Decellularized matrix for repairing intervertebral disc degeneration: Fabrication methods, applications and animal models.

Intervertebral disc degeneration (IDD)-induced low back pain significantly influences the quality of life, placing a burden on public health systems worldwide. Currently available therapeutic strategies, such as conservative or operative treatment, cannot effectively restore intervertebral disc (IVD) function. Decellularized matrix (DCM) is a tissue-engineered biomaterial fabricated using physical, chemical, and enzymatic technologies to eliminate cells and antigens. By contrast, the extracellular matrix (ECM), including collagen and glycosaminoglycans, which are well retained, have been extensively studied in IVD regeneration. DCM inherits the native architecture and specific-differentiation induction ability of IVD and has demonstrated effectiveness in IVD regeneration in vitro and in vivo. Moreover, significant improvements have been achieved in the preparation process, mechanistic insights, and application of DCM for IDD repair. Herein, we comprehensively summarize and provide an overview of the roles and applications of DCM for IDD repair based on the existing evidence to shed a novel light on the clinical treatment of IDD.

Intradiscal injection of human recombinant BMP-4 does not reverse intervertebral disc degeneration induced by nuclectomy in sheep.

Background: Intervertebral disc (IVD) degeneration is suggested as a major cause of chronic low back pain (LBP). Intradiscal delivery of growth factors has been proposed as a promising strategy for IVD repair and regeneration. Previously, BMP-4 was shown to be more potent in promoting extracellular matrix (ECM) production than other BMPs and TGF-ß in human nucleus pulposus (NP) cells, suggesting its applicability for disc regeneration. Methods: The effects of BMP-4 on ECM deposition and cell proliferation were assessed in sheep NP and annulus fibrosus (AF) cells in a pellet culture model. Further, a nuclectomy induced sheep lumbar IVD degeneration model was used to evaluate the safety and effects of intradiscal BMP-4 injection on IVD regeneration. Outcomes were assessed by magnetic resonance imaging, micro-computed tomography, histological and biochemical measurements. Results: In vitro, BMP-4 significantly increased the production of proteoglycan and deposition of collagen type II and proliferation of NP and AF cells. Collagen type I deposition was not affected in NP cells, while in AF cells it was high at low BMP-4 concentrations, and decreased with increasing concentration of BMP-4. Intradiscal injection of BMP-4 induced extradiscal new bone formation and Schmorl's node-like changes in vivo. No regeneration in the NP nor AF was observed. Conclusion: Our study demonstrated that although BMP-4 showed promising regenerative effects in vitro, similar effects were not observed in a large IVD degeneration animal model. The Translational Potential of This Article: The contradictory results of using BMP-4 on IVD regeneration between in vitro and in vivo demonstrate that direct BMP-4 injection for disc degeneration-associated human chronic low back pain should not be undertaken. In addition, our results may also shed light on the mechanisms behind pathological endplate changes in human patients as a possible target for therapy.

The expression of metalloproteinases in the lumbar disc correlates strongly with Pfirrmann MRI grades in lumbar spinal fusion patients.

Introduction: Increased catabolism of the extracellular matrix is observed under degenerative disc disease (DDD). The cleavage of extracellular matrix proteins in the nucleus pulposus (NP) by either matrix metalloproteinases (MMPs) or a disintegrin and metalloproteinases with thrombospondin motifs (ADAMTSs) is believed to be involved in the degeneration, but the mechanisms are not known. Research question: Here, we examine the correlation between expression of several MMPs and ADAMTSs subtypes in lumbar discs from 34 patients with low back pain (LBP) undergoing 1-2 level lumbar fusion surgery (L4/L5 and/or L5/S1) for DDD with or without spondylolisthesis. Materials and Methods: The mRNA levels of MMPs (subtypes 1, 2, 3, 10, and 13) and ADAMTSs (subtypes 1, 4, and 5) were analyzed using quantitative real-time polymerase chain reaction (RT-qPCR) and correlated to the Pfirrmann magnetic resonance imaging classification system (grade I-V) of lumbar DDD. Results: We find a highly significant positive correlation between Pfirrmann grades and the gene expression of MMP1 (r=0.67, p=0.0001), MMP3 (r=0.61, p=0.0002), MMP10 (r=0.6701, p=0.0001), MMP13 (r=0.48, p=0.004), ADAMTS1 (r=0.67, p=0.0001), and ADAMTS5 (r=0.53, p=0.0017). The similar regulation of these transcript suggests their involvement in disc degeneration. Interestingly, a post hoc analysis (uncorrected p-values) also demonstrated a positive correlation between expression of TNF-α, IL-6 and both ADAMTSs/MMPs and the Pfirrmann grades. Discussion and Conclusion: These findings show that disc degradation in DDD is strongly associated with the expression of some metalloproteinases.

