>
	<li>Pre-label at least three cryovials with de-identified sample name and aliquot number for each tissue (e.g., TKA-1 Cartilage 1).
	<ol>
		<li>Identify each tissue type from the specimen based on the differences in size, shape, color, and texture as shown and described in Figure 2. Identify the cartilage (Figure 2A arrow), bone (Figure 2B arrow), meniscus (Figure 2C), infrapatellar fat pad (Figure 2D), anterior cruciate ligament (Figure 2E), synovium (Figure 2F), and the vastus medialis oblique muscle (Figure 2G).</li>
	</ol>
	</li>
	<li>Isolate the articular cartilage.
	<ol>
		<li>Select a bone portion with minimal cartilage degradation.</li>
		<li>Using a No.10 scalpel, cut through the cartilage depth as far as possible to dissect the full thickness of the cartilage layer. 
		NOTE: A No.10 scalpel will only penetrate cartilage, not bone.</li>
		<li>Using the full thickness of the cartilage, dissect three 5 mm cubes.</li>
	</ol>
	</li>
	<li>Isolate the subchondral bone.
	<ol>
		<li>Use the same bone section from which cartilage was collected.</li>
		<li>Using a No.10 scalpel, scrape any remaining cartilage and residual tissues from the bone surface.</li>
		<li>Hold the bone portion to be cut with forceps and use the bone cutters to cut three 5 mm cubes.</li>
	</ol>
	</li>
	<li>Isolate the meniscus.
	<ol>
		<li>Identify a relatively undamaged portion of either medial or lateral meniscus.</li>
		<li>Using a No.10 scalpel and forceps, dissect three 5 mm cubes.</li>
	</ol>
	</li>
	<li>Isolate the infrapatellar fat pad.
	<ol>
		<li>Using a No.10 scalpel and forceps, cut the yellow portion of the tissue into three equal-sized and homogenous portions (~500 mg each).</li>
	</ol>
	</li>
	<li>Isolate the anterior cruciate ligament (ACL).
	<ol>
		<li>Using a No.10 scalpel and forceps, dissect three 5 mm cubes.</li>
	</ol>
	</li>
	<li>Isolate the synovium.
	<ol>
		<li>Using a No.10 scalpel and forceps, isolate the pink cellular portion of the membrane as much as possible by scraping away fat tissue.</li>
		<li>Dissect three equal-sized and homogenous portions (~200 mg each).</li>
	</ol>
	</li>
	<li>Isolate the vastus medialis oblique muscle (VMO).
	<ol>
		<li>Using a No.10 scalpel and forceps, remove any fat tissue from the specimen, leaving only the red muscle tissue.</li>
		<li>Dissect three 5 mm cubes. 
		NOTE: The initial size of the tissue limits the size of the portions.</li>
	</ol>
	</li>
	<li>Rinse the tissue portions with sterile PBS to remove any residue or debris.</li>
	<li>To perform histology, fix each tissue portion as described in Part 3.</li>
	<li>To perform RNA extraction, cut each of the three tissue portions into smaller pieces (~1-2 mm&#160;cubes).
	<ol>
		<li>Transfer the smaller pieces to a 2 mL cryovial, secure caps tightly, flash freeze by submerging in liquid nitrogen for 30 s, and then transfer to a -80 &#176;C freezer for long-term storage (up to 4 months tested in the current protocol). Repeat this for all the aliquots of all the tissues.</li>
		<li>Continue to Tissue homogenization as described in Part 4.</li>
	</ol>
	</li>
</ol>
3. Sample processing (for histology)
CAUTION: Formalin is a hazardous chemical, only use in a chemical fume hood.
<ol>
	<li>Fill pre-labeled 15 mL conical tubes with 10% formalin solution.</li>
	<li>Using forceps, transfer the tissue section to the formalin-filled tubes.</li>
	<li>Fix the tissues in formalin for 1 week at room temperature while shaking/agitating if possible.</li>
	<li>After 1 week, discard the formalin into proper chemical waste disposal, rinse the tissues with PBS, and then transfer to a fresh 15 mL conical tube containing 70% ethanol for long-term storage at 4 &#176;C until samples are embedded for sectioning. 
	NOTE: For bone/cartilage only, perform decalcification as follows.</li>
	<li>After 1 week, discard the formalin into proper chemical waste disposal, and rinse the tissues with PBS.</li>
