One strike loading organ culture model to investigate the post-traumatic disc degenerative condition

Abstract

Background: Acute trauma on intervertebral discs (IVDs) is thought to be one of the risk factors for IVD degeneration. The pathophysiology of IVD degeneration induced by single high impact mechanical injury is not very well understood. The aim of this study was using a post-traumatic IVD model in a whole organ culture system to analyze the biological and biomechanical consequences of the single high-impact loading event on the cultured IVDs.

Methods: Isolated healthy bovine IVDs were loaded with a physiological loading protocol in the control group or with injurious loading (compression at 50% of IVD height) in the one strike loading (OSL) group. After another 1 day (short term) or 8 days (long term) of whole organ culture within a bioreactor, the samples were collected to analyze the cell viability, histological morphology and gene expression. The conditioned medium was collected daily to analyze the release of glycosaminoglycan (GAG) and nitric oxide (NO).

Results: The OSL IVD injury group showed signs of early degeneration including reduction of dynamic compressive stiffness, annulus fibrosus (AF) fissures and extracellular matrix degradation. Compared to the control group, the OSL model group showed more severe cell death (P ​< ​0.01) and higher GAG release in the culture medium (P ​< ​0.05). The MMP and ADAMTS families were up-regulated in both nucleus pulposus (NP) and AF tissues from the OSL model group (P ​< ​0.05). The OSL injury model induced a traumatic degenerative cascade in the whole organ cultured IVD.

Conclusions: The present study shows a single hyperphysiological mechanical compression applied to healthy bovine IVDs caused significant drop of cell viability, altered the mRNA expression in the IVD, and increased ECM degradation. The OSL IVD model could provide new insights into the mechanism of mechanical injury induced early IVD degeneration.

The translational potential of this article: This model has a high potential for investigation of the degeneration mechanism in post-traumatic IVD disease, identification of novel biomarkers and therapeutic targets, as well as screening of treatment therapies.

Keywords: Degeneration; Extracellular matrix; Intervertebral disc; Mechanical injury; One strike loading; Post-traumatic.

Fig. 1

(A) The bioreactor for physiological loading, and MTS machine with custom designed chamber for one strike loading on bovine IVDs. (B) The experiment design flow chart of short term and long term studies.

Fig. 2

Disc height change (A) and Dynamic compressive stiffness (B) of the IVDs measured on day 0 after dissection, on day 1 after overnight free swelling (Day1 swelling), on day 2 after loading (Day2 after model), on day 3 and day 10 after overnight free swelling recovery (Day3/Day10 after swelling). Data were normalized to original height and dynamic compressive stiffness of each disc on day 0 after dissection, Mean ​+SD, n ​= ​5, ∗p ​< ​0.05, ∗∗∗P ​< ​0.001.

Fig. 3

Representative LDH/Ethidium homodimer staining images of IVDs collected on day 0 after dissection (Day 0, A, D, G), and on day 10 after physiological loading (Control, B, E, H) or one strike loading (Model, C, F, I). Cell viability was assessed using LDH/Ethidium homodimer staining (blue and blue/red ​= ​alive cell; red only ​= ​dead cell). Figures A, B, C represent NP tissue. Figures D, E, F represent inner AF (IAF) tissue. Figures G, H, I represent outer AF (OAF) tissue. Scale bar: 100 ​μm.

Fig. 4

The numbers of alive cells, dead cells (per 40,000 ​μm²) and cell viability from different groups. A1, B1, C1 represent NP region; A2, B2, C2 represent inner AF (IAF) region; A3, B3, C3 represent outer AF (OAF) region. Mean ​ ​+ ​ ​SD, ∗p ​< ​0.05, the data were normalized to day0 group (n ​= ​8).

Fig. 5

Representative Safranin O/Fast green staining images of cross histological sections from IVDs on day 0 after dissection (Day 0, A, D, G), on day 10 after physiological loading (Control, B, E, H), and on day 10 after one strike loading (Model, C, F, I). Scale bar: 500 ​μm. (J) Histological scoring values based on Safranin O/Fast green staining (n ​= ​3).

Fig. 6

Accumulative NO (A) and GAG (B) release in conditioned media of IVDs cultured with physiological loading (Control Group) or one strike loading (Model Group) during culture. Mean ​ ​+ ​ ​SD, n ​= ​3, as ​= ​after free swelling overnight recovery, al ​= ​after loading, NO ​= ​nitric oxide, GAG ​= ​glycosaminoglycan. Gene expression in NP (C, E) and AF (D, F) tissue of Model group normalized to Control group as 1, on day 3 in short term experiment (C, D, n ​= ​8), or on day 10 in long term experiment (E, F, n ​= ​14). ∗p ​< ​0.05; ↑ indicates the gene expression was significantly upregulated in Model group compared to Control group, ↓ indicates the gene expression was significantly downregulated in Model group compared to control group.