Contrast-enhanced microCT evaluation of degeneration following partial and full width injuries to mouse lumbar intervertebral disc

Study Design: Preclinical animal study. Objective: Evaluation of the degenerative progression resulting from either a partial- or full- width injury to the mouse lumbar intervertebral disc (IVD) using contrast-enhanced micro-computed tomography and histological analyses. We utilized a lateral-retroperitoneal surgical approach to access the lumbar IVD, and the injuries to the IVD were induced by either incising one side of the annulus fibrosus or puncturing both sides of the annulus fibrosus. The full-width injury caused dramatic reduction in nucleus pulposus hydration and significant degeneration. A partial-width injury produces localized deterioration around the annulus fibrosus site that resulted in local tissue remodeling without gross degeneration to the IVD. Methods: Female C57BL/6J mice of 3-4 months age were used in this study. They were divided into three groups to undergo a partial-width, full-width, or sham injuries. The L5/L6 and L6/S1 lumbar IVDs were surgically exposed using a lateral-retroperitoneal approach. The L6/S1 IVDs were injured using either a surgical scalpel (partial-width) or a 33G needle (full-width), with the L5/L6 serving as an internal control. These animals were allowed to recover and then sacrificed at 2-, 4-, or 8- weeks post-surgery. The IVDs were assessed for degeneration using contrast-enhanced microCT (CEμCT) and histological analysis. Results: The high-resolution 3D evaluation of the IVD confirmed that the respective injuries localized within one side of the annulus fibrosus or spanned the full width of the IVD. The full-width injury caused deteriorations in the nucleus pulposus after 2 weeks that culminated in significant degeneration at 8 weeks, while the partial width injury caused localized disruptions that remained limited to the annulus fibrosus. Conclusion: The use of CEμCT revealed distinct IVD degeneration profiles resulting from partial- and full- width injuries. The partial width injury may serve as a better model for IVD degeneration resulting from localized annulus fibrosus injuries in humans.

Anti-degenerative effect of melatonin on intervertebral disc: protective contribution against inflammation, oxidative stress, apoptosis, and autophagy

Intervertebral disc (IVD) degeneration is a leading cause of lower back pain. Although the etiology of IVD degeneration (IVDD) is unclear, excessive oxidative stress, inflammation and apoptosis and disruption of autophagy play important role in the pathogenesis of IVDD. Therefore, finding a solution to mitigate these processes could stop or reduce the development of IVDD. Melatonin, a powerful antioxidant, plays an important role in regulating cartilage tissue hemostasis. Melatonin inhibits destruction of extracellular matrix (ECM) of disc. Melatonin preserves ECM contents including sox-9, aggrecan, and collagen II through inhibiting matrix degeneration enzymes such as MMP-13. These protective effects may be mediated by the inhibition of oxidative stress, inflammation and apoptosis, and regulation of autophagy in IVD cells.

Computational Modeling Intervertebral Disc Pathophysiology: A Review

Lower back pain is a medical condition of epidemic proportion, and the degeneration of the intervertebral disc has been identified as a major contributor. The etiology of intervertebral disc (IVD) degeneration is multifactorial, depending on age, cell-mediated molecular degradation processes and genetics, which is accelerated by traumatic or gradual mechanical factors. The complexity of such intertwined biochemical and mechanical processes leading to degeneration makes it difficult to quantitatively identify cause–effect relationships through experiments. Computational modeling of the IVD is a powerful investigative tool since it offers the opportunity to vary, observe and isolate the effects of a wide range of phenomena involved in the degenerative process of discs. This review aims at discussing the main findings of finite element models of IVD pathophysiology with a special focus on the different factors contributing to physical changes typical of degenerative phenomena. Models presented are subdivided into those addressing role of nutritional supply, progressive biochemical alterations stemming from an imbalance between anabolic and catabolic processes, aging and those considering mechanical factors as the primary source that induces morphological change within the disc. Limitations of the current models, as well as opportunities for future computational modeling work are also discussed.

