Calcium Microcrystal Formation in Recurrent Herniation Patients After Autologous Disc Cell Transplantation

Abstract

Autologous disc cell transplantation (ADCT) is a cell-based therapy aiming to initiate regeneration of intervertebral disc (IVD) tissue, but little is known about potential risks. This study aims to investigate the presence of structural phenomena accompanying the transformation process after ADCT treatment in IVD disease. Structural phenomena of ADCT-treated patients (Group 1, n = 10) with recurrent disc herniation were compared to conventionally-treated patients with recurrent herniation (Group 2, n = 10) and patients with a first-time herniation (Group 3, n = 10). For ethical reasons, a control group of ADCT patients who did not have a recurrent disc herniation was not possible. Tissue samples were obtained via micro-sequestrectomy after disc herniation and analyzed by micro-computed tomography, scanning electron microscopy, energy dispersive spectroscopy, and histology in terms of calcification zones, tissue structure, cell density, cell morphology, and elemental composition. The major differentiator between sample groups was calcium microcrystal formation in all ADCT samples, not found in any of the control group samples, which may indicate disc degradation. The incorporation of mineral particles provided clear contrast between the different materials and chemical analysis of a single particle indicated the presence of magnesium-containing calcium phosphate. As IVD calcification is a primary indicator of disc degeneration, further investigation of ADCT and detailed investigations assessing each patient's Pfirrmann degeneration grade following herniation is warranted. Structural phenomena unique to ADCT herniation prompt further investigation of the therapy's mechanisms and its effect on IVD tissue. However, the impossibility of a perfect control group limits the generalizable interpretation of the results.

Keywords: Disc calcification; Intervertebral disc; Recurrent disc herniation; Regenerative therapy.

Fig. 1

A T2-MRI images of a representative group-1 patient: (left) the situation before micro-sequestrectomy of L4/5; (middle) slight increase in volume and water content by ADCT injection in L4/5; (right) recrudescence disc herniation of L4/5. B T2-MRI images of a group-1 patient with additional Modic changes: (left) the situation before micro-sequestrectomy of L5/S1 Modic Type I; (middle) slight increase in volume and water content by ADCT injection in L5/S1 Modic Type I; (right) recrudescence disc herniation of L5/S1 Modic Type I and increased zone of edema; C Representative T2-MRI images of an group-2 patient: (left) the situation before micro-sequestrectomy of L5/S1; (right) recrudescence without ADCT treatment. D T2-MRI image of a representative group-3 patient, the situation before micro-sequestrectomy L5/S1

Fig. 2

μCT images of osmium contrasted disc tissue samples, A μCT cross-section of ADCT- treated patient, marked by I small particles with a diameter of 1 μm; B 3D reconstruction of ADCT-treated patient, marked by II large particles with a diameter of 10 μm; C μCT cross-section of conventionally treated patient–first herniation; D μCT cross-section of conventionally treated patient–second herniation

Fig. 3

Light microscopy image of von Kossa staining of disc tissue removed by sequestrectomy after: A Recurrent disc herniation of an ADCT-treated patient (group-1); B Recurrent disc herniation of an ADCT-treated patient (group-1); C Recurrent disc herniation of a conventionally treated patient (group-2); D Single disc herniation of a conventionally treated patient (group-3)

Fig. 4

Scanning electron microscopy of herniated disc tissue: A Single cell I with surrounding, newly formed extracellular matrix II of the recurrent sequestration tissue of a patient after conventional treatment (group-3). At III strong material contrast due to fat accumulation; B Cell agglomerate with spindle-shaped cells IV embedded in dense matrix tissue V in recurrent sequester tissue after ADCT (group-1); C Cell agglomerate strongly contrasted with the surrounding area in recurrent sequester tissue after ADCT treatment (group-1); D Individual particles (VII small particles with a diameter of 1 µm and VIII large particles with a diameter of 10 µm) with no apparent substructure in recurrent sequester tissue after ADCT (group-1)

Fig. 5

Spot and mapping EDS analyses of different tissue regions of a group-1 patient in an SEM image: A, B SEM images with marked analysis positions 1–8 (elemental compositions shown in Table 2); C Spectra for the mapping analysis of the area of a particle 1; D Spectra for the mapping analysis of the surrounding fibrillar tissue area 2; E Spectra for the spot analysis of the particle at position 5