Poster Session - Intervertebral Disc - Biology - Hall E 0874 47th Annual Meeting, Orthopaedic Research Society, February 25 - 28, 2001, San Francisco, California

CELL DEATH AND MATRIX GENE EXPRESSION ARE UPREGULATED IN INTERVERTEBRAL DISCS DURING RECOVERY FROM SHORT DURATIONS OF MODERATE STATIC COMPRESSION

*Chin, J (A-NIH); +*Lotz, J (A-NIH) +*University of California San Francisco, San Francisco, CA. Orthopaedic Bioengineering Lab, Department of Orthopaedic Surgery, University of California, San

Francisco, CA 94143, 415-476-7881, Fax: 415-476-1128, jlotz@itsa.ucsf.edu Introduction: The degenerated human disc is characterized by decreases in both cell activity and density, resulting in a compromised extracellular matrix and altered biomechanical behavior. Animal experiments demonstrate that these features can be induced by physical force: cell death and altered gene expression are dependent on the magnitude and duration of in vivo compression (1,8). However, the mechanisms of this mechanobiologic interaction are unclear. In this study, we explored the temporal relationship between load and injury, and questioned whether the disc injury observed after continuous compression is due to a cumulative effect of load, or rather, is a delayed response to the initial loading event. To clarify this, we subjected mouse tail discs to short durations of compressive stress and allowed them to recover for varying periods. Methods: Tail discs between the 9th and 10th caudal vertebrae of 12 week old Swiss Webster mice were compressed with static axial loads using external fixators (approved by the University Committee on Animal Research). Two pairs of pins were drilled through the vertebral bodies on opposing sides of the test disc, and elastic bands calibrated to apply 0.8 MPa or 1.3 MPa were inserted over the pins. After specific durations of loading, the elastics were removed from some discs to allow recovery. Group 1 discs were compressed with 1.3 MPa for either 3 hours (h)(n=2), 6h (n=6) or 24h (n=8). Group 2 was similarly loaded to those in Group 1 but were allowed to recover after load removal for either 3 (n=3) or 6 days (d)(n=10). Group 3 was loaded with 0.8 MPa for 6h (n=3) and 24h (n=3). Group 4 was loaded like Group 3 but were allowed to recover for 6d (n=4). Sham operated control mice were subjected to pin insertion but were not loaded (n=26). Mice were sacrificed after the treatment period and test discs with adjacent vertebral bodies were fixed, decalcified and paraffin embedded. Apoptosis was assessed by the fluorescein-tunel method. Expression of type II collagen and aggrecan mRNA was determined by in situ hybridization. Results: Short durations of loading produced only modest adverse effects. Directly after compression with 1.3 MPa for 3-6h, cell death occurred sparsely in a few notochordal cells in the nucleus pulposus, comparable to levels seen in the sham controls and normal mice. Increasing the loading period to 24h (fig. a-c) resulted in a few more tunel-positive cells in the outer corners of the nucleus, annulus fibrosus and/or cartilage end plate. Aggrecan gene expression was maintained in the nucleus and type II collagen was still expressed in the cartilage end plate, but downregulated in the annulus. In contrast, extensive cell death was observed after the recovery period. In the 24h loaded and 3d recovered discs (fig. d-e), most of the nuclear cells were tunel positive, as were those in the middle and inner annulus where apoptotic bodies and tunel positive remnants were noted. The annulus displayed large regions of hypocellularity and exhibited various degrees of lamellar disorganization. Slight aggrecan signal remained in the nucleus, in contrast to the unrecovered discs. Expression for type II collagen was punctate and appeared upregulated in the cartilage end plate and remaining annular cells. When the recovery period was extended to 6d (fig. g-i), tunel-positive nuclear cells were generally in the peripheral regions and were less extensive compared to the 3d recovery. Collagen II was significantly elevated in the cartilage end plate and residual cells of the annulus where cell density remained low. These collagen-positive annular cells were morphologically similar to chondrocytes. Signal was punctate and intense in many nuclear cells. These cells co-existed among other notochordal cells that were smaller, compacted and stained punctately with hematoxylin, but did not express type II collagen. Aggrecan gene expression co-localized to the same regions, and was upregulated in the nucleus, which had been negative after the 3d recovery period. Peripheral nuclear apoptosis after 6d of recovery was also noted for the discs loaded for shorter durations (3h and 6h; similar results were observed when the load was reduced to 0.8 MPa for 6h and 24h). However, aggrecan expression was maintained in these short term loaded discs (before and after 6d of recovery) in contrast to the loss seen in 24h loaded and recovered discs.

Collagen expression was strong within the nucleus and/or nucleus periphery and cartilage end plate of recovered discs. Some regions of hypocellularity were noted in the annulus. Discussion: Our results demonstrate a delay between a loading event and adverse biologic consequences. Short durations (3h to 24h) of static loading induced major changes in disc cell function (viability, density, and expression of collagen and aggrecan genes) that were most notable only after a period of recovery with no load. In addition to the enhanced post-loading cell death, there was also evidence of a repair response (based on increased aggrecan and type II collagen transcription). This repair response was not observed in earlier studies of long duration continuous loading. The “activated” cells are likely those that remained viable during the short-duration loading event. Increased signal was often, but not exclusively, associated with large chondocytic cells within the annulus. These findings are consistent with cellular changes noted in four week post-operative rabbits whose discs were surgically denucleated (2). Delayed apoptosis after transient loading has been reported in human articular cartilage explants (3), in rat spinal cord after compression trauma (6) and in lymphoblast cultures (4). While we suspect the initiating event occurs during loading, we cannot exclude that the apoptotic trigger happens during recovery, similar to that noted in reperfusion injury following ischemia. The detrimental post-recovery response was minimal after the shortest compression period (3h). Since the time constant for disc creep is in this range (5), this observation suggests that excessive disc strain (and associated changes such as water content) mediates cell death. That is, loads removed before significant creep deformation has occurred may be less injurious. If true, this has important mechanistic implications, particularly with respect to dynamic loading regimens where beneficial effects of cyclic stress may be offset by the superimposition of large creep deformations. The results of this study suggest that load induced cell death is the consequence of transient, excessive strain. Furthermore, since cell death after recovery was comparable to that observed previously for continuous loading, impaired nutrition from diminished convective fluid flow may not be an important mediator. This result is supported by previous experiments that demonstrated immobilization alone did not result in apoptosis (9). We anticipate that this mechanistic perspective will be important for clarifying the tolerance of discs to more physiologic loading regimens.

Acknowledgements: Surgical expertise of Dr. Frank Kleinsteuck and Andrew Walsh is gratefully acknowledged. References: 1) Chin, JR et al., 45th ORS, 1999; 2) Takaishi H et al, J. Ortho. Res. 15:528, 1997; 3) D’Lima D et al,.46th ORS, 2000; 4) Takano KJ et al, Exp. Cell Res. 235:155, 1997; 5) Colliou OK, Thesis, 1998; 6) Li, GL et al, J. Neuropath. Exp. Neurology 55:280, 1996; 7) Piper, HM et al, Cardiovasc. Res. 38:291-300, 1998; 8) Lotz JC et al, Spine 23:2493-2506, 1998; 9) Court, C et al, 45th ORS, 1999