Engineering the Intervertebral Disc

Intervertebral disc degeneration (IVDD) is associated with lower back pain which affects millions around the globe annually. If untreated, or if allowed enough time, the severity of the disease can become extreme with few treatment options. Currently, the market has some synthetic IVD replacements, but they are not ideal solutions as they are prone wear particle generation. Furthermore, spinal fusion, a surgical treatment option, detrimentally alters spine biomechanics [1]. Therefore, there is a need for a viable, biochemically and micro-architecturally sound xenograft to be developed. In the past, the CI has developed a protocol to decellularize a bovine caudal IVD for scaffolding purposes. Decellularization serves to remove antigenic material in order to reduce any immune response in a human in this instance. Additionally, bovine caudal IVDs were decellularized because they have similar architecture and biochemical composition to the human lumbar discs [2]. However, this product suffered from altered structure due to swelling throughout the protocol as seen below [3].

In more recent efforts, the lab has  attempted to fully decellularize a IVD with bony endplates attached. The attached bone would potentially decrease the swelling of the soft tissue of the IVD, while also serving as a means of scaffold fixation in a human spinal unit. Preliminary results indicated that the bone decreased solution penetration and therefore limited decellularization. Compression was introduced to the protocol to aid solution imbibition which improved results but did not fully decellularize the scaffold. In this study a partial decalcification of the vertebral bodies was conducted in addition to the new compression decellularization protocol in order to promote bone porosity and solution penetration.

Hypothesis: Through the addition of partial decalcification of the intervertebral bodies, there will be increased solution flux leading to improved decellularization while maintaining microarchitecture throughout the intervertebral disc.

Bovine caudal IVDs [n=2] were isolated with a band saw cutting through the vertebral bodies. The IVDs were submerged in Immunocal, a diluted formic acid solution used for decalcification. Following the partial decalcification, the IVDs were run through a cycle of washes with detergents, freeze/thaws [3], and compression via a custom designed mechanical decellularization chamber as seen below.

The IVDs were then characterized via two histological stains: FAST and Hematoxylin & Eosin (H&E). FAST stain is a specialized stain for IVDs that allow the different regions to be stained in different colors illustrating the microarchitecture of the IVD. The H&E stain shows nuclei presence and extracellular matrix morphology. Semi-quantitative analysis on the cellularity of different regions of the IVD was conducted.