chondroitin-sulfates and Intervertebral-Disc-Degeneration

Topics: Animals; Antioxidants; Chondroitin Sulfates; Dopamine; Furans; Hyaluronic Acid; Hydrogels; Inflammation; Intervertebral Disc Degeneration; Nucleus Pulposus; Rats

Examination of intervertebral disc (IVD) regeneration in an ovine annular lesion model.. Sulfation motifs are important functional determinants in glycosaminoglycans (GAGs). Previous studies have correlated 3-B-3(-) and 7-D-4 chondroitin sulfate (CS) motifs in tissues undergoing morphogenetic transition in development. We hypothesize that these motifs may also be expressed in degenerate IVDs and may represent a reparative response.. Induction of disc degeneration by 5 mm or 6 × 20 mm lesions in the annulus fibrosus (AF) over 6 or 3 to 6 months postoperation (PO). Tissue sections were stained with toluidine blue-fast green, 3-B-3(-) and 7-D-4 CS-sulfation motifs were immunolocalized in 3-month PO 6 × 20 mm lesion IVDs. Sulfated glycosaminoglycan (GAG), 3-B-3(-), and 7-D-4 epitopes were quantitated by ELISIA (enzyme-linked immunosorbent inhibition assay) in extracts of AF (lesion site and contralateral half) and nucleus pulposus (NP) 0, 3, and 6 months PO.. Collagenous overgrowth of lesions occurred in the outer AF. Chondroid metaplasia in ~20% of the 6 × 20 mm affected discs resulted in integration of an outgrowth of NP tissue with the inner AF lamellae preventing propagation of the lesion. 3-B-3(-) and 7-D-4 CS sulfation motifs were immunolocalized in this chondroid tissue. ELISIA quantified CS sulfation motifs demonstrating an increase 3 to 6 months PO in the AF lesion and a reduction in sulfated GAG not evident in the contralateral AF.. (1) Outer annular lesions underwent spontaneous repair. (2) Chondroid metaplasia of the inner 6 × 20 mm defect prevented its propagation suggesting an apparent reparative response.

Topics: Animals; Annulus Fibrosus; Chondroitin Sulfates; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Epitopes; Glycosaminoglycans; Intervertebral Disc; Intervertebral Disc Degeneration; Regeneration; Sheep

Low back pain resulting from intervertebral disc (IVD) degeneration is a significant socioeconomic burden. The main effect of the degeneration process involves the alteration of the nucleus pulposus (NP) via cell-mediated enzymatic breakdown of key extracellular matrix (ECM) components. Thus, the development of injectable and biomimetic biomaterials that can instruct the regenerative cell component to produce tissue-specific ECM is pivotal for IVD repair. Chondroitin sulfate (CS) and type II collagen are the primary components of NP tissue and together create the ideal environment for cells to deposit de-novo matrix. Given their high matrix synthesis capacity potential post-expansion, nasal chondrocytes (NC) have been proposed as a potential cell source to promote NP repair. The overall goal of this study was to assess the effects of CS incorporation into disc derived self-assembled ECM hydrogels on the matrix deposition of NCs. Results showed an increased sGAG production with higher amounts of CS in the gel composition and that its presence was found to be critical for the synthesis of collagen type II. Taken together, our results demonstrate how the inclusion of CS into the composition of the material aids the preservation of a rounded cell morphology for NCs in 3D culture and enhances their ability to synthesise NP-like matrix.

Topics: Chondroitin Sulfates; Humans; Hydrogels; Intervertebral Disc; Intervertebral Disc Degeneration; Nucleus Pulposus; Regeneration

Topics: Actins; Adaptor Proteins, Signal Transducing; Animals; Cell Cycle Proteins; Cells, Cultured; Chondroitin Sulfates; Extracellular Matrix; Female; Glycosyltransferases; Humans; Intervertebral Disc; Intervertebral Disc Degeneration; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Nucleus Pulposus; Signal Transduction; Transcription Factors; YAP-Signaling Proteins