Inhibition of aberrant Hif1α activation delays intervertebral disc degeneration in adult mice

The intervertebral disc (IVD) is the largest avascular tissue. Hypoxia-inducible factors (HIFs) play essential roles in regulating cellular adaptation in the IVD under physiological conditions. Disc degeneration disease (DDD) is one of the leading causes of disability, and current therapies are ineffective. This study sought to explore the role of HIFs in DDD pathogenesis in mice. The findings of this study showed that among HIF family members, Hif1α was significantly upregulated in cartilaginous endplate (EP) and annulus fibrosus (AF) tissues from human DDD patients and two mouse models of DDD compared with controls. Conditional deletion of the E3 ubiquitin ligase Vhl in EP and AF tissues of adult mice resulted in upregulated Hif1α expression and age-dependent IVD degeneration. Aberrant Hif1α activation enhanced glycolytic metabolism and suppressed mitochondrial function. On the other hand, genetic ablation of the Hif1α gene delayed DDD pathogenesis in Vhl-deficient mice. Administration of 2-methoxyestradiol (2ME2), a selective Hif1α inhibitor, attenuated experimental IVD degeneration in mice. The findings of this study show that aberrant Hif1α activation in EP and AF tissues induces pathological changes in DDD, implying that inhibition of aberrant Hif1α activity is a potential therapeutic strategy for DDD.

Intradiscal injection of monosodium iodoacetate induces intervertebral disc degeneration in an experimental rabbit model

Background Establishing an optimal animal model for intervertebral disc (IVD) degeneration is essential for developing new IVD therapies. The intra-articular injection of monosodium iodoacetate (MIA), which is commonly used in animal models of osteoarthritis, induces cartilage degeneration and progressive arthritis in a dose- and time-dependent manner. The purpose of this study was to determine the effect of MIA injections into rabbit IVDs on the progression of IVD degeneration evaluated by radiographic, micro-computerized tomography (micro-CT), magnetic resonance imaging (MRI), and histological analyses. Methods In total, 24 New Zealand White (NZW) rabbits were used in this study. Under general anesthesia, lumbar discs from L1–L2 to L4–L5 had a posterolateral percutaneous injection of MIA in contrast agent (CA) (L1–L2: CA only; L2–L3: MIA 0.01 mg; L3–L4: 0.1 mg; L4–L5: 1.0 mg; L5–L6: non-injection (NI) control). Disc height was radiographically monitored biweekly until 12 weeks after injection. Six rabbits were sacrificed at 2, 4, 8, and 12 weeks post-injection and processed for micro-CT, MRI (T2-mapping), and histological analyses. Three-dimensional (3D) disc height in five anatomical zones was evaluated by 3D reconstruction of micro-CT data. Results Disc height of MIA-injected discs (L2–L3 to L4–L5) gradually decreased time-dependently (P < 0.0001). The disc height of MIA 0.01 mg-injected discs was significantly higher than those of MIA 0.1 and 1.0 mg-injected discs (P < 0.01, respectively). 3D micro-CT analysis showed the dose- and time-dependent decrease of 3D disc height of MIA-injected discs predominantly in the posterior annulus fibrosus (AF) zone. MRI T2 values of MIA 0.1 and 1.0 mg-injected discs were significantly decreased compared to those of CA and/or NI controls (P < 0.05). Histological analyses showed progressive time- and dose-degenerative changes in the discs injected with MIA (P < 0.01). MIA induced cell death in the rabbit nucleus pulposus with a high percentage, while the percentage of cell clones was low. Conclusions The results of this study showed, for the first time, that the intradiscal injection of MIA induced degenerative changes of rabbit IVDs in a time- and dose-dependent manner. This study suggests that MIA injection into rabbit IVDs could be used as an animal model of IVD degeneration for developing future treatments.

