Rat MMT Methods

Processing of Joints

Operated joints are cut into two approximately equal halves in the frontal plane and embedded in paraffin. Three sections are cut from each knee at approximately 160 µm steps and stained with toluidine blue. Left knees from animals 1-5 of the vehicle control group have a single section prepared and stained with T. Blue.


Evaluation of Joints

Tissues are analyzed microscopically. The worst-case scenario for the two halves on each slide is determined and used for evaluation. The values for each parameter are then averaged across the three sections to determine overall values for each animal.

In addition, for some parameters (noted below), regional differences across the tibial plateau are taken into consideration by dividing each section into three zones (1-outside, 2-middle, 3-inside). In the surgical OA model, the outside (z1) and middle (z2) thirds are most severely affected, and milder changes are present on the inside third (z3). When zones are scored individually, scores are assigned based on percent area of the zone affected. Zone areas are delineated using an ocular micrometer.

The following parameters are measured and/or scored:


Medial Tibial and Femoral General Cartilage Degeneration Score

General cartilage degeneration includes chondrocyte death/loss, proteoglycan (PG) loss, and collagen loss or fibrillation, and is scored as follows:

  • 0=no degeneration
  • 1=minimal degeneration, within the zone 5-10% of the matrix appears non viable as a result of significant chondrocyte loss (greater than 50% of normal cell density). PG loss is usually present in these areas of cell loss and collagen matrix loss may be present
  • 2=mild degeneration, within the zone 11-25% of the matrix appears non viable as a result of significant chondrocyte loss (greater than 50% of normal cell density). PG loss is usually present in these areas of cell loss and collagen matrix loss may be present
  • 3=moderate degeneration, within the zone 26-50% of the matrix appears non viable as a result of significant chondrocyte loss (greater than 50% of normal cell density). PG loss is usually present in these areas of cell loss and collagen matrix loss may be present
  • 4=marked degeneration, within the zone 51-75% of the matrix appears non viable as a result of significant chondrocyte loss (greater than 50% of normal cell density). PG loss is usually present in these areas of cell loss and collagen matrix loss may be present.
  • 5=severe degeneration, within the zone 76-100% of the matrix appears non viable as a result of significant chondrocyte loss (greater than 50% of normal cell density). PG loss is usually present in these areas of cell loss and collagen matrix loss may be present.

A 3-zone sum for cartilage degeneration is calculated in addition to expressing the data for each zone.


Medial Tibial Total Cartilage Degeneration Width

The width of the cartilage affected by any degeneration (cell loss, proteoglycan loss or collagen damage) is measured by ocular micrometer. This measurement extends from the origination of the osteophyte with adjacent cartilage degeneration (outside 1/3) across the surface to the point where tangential layer and underlying cartilage appear histologically normal.


Medial Tibial Substantial Cartilage Degeneration Width

Substantial Cartilage Degeneration is identified by chondrocyte and proteoglycan loss extending through greater than 50% of the cartilage thickness and is measured by ocular micrometer. In general, the collagen damage is mild (25% depth) or greater for this parameter but chondrocyte and proteoglycan loss extend to at least 50% or greater of the cartilage depth, indicating regions in which permanent structural changes have occurred.


Medial Tibia Depth Ratio of Any Matrix Change

The depth of any type of lesion (both chondrocyte and proteoglycan loss, but may have good retention of collagenous matrix and no fibrillation) and the depth to tidemark are measured by ocular micrometer in the area of greatest lesion severity in each of the 3 zones of the tibial plateau. A depth ratio of any matrix change is calculated by dividing the lesion depth by the total depth. This measurement is the most critical analysis of any type of microscopic change present. The total depth to tidemark can serve as an average measure of cartilage thickness in each of the three zones for comparison of anabolics when measures are taken at the midpoint of the zone.


Osteophyte Score and Measurement

Osteophyte thickness (tidemark to furthest point extending toward synovium) is measured with an ocular micrometer. Scores are assigned to the largest osteophyte in each section (typically found on the tibia) according to the following criteria:

  • 0=less than 200 µm
  • 1=small 200-299 µm
  • 2=medium 300-399 µm
  • 3=large 400-499 µm
  • 4=very large 500-599
  • 5=very large ≥600

Medial Tibial Bone Damage Score – Single score for all 3 sections

Damage to the calcified cartilage layer and subchondral bone (worst case scenario for all sections) is scored using the following criteria:

