Meniscus Tissue Response Following Tibia-Femoral Impact

1Lepinski, N.M.; 1Killian, M.L.; 2Haut, R. C.; 2Isaac, D.; +1Haut Donahue, T.L.1Michigan Technological University, Houghton, MI; 2Orthopaedic Biomechanics Laboratories, Michigan State University, East Lansing, MI

Senior author thdonahu@mtu.edu

IntroductionAtypical cell apoptosis has been observed in previous cartilage

wounding experiments and may play a role in the pathogenesis to osteoarthritis (OA).1 Studies show mechanical compression of articular cartilage may induce chondrocyte apoptosis, leading to increased synthesis of proteases, cytokines, and nitrous oxide (NO).1,3

Upregulation of these agents may perhaps contribute to the catabolism of the cartilage matrix in OA. In the case of overwhelming numbers ofcell death, loss of cells and reduction of extracellular matrix occurs by both apoptosis and secondary necrosis pathways.2,3 Previous studies have used anterior cruciate ligament (ACL) transections as an injury model to study apoptosis in the meniscus. The purpose of this study was to induce a traumatic injury through tibia-femoral impaction (TFI), either stretching or tearing the ACL, and analyze the cross sectional tissue divisions for differences in cell apoptosis, cell density, and tissue area based on zonal regions of the meniscus.

MethodsAdult Flemish Giant rabbits received TFI injuries to the left leg using

a device that promotes ACL injury yet, at the same time, retains the surrounding ligaments (Michigan State University’s Institutional Animal Care and Use Committee). One group was subjected to severe damage, completely tearing the ACL (TFI unconstrained), while another group experienced impaction without tibial translation, leaving the ACL intact(TFI constrained). An uninjured group of animals was used as controls. Both medial and lateral menisci were harvested at three months post-injury for unconstrained models and at six months for control and constrained models. Cross sectional divisions were made between anterior, central, and posterior regions of each meniscus (Figure 1).

Figure 1. Example of cross sectional divisions for meniscal regions.

Tissues were embedded in paraffin or Tissue-Tek OCT compound and sliced using a microtome or cryostat into six µm slices. Slices were placed on microscope slides pretreated with poly-L-lysine solution and misted with 60˚C ddH2O. Paraffin embedded tissues were incubatedovernight at 60˚C and OCT compound embedded tissues were allowed to dry at room temperature. Cell apoptosis was measured on paraffin-embedded samples using the DeadEnd Fluorometric TUNEL System (Promega). Briefly, tissue sections were deparaffinized in xylene, rehydrated in a decreasing ethanol series, washed in 1X phosphate buffered saline (PBS, pH 7.4) and then 0.85% NaCl. Slides were placed in Coplin jars and fixed in 4% paraformaldehyde (PFA), washed withPBS, digested with proteinase K (10mg/ml), and then washed and fixed again. The equilibration buffer was applied and slides were incubated at 37˚C with the rTdT incubation buffer for 90 minutes. The reaction was terminated in 2X SSC and unincorporated fluorescein-12-dUTP was washed away with PBS. Cell nuclei were stained with a propidium iodide dilution (1µg/ml in 1X PBS) and washed in ddH2O. Tissues embedded in OCT compound were only stained with propidium iodide and followed the procedure just after being placed in the Coplin jars. Deparaffinizing and rehydration steps were not necessary. To quantify total and apoptotic cell counts, CellC† was used to analyze all tissue sample images. Both total and specific cell counts were obtained for TUNEL samples while only total cell count was found for propidium iodide stains. The tissue area was found using MetaMorph (Molecular Imaging, Downingtown, PA, USA). Statistical analyses on control and experimental groups were done using a single factor ANOVA (significance of P<0.05). †http://www.cs.tut.fi/sgn/csb/cellc/index.html

Results Cell apoptosis between animal models varied greatly between samples and no significant difference was found between treatments (P>0.25). However, a general increase was seen in injured models (data not shown). When compared to the control, the tissue area decreased for both injuries while unconstrained impaction showed the largest decrease, suggesting a trend of greater tissue loss with more traumatic injuries(Figure 2A and Figure 3). As tissue area decreased, cell density was seen to increase in TFI unconstrained (Figure 2B).

Figure 2. Tissue area(A) and cell density(B) within the injured meniscal tissue of each animal model (n=2). Values shown represent the mean ± SD of meniscal regions within each meniscus.

Figure 3. Images showing decreasing tissue area amongst animal models: Control (A), TFI constrained (B), and TFI unconstrained (C) left leg, lateral meniscus, central region. Scale bar equals 2.0 mm.

Discussion Data collected for apoptotic cells produced a large variance between animal models. The counts for apoptotic cells were much lowercompared to previous studies when tissues were tested within days of the compression or injury.1 While early stages of apoptosis can be detected within minutes, the bodies produced by apoptosis will be present for hours before clearance by local phagocytes.2 Based on the time period between when the injury was given and when tissues were harvested, the low levels of apoptotic cell presence may indicate that cell clearance occurs during initial phases of injury.3,4

These data support previous studies done using ACL transections and the degeneration of the matrix within the meniscus, as the mean tissue area was smallest for TFI unconstrained. Also, TFI constrainedexperienced a mean decrease, suggesting a loss in the extracellular matrix as well. Even though the ACL was not torn during impact, the damaging effects of impaction can be seen in the slight decrease in the extracellular matrix. These injury models can serve as indicators as to which traumas are most harmful over time. However, measuring apoptosis after such time does not show distinguishing differences between injuries.

References 1. Loenig, A., et al., Arch Biochem Biophys. 381: 205-212, 20002. Roach, H., et al., Apoptosis. 9:265-277, 2004.3. Pelletier, J., et al., Arthritis Rheum. 43:1290-1299, 2000.4. Renvoize, C., et al., Cell Biol Toxicol. 14:111-120, 1998. 5. Nishida, M., et al. J Orthop Sci. 10:406-413, 2005.

Acknowledgements This research was supported by the Michigan Tech SURF program and in part by a grant from the CDC, National Center for Injury Prevention & Control (CE000623).

Poster No. 1198 • 55th Annual Meeting of the Orthopaedic Research Society