| 1. Lesion size and location |
Human: 2-mm diameter, ~1-cm deep biopsy from estimated geometric center of initial lesion, perpendicular to surface. Sample includes bone, full-thickness repair. Animal: sample block includes entire defect, flanking articular cartilage, subchondral bone encompassing potential regions of bone resorption. |
| 2. Timing of biopsy and sample recovery |
Human biopsy: 12-month or 24-month outcome, one biopsy per lesion. Macroscopic ICRS score. Video document and detailed description of biopsy site. Animal: acute defect (0-3 days) and long-term repair: ≥2 months (rabbit) or ≥6 months (large animal), with and without treatment. Exact endpoints can be tailored to individual studies and should provide information on the rate of implant incorporation or degradation in the joint. Macroscopic score (whole joint). Take any biochemistry or biomechanics samples prior to tissue fixation.
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| 3. Histoprocessing (for cartilage-bone analysis) |
Fixation in 10% normal buffered formalin or buffered 4% paraformaldehyde. Human: decalcify biopsies with bone for ~30 hours in 0.5 N HCl/0.1% glutaraldehyde115 or formic acid105 or ~2 weeks in 0.5 M EDTA at 4 °C. Animal: decalcify ~10 days (rabbit distal femur) or weeks (large animal samples) in 0.5 N HCl (with or without 0.1% glutaraldehyde)43 or longer in 10% EDTA/0.1% paraformaldehyde at 4 °C. Postdecalcification trimming step facilitates collecting at least 2 section levels per defect to take into account potential heterogeneity of repair. Postfixation after long periods of decalcification. Trim before embed. For each specimen, collect serial sections from at least 2 predetermined levels (fixed distance to each other in the repair tissue). 5-µm paraffin sections (human, sheep, horse) or 8- to 10-µm cryosections (rabbit) for collagen typing. Stain 2 serial sections from each level (for each stain). Tissue sections processed separately for electron microscopic analysis of matrix (optional).
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| 4. Staining |
H&E (cartilage-bone interface, cell morphology, tidemark, abnormal calcification). Safranin O or toluidine blue (glycosaminoglycan content). Collagen immunostaining for collagen type I and type II. Unstained sections (for polarized light microscopy [PLM]). Recut and stain any torn, folded, or poorly stained sections. Verify complete set of sections (use the best section free of folds, tears). Blind sections or digital scans prior to scoring.
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| 5. Evaluation methodsa
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Must be performed by 2 or 3 trained and blinded observers. Blinded consensus for outlier scores. Determine implant presence/absence. Histological scoring: ICRS-II (human, animal) or O’Driscoll (animal) that also assesses adjacent cartilage and cartilage-repair integration. Can use other scoring systems or evaluate predominant tissue type if want to compare results to literature. Histomorphometry: repair tissue thickness, total soft tissue volume above bone (for human biopsies where bone occupies ≥50% of section width) or above the projected tidemark (for animal sections with flanking cartilage). Analysis for area % glycosaminoglycan, % collagen type I, and % collagen type II staining. Optional: line measurements for defect width and % cartilage-subchondral bone integration (animal). PLM for collagen fibril orientation (semiquantitative scoring system).22 Optional: immunostain for specific markers of interest. Electron microscopy for cell morphology and collagen fibril diameter, orientation. Subchondral bone structure and repair (bone volume fraction). Subchondral cyst presence/absence (animal repair). Assess synovial histology.
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