Quality assessment of surgical disc samples discriminates human annulus fibrosus and nucleus pulposus on tissue and molecular level

A discrimination of the highly specialised annulus fibrosus (AF) and nucleus pulposus (NP) cells in the mature human intervertebral disc (IVD) is thus far still not possible in a reliable way. The aim of this study was to identify molecular markers that distinguish AF and NP cells in human disc tissue using microarray analysis as a screening tool. AF and NP samples were obtained from 28 cervical discs. First, all samples underwent quality sorting using two novel scoring systems for small-sized disc tissue samples including macroscopic, haptic and histological evaluation. Subsequently, samples with clear disc characteristics of either AF or NP that were free from impurities of foreign tissue (IVD score) and with low signs of disc degeneration on cellular level (DD score) were selected for GeneChip analysis (HGU1332P). The 11 AF and 9 NP samples showed distinctly different genome-wide transcriptomes. The majority of differentially expressed genes (DEGs) could be specifically assigned to the AF, whereas no DEG was exclusively expressed in the NP. Nevertheless, we identified 11 novel marker genes that clearly distinguished AF and NP, as confirmed by quantitative gene expression analysis. The novel established scoring systems and molecular markers showed the identity of AF and NP in disc starting material and are thus of great importance in the quality assurance of cell-based therapeutics in regenerative treatment of disc degeneration

Biomechanical testing of a polymer-based biomaterial for the restoration of spinal stability after nucleotomy

<p>Abstract</p> <p>Background</p> <p>Surgery for disc herniations can be complicated by two major problems: painful degeneration of the spinal segment and re-herniation. Therefore, we examined an absorbable poly-glycolic acid (PGA) biomaterial, which was lyophilized with hyaluronic acid (HA), for its utility to (a) re-establish spinal stability and to (b) seal annulus fibrosus defects. The biomechanical properties range of motion (ROM), neutral zone (NZ) and a potential annulus sealing capacity were investigated.</p> <p>Methods</p> <p>Seven bovine, lumbar spinal units were tested in vitro for ROM and NZ in three consecutive stages: (a) intact, (b) following nucleotomy and (c) after insertion of a PGA/HA nucleus-implant. For biomechanical testing, spinal units were mounted on a loading-simulator for spines. In three cycles, axial loading was applied in an excentric mode with 0.5 Nm steps until an applied moment of ± 7.5 Nm was achieved in flexion/extension. ROM and NZ were assessed. These tests were performed without and with annulus sealing by sewing a PGA/HA annulus-implant into the annulus defect.</p> <p>Results</p> <p>Spinal stability was significantly impaired after nucleotomy (p < 0.001). Intradiscal implantation of a PGA-HA nucleus-implant, however, restored spinal stability (p < 0.003). There was no statistical difference between the stability provided by the nucleus-implant and the intact stage regarding flexion/extension movements (p = 0.209). During the testing sequences, herniation of biomaterial through the annulus defect into the spinal canal regularly occurred, resulting in compression of neural elements. Sewing a PGA/HA annulus-implant into the annulus defect, however, effectively prevented herniation.</p> <p>Conclusion</p> <p>PGA/HA biomaterial seems to be well suited for cell-free and cell-based regenerative treatment strategies in spinal surgery. Its abilities to restore spinal stability and potentially close annulus defects open up new vistas for regenerative approaches to treat intervertebral disc degeneration and for preventing implant herniation.</p

A combinatorial relative mass value evaluation of endogenous bioactive proteins in three-dimensional cultured nucleus pulposus cells of herniated intervertebral discs: identification of potential target proteins for gene therapeutic approaches.

Painful degenerative disc diseases have been targeted by different biological treatment approaches. Nucleus pulposus (NP) cells play a central role in intervertebral disc (IVD) maintenance by orchestrating catabolic, anabolic and inflammatory factors that affect the extracellular matrix. IVD degeneration is associated with imbalances of these factors, resulting in a catabolic inflammatory metabolism. Therefore, accurate knowledge about their quantity and quality with regard to matrix synthesis is vital for a rational gene therapeutic approach. NP cells were isolated from 63 patients operated due to lumbar disc herniation (mean age 56 / range 29 - 84 years). Then, three-dimensional culture with low-glucose was completed in a collagen type I scaffold for four weeks. Subsequently cell proliferation evaluation was performed using 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide and intracellular concentration of 28 endogenously expressed anabolic, catabolic, inflammatory factors and relevant matrix proteins was determined by enzyme-linked immunosorbent assay. Specimen-related grades of degeneration were confirmed by preoperative magnetic resonance imaging. Independent from gender, age and grade of degeneration proliferation rates remained similar in all groups of NP cells. Progressive grades of degeneration, however, showed a significant influence on accumulation of selective groups of factors such as disintegrin and metalloproteinase with thrombospondin motifs 4 and 5, matrix metalloproteinase 3, metalloproteinase inhibitor 1 and 2, interleukin-1β and interleukin-1 receptor. Along with these changes, the key NP matrix proteins aggrecan and collagen II decreased significantly. The concentration of anabolic factors bone morphogenetic proteins 2, 4, 6 and 7, insulin-like growth factor 1, transforming growth factor beta 1 and 3, however, remained below the minimal detectable quantities. These findings indicate that progressive degenerative changes in NP may be problematic with regard to biologic treatment strategies. Hence, gene therapeutic interventions regulating relevant bioactive factors identified in this work might contribute to the development of regenerative treatment approaches for degenerative disc diseases

