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Objective: To catalog and characterize the proteome of normal human intervertebral disc (IVD). Methods: Nine magnetic resonance imaging (MRI) normal IVDs were harvested from 9 different brain dead yet alive voluntary organ donors and were subjected to electrospray ionization-liquid chromatography tandem mass spectrometry (ESI-LC-MS/MS) acquisition. Results: A total of 1,116 proteins were identified. Functional enrichment analysis tool DAVID ver. 6.8 categorized: extracellular proteins (38%), intracellular (31%), protein-containing complex (13%), organelle (9%), membrane proteins (6%), supramolecular complex (2%), and 1% in the cell junction. Molecular function revealed: binding activity (42%), catalytic activity (31%), regulatory activity (14%), and structural activity (7%). Molecular transducer, transporter, and transcription regulator activity together contributed to 6%. A comparison of the proteins obtained from this study to others in the literature showed a wide variation in content with only 3% of bovine, 5% of murine, 54% of human scoliotic discs, and 10.2% of discs adjacent to lumbar burst fractures common to our study of organ donors. Between proteins reported in scoliosis and lumbar fracture patients, only 13.51% were common, further signifying the contrast amongst the various MRI normal IVD samples. Conclusion: The proteome of “healthy” human IVDs has been defined, and our results show that proteomic data on IVDs obtained from scoliosis, fracture patients, and cadavers lack normal physiological conditions and should not be used as biological controls despite normal MRI findings. This questions the validity of previous studies that have used such discs as controls for analyzing the pathomechanisms of disc degeneration. [ABSTRACT FROM AUTHOR]

Intervertebral disc degeneration is accompanied by a loss of Extra-cellular matrix (ECM) due to an imbalance in anabolic and catabolic pathways. Identifying ECM proteins with anabolic and/or regenerative potential could be the key to developing regenerative therapies. Since human fetal discs grow and develop rapidly, studying these discs may provide valuable insights on proteins with regenerative potential. This study compares core matrisome of 9 fetal and 7 healthy adult (age 22–79) nucleus pulposus (NP), using a proteomic and bioinformatic approach. Of the 33 upregulated proteins in fetus NP's, 20 of which were involved in ECM assembly pathways: fibromodulin, biglycan, heparan sulfate proteoglycan 2, chondroitin sulfate proteoglycan 4, procollagen C-endopeptidase enhancer and Collagen—type 1a1, 1a2, 6a1, 6a3, 11a1, 11a2, 12a1, 14a1 and 15a1. Moreover, 10 of the upregulated proteins were involved in growth pathways 'PI3L-Akt signaling' and 'regulation of insulin like growth factor transport and uptake.' Thrombospondin 1,3 and 4, tenascin C, matrilin-3, and collagen- type 1a1, 1a2, 6a1, 6a3 and 9a1. Additionally, matrillin-2 and 'Collagen triple helix repeat containing 1' were identified as possible regenerative proteins due to their involvement in 'Regeneration' and 'tissue development' respectively. In conclusion, the consistency of human fetal NP's differs greatly from that of healthy adults. In view of these outcomes, the core matrisome of human fetal discs contains an abundant number of proteins that could potentially show regenerative properties, and their potential should be explored in future machinal experiments. [ABSTRACT FROM AUTHOR]

Study Design: Profiling proteins expressed in the nucleus pulposus of fetal intervertebral disc (IVD). Purpose: To evaluate the molecular complexity of fetal IVDs not exposed to mechanical, traumatic, inflammatory, or infective insults to generate improved knowledge on disc homeostasis. Overview of Literature: Low back pain is the most common musculoskeletal disorder, causing a significant reduction in the quality of life, and degenerative disc disorders mainly contribute to the increasing socioeconomic burden. Despite extensive research, the causative pathomechanisms behind degenerative disc disorders are poorly understood. Precise molecular studies on the intricate biological processes involved in maintaining normal disc homeostasis are needed. Methods: IVDs of nine fetal specimens obtained from medical abortions were used to dissect out the annulus fibrosus and nucleus pulposus under sterile operating conditions. Dissected tissues were transferred to sterile Cryovials and snap frozen in liquid nitrogen before transporting to the research laboratory for protein extraction and further liquid chromatography tandem mass spectrometry (LCMS/ MS) analysis. Collected data were further analyzed using Gene Functional Classification Tool in DAVID and STRING databases. Results: A total of 1,316 proteins were identified through LC-MS/MS analysis of nine fetal IVD tissues. Approximately 247 proteins present in at least four fetal discs were subjected to further bioinformatic analysis. The following 10 clusters of proteins were identified: collagens, ribosomal proteins, small leucine-rich proteins, matrilin and thrombospondin, annexins, protein disulfide isomerase family proteins and peroxiredoxins, tubulins, histones, hemoglobin, and prolyl 4-hydroxylase family proteins. Conclusions: This study provides fundamental information on the proteome networks involved in the growth and development of healthy fetal discs in humans. Systematic cataloging of proteins involved in various structural and regulatory processes has been performed. Proteins expressed most abundantly (collagen type XIV alpha 1 chain, biglycan, matrilin 1, and thrombospondin 1) in their respective clusters also elucidate the possibility of utilizing these proteins for potential regenerative therapies. [ABSTRACT FROM AUTHOR]

Study Design: Profiling proteins expressed in the nucleus pulposus (NP) of intervertebral discs (IVDs) in five different biological states. Purpose: To evaluate the molecular complexity of the collagen (COL) framework and its role in the health and disease of human IVDs. Overview of Literature: Changes in COL composition have been linked to degenerative disk disease (DDD). Despite the fact that humans have 28 different types of COLs, most of the literature focuses solely on COL-1 and COL-2. This study used high-end proteomic technology to examine the entire COL composition of the human IVD across fetal (developmental-FD), normal (healthy-ND), scoliotic (early degeneration-SD), herniated (degenerate-DH), and degenerated (DD) disk phenotypes. Methods: Forty NP tissues were snap-frozen in liquid nitrogen (-196°C) immediately before being subjected to proteomic and bioinformatic analyses from five different disk phenotypes (eight each). Results: Tandem mass spectrometric analysis revealed a total of 1,050 proteins in FDs, 1,809 in ND, 1,487 in SD, 1,859 in DH, and 1,538 in the DD group. Of 28 major collagens reported in the human body, this study identified 24 different collagens with 34 subtypes in NP. Fibril-forming collagens (COL-1, 2, and 11A1) and fibril-associated collagens with interrupted triple helices (COL-9A1, 12A1, and 14A1) were abundantly expressed in FDs, representing their role in the development of NP. Multiplexin (COL-15), a hybrid proteoglycan-collagen molecule, was discovered only in FDs. Degeneration was associated with COL2A1 downregulation and COL-10A1 upregulation. Conclusions: COL10 was discovered to be a new biomarker for disk degeneration. Besides COL-1 and 2, other important COLs (6, 9, 11, 12, 14, 15) with anabolic potential and abundant expression in the fetal phenotype could be investigated for tissue engineering and novel DDD therapy. [ABSTRACT FROM AUTHOR]