	<li>Transfer the tissue to a 50 mL conical tube with 45 mL of 10% EDTA solution (pH 7.4).</li>
	<li>If shaking/agitation is not possible, invert the tubes 10-15 times once daily.</li>
	<li>Discard and replace the EDTA solution once weekly.
	<ol>
		<li>Twice weekly, test the texture with an instrument (i.e., spatula) or gloved finger to confirm decalcification by observing deformation when pressure is applied. Time required to decalcify bone tissue will vary between samples from 4-6 weeks.</li>
	</ol>
	</li>
	<li>Once decalcified, rinse the tissue with PBS and transfer to a fresh 15 mL conical tube containing 70% ethanol for long-term storage at 4 &#176;C until samples are embedded for sectioning.</li>
</ol>
4. Tissue homogenization
CAUTION: The protocol uses the hazardous chemical phenol. Work with phenol must be performed in a chemical fume hood.
NOTE: Thoroughly clean all equipment and surfaces to be used with 70% ethanol (soak for a minimum of 10 min), followed by RNase decontaminant (soak for a minimum of 10 min), rinse with DEPC-treated water, wipe with a clean, lint-free paper towel, and then respray or soak with 70% ethanol.
<ol>
	<li>Hard tissue homogenization (articular cartilage, subchondral bone, meniscus)
	<ol>
		<li>Before homogenization, chill the mortar, pestle, and spatula using liquid nitrogen. These must be kept as cold as possible to prevent sample thaw.</li>
		<li>Process the samples one at a time, keeping the other samples at -80 &#176;C until used.</li>
		<li>Transfer the tissue sample to mortar using a chilled spatula; pour additional liquid nitrogen on top of the tissue and allow it to evaporate. Crush the tissue using the pestle. Repeatedly add more liquid nitrogen to the tissue sample, allow it to evaporate, and then continue grinding with the pestle to make a fine powder.</li>
		<li>After powdering the tissue as much as possible, transfer to a pre-chilled 1.5 mL microcentrifuge tube.
		<ol>
			<li>Pre-chill tubes by submerging in liquid nitrogen for 30 s prior to tissue transfer.</li>
		</ol>
		</li>
		<li>Add 1 mL of the acid-guanidinium-phenol solution to each tube and keep it on ice.</li>
		<li>Repeat steps 4.1.3-4.1.5 for each hard tissue sample.</li>
		<li>After each tissue, clean the mortar, pestle, and spatula with 70% ethanol, RNase decontaminant, DEPC-treated water, and an additional soak in 70% ethanol. Wipe any residual liquid with a clean, lint-free tissue.</li>
		<li>Incubate the samples on ice for an additional 20 min.</li>
	</ol>
	</li>
	<li>Soft tissue homogenization (infrapatellar fat pad, ACL, synovium, VMO)
	<ol>
		<li>Disinfect the homogenizer by running tubes of 70% ethanol, RNase decontaminant, DEPC-treated water, and an additional 70% ethanol wash, each for 30 s. Wipe any residual liquid with a clean, lint-free tissue. Repeat this between each sample.</li>
		<li>Pre-label 5 mL round bottom tubes for each sample. Add 1 mL of the acid-guanidinium-phenol solution to each tube.
		<ol>
			<li>Work on one sample at a time, keeping other samples to be processed at -80 &#176;C until use.</li>
		</ol>
		</li>
		<li>Transfer the tissue to a pre-labeled 5 mL tube with acid-guanidinium-phenol.</li>
		<li>Homogenize the tissues in 30 s pulses, keeping on ice during and between pulses. Repeat until the tissue is visually dissolved or for a maximum of five 30 s pulses. 
		NOTE: Some fibrous tissues (i.e., muscle) may not thoroughly homogenize.</li>
		<li>Incubate the dissolved tissue on ice and move to the next sample.
		<ol>
			<li>Clean the homogenizer as described in step 4.2.1. Ensure tissue chunks do not remain in the teeth of the probe; remove with sterile forceps if needed.</li>
		</ol>
		</li>
		<li>After homogenization of all the samples, incubate on ice in acid-guanidinium-phenol for an additional 20 min.</li>