Ultra-short Echo Time MR Imaging in Assessing Cartilage Endplate Damage And Association Between Its Lesion and Disc Degeneration For Chronic Low Back Pain Patients

Objective: To investigate the feasibility of ultra-short echo time (UTE) MRI in assessing cartilage endplate (CEP) damage and evaluating the relationship between total endplate score (TEPS) and lumbar intervertebral disc (IVD) degeneration.Materials and methods: 35 patients were measured for IVD using UTE imaging at 3T MR. Subtracted UTE images between short and long TEs were obtained to depict anatomy of CEP. The SNR and CNR were calculated to assess the image quality. A new grading criterion for endplate evaluation was developed based on Rajasekarank.S grading in this study. Two radiologists were employed to evaluate CEP and bony vertebral endplates (VEP) using new grading criterion and assess TEPS, independently. Cohen's kappa analysis was applied to evaluate the inter-observer agreement of endplate damage assessment between two radiologists, and the Kendall's TAU-B analysis was employed to determine the relationship between TEPS and IVD degeneration evaluated with Pfirrmann grading.Results: Well structural CEP was depicted on subtracted UTE images and confirmed by high SNR (33.0±2.92) and CNR values (9.4±2.08). Qualified subtracted UTE images were used by two radiologists to evaluate CEP and VEP damage. Excellent inter-observer agreement was confirmed by high value in Cohen's kappa test (0.839,P<0.001). Ensured by this, 138 endplates from 69 IVDs of 35 patients were classified into six grades based on the new grading criterion and TEPS of each endplate was calculated. In addition, the degeneration degree of IVDs were classified into five grades. Finally, using Kendall's TAU-B analysis, significant relationship was obtained between endplate damage related TEPS and IVD degeneration (r= 0.864,P<0.001).Conclusion: Ensured by high image quality, UTE imaging might be considered an effective tool to assess CEP damage. Additionally, further calculated TEPS has shown strong positive association with IVD degeneration, suggesting that the severity of endplate damage is highly linked with the degree of IVD degeneration.

Assessment of Radiological and Morphological Grading Systems of Intervertebral Disc Degeneration

Introduction: Intervertebral disc (IVD) degeneration is considered to be one of the main pathophysiological causes of low back pain. Several grading systems have been developed for both morphological and radiological assessment. The aim of this study was to assess the morphological and radiological characteristics of IVD degeneration and validate popular radiological Pfirrmann scale against morphological Thompson grading system. Methodology: Full spinal columns (vertebrae L1-S1 and IVD between them) were harvested from cadavers through an anterior dissection. MRI scans of all samples were conducted. Then, all vertebral columns were cut in the midsagittal plane and assessed morphologically. Result: A total of 100 lumbar spine columns (446 IVDs) were included in the analysis of the degeneration grade. Morphologic Thompson scale graded the majority of discs as grade 2 and 3 (44.2% and 32.1%, respectively), followed by grade 4 (16.8%), grade 1 (5.8%), and grade 5 (1.1%). The Radiologic Pfirrmann grading system classified 44.2% of discs as grade 2, 32.1% as grade 3, 16.8% as grade 4, 5.8% as grade 1, and 1.1% as grade 5. The analysis on the effect of age on degeneration revealed significant, although moderate, positive correlation with both scales. Analysis of the agreement between scales showed weighted Cohen’s kappa equal to 0.61 (p < 0.001). Most of the disagreement occurred due to a 1-grade difference (91.5%), whereas only 8.5% due to a 2-grade difference. Conclusion: With the increase in the prevalence of IVD disease in the population, reliable grading systems of IVD degeneration are crucial for spine surgeons in their clinical assessment. While overall there is an agreement between both grading systems, clinicians should remain careful when using Pfirmann scale as the grades tend to deviate from the morphological assessment.

