INTRODUCTION. Discectomy is the conventional treatment option for intervertebral disc (IVD) herniations but causes further degenerative progression of the IVDs due to low regenerative capacity. We’ve reported the regenerative mechanism and effect on the degenerated IVDs by implantation of the combination of bioresorbable ultra-purified alginate (UPAL) gel and allogeneic-bone marrow derived mesenchymal stem cells (BMSCs) in a rabbit model. On the other hand, rapidly expanding human allogeneic-BMSCs (RECs) are attractive candidates because of their highly-purified proliferating ability, multilineage potential, and cell uniformity compared with regular BMSCs. The purpose of this study is to compare the cellular properties of RECs and commercially available BMSCs, and to assess the effectiveness of the RECs combined with UPAL gel for severely degenerated IVDs in a large animal model as translational research.
METHODS. [In vitro test] RECs and commercially available human BMSCs were mono-cultured in a three-dimensional (3D) system in UPAL gel respectively for 7 days. Additionally, as control groups, we prepared the RECs and commercial BMSCs cultured in plane culture (2D culture). We evaluated cell proliferative capacity, viability, uniformity, and the expression of cell surface antigens (CD90 and CD45) in 4 types of the cells. [In vivo test] The L1/2-4/5 discs of 7 sheep were divided into four groups, Intact control, Discectomy, Gel, and REC + gel. First, we removed 20 mg nucleus pulposus (NP) tissue from the treated discs to create severely degenerated IVD. Four weeks later, 70 mg of NP were further removed from the degenerated IVDs and RECs combined with UPAL solution were filled into the IVD cavity. Twenty-four weeks after implantation, IVDs were qualitatively analyzed for assessment of IVD degeneration using MRI, histology, and immunohistochemistry (IHC). We performed tumorigenesis analysis additionally.
RESULTS. [In vitro test] Although there was no significant difference between RECs and BMSCs in cell viability, cell proliferative capacity and uniformity in RECs were higher than those in BMSCs. The percentages of CD90 positive cells in the RECs were higher than those in BMSCs. [In vivo test] In MRI analysis, Pfirrmann grades in the REC + gel groups were significantly lower than those in the Discectomy group. The histological grades of the REC + gel group were significantly lower than those of the Discectomy and Gel groups. In IHC, type II collagen positive cells were significantly higher in the REC group than in the two groups. In tumorigenesis analysis, invasive growth, karyomitosis, and nucleoli were not observed in REC group.
DISCUSSION. The superiority of RECs over commercial BMSCs in terms of cellular properties as MSCs was proven by direct comparison. Implantation of the REC and UPAL gel suppresses IVD degeneration after discectomy in severely degenerated IVDs, and type II collagen positive cells were significantly higher in the REC group without neoplastic changes, suggesting that the RECs with UPAL gel promoted spontaneous IVD regeneration and do not induce tumorigenesis. This study demonstrates the safety and effectiveness of this treatment strategy as a regenerative therapy. Based on these findings, we are currently conducting an investigator-initiated trials.