Quality Assessment of Surgical Disc Samples Discriminates Human Annulus Fibrosus and Nucleus Pulposus on Tissue and Molecular Level

A discrimination of the highly specialised annulus fibrosus (AF) and nucleus pulposus (NP) cells in the mature human intervertebral disc (IVD) is thus far still not possible in a reliable way. The aim of this study was to identify molecular markers that distinguish AF and NP cells in human disc tissue using microarray analysis as a screening tool. AF and NP samples were obtained from 28 cervical discs. First, all samples underwent quality sorting using two novel scoring systems for small-sized disc tissue samples including macroscopic, haptic and histological evaluation. Subsequently, samples with clear disc characteristics of either AF or NP that were free from impurities of foreign tissue (IVD score) and with low signs of disc degeneration on cellular level (DD score) were selected for GeneChip analysis (HGU1332P). The 11 AF and 9 NP samples showed distinctly different genome-wide transcriptomes. The majority of differentially expressed genes (DEGs) could be specifically assigned to the AF, whereas no DEG was exclusively expressed in the NP. Nevertheless, we identified 11 novel marker genes that clearly distinguished AF and NP, as confirmed by quantitative gene expression analysis. The novel established scoring systems and molecular markers showed the identity of AF and NP in disc starting material and are thus of great importance in the quality assurance of cell-based therapeutics in regenerative treatment of disc degeneration

Concentrations of endogenously expressed inflammatory cytokines, anabolic factors and matrix proteins in degenerative cervical NP cells.

<p>Concentrations of endogenously expressed inflammatory cytokines, anabolic factors and matrix proteins in degenerative cervical NP cells.</p

Proliferation rate of adult NP cells and expression levels of ADAMTS-4 and -5.

<p>NP cells were isolated from 63 samples of degenerative IVDs and 4 x 10⁵ cells from each sample were grown for four weeks in collagen I scaffold. Cell proliferation data (MTT assay) and data of ADAMTS-4 and -5 protein concentration (ELISA) from 100 µg total protein extracts were statistically analyzed on the bases of disc degeneration grade (DDG). Box plots with whiskers min. to max. show NP cell proliferation rates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081467#pone-0081467-g002" target="_blank">Figure 2a</a>), ADAMTS-4 protein expression levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081467#pone-0081467-g002" target="_blank">Figure 2b</a>) and ADAMTS-5 protein expression levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081467#pone-0081467-g002" target="_blank">Figure 2c</a>).</p

Proliferation rates of degenerative cervical NP cells and their endogenous protein expression levels of ADAMTS-4 and ADAMTS-5.

<p>Cervical NP cells were isolated from 15 cervical disc specimens of degenerative grade III and IV. 4×10⁵ cells from each specimen were grown for four weeks in collagen I scaffold. NP cell proliferation rates (MTT assay) and endogenous protein expression levels (ELISA) of ADAMTS-4 and ADAMTS-5 from 100 µg total protein extracts were analyzed on the basis of disc degeneration grade (DDG). Box plots with whiskers min to max show NP cell proliferation rates (Fig. 1a), ADAMTS-4 protein expression levels (Fig. 1b) and ADAMTS-5 protein expression levels (Fig. 1c).</p

Protein expression levels of endogenous inflammatory cytokines in cervical NP cells.

<p>15 degenerative grade III and IV herniated cervical discs were used to isolate NP specimens. Collagen I scaffold was used to culture 4×10⁵ NP cells from each specimen for four weeks. The endogenous expression levels of inflammatory cytokines were confirmed (ELISA) from 100 µg total protein extracts of each sample on the basis of disc degeneration grade (DDG). Box plots with whiskers min to max show IL-1β expression levels (Fig. 4a), IL-1 R expression levels (Fig. 4b), TNF-α expression levels (Fig. 4c) and TNF-α R1 expression levels (Fig. 4d).</p

Expression levels of anti-catabolic proteins TIMPs in degenerative NP cells.

<p>NP cells were isolated from 63 samples of degenerative IVDs and 4 x 10⁵ cells from each sample were cultured for four weeks in collagen I scaffold. Concentration data (ELISA) of TIMPs from 100 µg total protein extracts were statistically analyzed on the bases of disc degeneration grade (DDG). Box plots with whiskers min. to max. show TIMP-1 expression levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081467#pone-0081467-g004" target="_blank">Figure 4a</a>), TIMP-2 expression levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081467#pone-0081467-g004" target="_blank">Figure 4b</a>), TIMP-3 expression levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081467#pone-0081467-g004" target="_blank">Figure 4c</a>) and TIMP-4 expression levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081467#pone-0081467-g004" target="_blank">Figure 4d</a>).</p

Expression levels of endogenous MMPs in degenerative cervical NP cells.

<p>Cervical NP specimens of degenerative grade III and IV were isolated from 15 patients. From each specimen 4×10⁵ cells were grown in collagen I scaffold for four weeks to determine the endogenous expression levels (ELISA) of MMPs on the basis of disc degeneration grade (DDG). For each sample 100 µg total protein extracts were applied per experiment. Whiskers min to max of box plots show MMP-1 expression levels (Fig. 2a), MMP-2 expression levels (Fig. 2b), MMP-3 expression levels (Fig. 2c), MMP-7 expression levels (Fig. 2d) and MMP-13 expression levels (Fig. 2e).</p