Nucleus pulposus (NP) degeneration is usually the origin of intervertebral disc degeneration and consequent lower back pain. Although adipose-derived stem cell (ADSC)-based therapy is regarded to be promising for the treatment of degenerated NP, there is a lack of viable cell carriers to transplant ADSCs into the NP while maintaining cell function. In this study, we developed a type II collagen/chondroitin sulfate (CS) composite hydrogel-like ADSC (CCSA) delivery system with genipin as the cross-linking agent. The induction effect of the scaffold on ADSC differentiation was studied in vitro, and a rat coccygeal vertebrae degeneration model was used to investigate the regenerative effect of the CCSA system on the degenerated NP in vivo. The results showed that the CCSA delivery system cross-linked with 0.02% genipin was biocompatible and promoted the expressions of NP-specific genes. After the injection of the CCSA system, the disc height, water content, extracellular matrix synthesis, and structure of the degenerated NP were partly restored. Our CCSA delivery system uses minimally invasive approaches to promote the regeneration of degenerated NP and provides an exciting new avenue for the treatment of degenerative disc disease.. Nucleus pulposus (NP) degeneration is usually the origin of intervertebral disc degeneration and consequent lower back pain. Stem cell-based tissue engineering is a promising method in NP regeneration, but there is a lack of viable cell carriers to transplant ADSCs into the NP while maintaining cell function. In this study, we developed a type II collagen/chondroitin sulfate (CS) composite hydrogel-like ADSC (CCSA) delivery system with genipin as the cross-linking agent. Although several research groups have studied the fabrication of injectable hydrogel with biological matrix, our study differs from other works. We chose type II collagen and CS, the two primary native components in the NP, as the main materials and combined them according to the natural ratio of collagen and sGAG in the NP. The delivery system is preloaded with ADSCs and can be injected into the NP with a needle, followed by in situ gelation. Genipin is used as a cross-linker to improve the bio-stability of the scaffold, with low cytotoxicity. We investigated the stimulatory effects of our scaffold on the differentiation of ADSCs in vitro and the regenerative effect of the CCSA delivery system on degenerated NP in vivo.

Topics: Adipose Tissue; Animals; Cells, Immobilized; Chondroitin Sulfates; Collagen Type II; Hydrogels; Intervertebral Disc Degeneration; Nucleus Pulposus; Rats; Rats, Sprague-Dawley; Stem Cell Transplantation; Stem Cells

Intervertebral disc degeneration (IDD) is characterized by dehydration and loss of extracellular matrixes in the nucleus pulposus region. Chondroitin sulfate has been found to be the water-binding molecule that played a key role in IDD. Although investigators have reported that inflammatory cytokines are involved in the reduction of chondroitin sulfate in IDD, but the underlying mechanism is unrevealed. Since chondroitin sulfate synthesis is controlled by chondroitin sulfate glycosyltransferases CHSY-1/2/3 and CSGALNACT-1/2, their functional role and regulatory mechanism in IDD is not fully studied. Here, we set out to investigate the function and regulatory roles of these factors during IDD development. We found that among these chondroitin sulfate glycosyltransferases, CHSY-1/2/3 are significantly down-regulated in severe IDD samples than mild IDD samples. In vitro experiments revealed that Interleukin-1β and Tumor Necrosis Factor-α stimulation led to significant reduction of CHSY-1/2/3 at protein level than mRNA level in NP cells, indicating a post-transcriptional regulatory mechanisms are involved. By computational prediction and analysis, we found that inflammatory cytokines stimulated microRNA-194 and -515 target CHSY-1/2/3 mRNA and significantly interrupt their translation and downstream chondroitin sulfate deposition. Inhibition of microRNA-194 and -515 however, significantly rescued CHSY-1/2/3 expressions and chondroitin sulfate deposition. These findings together demonstrated a vital role of inflammatory stimulated microRNAs in promoting intervertebral disc degeneration by interrupt chondroitin sulfate synthesis, which may provide new insights into the mechanism and therapeutic approaches in IDD.