		<li>Transfer the samples from round bottom tubes to pre-labeled and pre-chilled 1.5 mL microcentrifuge tubes.</li>
	</ol>
	</li>
</ol>
5. RNA extraction from tissues
CAUTION: This protocol uses hazardous chemicals such as phenol, chloroform, and isopropanol. Perform all the work in a chemical fume hood.
NOTE: Equipment and reagents are reserved for RNA work only and must be of proper chemical grade for molecular applications (i.e., sterile, nuclease-free). This protocol succeeds both Parts 4.1 and 4.2 tissue homogenization protocols. Thoroughly clean all equipment and surfaces to be used with 70% ethanol (soak for a minimum of 10 min), followed by RNase decontaminant (soak for a minimum of 10 min). Rinse with DEPC-treated water, wipe the residual liquid with a clean, lint-free paper towel, and then respray or soak with 70% ethanol.
<ol>
	<li>Centrifuge the microcentrifuge tubes at 10000 x g for 10 min&#160;at 4 &#176;C to pellet the debris.</li>
	<li>Transfer the supernatant to a fresh 1.5 mL microcentrifuge tube. 
	NOTE: For fatty tissues, a lipid layer will sometimes be present on the top. Avoid transferring this by piercing the layer on the side of the tube with a pipette tip.</li>
	<li>Add 200 &#181;L of chloroform per 1 mL of the acid-guanidinium-phenol solution to each sample. Shake the tubes vigorously by hand for 30 s to mix. Then, incubate on ice for 2 min.</li>
	<li>Centrifuge samples at 10000 x g for 12 min at 4 &#176;C. 
	NOTE: After centrifugation, three layers will have formed: an aqueous phase containing RNA, a white DNA interphase, and a pink protein phase at the bottom.</li>
	<li>Transfer ~500 &#181;L of the upper, aqueous phase to a fresh 1.5 mL microcentrifuge tube. 
	NOTE: Do not disturb the interphase and the protein fractions. Store these phases at -80 &#176;C for future DNA or protein isolation.</li>
	<li>Add an equal volume of acid-guanidinium-phenol solution to the transferred aqueous phase, mix by inverting the tube 8-10 times. Incubate the tube on ice for 20 min.</li>
	<li>Add 200 &#181;L of chloroform per 1 mL of the acid-guanidinium-phenol solution to each sample. Shake vigorously by hand for 30 s to mix and then incubate on ice for 2 min.</li>
	<li>Centrifuge samples at 10000 x g for 12 min at 4 &#176;C.</li>
	<li>Transfer &#60;500 &#181;L of the aqueous phase to a fresh microcentrifuge tube, careful not to contaminate the sample with the other phases. 
	CAUTION: Discard the remaining phases with appropriate hazardous material disposal methods.</li>
	<li>Add an equal volume (as aqueous phase) of 100% isopropanol to each sample. Mix by inverting the tube 8-10 times. Incubate on ice for 5 min.
	<ol>
		<li>Add 1 &#181;L of glycogen coprecipitant to each sample to aid in locating the RNA pellet post-centrifugation.</li>
	</ol>
	</li>
	<li>Centrifuge the samples at 12000 x g for 25 min at 4 &#176;C.</li>
	<li>Locate the pellet in the tube (if using coprecipitant, it will appear blue). Carefully pour off the supernatant.</li>
	<li>Wash the pellet by adding 1 mL of ice-cold 75% ethanol to each sample, vortex to dislodge the pellet from the bottom of the tube. 
	NOTE: Prepare 75% ethanol using molecular grade pure ethanol and nuclease-free water.</li>
	<li>Centrifuge at 7000 x g for 5 min at 4 &#176;C. Carefully pour off the supernatant.</li>
	<li>Repeat steps 5.13 and 5.14 two more times.</li>
	<li>Quick spin (&#60;2000 x g for 5 s) to bring any residual liquid to the bottom of the tube. Use a P20 pipette to remove any residual ethanol from the bottom of the tubes.
	<ol>
		<li>Avoid touching the RNA pellet with a pipette tip. Change tips between samples.</li>
	</ol>
	</li>
	<li>With tube caps open, air dry samples at room temperature for 10 min. 
	NOTE: Pellet may become translucent as it dries. Ensuring all residual ethanol has evaporated will improve RNA purity.</li>
