This research was primarily supported by funding from the National Institutes of Health (UG3/UH3TR002136, UG3/UH3TR003090). In addition, we thank Dr. Paul Manner (University of Washington) for providing the human tissue samples and Dr. Jian Tan for their help in isolating the MSCs and creating the cell pool.

The following protocol follows the ethical guidelines of the University of Pittsburgh and the human research ethics committee of the University of Pittsburgh. Information on the materials used in this study is listed in the Table of Materials.
1. Manufacturing 3D-printed bioreactors
<ol>
	<li>Use a computer software to design osteochondral (Figure 2A) and miniJoint bioreactors (Figure 2B) that include chambers, inserts, and lids. The dimension information for each part is shown in Figure S1.</li>
	<li>Transfer the design to a 3D printer, and print with a photopolymer ink.</li>
	<li>Rinse the 3D-printed parts (Figure 2C-F) with 15 mL of 95% ethanol three times. Then, fully crosslink the printed pieces for 200 s in a flashlight polymerization device.</li>
	<li>Add O-rings to inserts and lids (Figure 2C, D), and test if the parts fit (Figure 2G, H).</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64186/64186fig02.jpg" /> 
Figure 2: Fabrication of the different components to make the miniJoint bioreactor. (A,B), 3D models of bioreactors for creating (A) osteochondral and (B) miniJoint chips. (C,D) 3D printed (C) lids and (D)&#160;inserts with the O-ring installed. (E,F) 3D printed chambers for (E) osteochondral and (F) miniJoint tissue culture. (G,H) Assembly of (G) osteochondral and (H) miniJoint chips. <a href="https://www.jove.com/files/ftp_upload/64186/64186fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Engineering the tissue components
NOTE: The processes for the fabrication of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and methacrylated gelatin (GelMA) are described in previous studies8,9.
<ol>
	<li>To create GelMA, follow the steps below.
	<ol>
		<li>Add 17 g of gelatin Type B to 500 mL of distilled water, and then mix on a shaker for 30 min at 37 &#176;C.</li>
		<li>Then, add 13 mL of methacrylic anhydride, place back on the 37 &#176;C shaker, and allow to shake overnight.</li>
		<li>The following day, aliquot the GelMA into individual dialysis bags, with ~60 mL in each bag.</li>
		<li>Place all the dialysis bags in distilled water with a stir bar, and allow for 7 days of dialysis. Change the water multiple times per day, and leave the bags at 4 &#176;C overnight.</li>
		<li>On day 7, freeze the GelMA at &#8722;80 &#176;C. Once completely frozen, proceed to lyophilization.</li>
		<li>Place the GelMA in a dish in the vacuum chamber of a lyophilizer, and allow for freeze-drying. Ensure the GelMA is completely dried prior to removal from the lyophilizer.</li>
	</ol>
	</li>
	<li>Dissolve the GelMA in Hank&#39;s balanced salt solution (HBSS with Ca2+ and Mg2+) at 15% (w/v). To ensure the pH is at 7.4, add NaOH in small amounts until the pH reaches 7.4. Supplement the solution with 1x antibiotic-antimycotic and 0.15% (w/v) LAP based on the volume acquired. Store the 15% GelMA solution at &#8722;20 &#176;C until use, and protect it from light.</li>
	<li>Place 3D-printed dual-flow bioreactor chambers, lids, and inserts into autoclave bags, and autoclave at 121 &#176;C for 20 min with steam and then for 20 min with dry heat.</li>
	<li>Inside the biological safety cabinet, soak the bioreactor chambers, lids, and inserts in 15 mL of sterile phosphate-buffered saline overnight, after which allow them to dry.</li>
	<li>Isolate human bone marrow-derived MSCs from total joint arthroplasty surgical waste with IRB approval (University of Pittsburgh and University of Washington).
	<ol>
		<li>Specifically, flush out the bone marrow from the trabecular bone of the femoral neck and head, and resuspend it in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM).</li>
		<li>Filter the suspension through a 40 &#181;m strainer, and centrifuge the flow-through at 300 x g for 5 min.</li>
		<li>Remove the supernatant, resuspend the pellets using growth medium [DMEM, 10% fetal bovine serum (FBS), and 1x antibiotic-antimycotic], and then place into tissue culture flasks.</li>
		<li>Change the culture medium every 3 days to 4 days. Ensure that a confluence of 70% to 80% is reached before proceeding further.</li>
		<li>Detach the cells by incubating with trypsin-EDTA for 2-3 min, and passage at a ratio of 1 million cells per T150 flask.</li>
		<li>Expand the cells to P5. After trypsinization, suspend the cells, count them, and then pellet by centrifuging at 300x g for 5 min.</li>
	</ol>
	</li>
	<li>With a 1,000 &#181;L pipette, resuspend the cells at 20 x 106 cells/mL in 15% GelMA solution. 
	NOTE: Turn off the light of the biological safety cabinet.</li>
	<li>Using sterile gloves, press a sterile, dry silicone mold against a Petri dish. Then, put one insert into each hole of the silicone mold with forceps, with the hole side of the insert facing down.</li>
	<li>Using a 200 &#181;L pipette, add the cell suspension to fill up the insert (~50 &#181;L per insert).</li>
	<li>Use a UV flashlight (LED light with a wavelength of 395 nm) to crosslink the top of the gel/insert for 1.5 min. Then, illuminate the other side for 30 s. Crosslinking occurs when the LAP photoinitiator is exposed to UV light. 
	NOTE: The cell suspension may be kept in the incubator during this period or protected from light.</li>
	<li>With sterile forceps, immediately transfer the inserts into 8 mL of growth medium in a non-tissue culture 6-well plate (DMEM supplemented with 10% [v/v] FBS and 1x antibiotic-antimycotic) to allow the cells to recover overnight.</li>
	<li>Differentiate the cells toward four lineages.
	<ol>
		<li>To engineer adipose tissue (AT), transfer the inserts to 8 mL of adipogenic medium (AM; Alpha-MEM, 10% FBS, 0.2 mM indomethacin, 1x insulin-transferrin-selenium (ITS), 0.45 mM 3-isobutyl-1-methylxanthine,&#160;0.1 &#956;M&#160;dexamethasone, and 1x antibiotic-antimycotic) to initiate differentiation. Ideally, four inserts are placed in a single well of a non-tissue culture well plate with 8 mL of adipogenic medium. Culture the cells in the well plates for 28 days, with medium changes every other day.</li>
		<li>To engineer the osteochondral units (OC), place the inserts into dual-flow bioreactor chambers, cap the wells, and infuse the two streams separately at a flow rate of 5 &#181;L/min with 35 mL of osteogenic medium (OM; DMEM, 10% FBS, 1x antibiotic-antimycotic, 0.1&#160;&#956;M dexamethasone, 0.01 M &#946;-glycerophosphate, 100 ng/mL bone morphogenic protein 7 (BMP7), 50 &#181;g/mL ascorbic acid, and 10 nM vitamin D3) and 35 mL of chondrogenic medium (CM; DMEM, 1x antibiotic-antimycotic, 1x&#160;ITS, 0.1&#160;&#956;M&#160;dexamethasone, 40 &#181;g/mL proline, 50 &#181;g/mL ascorbic acid, and 10 ng/mL transforming growth factor &#946;3)10. Maintain cell differentiation for 28 days by performing biweekly medium changes.</li>
		<li>To derive the fibroblasts, differentiate the MSCs in 2D culture over 21 days in a T150 cm2 tissue culture flask using 20 mL of fibrogenic medium (FM; advanced DMEM, 5% FBS, 1x GlutaMAX, 1x antibiotic-antimycotic, and 50 &#181;g/mL ascorbic acid). Change the medium every week. Use 4 mL of trypsin to detach the cells, and encapsulate the 3D gels within the inserts, following the protocol described above, to obtain synovial like-fibrous tissue (SFT). 
		NOTE: The compositions of all the differentiation media can be found in Table 1.</li>
	</ol>
	</li>
</ol>
3. Establishing the miniJoint chip
<ol>
	<li>Autoclave the 3D miniJoint bioreactor chambers, silicone tubing with a 0.062 inch inner diameter and a 0.125 inch outer diameter, and F 1/16 Luer-lock connectors. Connect the silicone tubing to the miniJoint bioreactor barb at one end, and connect the Luer lock at the other end.</li>
	<li>Prepare the AM, OM (removing BMP7), and FMCM mentioned in step 2.11. Additionally, prepare the common shared medium (SM; phenol red-free DMEM, 1x antibiotic-antimycotic,&#160;1x Na-pyruvate, 1x ITS, 40 &#181;g/mL proline, 50 &#181;g/mL ascorbic acid, and 0.5 ng/mL transforming growth factor &#946;3) to be used for the miniJoint culture. Load 35 mL of each medium into the medium reservoirs.</li>
	<li>Use straight forceps to transfer the osteochondral unit from the dual-flow bioreactor to the right well of the miniJoint bioreactor. Transfer the adipose tissue insert and fibrous tissue insert into the left and middle wells, respectively. Cap all the wells with sterilized lids.</li>
	<li>Connect the inlets of the miniJoint chip to the medium reservoirs and the outlets to syringes (Figure 3A-C).</li>
	<li>Mount the syringes onto a syringe pump (Figure 3C), and transfer the pump and chips to an incubator. The medium reservoirs are kept on ice outside the incubator.</li>
	<li>Operate the pump in the withdraw mode, drawing the medium from the medium reservoir into the miniJoint bioreactor chamber. This miniJoint culture process lasts 28 days.</li>
	<li>To model joint inflammation and cartilage degeneration, add interleukin 1&#946; (IL-1&#946;) to the fibrogenic medium stream at 10 ng/mL. Replace the syringes on the third day of IL-1&#946; treatment, and provide fresh IL-1&#946; to the fibrogenic medium. The treatment lasts for 7 days.</li>
	<li>During the drug testing step, after 3 days of IL-1&#946; treatment, administer the drug either in the shared medium, simulating an &#34;intraarticular administration&#34; (Figure 3D) when the drug is used locally in the knee joint, or in all the medium types, simulating a &#34;systemic administration&#34; (Figure 3E) when the drug acts on the knee joint through the circulation.</li>
	<li>Collect individual tissues for analysis after 7 days of IL-1&#946; treatment whether or not the samples underwent drug treatment for the previous 4 days.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64186/64186fig03.jpg" /> 
Figure 3: Assembly of the miniJoint. (A,B) Tissue-specific media are introduced from inlets 1-3 (I1-3) and moved out from outlets 1-3 (O1-3). The shared medium is perfused from I4 to O4. (C) The full setup of the miniJoint culture. Drugs (green sun like shapes) can be either introduced into (D) the shared medium only or (E) all the mediums to respectively simulate &#34;intraarticular administration&#34; or &#34;systemic administration&#34;. <a href="https://www.jove.com/files/ftp_upload/64186/64186fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Individual tissue collection
<ol>
	<li>Use sterile curved forceps to remove the inserts.</li>
	<li>Push a biopsy punch through the center of the insert to remove the gel, and place the gel in PBS.</li>
	<li>Cut the osteochondral gels in half when assessing the gene expression. 
	NOTE: Since the osteochondral gel consists of two tissue types, it is important to separate the osteogenic and chondrogenic cells.</li>
	<li>Collect the conditioned media and tissues for various experiments.
	<ol>
		<li>Collect around 1.5 mL from each medium source.</li>
		<li>Flash-freeze the conditioned media in liquid nitrogen after centrifuging at 14,000 x g for 10 min and discarding the sediment.</li>
		<li>For histological staining and immunostaining, first fix the OC and SFT samples in 10% buffered formalin, dehydrate them in ethanol of ascending concentrations, clear them in xylene, embed them in paraffin, and finally section them at a thickness of 6 &#181;m.</li>
		<li>For the AT microtissues, fix the samples in 10% buffered formalin, and directly stain them with Oil Red O solution or BODIPY.</li>
	</ol>
	</li>
</ol>

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The authors declare no competing interests.

In this article, we present a protocol for creating a knee joint-on-a-chip system, in which bone, cartilage, adipose tissue, and synovium-like tissues are formed from MSCs and co-cultured within a customized bioreactor. This multi-component, human cell-derived system with plug-and-play features represents a new tool for studying the pathogenesis of joint diseases and developing drugs. 
Given that different tissues favor specific culture media, it is critical to provide the respective medium fo...

Joint diseases like osteoarthritis (OA) are highly prevalent and debilitating and represent a leading cause of disability worldwide1. It is estimated that in the US alone, OA affects 27 million patients and occurs in 12.1% of adults aged 60 and above2. Unfortunately, most drugs currently used to manage joint diseases are palliative, and no effective disease-modifying OA drugs (DMOADs) are available3. This unmet medical need primarily stems from the absence of an effective model for studying the disease mechanisms and developing potential DMOADs. The conventional two-dimensional (2D) cell culture does not reflect the 3D nature of joint tissues, and the culture of tissue explants is often hindered by significant cell death and usually fails to replicate the dynamic tissue interconnections4. In addition, genetic and anatomical differences significantly reduce the physiological relevance of animal models4.
Organs-on-chips (OoCs), or microphysiological systems, are a promising research field at the interface of engineering, biology, and medicine. These in vitro platforms are minimal functional units that replicate defined healthy or pathological features of their in vivo counterparts5. Furthermore, these miniaturized systems can host diverse cells and matrices and simulate the biophysical and biochemical interactions between different tissues. Therefore, a microphysiological system that can faithfully recapitulate the native synovial joint promises to offer an effective platform for modeling joint diseases and developing potential DMOADs.
Human mesenchymal stem cells (MSCs) can be isolated from many tissues throughout the body and differentiated into osteogenic, chondrogenic, and adipogenic lineages6. MSCs have been successfully used to engineer various tissues, including bone, cartilage, and adipose tissue6, thus meaning they represent a promising cell source for engineering the tissue components of the knee joint. We recently developed a miniature joint-mimicking microphysiological system, named miniJoint, that comprises MSC-derived bone, cartilage, fibrous, and adipose tissues7. In particular, the novel design enables tissue crosstalk by microfluidic flow or permeation (Figure 1). Herein, we present the protocols for the fabrication of the chip components, the engineering of the tissue components, the culture of the engineered tissues in the chip, and the collection of tissues for downstream analyses.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64186/64186fig01.jpg" /> 
Figure 1: Schematic of the miniJoint chip showing the arrangement of the different tissue components and medium flows. OC = osteochondral tissue. <a href="https://www.jove.com/files/ftp_upload/64186/64186fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>3-isobutyl-1-methylxanthine</td><td>Sigma -Aldrich</td><td>I17018-1G</td><td></td></tr><tr><td>6 well non-tissue culture plate</td><td>Corning Falcon&amp;reg; Plates</td><td>351146</td><td></td></tr><tr><td>24 well non-tissue culture plate</td><td>Corning Falcon&amp;reg; Plates</td><td>351147</td><td></td></tr><tr><td>30 mL syringes</td><td>BD Syringe Luer Lock Cascade Health</td><td>302832</td><td></td></tr><tr><td>Alcian blue stain</td><td>EK Industries</td><td>1198</td><td>1% w/v, pH 1.0</td></tr><tr><td>Advanced DMEM</td><td>Gibco</td><td>12491-015</td><td></td></tr><tr><td>&amp;alpha;MEM</td><td>Gibco</td><td>12571-063</td><td></td></tr><tr><td>Antibiotic-antimycotic</td><td>Gibco</td><td>15240-062</td><td></td></tr><tr><td>Biopsy punch</td><td>Integra Miltex</td><td>12-460-407</td><td></td></tr><tr><td>BODIPY&amp;reg; fluorophore</td><td>Molecular Probes</td><td></td><td></td></tr><tr><td>Bone morphogenic protein 7 (BMP7)</td><td>Peprotech</td><td></td><td></td></tr><tr><td>Curved forceps</td><td>Fisher Brand</td><td>16100110</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>11995-065</td><td>Dulbecco&amp;rsquo;s Modified Eagle Medium</td></tr><tr><td>Dexmethasome</td><td>Sigma -Aldrich</td><td>02-05-2002</td><td></td></tr><tr><td>E-Shell 450 photopolymer in</td><td>EnvisionTec</td><td>RES-01-4022</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gemini-Bio Products</td><td>900-208</td><td></td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>3505-061</td><td></td></tr><tr><td>gelatin from bovine skin</td><td>Hyclone</td><td>1003372809</td><td></td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution</td><td>Sigma -Aldrich</td><td>SH30588.02</td><td></td></tr><tr><td>indomethacin</td><td>Sigma -Aldrich</td><td>I7378-56</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium-Ethanolamine (ITS)</td><td>Gibco</td><td>51500-056</td><td></td></tr><tr><td>interleukin 1&amp;beta;</td><td>Peprotech</td><td>200-01B</td><td></td></tr><tr><td>Leur-loc connectors</td><td>Cole-Parmer Instruments</td><td>45508-50</td><td></td></tr><tr><td>L-proline</td><td>Sigma -Aldrich</td><td>115388-93-7</td><td></td></tr><tr><td>&amp;beta;-glycerophosphate</td><td>Sigma -Aldrich</td><td>1003129352</td><td></td></tr><tr><td>Medium bags</td><td>KiYATEC</td><td>FC045</td><td></td></tr><tr><td>Methacrylic Anhydride</td><td>Sigma -Aldrich</td><td>102378580</td><td></td></tr><tr><td>Phosphate buffered Saline</td><td>Corning</td><td>21-040-CM</td><td></td></tr><tr><td>Pointed forceps</td><td>Fisher Brand</td><td>12000122</td><td></td></tr><tr><td>Silicon mold</td><td>McMaster-Carr</td><td>RC00114P</td><td></td></tr><tr><td>Silicon o-rings</td><td>McMaster-Carr</td><td>ZMCCs1X5</td><td>1mm x 5mm</td></tr><tr><td>SolidWorks</td><td>Dassault Syst&amp;egrave;mes SE, V&amp;eacute;lizy-Villacoublay, France</td><td></td><td></td></tr><tr><td>Surgical Blades</td><td>Integra Miltex</td><td>4-122</td><td></td></tr><tr><td>Syringe pump</td><td>Lagato210P, KD Scientific</td><td>Z569631</td><td>10 syringe racks</td></tr><tr><td>T-182 tissue culture flasks</td><td>Fisher Brand</td><td>FB012939</td><td></td></tr><tr><td>Tissue Culture Dish 150 mm</td><td>Fisher Brand</td><td>FB012925</td><td></td></tr><tr><td>Transforming Growth Factor Beta (TGF-&amp;beta;3)</td><td>Peprotech</td><td>100-36E</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>UV Flashlight</td><td>KBS</td><td>KB70109</td><td>395 nm</td></tr><tr><td>Vida Desktop 3D Printer</td><td>EnvisionTec</td><td></td><td></td></tr><tr><td>Vitamin D3</td><td>Sigma -Aldrich</td><td>32222-06-3</td><td>1,25-dihydroxyvitamin D3</td></tr></tbody></table>

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.

All the tissues of the miniJoint were collected to analyze their phenotypes following 28 days of culture in the miniJoint (Figure 4A). This has been detailed in our previous publication7.
Through the use of RT-qPCR, immunostaining, and histological staining, it was confirmed that the tissue-specific phenotypes were well maintained for the individual microtissues (Figure 4). For example, the osseous component of...

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

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 ...

creation-knee-joint-on-chip-for-modeling-joint-diseases-testing

Research

JoVE Journal

Bioengineering

3.7K Views.  University of Pittsburgh School of Medicine. Ons vermogen om nauwkeurig te voorspellen hoe mensen reageren op geneesmiddelen tijdens klinische onderzoeken is beperkt vanwege het gebrek aan fysiologisch relevante preklinische modellen. Dit microfysiologische systeem kan die ongelijkheid aanpakken. Dit van menselijke cellen afgeleide systeem bootst nauwkeurig de hele gewrichtsziekte na aard van artrose in de menselijke knie, wat het mogelijk maakt om ziektemodificerende artrosegeneesmiddelen te testen voorafgaand aan klinische proeven, wa...

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

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...

A Lab-On-A-Chip Platform for Stimulating Osteocyte Mechanotransduction and Analyzing Functional Outcomes of Bone Remodeling

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical environment. During this process, mechanosensitive osteocytes regulate the opposing responses between the catabolic osteoclasts and anabolic osteoblasts. To better understand the highly intricate signaling pathways that regulate this process, our lab has developed a foundationary lab-on-a-chip (LOC) platform for analyzing functional outcomes (formation and resorption) of bone remodeling within a small scale system. As bone remodeling is a lengthy process that occurs on the order of weeks to months, we developed long-term cell culturing protocols within the system. Osteoblasts and osteoclasts were grown on functional activity substrates within the LOC and maintained for up to seven weeks. Afterward, chips were disassembled to allow for the quantification of bone formation and resorption. Additionally, we have designed a 3D printed mechanical loading device that pairs with the LOC platform and can be used to induce osteocyte mechanotransduction by deforming the cellular matrix. We have optimized cell culturing protocols for osteocytes, osteoblasts, and osteoclasts within the LOC platform and have addressed concerns of sterility and cytotoxicity. Here, we present the protocols for fabricating and sterilizing the LOC, seeding cells on functional substrates, inducing mechanical load, and disassembling the LOC to quantify endpoint results. We believe that these techniques lay the groundwork for developing a true organ-on-a-chip for bone remodeling.

Here, we present protocols for analyzing bone remodeling within a lab-on-a-chip platform. A 3D printed mechanical loading device can be paired with the platform to induce osteocyte mechanostransduction by deforming the cellular matrix. The platform can also be used to quantify bone remodeling functional outcomes from osteoclasts and osteoblasts (resorption/formation).

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Biology

A Friction Testing-Bioreactor Device for Study of Synovial Joint Biomechanics, Mechanobiology, and Physical Regulation

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication functions, fostering a deleterious physical environment. The frictional interactions of articular cartilage and synovium may influence joint homeostasis through tissue level wear and cellular mechanotransduction. To study these mechanical and mechanobiological processes, a device capable of replicating the motion of the joint is described. The friction testing device controls the delivery of reciprocal translating motion and normal load to two contacting biological counterfaces. This study adopts a synovium-on-cartilage configuration, and friction coefficient measurements are presented for tests performed in a phosphate-buffered saline (PBS) or synovial fluid (SF) bath. The testing was performed for a range of contact stresses, highlighting the lubricating properties of SF under high loads. This friction testing device can be used as a biomimetic bioreactor for studying the physical regulation of living joint tissues in response to applied physiologic loading associated with diarthrodial joint articulation.

The present protocol describes a friction testing device that applies simultaneous reciprocal sliding and normal load to two contacting biological counterfaces.

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...

Medicine

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.

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 ...

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 ...

Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired movement, and eventually, knee osteoarthritis (kOA). However, there is little effective treatment for cartilage defects or even kOA. Animal models are important for developing therapeutic drugs, but the existing models for cartilage defects are unsatisfactory. This work established a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats, and the subsequent pain behavior and histopathological changes were used as readout experiments. After surgery, the mechanical withdrawal threshold was decreased, chondrocytes at the injured site were lost, matrix metalloproteinase MMP13 expression was increased, and type II collagen expression decreased, consistent with the pathological changes observed in human cartilage defects. This methodology is easy and simple to perform and enables gross observation immediately after the injury. Furthermore, this model can successfully mimic clinical cartilage defects, thus providing a platform for studying the pathological process of cartilage defects and developing corresponding therapeutic drugs.

This protocol establishes a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats and measuring the subsequent pain behavior and histopathological changes.

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired ...

Impact of Non-Invasive ACL Injury and Surgery on Protease Activity in Mice

Non-Invasive Compression-Induced Anterior Cruciate Ligament (ACL) Injury and In Vivo Imaging of Protease Activity in Mice

Traumatic joint injuries such as anterior cruciate ligament (ACL) rupture or meniscus tears commonly lead to post-traumatic osteoarthritis (PTOA) within 10-20 years following injury. Understanding the early biological processes initiated by joint injuries (e.g., inflammation, matrix metalloproteinases (MMPs), cathepsin proteases, bone resorption) is crucial for understanding the etiology of PTOA. However, there are few options for in vivo measurement of these biological processes, and the early biological responses may be confounded if invasive surgical techniques or injections are used to initiate OA. In our studies of PTOA, we have used commercially available near-infrared protease activatable probes combined with fluorescence reflectance imaging (FRI) to quantify protease activity in vivo following non-invasive compression-induced ACL injury in mice. This non-invasive ACL injury method closely recapitulates clinically relevant injury conditions and is completely aseptic since it does not involve disrupting the skin or the joint capsule. The combination of these injury and imaging methods allows us to study the time course of protease activity at multiple time points following a traumatic joint injury.

Author Spotlight: Investigating Early Events and Long-Term Effects of ACL Injuries for Osteoarthritis Progression

Non-invasive ACL injury is a reliable and clinically relevant method for initiating post-traumatic osteoarthritis (PTOA) in mice. This injury method also allows for in vivo quantification of protease activity in the joint at early time points post-injury using protease-activatable near infrared probes and fluorescence reflectance imaging.

Traumatic joint injuries such as anterior cruciate ligament (ACL) rupture or meniscus tears commonly lead to post-traumatic osteoarthritis (PTOA) within ...

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.

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 ...

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

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