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

The main questions we want to answer are what is the native synovial immune response to infectious and inflammatory stimuli, and how is that reflected in alterations to synovial structure? My main clinical and research focus is on treatment of morbidly obese patients with end-stage RC arthritis. Frequently in this patient population, they have been medically neglected given their difficulty in finding surgical treatment options for their disease.

To begin, perform arthrotomy and dissection in the operating room. Use a standard medial parapatellar approach and perform a more extensile exposure when operating on a patient with morbid obesity and end-stage osteoarthritis. Identify the anterior synovium and use electrocautery to dissect the tissue.

Observe the knee for signs of inflammation, as patients with obesity may demonstrate a local inflammatory response that correlates with the broader inflammatory effects of obesity. Pass the synovium to the research team member in a sterile manner. Place the specimen container filled with cold Dulbecco's PBS or DPBS on ice and transfer the human anterior synovial tissue acquired during the total knee arthroplasty into the container.

Wash the tissue two times in cold DPBS for 10 minutes on a plate rocker. Next, transfer the synovium to a 10 centimeter or larger sterile Petri dish under a Biosafety Level 2 cabinet. Add cold sterile low glucose DMEM containing sodium pyruvate and L-glutamine to fill the dish halfway.

Identify the synovial lining by the pearlescent intimal layer without peeling it away. Arrange the required tools to obtain three millimeter biopsy cores for a minimum of two technical replicates. Using iris scissors or a scalpel, dissect away the extraneous adipose and stromal tissue from the cauterized side to isolate the synovium, maintaining a tissue depth of approximately four to five millimeters throughout.

Change the Petri dish media as necessary to maintain the field of view. Position the tissue with the synovial lining facing the coring tool. Stabilize the tissue with the non-dominant hand using forceps or hemostats, and core the tissue by applying a firm perpendicular pressure with gentle twisting.

Replace the biopsy punch after approximately 10 to 15 uses, or when the tissue twists during coring to avoid damaging the intimal lining structure. Open a second Petri dish and fill it with five to 10 milliliters of media containing 10%FBS tempered at 37 degrees Celsius. With flat or non-toothed forceps, transfer the cores to the dish and gently agitate for one to two seconds to wash away debris without pinching them tightly.

To prepare the bottom well of the Joint Space Analysis System, add 600 microliters of low glucose DMEM containing 10%FBS tempered at 37 degrees Celsius. Add the vehicle control or treatment reagent of choice, such as LPS or MCP-1, to the bottom well. Select the transwell pore size based on the desired experimental outcome, and add 300 microliters of low glucose DMEM containing 10%FBS to the transwell.

Using sterile forceps, gently transfer individual tissue cores into the media of the transwell, avoiding frayed and floating cores. Next, place the assembly in an incubator at 37 degrees Celsius with 5%carbon dioxide for eight to 72 hours depending on the downstream assays to be performed. Fix the synovial tissue in 10%neutral buffered formalin.

Then embed and section it for histologic analysis. After staining with hematoxylin and eosin, image the tissue sections at 10X and 40X using bright-field microscopy. Use Photoshop to outline the intimal lining and sublining regions in the 40X TIFF image, masking non-target areas, erythrocytes, and large vasculature for subsequent quantitative analysis.

Analyze the final masks to quantify intimal lining and sublining thickness, integrity, and cellularity. Finally, assess the migratory cells by flow cytometry and the soluble factors in the collected media by enzyme-linked immunosorbent assay. The synovial tissue cultured with increasing doses of MCP-1 or LPS showed distinct intimal lining morphology changes including friable intimal lining and a thickened intimal lining with decreased cellular density when compared to the control.

MCP-1 and LPS treatment altered the synovial cytokine response. MCP-1 induced pathogenic fibroblast marker podoplanin expression in the sublining, whereas LPS increased podoplanin in both the intimal lining and the sublining. Migratory immune cells in the bottom well included neutrophils, NK cells, NKT cells, T cells, B cells, or dendritic cells, monocytes, or macrophages, and M2 macrophages.

T cells were the most abundant migratory immune cells. Statistically, there was no difference in cell migration based on bottom well stimulus. Osteoprotegerin, an arthritis relevant protein, was detected at 24, 48, and 72 hours in untreated cultured arthritic synovium and was significantly higher at 72 hours than at 24 hours.

Inducible nitric oxide synthase expression in the intimal lining was high in controls at zero hours and was nearly absent after 24 hours. The expression of nitric oxide synthase was maintained when treated with N-acetyl cysteine. Conversely, N-acetyl cysteine did not alter matrix metalloproteinase-9 expression, a marker of both synovitis and potential cartilage damage.

This protocol allows researchers to study the alterations in synovial intimal and sublining layers, including depth, cellularity, and intactness, which is all indicative of inflammation. The most important considerations while performing this protocol are viable tissue acquisition from appropriate donors, sterility, and intimal lining identification for biopsy selection. Future studies will evaluate improving the length of viable tissue culture to evaluate regeneration of the synovial intimal lining.