  • 0=No changes
  • 1=Increased basophilia at tidemark, no fragmentation of tidemark, no marrow changes or if present minimal and focal, affects less than 10% of linear width of tidemark
  • 2=Increased basophilia at tidemark, minimal to mild focal fragmentation of calcified cartilage of tidemark affects approximately 1-10% of the linear width of the tidemark, mesenchymal change in marrow involves up to 1/4 of total area but generally is restricted to subchondral region under lesion, no collapse of cartilage into epiphyseal bone
  • 3=Increased basophilia at tidemark, mild to moderate focal or multifocal fragmentation or loss of calcified cartilage/subchondral bone (multifocal) affects 11-25% of linear width of tidemark, mesenchymal change in marrow is up to 3/4 of total area, areas of marrow chondrogenesis may be evident with focal collapse of articular cartilage into epiphyseal bone less than 200 µm deep
  • 4=Increased basophilia at tidemark, marked to severe fragmentation and or loss of calcified cartilage/subchondral bone affects 26-50% of linear width of tidemark, marrow mesenchymal change involves up to 3/4 of area and articular cartilage may have collapsed into the epiphysis to a depth of 200-350 µm or less from tidemark
  • 5=Increased basophilia at tidemark, marked to severe fragmentation and or loss of calcified cartilage/subchondral bone affects greater than 50% of the width of the tidemark, marrow mesenchymal change involves up to 3/4 of area and articular cartilage may have collapsed into the epiphysis to a depth of greater than 350 µm from tidemark

Medial Tibial Bone Sclerosis Score – Single score for all 3 sections

Medial tibial subchondral/epiphyseal bone thickening/sclerosis is scored based on the following criteria using a comparison to the lateral tibia and/or normal left medial tibias (if present and age matched):

  • 0=Normal, no observable difference in subchondral or epiphyseal trabecular bone thickness in medial vs. lateral
  • 1=5-10% increase in subchondral or epiphyseal trabecular bone thickness in medial vs. lateral
  • 2=11-25% increase in subchondral or epiphyseal trabecular bone thickness in medial vs. lateral
  • 3=26-50% increase in subchondral or epiphyseal trabecular bone thickness in medial vs. lateral, obvious reduction in marrow spaces in outer ¾ of medial tibia
  • 4=51-75% increase in subchondral or epiphyseal trabecular bone thickness in medial vs. lateral, generally has very little marrow space in outer ¾ of medial tibia, marrow spaces remain adjacent to cruciates.
  • 5=76-100% increase in subchondral or epiphyseal trabecular bone thickness in medial vs. lateral, generally has very little marrow space remains in medial tibia

Total Joint Scores

The three-zone sums of the tibial and femoral cartilage degeneration scores, and the osteophyte score were summed to determine a total joint score. A sum of tibial cartilage degeneration and osteophyte scores without the femur was also calculated.


Synovitis Score

Synovial inflammation (mainly mononuclear cell infiltration concentrated on the medial side) was scored as follows. Descriptions of other changes (typically fibrosis, or acute inflammation/neutrophil infiltration extending into the lateral compartment-usually associated with IA treatments) are also provided, if present:

  • 0=normal synovium
  • 5=very minimal synovitis (generally focal or scattered minimal diffuse)
  • 1=minimal synovitis (generally focal or scattered minimal diffuse)
  • 2=mild synovitis (multifocal to confluent areas of mild mononuclear cell infiltration)
  • 3=moderate synovitis (confluent areas of moderate mononuclear cell infiltration)
  • 4=marked synovitis (confluent areas of marked mononuclear cell infiltration)
  • 5=severe synovitis (confluent areas of severe mononuclear cell infiltration)

Medial Tibial Collagen Degeneration

Collagen damage across the medial tibial plateau (most severely affected section of the two halves) is quantified by measuring the total width of the following using an ocular micrometer. Measurements are expressed as a percentage of the total tibial surface width:

  • Any damage (fibrillation ranging from superficial to full thickness loss).
  • Severe damage (total or near total loss of collagen to tidemark, >90% thickness)
  • Marked damage (extends through 61-90% of the cartilage thickness)
  • Moderate damage (extends thru 31-60% of the cartilage thickness)
  • Mild damage (extends through 11-30% of the cartilage thickness)
  • Minimal damage (very superficial, affecting upper 10% only)

Medial Tibial Cartilage Degeneration Image Analysis

Image analysis of cartilage degeneration areas may be performed if justified by results of the general pathology evaluation (at the pathologist’s discretion).

Photomicrographs of the single most severely affected section from each animal are taken at 50x with a MOTIC microscope camera. The following are marked on a 10.3 x 7.7 hard copy of these photomicrographs, then measured in Adobe Photoshop on the original photos:

  • Total area from the tidemark to the surface (or projected surface in degenerated areas) over the outer two zones (5.8 cm on the printed photo) or all three zones (8.6 cm) of the tibial plateau, measured from the inner edge of the osteophyte
  • Area of non-viable matrix (cartilage with less than 50% chondrocytes, proteoglycan, and intact collagen) and no matrix within the total area
  • Area of no matrix within the total area

These two matrix loss measurements were then compared back to the total area to derive the percent loss of viable matrix area and the percent loss of any matrix area. Determination of whether to evaluate two zones or all three is made by the pathologist based on lesion severity and apparent treatment effects.


Growth Plate Thickness

Growth plate thickness is measured in all knees on medial and lateral sides (2 measures/joint) at the approximate midpoint of the medial and lateral physis (assuming a non tangential area of the section) using an ocular micrometer. The lateral thickness is also subtracted from the medial to determine the difference between the two.


Medial Collateral Ligament/Synovial Repair

Measurements are made of the thickness of the medial synovial/collateral ligament repair in a non-tangential area of the section using an ocular micrometer.

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