Computed tomography osteoabsorptiometry for imaging of degenerative disc disease.

BACKGROUND: Lower back pain is a common condition with significant morbidity and economic impact. The pathophysiology is poorly understood but is in part attributable to degenerative disc disease (DDD). The healthy intervertebral disc ensures spine functionality by transferring the perceived load to the caudally adjacent vertebrae. The exposure to recurring mechanical load is mirrored in the mineralization pattern of the subchondral bone plate (SBP), where increased bone density is a sign of repetitive localized high stress. Computed tomography -osteoabsorptiometry (CT-OAM) is a technique based on conventional CT scans that displays the mineral density distribution in the SBP as a surface-color map. The objective of this study was to measure and analyze the SBP mineral density patterns of healthy lumbar intervertebral disc (IVDs) and those suffering DDD using CT-OAM densitograms. These findings should provide in vitro insight into the long-term morphological properties of the IVD and how these differ in the state of disc degeneration. METHODS: The CT-data sets of spines from 17 healthy individuals and 18 patients displaying DDD in the lumbar spine were acquired. Individual vertebrae of both cohorts were 3D reconstructed, processed using image analysis software, and compared to one another. Maximum intensity projection of the subchondral mineralization provided surface densitograms of the SBP. The relative calcium concentration, the local maxima of mineralization, and a mean surface projection of level-defined SBPs were calculated from the densitogram and statistically compared. RESULTS: The inferior SBP, adjacent to degenerating disc, display an 18-41 % higher relative calcium concentration than their healthy counterparts. In the opposing superior SBPs the relative calcium content is significantly increased. Whereas it is reasonably consistent for L1-L3 (L1: 132 %, L2: 127 %, L3: 120 %), the increase grows in caudal direction (L4: 131 %, L5: 148 %, S1: 152 %). Furthermore, a change in the areal distribution of excessive mineralization can be differentiated between healthy and diseased motion segments. CONCLUSIONS: The acquired data provide in vitro proof of the mechanical and anatomical properties of the SBP in relation to the state of disc degeneration. In conjunction with the diagnostic use of CT-osteoabsorptiometry, our data provide a basis for a non-invasive and sensitive technique that correlates with disc functionality. This could be promising in various cases, from early identification of early stages of DDD, tracking disease progression, and assessing the repercussions of surgical procedures or experimental therapies.

PRIMUS: Comprehensive proteomics of mouse intervertebral discs that inform novel biology and relevance to human disease modelling.

Mice are commonly used to study intervertebral disc (IVD) biology and related diseases such as IVD degeneration. Discs from both the lumbar and tail regions are used. However, little is known about compartmental characteristics in the different regions, nor their relevance to the human setting, where a functional IVD unit depends on a homeostatic proteome. Here, we address these major gaps through comprehensive proteomic profiling and in-depth analyses of 8-week-old healthy murine discs, followed by comparisons with human. Leveraging on a dataset of over 2,700 proteins from 31 proteomic profiles, we identified key molecular and cellular differences between disc compartments and spine levels, but not gender. The nucleus pulposus (NP) and annulus fibrosus (AF) compartments differ the most, both in matrisome and cellularity contents. Differences in the matrisome are consistent with the fibrous nature required for tensile strength in the AF and hydration property in the NP. Novel findings for the NP cells included an enrichment in cell junction proteins for cell-cell communication (Cdh2, Dsp and Gja1) and osmoregulation (Slc12a2 and Wnk1). In NP cells, we detected heterogeneity of vacuolar organelles; where about half have potential lysosomal function (Vamp3, Copb2, Lamp1/2, Lamtor1), some contain lipid droplets and others with undefined contents. The AF is enriched in proteins for the oxidative stress responses (Sod3 and Clu). Interestingly, mitochondrial proteins are elevated in the lumbar than tail IVDs that may reflect differences in metabolic requirement. Relative to the human, cellular and structural information are conserved for the AF. Even though the NP is more divergent between mouse and human, there are similarities at the level of cell biology. Further, common cross-species markers were identified for both NP (KRT8/19, CD109) and AF (COL12A1). Overall, mouse is a relevant model to study IVD biology, and an understanding of the limitation will facilitate research planning and data interpretation, maximizing the translation of research findings to human IVDs.