Engineering the Intervertebral Disc

Mario Krussig & Dr. Jeremy Mercuri
The Laboratory of Orthopaedic Tissue Regeneration and Orthobiologics


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.

HypothesisThrough 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.

Materials and Methods

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.


Intervertebral disc degeneration (IVDD) is a cell-mediated process that may result in low back pain due to the degradation of biomechanical integrity within the spine. Although a total disc replacement would be the best solution to advanced IVDD, there is no viable full disc replacement model that is biocompatible and fully mimics the human IVD (hIVD). The biomechanics and functionality of the IVD largely depends on the nucleus pulposus (NP) core and exterior annulus fibrosus (AF). It has been previously hypothesized that a decellularized bovine caudal disc (bIVD) could serve as a viable scaffold because the bIVD is similar to that of the hIVD in structure and chemical composition. Previously the CI successfully decellularized bIVDs without endplates attached but observed significant swelling within the NP. In order to combat this, the CI has transitioned to decellularizing a full bIVD with endplates attached to alleviate swelling, however this limits the permeability of decellularization solutions. Recent efforts determined that a series of compression cycles incorporated with the previous protocol assisted in decellularization, but did not result in complete decellularization. Therefore, this summer it was hypothesized that partially demineralizing the scaffolds prior to decellularization may increase the porosity of the bone and could aid in the decellularization of the bIVD along with the cyclical loading. The decellularization of the bIVD would be determined by quantifying histological samples via a Hematoxylin and Eosin stain for cellularity whereas the microarchitecture and structural integrity would be determined by a FAST stain.


FAST stain Analysis:

The first images below (click to rotate images) show a fresh control where three key regions are shown in different colored boxes: the nucleus pulposus (NP), annulus fibrosis (AF), and endplate (EP). The latter two images show two IVDs that were processed with the partial decalcification prior to the decellularization protocol.

Our results showed similar coloration and microarchitecture of the IVD post our protocol. Therefore, the disc shape and structure was likely maintained.

FAST Stain key colors

Green/blue = glycosaminoglycan

Red = proteoglycan

Yellow = collagen

Hematoxylin and Eosin stain Analysis:

The results were compared to fresh controls [n=4], and previously quantified samples [n=4] which were decellurized with the compression protocol without decalcification.


Through the incorporation of the partial decalcification of the bony endplates, there was a significant reduction in cell nuclei in both the NP and EP relative to the fresh controls. The AF did not show a reduction in cell nuclei; however, this may be due to the difficulty of counting the nuclei accurately. In this region, the nuclei and ECM were difficult to differentiate. Furthermore, when compared to the previous compression protocol there was a significant decrease in nuclei in the NP region.

Additionally, the FAST stain showed that the architecture of the IVD was not compromised. Therefore, it can be concluded that although not yet fully decellularized the partial decalcification of vertebral bone acts as a stepping stone toward a full decellularization while still not damaging the integrity of the microarchitecture.

Future Outlooks

In order to continue on the track to a fully decellularized IVD, the team has come up with numerous new methods. In the near future there are 4 more samples that underwent partial decalcification that need to be processed further. These samples will undergo a higher compressive load applied throughout the protocol due to an updated compression chamber design.

Additionally, one new step that could be incorporated is the dehydration through lyophilization. This would allow for better fluid reabsorption.

Furthermore, increasing chemical concentrations of active reagents in the decellularization solution could also aid in decellularization.


[1] Kim et al. Eur Spine. 2015.

[2] Oshima et al. J. Orthop Res. 1993

[3] Hensley et al. J. Biomed. Mater. Res. A. 2018.


Research support for our lab has been provided in part by the National Institute of General Medical Sciences of the National Institutes of Health (award number: 5P20GM103444) and the Clemson University Creative Inquiry Program.