Topics: 3' Untranslated Regions; Adult; Aged; Biomarkers; Biosynthetic Pathways; Chondroitin Sulfates; Computational Biology; Cytokines; Fluorescent Antibody Technique; Gene Expression Profiling; Gene Expression Regulation; Glycosyltransferases; Humans; Immunohistochemistry; Inflammation Mediators; Intervertebral Disc; Intervertebral Disc Degeneration; MicroRNAs; Middle Aged; Nucleus Pulposus; RNA Interference; RNA, Messenger; Severity of Illness Index

The objective of this study is to assess the correlation between the degree of degeneration of lumbar discs according to the Pfirrmann classification system and the concentrations of metabolites determined by means of 1H high-resolution magic angle spinning nuclear magnetic resonance (1H HR MAS NMR) spectroscopy.. Twenty-six human intervertebral lumbar discs that were operated on due to degenerative disease were analyzed. Routine preoperative 1.5T, T2-weighed magnetic resonance (MR) images were used to classify the cases according to the Pfirrmann classification system. In all the cases, during microdiscectomy, the fragments of the annulus fibrosus and nucleus pulposus were harvested and their metabolic profile was examined by means of 1H HR MAS. The grades of disc degeneration on the Pfirrmann scale were correlated with the metabolite concentrations.. Spectral analyses of the intervertebral discs with Pfirrmann grades IV and V demonstrated significantly higher concentrations of creatine, glycine, hydroxyproline, alanine, leucine, valine, acetate, isoleucine, α,β-glucose, and myo-inositol, and a lower intensity of the N-acetyl peak of chondroitin sulfate, compared to the spectra with Pfirrmann grade III.. Our results demonstrate correlations between metabolite concentrations and the degree of lumbar disc degeneration assessed using the Pfirrmann grading system and provide another step toward the potential use of in vivo MR spectroscopy for investigation of biomarkers in lumbar disc degeneration.

Topics: Adolescent; Adult; Aged; Amino Acids; Annulus Fibrosus; Chondroitin Sulfates; Glucose; Glycosaminoglycans; Humans; Inositol; Intervertebral Disc; Intervertebral Disc Degeneration; Lumbar Vertebrae; Magnetic Resonance Spectroscopy; Middle Aged; Nucleus Pulposus; Proteoglycans; Young Adult

Intervertebral disc (IVD) degeneration is strongly associated with low back pain, a major cause of disability worldwide. An in-depth understanding of IVD cell physiology is required for the design of novel regenerative therapies. Accordingly, aim of this work was the study of IVD cell responses to mitogenic growth factors in a three-dimensional (3D) organotypic milieu, comprising characteristic molecules of IVD's extracellular matrix. In particular, annulus fibrosus (AF) cells were cultured inside collagen type-I gels, while nucleus pulposus (NP) cells in chondroitin sulfate A (CSA) supplemented collagen gels, and the effects of Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF), and Insulin-Like Growth Factor-I (IGF-I) were assessed. All three growth factors stimulated DNA synthesis in both AF and NP 3D cell cultures, with potencies similar to those observed previously in monolayers. CSA supplementation inhibited basal DNA synthesis rates, without affecting the response to growth factors. ERK and Akt were found to be phosphorylated following growth factor stimulation. Blockade of these two signaling pathways using pharmacologic inhibitors significantly, though not completely, inhibited growth factor-induced DNA synthesis. The proposed culture systems may prove useful for further in vitro studies aiming at future interventions for IVD regeneration.

Topics: Animals; Cattle; Chondroitin Sulfates; Collagen; DNA; Fibroblast Growth Factor 2; Humans; Insulin-Like Growth Factor I; Intervertebral Disc; Intervertebral Disc Degeneration; Low Back Pain; Organ Culture Techniques; Platelet-Derived Growth Factor; Regenerative Medicine; Signal Transduction

