Biologic canine and human intervertebral disc repair by notochordal cell-derived matrix: from bench towards bedside

The socioeconomic burden of chronic back pain related to intervertebral disc (IVD) disease is high and current treatments are only symptomatic. Minimally invasive strategies that promote biological IVD repair should address this unmet need. Notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) during IVD maturation and degeneration. The regenerative potential of NC-secreted substances on CLCs and mesenchymal stromal cells (MSCs) has already been demonstrated. However, identification of these substances remains elusive. Innovatively, this study exploits the regenerative NC potential by using healthy porcine NC-derived matrix (NCM) and employs the dog as a clinically relevant translational model. NCM increased the glycosaminoglycan and DNA content of human and canine CLC aggregates and facilitated chondrogenic differentiation of canine MSCs in vitro. Based on these results, NCM, MSCs and NCM+MSCs were injected in mildly (spontaneously) and moderately (induced) degenerated canine IVDs in vivo and, after six months of treatment, were analyzed. NCM injected in moderately (induced) degenerated canine IVDs exerted beneficial effects at the macroscopic and MRI level, induced collagen type II-rich extracellular matrix production, improved the disc height, and ameliorated local inflammation. MSCs exerted no (additive) effects. In conclusion, NCM induced in vivo regenerative effects on degenerated canine IVDs. NCM may, comparable to demineralized bone matrix in bone regeneration, serve as ‘instructive matrix’, by locally releasing growth factors and facilitating tissue repair. Therefore, intradiscal NCM injection could be a promising regenerative treatment for IVD disease, circumventing the cumbersome identification of bioactive NC-secreted substances.


Supplementary File 3: Modic changes, periosteal bone formation, spondylosis and end plate (EP) lysis in the intervertebral discs (IVDs).
The development of Modic Changes (MC) was recorded during the study. In noNX-IVDs, only one MC type 3 was observed at T = 0, 3, and 6 months. In NX-IVDs, one and a half month after NX, and before the different treatments were applied (T = 0 months), several MCs were already present. During the study, i.e. after the first intradiscal injections, control NX-IVDs developed two type 1 MCs, while 1xNCM-treated NX-IVDs developed one additional type 1 MC and one type 1 MC progressed towards a type 3 MC, MSC-treated NX-IVDs developed one type 1 MC, and NCM+MSC-treated NX-IVDs developed four type 1 and two type 3 MCs. In contrast, 2xNCM-treated NX-IVDs did not develop any additional MCs, presumably (also) due to the less invasive percutaneous approach of the L7-S1 IVD.
Overview of the type of Modic changes observed by MRI analysis. -: no Modic changes observed. Red depicted Modic changes are different from T = 0 months (1.5 month after moderate degeneration was induced by partial NP removal (NX)). n = 5.
The development of periosteal bone formation and ventral spondylosis was determined at T = 6 months using CT. When these features were present, they were located at the left side of the spinal column, the side employed to induce IVD degeneration. No periosteal bone formation or ventral spondylosis was detected in control, 2xNCM and MSC-treated noNX-IVDs. In contrast, mild ventral spondylosis was observed in 1xNCM-treated (3/5) and NCM+MSC-treated (1/5) noNX-IVDs. Additionally, minimal (1/5) and distinct (1/5) periosteal bone formation was observed in NCM+MSC-treated noNX-IVDs. In NX-IVDs, more pathological features were detected. In 4/5 control NX-IVDs, distinct periosteal bone formation was detected. Furthermore, 1xNCM-treated NX-IVDs showed mild-distinct bone formation (5/5) and mild spondylosis (3/5), and MSC-and MSC+NCM-treated NX-IVDs showed distinct bone formation (2/5 and 4/5, respectively). 2xNCM-treated NX-IVDs showed none of these pathological processes on CT analysis, again presumably due to the less invasive approach of the L7-S1 IVD. EP lysis (determined by CT analysis at T = 6 months) was only present in NX-IVDs, most probably due to the NX procedure.
Benjamini & Hochberg False Discovery Rate post-hoc tests were performed to correct for multiple comparisons.

I. In vitro studies a. Sulphate incorporation assay
Shortly, chondrocyte-like cells (CLCs) from the six canine and human donors were cultured in micro-aggregates of 35,000 cells in duplicates in basal culture medium, basal culture medium supplemented with 10 ng/mL TGF-β 1 (positive control) or 10 mg/mL NCM. After 7 days, the micro-aggregates were pulsed with 20 mCi 35 SO 4 2 -. Four hours later, the micro-aggregates were washed twice with 500 µL PBS and frozen at −20° C. After one day, the micro-aggregates were digested for two hours in papain buffer. The next day, the amount of 35 SO 4 2 -labelled GAGs in the papain digest was measured by liquid scintillation analysis and normalized for the micro-aggregates' DNA content (measured using the dsDNA High Sensitivity Assay Kit, Invitrogen).
Computed tomography (CT) images were obtained postmortem with dogs positioned in dorsal recumbency to monitor extradiscal calcification and EP pathology. The CT scans were made using a 64 slice CT scanner (Siemens Somatom Definition AS, Siemens Healthcare) with the following parameters: 120 kV, 350 mas, 1000 ms tube rotation time, 0.6 mm slice thickness, 0.35 spiral pitch factor, 512 × 512 pixel matrix and 93 mm fixed field view. Reconstructions were made in transverse and sagittal planes using soft tissue and bone reconstruction kernels and images were reviewed in soft tissue/bone settings (window length 50, width 300, and window length 600, width 3000, respectively).

d. Sample collection
The vertebral column (T11-S1) was harvested using an electric multipurpose saw (Bosch). The muscles were removed and the vertebrae were transversely transected using a band saw (EXAKT tape saw). The ten spinal units (½ vertebra -endplate -IVD -endplate -½ vertebra) were sagitally transected into two identical parts using a diamond band saw (EXAKT 312). In one part the bisected IVD tissue was isolated from the endplate and vertebra with a surgical knife and was snap frozen in liquid nitrogen and stored at −70° C for biochemical analyses. The other part was used to photograph (Olympus VR-340) the other half of the IVD for macroscopic Thompson score evaluation. The samples were fixed in 4% buffered formaldehyde for 14 days, decalcified in PBS with 0.5M EDTA for two months and embedded in paraffin.

e.
RNA isolation, cDNA synthesis, and RT-qPCR