	<li>Add 25 &#181;L of nuclease-free water to each tube to dissolve the pellet.</li>
	<li>Incubate at room temperature for 5 min.</li>
	<li>Gently pipette up and down to mix the RNA.</li>
	<li>Aliquot 5 &#181;L of the sample into a fresh tube for quality control analysis (Part 6). Store the remaining 20 &#181;L at -80 &#176;C for gene expression assays.</li>
</ol>
6. Quality control
<ol>
	<li>Determine the concentration and purity of RNA using a spectrophotometer according to the manufacturer&#39;s instructions.</li>
	<li>Determine the RNA integrity using an electrophoresis device according to the manufacturer&#39;s instructions. 
	NOTE: Dilute the RNA to be within the range of the chip detection limits.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+science+of+articular+cartilage.">Basic science of articular cartilage.</a> Clinics in Sports Medicine. 36 (3), 413-425 (2017).</li><li>Le Bleu, H. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+high-quality+RNA+from+human+articular+cartilage.">Extraction of high-quality RNA from human articular cartilage.</a> Analytical Biochemistry. 518, 134-138 (2017).</li><li>Ali, S. A., Alman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+extraction+from+human+articular+cartilage+by+chondrocyte+isolation.">RNA extraction from human articular cartilage by chondrocyte isolation.</a> Analytical Biochemistry. 429 (1), 39-41 (2012).</li><li>Schroeder, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rin:+An+RNA+integrity+number+for+assigning+integrity+values+to+rna+measurements.">The Rin: An RNA integrity number for assigning integrity values to rna measurements.</a> BMC Molecular Biology. 7, 3 (2006).</li><li>Li, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-platform+assessment+of+transcriptome+profiling+using+RNA-seq+in+the+abrf+next-generation+sequencing+study.">Multi-platform assessment of transcriptome profiling using RNA-seq in the abrf next-generation sequencing study.</a> Nature Biotechnology. 32 (9), 915-925 (2014).</li><li>Nazarov, P. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+sequencing+and+transcriptome+arrays+analyses+show+opposing+results+for+alternative+splicing+in+patient+derived+samples.">RNA sequencing and transcriptome arrays analyses show opposing results for alternative splicing in patient derived samples.</a> BMC Genomics. 18 (1), 443 (2017).</li><li>Madissoon, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=scRNA-seq+assessment+of+the+human+lung,+spleen,+and+esophagus+tissue+stability+after+cold+preservation.">scRNA-seq assessment of the human lung, spleen, and esophagus tissue stability after cold preservation.</a> Genome Biology. 21 (1), (2019).</li><li>Scholes, A. N., Lewis, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+RNA+isolation+methods+on+RNA-seq:+implications+for+differential+expression+and+meta-analyses.">Comparison of RNA isolation methods on RNA-seq: implications for differential expression and meta-analyses.</a> BMC Genomics. 21 (1), 249 (2020).</li><li>Smith, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+normal+synovium.">The normal synovium.</a> Open Rheumatology Journal. 5, 100-106 (2011).</li><li>Kukurba, K. R., Montgomery, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+sequencing+and+analysis.">RNA sequencing and analysis.</a> Cold Spring Harbor Protocols. 2015 (11), 951-969 (2015).</li><li>Mobasheri, A., Kapoor, M., Ali, S. A., Lang, A., Madry, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+deep+phenotyping+in+osteoarthritis:+how+can+high+throughput+omics+technologies+advance+our+understanding+of+the+cellular+and+molecular+taxonomy+of+the+disease.">The future of deep phenotyping in osteoarthritis: how can high throughput omics technologies advance our understanding of the cellular and molecular taxonomy of the disease.</a> Osteoarthritis and Cartilage Open. 3 (2), 100144 (2021).</li></ol>