There is much interest in the development of a cellular therapy for the repair or regeneration of degenerate intervertebral discs (IVDs) utilising autologous cells, with some trials already underway. Clusters of cells are commonly found in degenerate IVDs and are formed via cell proliferation, possibly as a repair response. We investigated whether these clusters may be more suitable as a source of cells for biological repair than the single cells in the IVD.. Discs were obtained at surgery from 95 patients and used to assess the cell viability, growth kinetics and stem or progenitor cell markers in both the single and clustered cell populations.. Sixty-nine percent (±15) of cells in disc tissue were viable. The clustered cell population consistently proliferated more slowly in monolayer than single cells, although this difference was only significant at P0-1 and P3-4. Both populations exhibited progenitor or notochordal cell markers [chondroitin sulphate epitopes (3B3(-), 7D4, 4C3 and 6C3), Notch-1, cytokeratin 8 and 19] via immunohistochemical examination; stem cell markers assessed with flow cytometry (CD73, 90 and 105 positivity) were similar to those seen on bone marrow-derived mesenchymal stem cells.. These results confirm those of previous studies indicating that progenitor or stem cells reside in adult human intervertebral discs. However, although the cell clusters have arisen via proliferation, there appear to be no greater incidence of these progenitor cells within clusters compared to single cells. Rather, since they proliferate more slowly in vitro than the single cell population, it may be beneficial to avoid the use of clustered cells when sourcing autologous cells for regenerative therapies.

Topics: 5'-Nucleotidase; Adolescent; Adult; Antigens, CD; Biomarkers; Cell Proliferation; Cell Survival; Cells, Cultured; Chondroitin Sulfates; Endoglin; Epitopes; GPI-Linked Proteins; Humans; Intervertebral Disc; Intervertebral Disc Degeneration; Keratin-19; Keratin-8; Male; Middle Aged; Receptor, Notch1; Receptors, Cell Surface; Thy-1 Antigens; Young Adult

Clinical and radiological outcomes of lumbar interbody fusion using artificial fusion cages filled with calcium phosphate cements (CPCs) were retrospectively reviewed. Between 2002 and 2011, 25 patients underwent lumbar interbody fusion at Tokushima University Hospital, and 22 patients were enrolled in this study. Of these, 5 patients received autologous local bone grafts and 17 received CPC. Japan Orthopedic Association (JOA) score was used for clinical outcome assessments. Lumbar radiography and computed tomography (CT) were performed at 12, 24 months and last follow-up period to assess bony fusion. The mean JOA score of all patients improved from 9.3 before surgery to 21.0 at 24 months after surgery. Fusion had occurred in 5 of 5 patients in the local bone graft group and in 16 of 17 patients in CPC group at 24 months postoperatively. No surgically related complication was occurred in both groups. CPC is a useful and safe graft material for lumbar interbody fusion.

Topics: Aged; Bone Cements; Bone Transplantation; Chondroitin Sulfates; Female; Follow-Up Studies; Humans; Hydroxyapatites; Image Interpretation, Computer-Assisted; Intervertebral Disc Degeneration; Lumbar Vertebrae; Male; Middle Aged; Osseointegration; Pedicle Screws; Prosthesis Implantation; Retrospective Studies; Scoliosis; Spinal Fusion; Spondylolisthesis; Succinates; Tomography, X-Ray Computed

