Introduction: Cell transplantation has been explored as a potential therapy against intervertebral disc (IVD) degeneration¹. Nevertheless, intradiscal cell transplantation causes additional damage following needle puncture, potentially promoting further degeneration. In this study, we examined the potential of transplanting mesenchymal stromal cells (MSC) into the vertebrae as an alternative strategy for IVD repair and assess the potential of MSC homing into the degenerating IVD^2-4.

Methods: All animal experiments were approved by the Tokai University School of Medicine committee for safe animal experimentation (#214033). 24 female SD rats were subjected to coccygeal IVD degeneration through nucleus pulposus (NP) aspiration in two discs. Directly after, from a caudal approach, 10 uL of PBS (Sham) or 10 uL PBS containing 25x10⁴ rat MSC were transplanted into the vertebrae, neighboring either a healthy or degenerating disc. (Fig1A) The rats were followed for 4 weeks, with disc height (DHI) measurements⁵ every 2 weeks, upon which the rats were sacrificed and the degenerative state was examined using paraffin sections. For the second part, we aimed to trace the transplanted MSCs. Ubiquitously GFP-expressing mouse MSCs were obtained and expanded. 24 female rats were similarly subjected to disc degeneration in two coccygeal IVD. 10 uL of 25x10⁴ GFP⁺ MSC were transplanted into the vertebrae next to a healthy or degenerated disc. (Fig1A) As a positive control, 10x10⁴ GFP⁺ MSC were injected intradiscally into a degenerating IVD. DHI measurements were performed on 5, 10, and 14 days post transplantation. Discs were explanted and prepared for cryo-sectionings. GFP was detected through rabbit-anti-GFP (ab290), goat-anti-rabbit Alexa 633 (A21071), and confocal microscopy. Statistical analysis was performed using Two-way ANOVA, post-hoc Tukey’s multiple comparison test using Prism 9 (GraphPad).

Results: For the first part, DHI showed a significant decline for the degenerating IVD receiving PBS in the neighboring vertebrae, while DHI was largely maintained when MSC were transplanted.(Fig1B) Histological observations suggested a trend of improvement after MSC transplantation. For the second part, DHI was similarly maintained for degenerating IVD neighboring vertebra that received MSC. Surprisingly, intradiscal injection of MSC showed a significant decrease in DHI. MSC tracing showed GFP⁺ cells in IVD treated intradiscally with MSCs and a similar rate as in IVD neighboring a vertebra receiving MSCs, although overall numbers were low.(Fig1C)

Discussion: MSC transplantation into the vertebrae showed a clear benefit on maintaining DHI following degeneration induction, although this did not directly translate into clear improvements in histology. MSC tracing further revealed the capacity of MSC to migrate from the vertebrae into the IVD, which mainly occurred into degenerating IVDs. Nonetheless, the number of MSC stably integrating into the IVD revealed to be limited, as suggested by the limited numbers detected in the intradiscal injected IVD. Whether the observed effects result from MSC homing, paracrine signaling, or otherwise, remains to be determined. Our work suggests a potential alternative to intradiscal cell transplantation, although further examination and optimization are required.

Figure 1.) (A) Overview of the different transplantation conditions (B) DHI outcomes part 1 (C) Example of MSC tracing part 2