The authors declare no conflicts of interest.

The protocol presented has proved successful for collecting seven primary human OA tissues for RNA extraction (Table 1) and histological processing (Figure 4). Prior to collecting patient samples, it is necessary to establish an IRB-approved protocol, ideally in collaboration with a surgeon or surgical team. Applying a standardized protocol for specimen collection (e.g., resection from consistent in situ locations) is essential for maximizing experimental reproducib...

The knee is the largest synovial joint in the human body, comprising the tibiofemoral joint between the tibia and the femur and the patellofemoral joint between the patella and the femur1. The bones in the knee are lined with articular cartilage and supported by various connective tissues, including menisci, fat, ligaments, and muscle, and a synovial membrane encapsulates the whole joint to create a synovial fluid-filled cavity1,2,3 (Figure 1). A healthy knee functions as a mobile hinge joint that allows frictionless motion in the frontal plane1,3. Under pathological conditions, movement can become restricted and painful. The most common degenerative knee joint disease is osteoarthritis (OA)4. A variety of risk factors are known to predispose to OA development, including older age, obesity, female sex, joint trauma, and genetics, among others5,6. There are currently an estimated 14 million people in the USA with symptomatic knee OA, with the prevalence increasing due to rising population age and rates of obesity7,8. Initially considered to be a disease of the cartilage, OA is now understood as a disease of the whole joint9. Commonly observed pathological changes in OA include articular cartilage erosion, osteophyte formation, subchondral bone thickening, and inflammation of the synovium9,10. Since there is no known cure for OA, treatments primarily focus on symptom (e.g., pain) management11,12, and once OA has progressed to end-stage, joint replacement surgery is often indicated13.
Joint replacement surgeries can either be partial or total knee replacements, with total knee arthroplasty (TKA) including replacing the entire tibiofemoral articulation and the patellofemoral joint. As of 2020, approximately 1 million TKAs are performed in the USA each year14. During TKA, an orthopedic surgeon resects the upper portion of the tibial plateau and the lower femoral condyles (Figure 2A, 2B) to be fitted with prosthetic implants. Sometimes misinterpreted by patients, in a TKA, only 8-10 mm is resected from the end of each bone, which is subsequently capped or resurfaced, with metal. An interposed polyethylene liner forms the bearing surface (i.e., padding) between the two metal implants. In addition, several soft tissue components of the joint are fully or partially excised to achieve proper joint balance. Among these tissues are the medial and lateral menisci (Figure 2C), infrapatellar fat pad (Figure 2D), anterior cruciate ligament (ACL; Figure 2E), synovium (Figure 2F), and vastus medialis oblique muscle (VMO; Figure 2G)15. Though TKAs are generally successful for OA treatment, around 20% of patients report reoccurrence of pain post-surgery16. Along with the high cost and relative invasiveness of the procedure, these limitations point to the need for further research to identify alternative treatments to mitigate the progression of OA.
To explore disease mechanisms in OA that may present new avenues for therapeutic intervention, experimental systems, including cells, tissue explants, and animal models can be used. Cells are typically cultured in monolayer and are derived from primary human or animal tissues (e.g., chondrocytes isolated from cartilage) or immortalized cells (e.g., ATDC517 and CHON-00118). While cells can be useful for manipulating experimental variables in a controlled culture environment, they do not capture conditions of the natural joint which are known to impact cell phenotypes19. To better recapitulate the complex cascade of chemical, mechanical, and cell-to-cell communication underlying OA, an alternative is found in primary human or animal tissue samples, whether used fresh or cultured ex vivo as explants, to preserve tissue structure and the cell microenvironment20. In order to study the joint in vivo, small (e.g., mouse21) and large (e.g., horse22) animal models for OA (e.g., through surgical induction, genetic alteration, or aging) are also useful. However, translation from these models to human disease can be limited by anatomical, physiological, and metabolic differences, among others23. Considering the advantages and disadvantages of experimental systems, the key strengths of being species-specific and maintaining the extracellular niche offered by the primary human OA tissues maximize the translational potential of research findings.
Primary human OA tissues can be readily obtained following TKA, making the high frequency of TKAs a valuable resource for research. Among potential experimental applications are gene expression and histological analyses. To realize the potential of primary human OA tissues for these research approaches and others, outlined are the following key considerations. First, the use of patient specimens is subject to ethical regulation, and protocols must meet Institutional Review Board (IRB) approvals24. Second, the inherent heterogeneity of human primary diseased tissues and the influence of variables such as age and sex, among others, create the need for careful patient selection (i.e., application of eligibility criteria) and data interpretation. Third, the unique biological properties of different tissues in the joint (e.g., low cellularity of cartilage and meniscus25) can present challenges during experiments (e.g., isolating high quality and quantity of RNA). This report addresses these considerations and presents a protocol for patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (i.e., assessment of RNA purity and integrity; Figure 3) to encourage the use of primary human OA tissues in the research community.

