Fluoroscopy assisted minimally invasive transplantation of allogenic mesenchymal stromal cells embedded in HyStem reduces the progression of nucleus pulposus degeneration in the damaged ntervertebral [corrected] disc: a preliminary study in rabbits

Abstract

This study was conducted to develop a technique for minimally invasive and accurate delivery of stem cells to augment nucleus pulposus (NP) in damaged intervertebral discs (IVD). IVD damage was created in noncontiguous discs at L4-L5 level; rabbits (N = 12) were randomly divided into three groups: group I treated with MSCs in HyStem hydrogel, group II treated with HyStem alone, and group III received no intervention. MSCs and hydrogel were administered to the damaged disc under guidance of fluoroscopy. Augmentation of NP was assessed through histological and MRI T2 mapping of the NP after eight weeks of transplantation. T2 weighted signal intensity was higher in group I than in groups II and III (P < 0.05). Disc height index showed maximum disc height in group I compared to groups II and III. Histological score of the degenerative index was significantly (P < 0.05) lower in group I (8.6 ± 1.8) than that in groups II (11.6 ± 2.3) and III (18.0 ± 5.7). Immunohistochemistry staining for collagen type II and aggrecan staining were higher in group I as compared to other groups. Our results demonstrate that the minimally invasive administration of MSCs in hyaluronan hydrogel (HyStem) augments the repair of NP in damaged IVD.

Figure 1

Rabbits placed under the fluoroscopy machine (a). Image captured after the disc damage was created at IVD L4/L5 (b).

Figure 2

Immunohistochemical staining of MSCs. CD 44 positive marker (a), CD 29 positive marker (b), and CD 45 negative marker (c).

Figure 3

The functional assays for differentiation of culture MSCs. (a) Representative images of adipogenic differentiation demonstrated by formation of oil droplet. (b) Chondrogenic differentiation demonstrated by positive expression of proteoglycans matrix within the pellet stained with Safranin O/fast green staining. (c) Osteogenic differentiation demonstrated by positive bone matrix mineralization stained with Alizarin Red S staining.

Figure 4

SEM image of Hyaluron based hydrogel alone (a). Cell attached on the surface of hydrogel (b).

Figure 5

MRI images (T2 weighted sagittal views) before operation, 8 weeks, and 16 weeks after annular puncture was created. MSCs containing hydrogel group I baseline (a), 8 weeks (b) and 16 weeks (c), showing improvement in signal intensity suggesting regeneration of IVD. Hydrogel only group II baseline (d), 8 weeks (e) and 16 weeks (f), showing progression of disc degeneration. Untreated group III baseline (g), 8 weeks (h) and 16 weeks (i), showing progression of degeneration.

Figure 6

Magnetic resonance imaging results of normalized T2 values. Magnetic resonance imaging (MRI) analysis was performed before operation, 8 weeks after defects were created, and 8 weeks after transplantation (16 weeks). The normalized T2 value of group I after transplantation with MSC + hydrogel shows significant difference between group II and group III. ∗ indicates significance at P < 0.05.

Figure 7

Nucleus pulposus disc height index. Values obtained at each point are normalized to preoperative score at day “0” (100%). ∗ indicates significance at P < 0.05.

Figure 8

Typical histological changes in normal disc (a–d), MSC and hydrogel (e–h), hydrogel (i–l), and untreated (m–p). On Safranin O stained sections, a moderate-to-severe degeneration was observed in the hydrogel (k, l) and untreated (o, p) groups with formation of fibrocyte-like cells. In the MSC containing hydrogel group, the increasing number of chondrocyte-like cells which appeared as large cells encircled with pericellular matrix densely stained with Safranin O was found in the nucleus pulposus (g, h).

Figure 9

Quantitative histological evaluation of disc degeneration index using Boo's scoring. Significance is represented by *(P < 0.05).

Figure 10

Immunohistochemical staining of different groups stained for collagen type II and aggrecan. Normal group (a, e), MSC and hydrogel group (b, f), hydrogel group (c, g), and untreated defect group (d, h).