The Effect of Ageing and Degeneration on Glycosaminoglycan Concentration in the Intervertebral Disc

Introduction: The correct spatial distribution and high negative charge of glycosaminoglycans (GAGs) within the intervertebral disc (IVD) are responsible for discs water imbibition, proper osmotic pressure, and as such IVD’s physiological swelling behaviors and compressive properties. The aim of this study was to investigate the association of the concentration and distribution of GAG with IVD degeneration as measured by Pfirrmann et al. and Thompson et al. grading systems. Methodology: Full spinal columns (vertebrae L1-S1 and IVD between them) were harvested from fresh cadavers through an anterior dissection. MRI scans were taken of all spinal columns and were assessed using Pfirrmann grading system. All vertebral columns were cut in the midsagittal plane. The level of degeneration was assessed morphologically using Thompson et al. grading system. Samples from five regions of the L5/S1 IVDs were taken for GAG concentration analyses. Standard curve spectrophotometry was utilized for this purpose. Result: One hundred lumbar spine columns (L1-S1) were harvested from cadavers. Radiologic assessment using the Pfirrmann grading system and morphological Thompson grading system classified majority of discs as grade 3 and 4. A total of 478 samples from five regions of L5/S1 IVDs were included in the analysis of GAG content. The samples from the nucleus pulposus showed on average the highest concentration of GAG, although the differences were not statistically significant. The one-way analysis of variance (ANOVA) showed no statistically significant differences in the mean GAG mass between different Pfirrmann grades (F = 1.85, p = 0.13) and between different Thompson grades (F = 1.17, p = 0.33). Conclusion: Our study showed no association between GAG concentration levels and degeneration grade of the IVD as measured by radiological Pfirrmann and morphological Thompson grading systems.

Exosomes Derived from Bone Mesenchymal Stem Cells Alleviate Compression-Induced Nucleus Pulposus Cell Apoptosis by Inhibiting Oxidative Stress

Oxidative stress is relevant in compression-induced nucleus pulposus (NP) cell apoptosis and intervertebral disc (IVD) degeneration. Exosomes derived from bone mesenchymal stem cells (BMSCs-Exos) are key secretory products of MSCs, with important roles in tissue regeneration. This research is aimed at studying the protective impact of BMSCs-Exos on NP cell apoptosis caused by compression and investigating the underlying mechanisms. Our results indicated that we isolated BMSCs successfully. Exosomes were isolated from the BMSCs and found to alleviate the inhibitory effect that compression has on proliferation and viability in NP cells, decreasing the toxic effects of compression-induced NP cells. AnnexinV/PI double staining and TUNEL assays indicated that the BMSCs-Exos reduced compression-induced apoptosis. In addition, our research found that BMSCs-Exos suppressed compression-mediated NP oxidative stress by detecting the ROS and malondialdehyde level. Furthermore, BMSCs-Exos increased the mitochondrial membrane potential and alleviated compression-induced mitochondrial damage. These results indicate that BMSCs-Exos alleviate compression-mediated NP apoptosis by suppressing oxidative stress, which may provide a promising cell-free therapy for treating IVD degeneration.

Single-cell Atlas Unveils Cellular Heterogeneity and Novel Markers in Human Neonatal and Adult Intervertebral Discs

The origin, composition, distribution, and function of cells in the human intervertebral disc (IVD) has not been fully understood. Here, cell atlases of both human neonatal and adult IVDs have been generated and further assessed by gene ontology pathway enrichment, pseudo-time trajectory, histology, and immunofluorescence. Comparison of cell atlases revealed the presence of several sub-populations of notochordal cells (NC) in the neonatal IVD and a small quantity of NCs and associated markers in the adult IVD. Developmental trajectories predicted that most neonatal NCs develop into adult nucleus pulposus cells (NPCs) while some keep their identity throughout adulthood. A high heterogeneity and gradual transition of annulus fibrosus cells (AFCs) in the neonatal IVD was detected and their potential relevance in IVD development was assessed. Collectively, comparing single-cell atlases between neonatal and adult IVDs delineates the landscape of IVD cell biology and may help discover novel therapeutic targets for IVD degeneration.