Advancement in tissue engineering provides a promising approach to recover the functionality of the degenerated intervertebral disc. In our study, a nucleus pulposus (NP) cell-seeded collagen II/hyaluronan/chondroitin-6-sulfate (CII/HyA/CS) tri-copolymer construct was implanted into the disc space directly after nucleotomy in a rabbit model.. The aim of this study was to investigate whether the NP cell-seeded CII/HyA/CS tri-copolymer constructs could regenerate the degenerated disc in vivo after implantation into the rabbit nucleotomy model.. Nucleotomy is one of the most prevalent surgical modalities to treat degenerative disc disease, which could achieve good short-term effects of pain relieve, whereas removal of the entire or partial NP changes the biomechanical characteristics of the remaining disc and the adjacent vertebral segments and a series of long-term complications such as accelerated annulus and the facet joints degeneration may ensue. Therefore, it is necessary to think about possible procedures immediately after the primary nucleotomy surgery to avoid these complications.. NP cells isolated from thoracic and lumbar spines of New Zealand White rabbits of approximately 3 weeks of age and 1 kg in weight were labeled with a 5- (and-6) -carboxyflurescein diacetate succinimidyl ester (CFDA-SE) fluorescent dye and seeded within the CII/HyA/CS scaffold by a centrifugation method. After in vitro culture for 1 week, NP cell-seeded CII/HyA/CS tri-copolymer constructs were allografted into the disc defects of recipient rabbit immediately after nucleotomy of the lumbar spine. The Bradner Disc Index and the T2-weighted signal intensity index were determined using lateral plane radiographs and magnetic resonance imaging at 4, 12, and 24 weeks after the operation. Finally, the operated discs were explanted for gross morphological observation, histological evaluation, and cell viability assessment. Animals with only nucleotomy and cell-free CII/HyA/CS scaffold implantation served as controls.. In our study, we could demonstrate that the T2-weighted signal intensity index of the operated discs decreased in all three groups 1 month after surgery and the index of the cell-containing scaffold insertion group was significantly higher than that of the other two groups. After 24 weeks, the index of the cell-containing scaffold insertion group increased significantly. However, further decline was observed in both the noninsertion group and the scaffold insertion group. In radiographic analysis, the narrowing of the intervertebral disc space was significantly retarded by the cell-scaffold hybrids implantation up to 24 postoperative weeks. Furthermore, the gross morphology and histological evaluation indicated that the allografted NP cells were viable and showed extracellular matrix production.. In our study, we had constructed rabbit NP cell-seeded CII/HyA/CS tri-copolymer implants in vitro. Immediately after nucleotomy of the recipient rabbit, we allografted the precultured cell-scaffold hybrids into the lacuna of the disc. Results documented survival of the allografted NP cells and extracellular matrix deposition, which finally resulted in maintenance of disc height and restoration of T2-weighted signal intensity on magnetic resonance imaging.

Topics: Animals; Cell Culture Techniques; Cell Survival; Cell Transplantation; Cells, Cultured; Chondroitin Sulfates; Collagen Type II; Disease Models, Animal; Female; Graft Survival; Hyaluronic Acid; Intervertebral Disc; Intervertebral Disc Degeneration; Lumbar Vertebrae; Magnetic Resonance Imaging; Male; Polymers; Rabbits; Radiography; Regeneration; Tissue Engineering; Tissue Scaffolds; Transplantation, Homologous; Treatment Outcome

Whole ovine caudal intervertebral discs were cultured under simulated-physiologic or high-frequency loading and either sufficient or limited nutrition for 7 days.. To study the effect of high-frequency loading under sufficient or limited glucose conditions and to investigate the additive effects of load and nutrition on cell survival, gene expression, and cell activity after 7 days of culture.. Limited nutrition and certain mechanical stimuli are generally believed to be etiologic factors for disc degeneration. Although these effects and their interactions have been demonstrated in cell culture, no investigations have been reported in entire discs.. Discs were maintained in a whole organ culture bioreactor system under simulated-physiologic (0.2 Hz) or high-frequency (10 Hz) loading, in media with either limited (2 g/L) or sufficient (4.5 g/L) glucose concentration. After 7 days, cell viability, relative gene expression, newly synthesized chondroitin sulfate content, glycosaminoglycan synthesis rate, and disc morphology were assessed after culture and compared with fresh tissue.. Culture under either limited glucose or high-frequency loading conditions led to a significant drop in cell viability. Combined treatment with limited glucose and high-frequency loading resulted in an additive increase in cell death in both the anulus fibrosus and nucleus pulposus and in an increase in MMP13 gene expression.. Supporting in vivo studies and cell culture experiments, high-frequency loading simulating vibration conditions shows detrimental effects on intervertebral disc cells in whole organ culture. The effect on cell viability was exacerbated by limited nutrition culture. However, neither frequency nor limited glucose affected cell metabolism, measured by glycosaminoglycan synthesis rate. Longer culture periods may be required to detect changes at the extracellular matrix level.

Topics: Animals; Bioreactors; Cell Survival; Chondroitin Sulfates; Gene Expression Regulation, Enzymologic; Glucose; Glycosaminoglycans; Intervertebral Disc; Intervertebral Disc Degeneration; Matrix Metalloproteinase 13; Nutritional Status; Organ Culture Techniques; Sheep; Time Factors; Vibration