Department of Traumatology and Sports medicine, Med. Univ. of Innsbruck, Austria; 2

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1 Differences in Morphology of Intervertebral Disc Cells after Traumatic Injury related to the Pathology of Soft Disc Herniation. An Ultrastructural Investigation of the Lower Cervical Spine. I. Sitte 1, A. Kathrein 2, F. Pedross 3, K. Pfaller 4, S. Roberts 5 1 Department of Traumatology and Sports medicine, Med. Univ. of Innsbruck, Austria; 2 Department for Traumatology and Sports medicine, St. Vinzenz Krankenhaus, Zams, Austria; 3 Department for Medical Statistics, Informatics und Health Economics; Med. Univ. of Innsbruck, Austria; 4 Division of Histology and Embryology, Med. Univ. of Innsbruck, Austria; 5 Centre for Spinal Studies, RJAH Orthopaedic Hospital, Oswestry, Shropshire and ISTM, Keele University, UK.

2 Introduction Disc cell death following traumatic injury to the human cervical spine shows up to 70% cell death by necrosis. Compression fractures (A) have higher levels of apoptosis/chondroptosios than fractures with less compression (C); A-discs also contain cells with a novel morphology, balloon cells. The present study was undertaken to examine differences in cell morphologies and architecture in discs from patients with injuries in comparison to soft disc herniation (DH). A-Fracture disc herniation

3 Materials & Methods Anterior discs (n=40) from 28 patients with either traumatic injuries to their cervical spines (C4-Th1) between 0 days and 3 days post-injury or disc prolapse were obtained. Additionally 8 discs from 2 patients were taken for control (C5-Th3). Roberts et al., Spine 1991 Trauma group (n=8 discs each group): - A-Fractures (DGI,II): 0-3 days pt (n=5 patients; yrs) - C-Fractures (DGI,II,III): 0-3 days pt (n=8 patients; yrs). Injuries were classified via Magerl s system. Disc prolapse group (n=8 discs each group): Magerl et al., Eur Spine J disc prolapse DGII (n=8 patients, yrs); Pain onset 4 month -4 yrs - disc prolapse DGIII (n=7 patients, yrs); Pain onset 3 weeks -2 yrs Degeneration was scored radiologically (grade I-V according to Benneker et al., Eur Spine J 2006). Control discs (n=8) from 2 patients (age 23/49) died by a cerebral aneurism bleeding (n=7, DG II; n=1, DG III; without symptoms of a degenerative disc disease or an history of traumatic injury of the spine). Statistical analyses: SPSS version 16 Acknowledgements: Supported by ÖNB (Project numbers: ÖNB 8590; ÖNB 10032; Ethics commission: UN 1052; UN 1653)

4 Materials & Methods Histological Investigations - MMC Goldner stain - Paraffin - HE Ultrastructural Investigations - Cell morphology was examined ultrastructurally to identify and quantify: (a) healthy cell (b) balloon cell A3 Fracture DGII C4/5 0d pt MMC Goldner 5X A3.3.Fx 0d pt C6/7 DG II A3.3.-Fx 0d pt C6/7 DG II iaf 8000X (25 yrs) oaf 8000X (25 yrs) (c) chondroptotic cell (d) necrotic cell Disc Prolapse DGII C6/7 pain onset >2 yrs MMC Goldner 5X A3.3.-Fx 0d pt C5/6 DG I C2.2-Fx 0d pt C6/7 DG III oaf 8000X (17 yrs) oaf-iaf 8000x (66 yrs)

5 CT and MRI: Trauma Disc Prolapse - Control Trauma Disc Prolapse Control A-Fracture C-Fracture 30 yrs (DGI) 58 yrs (DGII) 36 yrs (DGII) 49 yrs (C5/6 DGIII - C6/7,C7/Th1 DGII)

6 Histology: Trauma Disc Prolapse - Control Trauma Disc Prolapse Control A-Fract. 36 yrs (DGII) 23 yrs (C5/6 DGIII) 30 yrs (DGI) No cell clusters were seen in patients with A-fractures (n=3 GI; n=5 GII), whereas some were present (up to 9 cells/cluster) in discs from patients with C-fractures (n=2 GI; n=4 GII; n=2 GIII); many were seen in DH, with 6 DGII discs and 4 DGIII having large clusters (>10 cells). Most of these discs had vascularisation in the outer annulus fibrosus (oaf), but it was in only 4 discs of the trauma group. Control discs with GII presented some cell clusters.

7 TEM: Trauma Disc prolapse - Control Trauma Disc Prolapse Control Healthy cell (Pat. 30 yrs) Healthy cell (Pat. 33 yrs) Healthy cell cluster (Pat. 23 yrs) Balloon Cell Balloon Cell Balloon Cell

8 RESULTS: TEM Investigations Balloon cells: Characteristic: blown up with an homogenous nucleus, mostly with a nucleulus. Found in all discs with prolapse (GII,GIII), mostly in iaf with more than mean 30%. Often hyperthroph Proliferate in clusters Ghadially FN 1997 Ultrastructural Pathology of Cell and Matrix Butterworth Heinemann First described in traumatic injured discs, there related to compression loads. (Sitte et al, Spine 2009) Where else to find these kind of nucleus morphology? p 475 Plasma cells Multiple Myeloma

9 Results Statistics: Trauma Disc prolapse - Control Significantly more necrotic cells were seen in both trauma groups in all disc regions (mean in oaf: 60%) compared to the control (A/C-oAF, p=0.002/0,001) or the DH GIII group (oaf; p=0.011). More apoptotic/chondroptotic disc cells (<20%) were seen in A than any other group (p=0.007 control oaf). Balloon cells were not found in C-fractures, but were in those with A-fractures and throughout all herniated discs. Surprisingly, all control discs with GII had a similar incidence of balloon cells to that seen in DH GII.

10 Trauma Disc prolapse - Control Diagnosis Mechanism Healthy Cells Trauma A-Fracture compression load Mean 21% iaf (0-37%) Balloon Cells Mean 13% iaf (0-48%) Trauma tensile and C-Fracture shear load Mean 18% iaf (0-45%) Ø Disc prolapse??? DGII genetic influence Mean 38% iaf (21-57%) Mean 17% iaf (5-40%) Disc prolapse??? DGIII genetic influence Mean 36% iaf (8-62%) Mean 37% iaf (14-63%) Control (Patients died by a cerebral aneurism bleeding; no symptoms, no trauma) Collagen aberration - Mutation of COL3A1 in arterial dissection Grond-Ginsbach 2010 Mean 45% iaf (32-81%) Mean 16% iaf (0-41%)

11 Discussion and Conclusion The changes in cell morphology differed in discs with injuries compared to those seen in DH. Disc injury led to cell death via necrosis; chondroptosis/apoptosis was most common in discs with most compressive loading. Balloon cells were found in A-discs, DH and the control. Vessel ingrowth was rare in the trauma group. Most vascularisation and clustering of cells was seen in herniated discs. It is suggested that different mechanisms are involved in these pathologies and that the presence of balloon cells and clusters in the control could indicate the beginning disc degeneration.

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