<table><tbody><tr><td>1.5 mL microcentrifuge tubes</td><td>Eppendorf</td><td>05 402</td><td>Sterile, nuclease-free. Reserved for RNA work only.</td></tr><tr><td>10% Formalin</td><td>Cardinal Health</td><td>C4320-101</td><td>Store in chemical cabinet when not in use.</td></tr><tr><td>100% Chloroform (Molecular Biology Grade)</td><td>Fisher Scientific</td><td>ICN19400290</td><td>Sterile, nuclease-free. Reserved for RNA work only, store in chemical cabinet when not in use.</td></tr><tr><td>100% Ethanol (Molecular Biology Grade)</td><td>Fisher Scientific</td><td>BP2818500</td><td>Sterile, nuclease-free. Reserved for RNA work only, when diluting use DEPC/nuclease-free water.</td></tr><tr><td>100% Isopropanol (Molecular Biology Grade)</td><td>Fisher Scientific</td><td>AC327272500</td><td>Sterile, nuclease-free. Reserved for RNA work only, store in chemical cabinet when not in use.</td></tr><tr><td>100% Reagent Alcohol</td><td>Cardinal Health</td><td>C4305</td><td>Diluted to 70% with dH&lt;sub&gt;2&lt;/sub&gt;O for cleaning purposes.</td></tr><tr><td>15 cm sterile culture dishes</td><td>Thermo Scientific</td><td>12-556-003</td><td>Sterile, nuclease-free.</td></tr><tr><td>15 mL polypropylene (Falcon) tubes</td><td>Fisher Scientific</td><td>14 959 53A</td><td>Sterile, nuclease-free.</td></tr><tr><td>2 mL cryovials (externally threaded)</td><td>Fisher Scientific</td><td>10 500 26</td><td>Sterile, nuclease-free.</td></tr><tr><td>5 mL round-bottom tubes</td><td>Corning</td><td>352052</td><td>Sterile, nuclease-free. Reserved for RNA work only.</td></tr><tr><td>50 mL polypropylene (Falcon) tubes</td><td>Fisher Scientific</td><td>12 565 271</td><td>Sterile, nuclease-free.</td></tr><tr><td>Bioanalyzer</td><td>Agilent</td><td>G2939BA</td><td>For RNA integrity measurement.</td></tr><tr><td>Biosafety Cabinet</td><td>General lab equipment</td><td></td><td></td></tr><tr><td>Bone Cutters</td><td>Fisher Scientific</td><td>08 990</td><td>Sterilized with 70% EtOH.</td></tr><tr><td>Chemical Fume Hood</td><td>General lab equipment</td><td></td><td></td></tr><tr><td>Disposable Scalpels (No.10)</td><td>Thermo Scientific</td><td>3120032</td><td>Sterile, nuclease-free.</td></tr><tr><td>EDTA</td><td>Life Technologies</td><td>15-576-028</td><td>10% solution with dH&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>Forceps</td><td>Any vendor</td><td></td><td>Sterilized with 70% EtOH.</td></tr><tr><td>Glycoblue Coprecipitant</td><td>Fisher Scientific</td><td>AM9516</td><td>Reserved for RNA work only, store at -20 &amp;deg;C.</td></tr><tr><td>Kimwipes</td><td>Fisher Scientific</td><td>06-666</td><td></td></tr><tr><td>Liquid Nitrogen</td><td>Any vendor</td><td></td><td></td></tr><tr><td>Liquid Nitrogen Dewar</td><td>General lab equipment</td><td></td><td></td></tr><tr><td>Mortar and Pestle</td><td>Any vendor</td><td></td><td>Reserved for RNA work only, sterilzed per protocol.</td></tr><tr><td>Nanodrop Spectrophotometer</td><td>Thermo Scientific</td><td>ND-2000</td><td>For RNA purity and yield measurements.</td></tr><tr><td>Nuclease-free/DEPC-treated water</td><td>Fisher Scientific</td><td></td><td>Sterile, nuclease-free. Reserved for RNA work only.</td></tr><tr><td>PBS (Sterile)</td><td>Gibco</td><td>20 012 050</td><td>Sterile, nuclease-free.</td></tr><tr><td>Pipettes (2 &amp;micro;L, 20 &amp;micro;L, 200 &amp;micro;L, 1000 &amp;micro;L) &amp;amp; tips</td><td>Any vendor</td><td></td><td>Sterile, nuclease-free.</td></tr><tr><td>Plasma/Serum Advanced miRNA kit</td><td>Qiagen</td><td>217204</td><td></td></tr><tr><td>Refrigerated Centrifuge 5810R</td><td>Eppendorf</td><td>22625101</td><td></td></tr><tr><td>RNAlater</td><td>Thermo Scientific</td><td>50 197 8158</td><td>Sterile, nuclease-free.</td></tr><tr><td>RNAse Away/RNAseZap</td><td>Fisher Scientific</td><td>&lt;br/&gt; 7002</td><td></td></tr><tr><td>Spatula (semimicro size)</td><td>Any vendor</td><td></td><td>Reserved for RNA work only.</td></tr><tr><td>Tissue homogenizer</td><td>Pro Scientific</td><td>01-01200</td><td>Reserved for RNA work only, sterilzed per protocol.</td></tr><tr><td>TRIzol Reagent</td><td>Fisher Scientific</td><td>15 596 026</td><td>Sterile, nuclease-free. Reserved for RNA work only.</td></tr></tbody></table>

