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Mar 2017

One step closer to halting the “relentless cascade” of disc degeneration

Reproduced from original publication on Spinal News International.

According to a “Best Paper” presentation at the North American Spine Society annual meeting (NASS; 26–29 October 2016, Boston, USA), it may be possible to develop a minimally-invasive, percutaneously-delivered combination of “cell-based bioactive factors” which can “mediate the progression of degeneration of the disc.”

Researchers based in Toronto, Canada, sought to identify which factors secreted by the notochochordal cell-rich intervertebral disc nucleus pulposus could be used to mediate disc degeneration via a rat-tail model of degenerative disc disease (DDD).

Presenting the team’s research, W Mark Erwin (Department of Surgery, Divisions of Orthopaedic and Neurological Surgery, Toronto, Canada) hailed the ability to cause “any kind of biological effect to change the progression of disease” as the “Holy Grail” of research into DDD. He told Spinal News International, “Currently, no treatment has been shown to definitively influence the course of DDD. Patients suffering from painful, non-operative DDD are treated symptomatically using approaches including exercise, cognitive behavioural therapy, medication, injections and various other physical modalities—none of which can influence the course of the malady.

"Approximately 40% of chronic back pain (plus neck pain and neurological disorders secondary to nerve or even spinal cord compression) are associated with DDD. Therefore, the ability to influence the otherwise relentless, degenerative cascade—or even to promote ‘regeneration’—would be a revolution in the management of this painful, disabling and costly condition.”

Research into the differences in the development of DDD in non-chondrodystrophic and chondrodystrophic dogs forms the foundation of the team’s current work. “Non-chondrodystrophic (mongrel) dogs are resistant to developing DDD, in contrast to chondrodystrophic animals such as beagles that develop DDD early and, significantly, in a similar fashion to humans,” Erwin explained to Spinal News International. “This unique phenomenon is the ongoing subject of international investigation. We have published a number of studies demonstrating that tissue culture media conditioned by incubating the non-chondystrophic notochordal cell-rich nucleus pulposus within tissue culture media, and therefore developing ‘notochordal cell-conditioned medium’ (NCCM) protects disc cells from apoptotic cell death and degeneration in various species.”

Figure 1: X20 Safranin-O stained rat intervertebral disc nucleus pulposus (IVD NP). Rat-tail IVD NP initially injured and four weeks later injected with (a) saline (b) notochordal cell-derived factors, harvested six weeks later.

Erwin’s team has demonstrated the ability of a single injection of notochordal cell-conditioned medium (NCCM) into a DDD rat-tail model—simulated by a needle puncture injury—to suppress both apoptotic signalling and the catabolic effects of inflammatory cytokines on nucleus pulposus (manuscript currently under review). Similarly, work with human disc cells has demonstrated the ability of NCCM to mediate other kinds of cell death and degeneration.

“Our recent publication in The Spine Journal detailed our findings that NCCM can suppress chemotherapy-induced cell death in human disc cells, and we have determined the mechanism through which these processes occur. We have also demonstrated that the extracellular matrix of the beagle disc is quite similar to the degenerative human disc (Arthritis Research and Therapy, 2015),” Erwin explained to Spinal News International, “These studies clearly indicated that the factors secreted by the notochordal cell-rich disc have a profound anti-degenerative/pro-cell survival and anabolic effect upon disc cells.”

The purpose of the research presented at NASS was to find the necessary and sufficient factors secreted by the NCCM for the mediation of DDD.

“We next needed to identify the nature of the mysterious factors secreted by the notochordal cell-rich disc and see if the introduction of the unidentified molecules could restore the injured disc,” Erwin said. “Therefore, we used sophisticated biochemical, molecular, proteomic and immunohistochemical methods to determine the identity of the notochordal cell-secreted candidate factors. In parallel, the team validated the normal progression of cellular, biomechanical and molecular events post-disc injury in a rat-tail model of DDD, which was used as a control. At eight to ten weeks post-needle puncture, the team observed significant fibrocartilaginous change in the disc, as well as a sudden onset of inflammatory markers. According to the presentation, these were associated with “a suppression of the inhibitors of metalloproteases, an increase in metalloprotease activity and a complete loss of stem cells and notochordal markers.”

