Use of non-degenerate human osteochondral tissue and confocal laser scanning microscopy for the study of chondrocyte death at cartilage surgery

Abstract

Although autologous osteochondral grafting has been widely applied in humans, most in vitro work has been on animal models. The aims of this study were to: (i) elaborate a full thickness human femoral condylar model using discard material from knee arthroplasty resections, and (ii) use this model to assess chondrocyte viability in response to surgical trauma. Homogeneous regions of human lateral femoral condyle bone-cartilage were procured from knee arthroplasty resections. These were graded prospectively, firstly by visual inspection, and then by confocal laser scanning microscopy (CLSM). Samples were subjected to tests of tissue hydration, including analysis of water content and swelling after excision from underlying bone. Surgical cuts were made in explants that were macroscopically and microscopically normal (i.e. Grade 0). Associated margins of death were assessed from both transverse and surface perspectives. Thirty-nine samples were obtained from anterior and distal femoral cuts (16 knees from 13 patients) for (1) macroscopic grading, (2) microscopic analysis, (3) analyses of water content as cut and on re-equilibration after excision from bone. Thirteen were Grade 0 on macroscopic viewing - however one showed fibrillation on microscopy and was therefore reassigned Grade 1. Grade 0 tissue had a water content of 73.8±0.38%, in agreement with control values from the literature. Tissues of Grades 2 and 3 were significantly (P=0.03, and P=0.004) more hydrated (76.0±0.59%, 76.7±0.99%) than Grade 0 tissue. Grade 0 tissue from the anterior cut did not swell significantly following excision from subchondral bone. However Grade 0 tissue from the distal cut showed a small but statistically significant (P=0.019) increase in water content (1.68±0.39%) following excision. With increasing grade there was increased tendency to swell off the bone (P<0.0001). Transverse imaging showed that the Acufex MP surgical harvester caused a greater margin of cell death (211±18.3μm) in the superficial zone (SZ) than the midzone (50.5±13.6μm; P=0.022), or SZ death from a scalpel cut (33.0±8.5μm; P=0.0009). Similarly, in unfixed samples viewed from the surface perspective, the margin of death for the surgical harvester (277±7.2μm) was significantly (P<0.0001) greater than that for the scalpel (38.8±7.2μm). If macroscopically and microscopically non-degenerate, then human lateral femoral condylar cartilage from the anterior cut of knee resections has normal biophysical parameters (water content and lack of swelling on excision). The surgical harvester (Acufex), used in human osteochondral grafting, caused a substantial margin of cell death at the periphery of the graft, and the SZ appeared to be especially vulnerable. This effect may be important in terms of limiting the reparative capacity of the SZ. The harmful effect on viability is likely to impede lateral repair which is fundamental to subsequent structural and functional integrity.