tissue collection and rna extraction from the human osteoarthritic knee joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Seven unique human knee joint tissues are available for collection from patients undergoing TKA for OA (Figure 1). In this protocol, each of these tissues were identified and processed within 4 h of surgical removal (Figure 2). Following the steps outlined in Figure 3, portions of each tissue were formalin-fixed for histological assessment (Figure 4), while other portions were flash-frozen for RNA isola...

Watch this Scientific Journal Video about Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint at JoVE.com

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...

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Research

JoVE Journal

Biology

5.8K Views.  Henry Ford Health System. Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

Video: Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Developmental Biology

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

Novel Diagnostics in Revision Arthroplasty: Implant Sonication and Multiplex Polymerase Chain Reaction

In orthopedic patients, foreign body-associated infections, especially periprosthetic joint infections (PJIs), are a devastating complication of arthroplasty. Infection requires complex treatment, may result in long hospitalization and causes considerable costs. Multiple surgical revisions can be necessary in these patients, with a loss in function as well as in quality of life.
The routine preoperative diagnostics include blood examination for C-reactive protein (CRP) and other biomarkers, as well as joint aspirate analysis for cell count, differentiation, and culture. Intraoperative specimens for histology and microbiology are also standard procedure. The microbiological examination of removed implants with sonication, in combination with the implementation of molecular biology techniques in microbiology, represent two novel techniques currently employed to enhance the differential diagnostics of PJI.
We present here the step-wise procedure of analyzing joint aspirate and sonication fluid, using a cartridge-based multiplex polymerase chain reaction (PCR) system. Results were matched against conventional cultures and consensus criteria for PJI. Conventional microbiological cultures from tissue biopsies, joint aspirate and sonication fluid showed a sensitivity of 66.7%, 66.7%, and 88.9%, respectively, and a specificity of 82.3%, 54.6%, and 61.5%, respectively. The PCR diagnostic of the sonication fluid and the joint fluid showed a sensitivity of 50.0% and 55.6%, respectively, and both a specificity of 100.0%. Both PCR diagnostics combined had a sensitivity of 66.7% and a specificity of 100.0%. The multiplex PCR therefore presents a rapid diagnostic tool with moderate sensitivity but high specificity in diagnosing PJI.

Differential diagnostics in painful arthroplasty is crucial for treatment success. Joint aspiration is routinely performed preoperatively. Multiplex polymerase chain reaction of the joint aspirate and the sonication fluid, a feasible tool for quick pathogen detection from these samples is described.

In orthopedic patients, foreign body-associated infections, especially periprosthetic joint infections (PJIs), are a devastating complication of ...

Medicine

A Cryo-pulverization Protocol for Processing Mouse Paws to Evaluate Molecular Pathways of Tissue Inflammation in a Collagen Induced Arthritis Model

Profiling molecular changes in local tissues is crucial to understand the mechanism(s) of action of therapeutic candidates in vivo. In the field of arthritis research, many studies are focused on inflamed joints that are composed of a complex mixture of bone, cartilage, muscle, stromal cells and immune cells. Here, we established a reliable and robust mechanical method to disrupt inflamed mouse paws into homogeneous pulverized samples in a cryogenically controlled environment. Protein and RNA lysates were processed to enable proteomic and transcriptional endpoints and molecular characterization of relevant disease pathways in local tissue.

A cryogenic pulverization method to process murine paws using a liquid nitrogen freezer mill was developed to improve the yield and quality of RNA or protein extracted from the tissues and enable the analysis of molecular profiles associated with inflammatory responses.

Profiling molecular changes in local tissues is crucial to understand the mechanism(s) of action of therapeutic candidates in vivo. In the field of ...

Immunology and Infection

Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target joint disorders are mostly palliative. The unmet need for effective disease-modifying OA drugs (DMOADs) has been primarily caused by the absence of appropriate models for studying the disease mechanisms and testing potential DMOADs. Herein, we describe the establishment of a miniature synovial joint-mimicking microphysiological system (miniJoint) comprising adipose, fibrous, and osteochondral tissue components derived from human mesenchymal stem cells (MSCs). To obtain the three-dimensional (3D) microtissues, MSCs were encapsulated in photocrosslinkable methacrylated gelatin before or following differentiation. The cell-laden tissue constructs were then integrated into a 3D-printed bioreactor, forming the miniJoint. Separate flows of osteogenic, fibrogenic, and adipogenic media were introduced to maintain the respective tissue phenotypes. A commonly shared stream was perfused through the cartilage, synovial, and adipose tissues to enable tissue crosstalk. This flow pattern allows the induction of perturbations in one or more of the tissue components for mechanistic studies. Furthermore, potential DMOADs can be tested via either &#34;systemic administration&#34; through all the medium streams or &#34;intraarticular administration&#34; by adding the drugs to only the shared &#34;synovial fluid&#34;-simulating flow. Thus, the miniJoint can serve as a versatile in vitro platform for efficiently studying disease mechanisms and testing drugs in personalized medicine.

We provide detailed methods for generating four types of tissues from human mesenchymal stem cells, which are used to recapitulate the cartilage, bone, fat pad, and synovium in the human knee joint. These four tissues are integrated into a customized bioreactor and connected through microfluidics, thus generating a knee joint-on-a-chip.