NCCM was then introduced into the rat-tail model, which was evaluated for markers of degeneration. “Once we had detailed the normal degenerative course, we chose to deliver the candidate factors alone and in combination mid-way through the degenerative cascade that we had previously determined,” Erwin told Spinal News International. “We then compared the results to no treatment at all, or an injection of phosphate buffered saline (PBS). We were delighted and surprised at the robust anti-degenerative/regenerative effect of the single injection (Figure 1).”

On deeper evaluation using methods including fast protein liquid chromatography, centrifugation and mass spectroscopy, the team were able to isolate specific molecules from the notochordal cell “secretome”. Ingenuity Pathway Analysis revealed that these factors “figure prominently in molecular protein-signalling networks.” Analysis of healthy rodent discs showed these factors to be “abundantly present,” as they are in the mongrel dog. However, these factors are expressed in far lower amounts in the beagle disc, and are undetectable in the injured rat or degenerative human disc. Testing the combination of these factors in human discs showed a “significant increase in cell proliferation.”

Erwin (back middle) and his lab team

Injection of these factors into degenerative rat-tail discs resulted in a particularly significant increase in in collagen type 2 and aggrecan, as well as suppression of  activated caspase-3 activity.

“Every scientific study comes with its own limitations,” Erwin told Spinal News International, when queried about the scope of the study. “In order to circumvent at least part of the small animal limitations, we emulated treating a degenerating disc and therefore delivered treatment mid-way through the degenerative process. The rat-tail does bear weight to some degree as the animal normally explores its environment including standing bipedal for much of the day. However, the loads are nowhere near comparable to the human, and the disc is orders of magnitude smaller than is the human. But, the signalling pathways involved are quite similar.” As a pre-clinical proof-of-concept study, he said, the rodent model limitations are far outweighed by the advantages.

“Currently we are actively investigating important cellular changes and molecular signalling pathways involved with the development and progression of DDD,” Erwin said, “In a recent publication in The Spine Journal, we report that NCCM suppresses cell death by stabilising the mitochondrial membrane within disc cells and by suppressing the caspase system of enzymes involved in cell death.

“For the first time, we also showed that under pro-cell death conditions, NCCM upregulates the expression of the most powerful anti-cell death protein—X-linked inhibitor of apoptosis proteins (XIAP)—within disc cells that further assists with the anti-cell death effect. A significant percentage of the donors responded to the in vitro treatments, and we think that there may be a link between the response to treatment and inflammatory cytokine expression by disc cells.”

Erwin (left) with Mehrkens and his “Young Investigator” award

Erwin’s team for the present study includes doctors Ajay Matta, Zia Karim, Xiaomei Wang, technician Hoda Geramai, collaborator David Isenman, and former students and surgeon/scientist trainees including Arne Mehrkens and Sebastian Müller (University of Basel, Basel, Switzerland). Merhkens was awarded a NASS “Young Investigator” award for his work on the NCCM-XIAP study.

“Like many issues in science and research, ‘the devil is in the details’. While the human condition shares many similarities to DDD induced in animal models, the conclusions must be interpreted appropriately,” Erwin said, when asked how long it might take to see disc regeneration in clinical practice. “With respect to the delivery of recombinant forms of notochordal cell-secreted factors,” he concluded, “the proper selection of factors, delivery method, appropriate patient selection and outcome measures are important decisions that remain to be resolved. Furthermore, it is likely that multiple factors may need to be introduced for a satisfactory outcome.”

According to Erwin, “The next step will be to study the effects of specific NCCM-based molecules in a suitable large animal model of DDD, before moving on to a human clinical trial.”

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