Proceedings of the NASS 19 th Annual Meeting / The Spine Journal 4 (2004) 3S–119S 10S Fig. 1. PG and Collagen Synthesis. METHODS: Platelet-poor plasma (PPP) and PRP were isolated from porcine blood using the Platelet Concentration System (Symphony, Depuy Acromed). Nucleus pulposus (NP) and annulus fibrosus (AF) cells, en- zymatically isolated from porcine IVDs, were pre-cultured in alginate beads for 7 days in DMEM/F12 with 10% FBS and 25 μg/ml ascorbate [3]. The beads were then cultured for another 3 days either in 10%FBS (control condition) or with 10% PPP or 10% PRP. The synthesis of PGs and collagen was assessed as 35S-Sulfate and 3H-Proline incorporation as previously described [3]. PG and DNA content were measured by the DMMB method and Hoechst method, respectively. The two compartments [cell-associ- ated matrix (CM) and further removed matrix (FRM)] were separately as- sessed for PG metabolism [3]. RESULTS: DNA Content: PRP showed a mild stimulatory effect on the DNA content of alginate beads containing AF cells. PG Synthesis: PG synthesis was significantly upregulated in the PRP groups (Fig). The addi- tion of PRP significantly increased the distribution of PGs into the CM of AF cells (p0.01 vs FBS, PPP) although no significant difference was observed in NP cells (Fig). PG Content: PRP significantly stimulated PG accumulation in alginate beads (as % of FBS group; NP: PRP 140%, p0.01; AF: PRP 157%, p0.01). Collagen Synthesis: PRP significantly stimulated collagen synthesis, (Fig). No significant differences in the distri- bution of collagen in the CM and FRM were observed among the three groups. CONCLUSIONS: PRP significantly stimulated ECM synthesis by both porcine IVD cells cultured in alginate beads; the response to PRP was greater in AF cells than in NP cells. Although experimental conditions limited us to the use of only allogenic PRP, it can be predicted that autologous PRP has a similar effect on matrix metabolism. The possibility of applying PRP locally, such as an injection into the NP and/or AF, should be examined. In addition, given the risks of using animal serum for tissue engineering, autologous blood may gain favor as a source of PRP growth factors and serum supplements needed for stimulating cells to engineer IVD tissues. DISCLOSURES: No disclosures. CONFLICT OF INTEREST: Author (HA) Grant Research Support: DePuy. doi: 10.1016/j.spinee.2004.05.016 15. Load induced annular degradation is inhibited by tension Adam H. Hsieh, Tamer Hadi, Jeffrey C. Lotz; University of California, San Francisco, San Francisco, CA, USA BACKGROUND CONTEXT: Previous studies have demonstrated that chronic uniform compression applied to mouse discs in vivo leads to disc degeneration. Finite element analyses have suggested that loss of tension in lamellar collagen coupled with significant hydrostatic stress generated in the annulus may contribute to degenerative changes. PURPOSE: This study tests our hypothesis that load-induced degenerative changes in the annulus can be inhibited by tension. Specifically, after injurious loading motion segments were placed at fixed bending angles to generate annular tension on one side of the disc. STUDY DESIGN/SETTING: An established mouse tail model of load- induced disc degeneration was used to apply injurious levels (7 day, 1.3 MPa) of uniform compression. After compression, motion segments were Fig. 1. Effects of recovery or bending after injurious loading on histology (top) and collagen birefringence (bottom). subjected to one of four treatments consisting of combinations of unloaded recovery and fixed-angle bending. PATIENT SAMPLE: N/A OUTCOME MEASURES: We evaluated the effects of annular tension both on disc health and on collagen conformational stability against enzy- matic degradation. METHODS: Apoptosis, disc morphology, and collagen birefringence were assessed, respectively, by TUNEL, histology, and polarized light micros- copy. Resistance to enzymatic degradation was determined by incubating devitalized and denucleated murine motion segments with collagenase (100ng/mL) for 24 hours either in uniform tension or while slack. Bio- mechanical testing was used to measure changes in stiffness and failure load. RESULTS: Loaded discs allowed only to recover exhibited highly disorga- nized annular morphology with radial fissures apparent in birefringence images (Fig. 1). Interactions between recovery and bending were important in modulating different measures of disc health. Discs allowed to recover before bending had greater cellularity, but those placed immediately in bending possessed better annular morphology (not shown). Cellularity dif- ferences were independent of apoptosis. Collagen conformation signifi- cantly affected resistance to enzymatic degradation. Tensile-loaded discs incubated in collagenase failed at higher loads and were stiffer than slack specimens (Fig. 2). CONCLUSIONS: This study demonstrates that tensional stress inhibits collagen degradation in vivo even after exposure to a mechanical insult known to induce annular degenerative changes. In particular, tension pre- vented formation of annular fissuring, a hallmark of disc degeneration in humans. Moreover, we show that inhibition of degradation is partially due to increased conformational resistance of collagen to enzymatic degradation. However, tension also negatively affected annular cellularity, which was found not to involve further apoptosis. These opposing effects on lamellar structure and cellularity, both customarily used to define disc health, suggest Fig. 2. Mechanical properties of discs in tension or slack after collagen- ase incubation.