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Biotribological Testing and Analysis of Articular Cartilage Sliding against Metal for Implants

Osteochondral defects in middle-aged patients might be treated with focal metallic implants. First developed for defects in the knee joint, implants are now available for the shoulder, hip, ankle and the first metatarsalphalangeal joint. While providing pain reduction and clinical improvement, progressive degenerative changes of the opposing cartilage are observed in many patients. The mechanisms leading to this damage are not fully understood. This protocol describes a tribological experiment to simulate a metal-on-cartilage pairing and comprehensive analysis of the articular cartilage. Metal implant material is tested against bovine osteochondral cylinders as a model for human articular cartilage. By applying different loads and sliding speeds, physiological loading conditions can be imitated. To provide a comprehensive analysis of the effects on the articular cartilage, histology, metabolic activity and gene expression analysis are described in this protocol. The main advantage of tribological testing is that loading parameters can be adjusted freely to simulate in vivo conditions. Furthermore, different testing solutions might be used to investigate the influence of lubrication or pro-inflammatory agents. By using gene expression analysis for cartilage-specific genes and catabolic genes, early changes in the metabolism of articular chondrocytes in response to mechanical loading might be detected.

This protocol describes the preparation, biotribological testing, and analysis of osteochondral cylinders sliding against metal implant material. Outcome measures included in this protocol are metabolic activity, gene expression and histology.

Osteochondral defects in middle-aged patients might be treated with focal metallic implants. First developed for defects in the knee joint, implants are ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Establishment of the KOA Model in Rabbits Using the Modified Videman Method

Application of Acupotomy in a Knee Osteoarthritis Model in Rabbit

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of people with KOA. Among several pathogenic factors, the biomechanical imbalance of the knee joint is one of the main causes of KOA. Acupotomology believes that restoring the mechanical balance of the knee joint is the key to treating KOA. Clinical studies have shown that acupotomy can effectively reduce pain and improve knee mobility by reducing adhesion, contracture of soft tissues, and stress concentration points in muscles and tendons around the knee joint. 
In this protocol, we used the modified Videman method to establish a KOA model by immobilizing the left hindlimb in a straight position. We have outlined the method of operation and the precautions related to acupotomy in detail and evaluated the efficacy of acupotomy in conjunction with the theory of "Modulating Muscles and Tendons to Treat Bone Disorders" through the detection of the mechanical properties of quadriceps femoris and tendon, as well as cartilage mechanics and morphology. The results show that acupotomy has a protective effect on cartilage by adjusting the mechanical properties of the soft tissues around the knee joint, improving the cartilage stress environment, and delaying cartilage degeneration.

Author Spotlight: Investigating the Mechanism of Action of Acupotomy in Treating Knee Osteoarthritis

In this protocol, a knee osteoarthritis model was prepared using the modified Videman method, and the operation procedures and precautions of acupotomy are detailed. The effectiveness of acupotomy has been demonstrated by testing the mechanical properties of quadriceps femoris and tendon and the mechanical and morphological properties of cartilage.

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of ...

An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique tissue important for proper function of the synovial joint and is constituted by a dense extracellular matrix (ECM), rich in proteoglycan and type II collagen. Chondrocytes are the only cell type present within cartilage and are widespread and relatively low in number. Altered external stimuli and cellular signalling can lead to changes in ECM composition and deterioration, which are important pathological hallmarks in diseases such as osteoarthritis (OA) and rheumatoid arthritis.
Ex vivo cartilage models allow 1) profiling of chondrocyte mediated alterations of cartilage tissue turnover, 2) visualizing the cartilage ECM composition, and 3) chondrocyte rearrangement directly in the tissue. Profiling these alterations in response to stimuli or treatments are of high importance in various aspects of cartilage biology, and complement in vitro experiments in isolated chondrocytes, or more complex models in live animals where experimental conditions are more difficult to control.
Cartilage explants present a translational and easily accessible method for assessing tissue remodeling in the cartilage ECM in controllable settings. Here, we describe a protocol for isolating and culturing live bovine cartilage explants. The method uses tissue from the bovine knee, which is easily accessible from the local butchery. Both explants and conditioned culture medium can be analyzed to investigate tissue turnover, ECM composition, and chondrocyte function, thus profiling ECM modulation.

Here, we present a protocol describing isolation and culturing of cartilage explants from bovine knees. This method provides an easy and accessible tool to describe tissue changes in response to biological stimuli or novel therapeutics targeting the joint.

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique ...