A Pre-Clinical Model of Synovitis Using Ex vivo Human Synovial Tissue with Preserved Function and Architecture

Summary

We developed a human ex vivo transwell system capable of evaluating acute inflammatory, infectious, and structural changes in synovium.

Abstract

Arthritis is an inflammatory state within joints resulting in cartilage damage, pain, and loss of mobility. Recent advances in arthritis research specifically demonstrate that the joint capsule (e.g., synovium) is an important source of this inflammation, but there are no human models that replicate essential synovial architecture. To address this, the Joint Space Analysis System, or JSAS, was created. Anterior synovium was obtained intra-operatively from patients undergoing Total Knee Arthroplasty (TKA). Synovium was dissected and sectioned into 3 mm biopsy cores. Cores were placed in the upper well of a 5 µm or 0.4 µm transwell with 300 µl of DMEM with10% FBS. In the bottom well, 600 µL of media was added, and exchanged every 2-3 days. Viability was assessed up to 7 days in hyperoxic (50%), atmospheric/standard (~21%), and physiologic (5%) incubation conditions. Stimuli in the bottom well included monocyte chemoattractant protein 1 (MCP-1/CCL2), lipopolysaccharide (LPS), N-acetyl cysteine, S. aureus, and B. burgdorferi. Media was stored for ELISA, and tissue was stored for formalin fixed paraffin embedded (FFPE) analysis. In standard conditions, synovium remained fully viable for 3 days. Stimulus modified the structure and function of intimal lining and sublining synovial cells, including loss of the resident macrophage border, sublining expansion, upregulation of pathogenic fibroblasts, and production of cytokines IL-1β and TNFα. Immune cells and fibroblasts migrated to the bottom chamber (5 µm pores) per flow cytometry analysis. Mobile B. burgdorferi migrated into tissue at the 0.4 µm pore size while non-motile S. aureus did not. Relevant cytokines were expressed in sufficient quantity for ELISA. JSAS is a modular system capable of studying acute alterations to human synovium, allowing for the complexity of 3D structures in a pre-clinical model while maintaining biologically relevant structure and function.

Introduction

Arthritis afflicts over 20% of all U.S. adults and 50% of those over age 65 with chronic conditions¹. It is also a leading cause of disability worldwide². Arthritis includes not only cartilage damage, but also inflammation of the joint capsule or synovium (e.g., synovitis). Chronic synovitis is likely a major source of pro-inflammatory cytokines that potentiate bone and cartilage damage in osteoarthritis and rheumatoid arthritis^3,4. Therefore, understanding the immune-mediated changes within the synovium is likely essential for developing new therapeutic and possibly preventative options for patients with arthritis.

Synovium is an organized structure composed of two major layers: the cellular intimal lining (IntL) and the sublining (SubL). The IntL interfaces with synovial fluid (SF), and the SubL makes SF components and contains vascular structures⁵. Within the IntL, there are both macrophages and fibroblast-like synoviocytes (FLS). The macrophages of the IntL are resident cells, locally renewed by interstitial macrophages within the SubL⁶. These resident synovial macrophages (RSMs) are M2-skewed by expression of CD206 and TREM2, epithelialized, and express tight junction markers. This sub-structure of the IntL may be key in functionally and physically maintaining joint space homeostasis^5,6. This has been observed in rheumatoid arthritis (RA). For example, when the IntL breaks down, patients with rheumatoid arthritis experience flare symptoms, which resolve when the lining rebuilds^6,7. Spontaneous RA remission is specifically associated with MerTK ⁺CD206⁺ synovial macrophages⁷. RSMs were also identified in SF of patients with acute joint pain, finding that the severity of infectious or inflammatory disease correlated with the quantity of RSMs and inflammatory cytokines⁸. Unfortunately, the relationship between the IntL and SubL structures, functional alterations of RSMs and FLS, and the production of inflammatory mediators that may go on to damage cartilage and/or bone is difficult to query, especially in humans.

To query underlying mechanisms of synovitis in multiple clinically relevant settings, a human ex vivo model called the Joint Space Analysis System (JSAS) was developed. This system produces reproducible results with modular elements to allow broad evaluation of inflammatory and infectious arthritis. This article describes the processes of tissue acquisition from common open-joint surgeries, such as total joint replacement, JSAS design, the histomorphometry necessary to quantify acute structural alterations in synovium, and the functional responses of the tissue, including by immunofluorescence (IF), RNAscope, and enzyme-linked immunosorbent assay (ELISA). JSAS differs from common human in vitro models by maintaining the IntL and SubL structures, allowing for near in vivo insights into acute or acute on chronic synovitis that leads to cartilage damage. By maintaining the native structure of synovium, it is possible to dissect the RSM and FLS intrinsic capabilities to dampen or perpetuate inflammation. As an ex vivo model, it neither requires differentiation of peripheral immune cells into synovial macrophages nor oversimplification of synovium to 1-2 discrete cell types only. It differs from animal models such as Collagen Induced Arthritis (CIA) by using naturally diseased human tissue, allowing pathogen-specific evaluation that is human-relevant. Finally, multiple infections can be simulated, allowing for a model of septic arthritis in addition to osteoarthritis and inflammatory arthritis. The main limitation is the culture for 3 days in standard conditions prior to the onset of apoptosis.

Protocol

Patient consent and tissue processing protocol were approved by the local Institutional Review Board (IRB). The reagents and the equipment used are listed in the Table of Materials.

1. Tissue acquisition and biopsy

1. Identify patients pre-operatively based on the surgeon's schedules. Recruit patients aged 18-65 who are undergoing open joint surgery, such as total knee arthroplasty (TKA) or Tibial Tubercle Osteotomy (TTO, also known as Fulkerson's), and who are not on systemic immunomodulatory medication.
   NOTE: Ideally, exclude patients who have more than one arthroscopy or more than one glucocorticoid injection, as these patients had a higher intra-operative failure of tissue acquisition than other populations due to fibrosis. Given the risk of infection transfer, defer recruitment of patients with a history of Hepatitis C or Human Immunodeficiency Virus (HIV) unless pertinent to outcomes. Practice safe handling practices at all times with fresh human tissue, regardless of infectious disease status.
2. Orthopedic surgeons perform arthrotomy and dissection as part of the standard operating procedure. Identify the anterior synovium immediately proximal to the trochlear groove and use electrocautery to excise the synovium en bloc while avoiding dissection of the articularis genu muscle or near the medial and lateral epicondyles. Synovectomy is relatively neutral for TKA outcomes, and in inflammatory conditions, may be beneficial^9,10. Pass the synovium sterilely to the research team member and place it in a sterile specimen container with cold Dulbecco's phosphate-buffered saline (DPBS) on ice.
   NOTE: Terminate tissue acquisition if there are abnormalities noted by the surgical team, or if synovectomy would impair closure or surgical outcome. The latter in particular would be highly uncommon in TKA, as synovium excision may be utilized as an extensile procedure to loosen tight joints.. The quantity of excision depends upon the size of the experiment. It is possible to obtain approximately four 3 mm biopsy cores per 1 cm² of tissue. In many cases, it is possible to obtain 24 cm² or more of tissue. Tissue weight is highly dependent on surgical dissection, thus, it is not as reliable as surface area for determining adequacy for the experiment. Figure 1 illustrates steps 1.2-1.7.
3. Return the synovium to the lab and wash twice in cold DPBS for 10 min on a plate rocker.
4. Transfer the synovium to a 10 cm or larger sterile Petri dish under sterile conditions in a biosafety level 2 (BSL2) certified biosafety cabinet. Add cold, sterile, low-glucose Dulbecco's Modified Eagle Medium (DMEM) media containing sodium pyruvate and L-glutamine, enough to fill the dish halfway. Identify the synovial lining by the pearlescent IntL layer (Figure 2A), as opposed to the cauterized side (Figure 2B).
   NOTE: The IntL is fragile. Careful not to peel it away, or it will dissociate from the sublining (Figure 2A, gripped by hemostat). Pearlescence may be altered by prior history of hemarthrosis (appearing yellow-orange due to hemosiderin), high BMI (covered in adipose), or inflammation (red, friable, and villous). It is absolutely essential to correctly identify the IntL prior to proceeding. Examine carefully for signs of surgical excision, cautery, bleeding, and coagulation to determine that the following steps are not occurring on the IntL.
5. Dissect away extraneous adipose and stromal tissue on the cauterized side to isolate synovium, maintaining approximately 4-5 mm of tissue depth in all areas. Iris scissors or scalpels may be used per personal preference. Change the Petri dish media as necessary to maintain the field of view.
   NOTE: Posterior stromal or adipose tissue is easily excised. However, difficulty with dissection or feeling great resistance is indicative of retained ligament, tissue metaplasia such as inappropriate cartilage deposition, or cutting through the IntL.
6. Once dissected to appropriate depth, obtain 3 mm biopsy cores as needed for a minimum of 2 technical replicates per tested condition. Perform with the synovial lining facing the coring tool rather than against the surface of the Petri dish. A quality biopsy punch may be used approximately 10-15 times before needing replacement due to dulling, as a dull biopsy punch will lead to damage of the intimal lining structure.
   1. Perform coring with forceful perpendicular load and gentle twisting. If this twists the tissue, the biopsy tool has become dull and needs replacement. Stabilize with the non-dominant hand using forceps or hemostats.
      NOTE: 3 mm cores were used to maximize tissue perfusion as well as the number of cores per patient and guarantee enough technical replicates for these experiments. Alternative size cores may be used, but only 3 mm was tested for this protocol.
      NOTE: Excess retained adipose from insufficient dissection may cause the core to float in JSAS, which is suboptimal. If the cores float to the surface rather than remaining submerged, return to dissection or choose a submerged core.
7. Open a second Petri dish and fill with 5-10 mL of warmed (37 °C) media with 10% FBS. With flat or non-toothed forceps, transfer cores to this dish to wash away debris with 1-2 s of gentle mechanical agitation. Do not pinch the cores tightly, or this may damage the intimal lining.
   NOTE: May pause here for ≤24 h by placing tissue cores in the incubator at standard setting of 37 °C and 5% CO₂.

2. Set up of Joint Space Analysis System (JSAS)

1. Prepare the bottom well of JSAS with 600 µL of warmed (37 °C) low glucose DMEM with 10% FBS. Though 1% penicillin-streptomycin has been tested without noticeable negative effect in this system, antibiotics are not typically added to media if sterility has been adequately maintained.
   NOTE: If co-culturing with bacteria or a cell line that requires a different media, the bottom well may be a different medium. Avoid antibiotic media if co-culturing with bacteria.
2. To the bottom well, add the treatment of choice. For example, lipopolysaccharide (LPS). To avoid osmolarity differences, it is recommended that the volume of treatment not exceed 2.5% of the total volume (e.g., ≤15 µL for 600 µL). Buffer any pH changes from treatments to maintain pH 7.0-7.4. Otherwise, utilize vehicle controls.
3. Choose the transwell pore size depending on the desired experimental outcome (Figure 3).
4. Add 300 µL of low-glucose DMEM with 10% FBS to the transwell.
5. Gently transfer individual cores with sterile forceps into the media of the transwell. The orientation of the tissue cannot be controlled; however, the adiposity and natural buoyancy of the stromal tissue naturally make the intimal lining face down for the majority of tissue cores.
   1. Carefully visualize the cores as the process of coring may disrupt the intimal lining, and avoid using cores that appear frayed at the start of the experiment. If the core is floating, improve the dissection and choose an alternate core.
      NOTE: Consider a collagen matrix if tissue orientation is important; however, this will impact the time to develop a chemical gradient with the bottom well.
6. Place in incubator at 37 °C and 5% CO₂ for the desired length of time.
   NOTE: Earliest time point recommended for biologic changes is 8 h. The latest validated time point for biologic activity is 3 days.

3. Experimental takedown and analysis

1. For histology, use sterile, non-toothed forceps to transfer tissue to 3-5 mL of 10% neutral buffered formalin (NBF) for formalin-fixed, paraffin-embedded (FFPE) analysis. Store in NBF for 3-7 days prior to embedding and sectioning.
   1. Do not let the tissue sit for extended periods, as this worsens autofluorescence. Perform histologic analysis, including hematoxylin and eosin (H&E)¹¹ staining for histomorphometry, immunofluorescence (IF)¹², and/or RNAscope with IF¹³ according to standard methods.
      NOTE: Careful not to puncture the transwell with forceps when removing the tissue. Be extremely gentle in transferring the core, or the intimal lining may dissociate. Gently place the core in fixative and do not agitate. NBF should be used inside a certified chemical fume hood, must be disposed of according to local environmental health and safety (EHS) guidelines, and may not be poured down sinks. Unused human tissue from the experiment is placed in excess 10% bleach for 30 min, then placed in secondary containment per local EHS requirements, then disposed of in labeled biohazard bins for incineration. The bleach solution may be poured down the sink if approved by local EHS. Follow local guidelines, which may differ based on the institution.
2. Embed technical replicates in the same FFPE block. Use screened cassettes to avoid loss of sample. Section in the longitudinal axis at 5 µm thickness. Place three serial sections on one slide (Supplementary Figure 1).
3. Image H&E slides at 10x and 40x using brightfield microscopy. Identify the IntL as the organized cellular layer at the interface between tissue and empty space.
   1. Acquire 3 representative 10x sections and 10 representative 40x sections across a minimum of 2 technical replicates per treatment condition, maintaining the IntL in the field of view. Capture images as high-resolution TIFFs. Perform histomorphometry analysis:
   2. Perform measurements in ImageJ/FIJI. First, set the scale using the Analyze menu → Set Scale. Set Scale requires the use of a calibration slide imaged on each respective microscope at each objective. First, use the straight line tool to create a line of known length based on the calibration slide image. Assign the scale globally.
      NOTE: At 10x, the scale is 3 px/µm. At 40x, the scale is 12 px/µm.
   3. Measure SubL thickness using the 10x images with the straight line tool. Measure thickness as a plumb depth from the IntL surface to the bottom of the SubL (µm). After drawing each line, use the keyboard shortcut m to measure the length of the line.
      1. Average a minimum of 5 measurements for each 10x image (Figure 4A,B). The transition between SubL and stromal tissue is demarcated by a transition to less organized, less cellular areas containing collagen and adipose (Figure 4B). Bisect the transition point between tissue compartments.
   4. For 40x measurements of IntL thickness, IntL cellularity, IntL integrity, and SubL cellularity, begin by opening the 40x TIFF in Adobe Photoshop (or any similar image editor). The IntL is defined as a layer ~1-5 cells thick, organized into a distinct cellular layer at the interface between tissue and empty space (e.g., synovial fluid) (Figure 4C).
   5. Create a new layer. Select the Polygonal Lasso tool. Using a mouse click to initiate the lasso as well as anchor inflections in the line, bisect the transition between the IntL and the SubL across the entirety of the image. Then, continue the lasso just outside the IntL in the empty space.
      1. Close the lasso with a double click. Invert the selected area (select → inverse), then fill the space with black using the paint bucket tool. This will cover all areas that are not the IntL. Save this image as a high resolution JPG representing the IntL, then hide the layer.
   6. Create a new layer. Under polygonal lasso settings, select the intersection feature, which will only select where the new lasso area and old lasso areas intersect. Lasso around the sublining layer by clicking through the middle of the IntL, around the left and right edges of the image into the background canvas, and then bisect the transition between SubL and acellular tissue behind it.
      1. If the bottom of the SubL exceeds the dimensions of the 40x image, simply select the background canvas around the remaining edge. Again, select the inverse of this area as above, then use the paint bucket tool to fill with black. Save this image as a high resolution JPG representing the SubL. Save this layered image as a PSD for future reference.
         NOTE: Erythrocytes are common in these slides and may interfere with future cell count measurements. If there are large areas of vasculature, use the polygonal lasso tool, the paintbrush tool, or other tools of choice to create masks on a separate layer covering these areas.
   7. In FIJI, open the IntL mask image. Measure IntL thickness (µm) as an average of a minimum of 5 measurements over the 40x image using the straight line tool as in step 3.3.2-3.3.3.
   8. Measure the total length (µm) of the IntL using the segmented line tool. Then measure the length of only the intact areas, also with the segmented line tool (Figure 4D,E). IntL sections that are not intact are demarcated by gaps between cells, a moth-eaten appearance, and breaking or flaking away of IntL from tissue.
      1. Calculate the % intact IntL border in as (intact IntL/total IntL)*100. This must be compared to the same time point controls to compensate for any sectioning artifact.
   9. Measure the cellularity of the IntL using the IntL mask image. First, transform to 8-bit (Image → Type → 8-bit). Then threshold (Image → Threshold) to minimize noise and maximize cell identification. In the threshold menu, deselect dark background and use the bottom toggle for adjustments.
      1. Manually determine threshold per image, aided by the disappearance of noise (e.g., background artifact) while retaining the original number of nuclear bodies. Compare frequently between the original image to maintain fidelity. When noise is minimized (false positive) but the hematoxylin signal is maximized (true nuclear positive), it is appropriate to set the threshold (Supplementary Figure 2).
         NOTE: Threshold will be consistent across a single slide scan, but will differ between slide scans and also between any individual 10x or 40x images. It will also differ between batches of H&E, as the depth of color is unlikely to be consistent. Thus, simple macros are inappropriate if there are differences in any of the following categories between images: patient, H&E batch, section thickness, lighting conditions (room or microscope light brightness/color temperature), software used to acquire the image, microscope used to acquire the image, and use of white balance or color balancing algorithms. This list is not exhaustive. Using the AmScope T390B-3M microscope with a compatible camera and software, with microscope light at maximum in a dimly lit room, utilizing automatic white balance settings, 10 random 40x images encompassing 3 patients, the threshold ranged from 96-182, with an average of 137. This broad range reflects the differences in the underlying 8-bit histograms, not user inconsistency. Thus, assuring visual fidelity between the original image and the threshold image is essential, not the exact number of the threshold. Automating this process is out of the scope of the typical bench scientist and would require programming from an image analysis professional.
   10. Use Analyze Particles (Analyze → analyze particles) with cell size 8-150 µm² to identify and count nuclei as a surrogate for cell number. Select overlay masks in the settings to visualize the identified cells, again comparing to the original image. As the IntL is primarily a linear, not volumetric, structure, normalize this 100 µm of IntL length (e.g., #cells/100 µm). This averaged approximately 13.19 (range 5.4-21.9, SD 3.9) cells per 100 µm in freshly retrieved synovial tissue.
       NOTE: The range 8-150 µm² was determined based on the Set Scale and iterative visual comparison of the thresholded image to the original image. The limits determined empirically through the dataset correspond to shape assumptions based on macrophages having a cell diameter that can range from approximately 8-20 µm^14,15. Larger sizes were excluded as this often represented multiple discrete nuclei in close proximity being read as one cell.
       NOTE: Similar workflows may be established in, for example, QuPath.
   11. Open the SubL mask with erythrocytes excluded (Figure 4G). Perform 8-bit transformation, threshold, and Analyze Particles as mentioned in steps 3.3.9-10. As the SubL is a volumetric layer, normalize the number of cells counted to 100 µm² of SubL area (e.g., measurement of non-masked areas) (Figure 4H).
4. For flow cytometry analysis of migratory cells in the bottom well, centrifuge at 300 x g at 4 °C for 10 min and keep the cell pellet on ice. Expect ~20,000 live, migratory cells per 3 mm biopsy core at 24 h. Flow cytometry methods have been published previously⁸.
5. For ELISA, centrifuge media at max speed (>5000 x g) for 5 min at 4 °C to pellet any cells or cell debris. Store at the desired temperature. ELISA methods have been published previously⁸.

Results

Optimizing oxygen culture conditions

Physiologic tissue perfusion is approximately 6%-8% oxygen, while standard incubation conditions for cell culture are at atmospheric oxygen levels (~21%)¹⁶. Comparatively, hyperoxygenation may improve cell culture viability or be toxic¹⁷. Adequate utilization of JSAS necessitated early determination of synovial tissue viability; therefore, apoptosis was assessed by caspase-3 immunohistochemist...

Discussion

This work demonstrated JSAS capabilities, specifically maintenance of living synovial tissue with intact, functionally responsive synovial architecture. There was minimal cell death out to 72 h of culture in most incubation conditions. To evaluate the microscopic changes of synovial structure, a unique histologic measuring system was described, quantifying structural alterations of the IntL and SubL. Such changes are indicative of acute or acute-on-chronic synovitis. This quantification may be particularly beneficial whe...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 cm Petri dishes	Corning	CLS430167
23srRNA RNAscope probe	ACD/Biotechne	468211
24 well plates	Life Sciences	3524
3 mm biopsy punches	Integra	12460406
50 mL conicals	N/A
70% ethanol (for sterilization)	N/A
Borrelia burgdorferi	ATCC	35210	must be utilized in the late exponential phase for maximum pathogenicity
BSK-H	Sigma-Aldrich	B8291
CD68 antibody	Invitrogen	14-0688-82	1:1000
CO2 tank (standard conditions)	N/A
Dulbecco's Phosphate buffered saline (Ca-/Mg-)	Thermofisher	14190144
fetal bovine serum	Biotechne	S11150	Heat inactivated Lot: F22100
forceps (non-toothed)	N/A
hemostat	N/A
Humidified cell incubator (standard conditions)	N/A
Hypoxia incubator	N/A
hypoxia tank (5% O2, 5% CO2, remainder N2, physiologic conditions)	N/A
IL-4 antibody	Thermofisher	MA5-42470	1:100
iNOS antibody	Thermofisher	PA1-036	1:100
Iris scissors	N/A
Lipopolysaccharide	Sigma-Aldrich	L2630
low glucose DMEM (+Sodium pyruvate, +L-glutamine)	Life Technologies	11885084
MMP9 antibody	Thermofisher	MA5-32705	1:1000
Modular incubator chamber (hyperoxic conditions)	Emrbient, Inc	MIC-101
Monocyte chemoattractant protein 1	Peptrotech	300-04-20UG
N-acetyl cysteine	Sigma-Aldrich	A9165	Must be made fresh for each experiment and buffered in equimolar sodium bicarbonate to a pH of 7-7.4
OPG ELISA	Millipore	RAB0484	1:1 dilution
Oxygen tank with liter per minute flow regulator	N/A
PDPN antibody	Invitrogen	14-9381-82	1:100
Propidium Iodide	Thermofisher	J66764.MC
RNAscope multiplex fluorescent V2 assay	ACD/Biotechne	323100
sRANKL ELISA	MyBioSource	MBS262624	1:1 dilution
Staphylococcus aureus USA 300 MRSA	ATCC	BAA-1717
SYBR Green	Thermofisher	S7563	10,000X stock
TNF antibody	Thermofisher	PA5-19810	1:100
transwell inserts - 0.4 um size	Millicell	PICM01250
transwell inserts - 5 um size	Corning	CLS3421