Aggrecan Gene Research Articles

Development and characterization of a novel non-surgical mouse model of intervertebral disc degeneration

Purpose: In the spinal column, the purpose of the intervertebral discs (IVD) is to provide the spine with flexibility and load-bearing capacity. Each IVD is comprised of a highly hydrated nucleus pulposus (NP) core that is enclosed by the concentric collagenous fibers of the annulus fibrosus (AF). Maintenance of this composite structure and its biomechanical function requires proper activity of disc-resident cells, with balanced synthesis and degradation of macromolecular components. Despite that, our understanding of the role of senescence and cell death on the progression of disc degeneration is limited. IVD degeneration is one of the main contributors to low back pain and is known to precede several prevalent and debilitating spinal diseases, such as disc herniation and spondylosis. Although the causes of IVD degeneration are poorly understood, dysfunction and progressive decline in the number of resident cells have been found with aging and disease. Most animal models currently used to investigate the pathophysiology of IVD degeneration rely on surgical intervention, where invasiveness coupled with an inherent inflammatory response, limits our ability to mimic the pathogenesis of non-traumatic human disc degeneration. Hence, we sought to develop a non-surgical mouse model of IVD degeneration using genetically-induced targeted ablation of disc-resident cells that express aggrecan (Acan), the main proteoglycan component found in the extracellular matrix of the IVDs. Methods: We crossed heterozygous mice that express Cre recombinase under the control of the aggrecan gene promoter (AcanCreERT2) with mice that express diphtheria toxin (DTA) following Cre recombination (Gt(ROSA)DTA). The Acan+/Cre ;DTA+/- offspring received tamoxifen (4-OHT) interperitoneally for 5 (five) consecutive days to promote the targeted ablation of Acan-expressing cells. Animals were euthanized, and spines were harvested at 1-, 5-, 9- and 12-weeks post-DTA induction for histology. Sagittal sections were stained with Safranin-O/Fast-green and compared to littermate controls lacking the Cre allele (Acan+/+;DTA+/-) who received the same treatment, to allow a detailed spatiotemporal analysis of changes in the IVDs structure and composition. Long-term histological assessment was also performed at 7-months post-DTA induction, to investigate IVD degenerative changes with aging. Results: Histological analysis revealed mild degenerative changes of the IVDs by 5-weeks post-DTA induction in the Acan+/Cre:DTA mice, with the cells in the NP demonstrating a hypertrophic phenotype by 12-weeks, with few to no notochordal cells identified. Lumbar IVDs exhibited profound histological changes by 7-months post-DTA induction when compared to controls (Figure 1). These changes included decreased cellularity, loss of NP/AF borders, altered AF lamellar fiber structure, and disruption of NP matrix with loss of proteoglycan staining, and are typical characteristics of severe disc degeneration. Conclusions: Targeted cell depletion within the IVD resulted in progressive histological changes in the structure and composition of the NP, with clear degeneration of the AF compared to controls, reaching severe IVD degeneration by 7-months post-DTA induction. This novel mouse model of IVD degeneration does not require direct injury to the IVD; thus, it may contribute to further understanding the mechanisms underlying spontaneous and/or age-related degeneration of IVD, which more closely mimic the progression of human disease. Considering the structure-function relationship of this tissue, future studies should explore the mechanical behavior of IVDs to assess how the overall structural degeneration seen histologically translates into the biomechanical properties of the tissue.

Actions of Prostaglandins on Human Nucleus Pulposus Metabolism Inferred by Cyclooxygenase 2 Inhibition of Cytokine Activated Cells.

ObjectiveLow back pain is frequently treated with nonsteroidal anti-inflammatory drugs (NSAIDs), but little is known about intervertebral disc metabolism of the prostaglandins that are diminished by these drugs. Hence, this study aimed at delineating prostaglandin actions in cytokine activated disc cells by comparing the response of nucleus pulposus (NP) cells to the pro-inflammatory cytokine interleukin (IL)-1β with and without cyclooxygenase 2 (COX-2) inhibition. MethodsNP cells cultured in alginate beads were activated with IL-1β ± the COX-2 inhibitor Sc-58125. Media harvested from cultured cells were analyzed for prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2α), IL-6, and matrix metalloproteinase (MMP)-3 by enzyme-linked immunosorbent assay and nitric oxide by Griess Reaction. Gene expression along with proteoglycan, collagen, and total protein synthesis were also measured. ResultsIL-1β increased culture media PGE2 and PGF2α, but decreased proteoglycan and collagen syntheses as well as mRNA expression of the matrix genes aggrecan, versican, collagen I, and collagen II. COX-2 inhibition partially rescued proteoglycan and collagen syntheses and collagen I mRNA, but decreased collagen II mRNA IL-1β activated NP cells. COX-2 inhibition initially enhanced and subsequently reduced IL-1β induced inducible nitric oxide synthase, without altering medium nitrite. IL-1β induction of MMP-3 mRNA was increased by COX-2 inhibition at 24 and 48 hours. ConclusionCOX-2 inhibition alters the response of NP cells to IL-1β, suggesting IL-1β action on disc cells is mediated at least in part through COX-2 and its prostaglandins. COX-2 inhibition produces minimal effects on several key catabolic mediators, with the exception of MMP-3. Blocking COX-2 might be beneficial for maintaining disc matrix since it provides an overall rescue of IL-1 induced loss of matrix protein synthesis.

Regulation of differentiation of annulus fibrosus-derived stem cells using heterogeneous electrospun fibrous scaffolds.

BackgroundTissue engineering of the annulus fibrosus (AF) shows promise as a treatment for patients with degenerative disc disease (DDD). However, it remains challenging due to the intrinsic heterogeneity of AF tissue. Fabrication of scaffolds recapitulating the specific cellular, componential, and microstructural features of AF, therefore, is critical to successful AF tissue regeneration.MethodsPoly-L-lactic acid (PLLA) fibrous scaffolds with various fiber diameters and orientation were prepared to mimic the microstructural characteristics of AF tissue using electrospinning technique. AF-derived stem cells (AFSCs) were cultured on the PLLA fibrous scaffolds for 7 days.ResultsThe morphology of AFSCs significantly varied when cultured on the scaffolds with various fiber diameters and orientation. AFSCs were nearly round on scaffolds with small fibers. However, they became spindle-shaped on scaffolds with large fibers. Meanwhile, upregulated expression of collagen-I gene happened in cells cultured on scaffolds with large fibers, while enhanced expression of collagen-II and aggrecan genes was seen on scaffolds with small fibers. The production of related proteins also showed similar trends. Further, culturing AFSCs on a heterogeneous scaffold by overlaying membranes with different fiber sizes led to the formation of a hierarchical structure approximating native AF tissue.ConclusionFindings from this study demonstrate that fibrous scaffolds with different fiber sizes effectively promoted the differentiation of AFSCs into specific cells similar to the types of cells at various AF zones. It also provides a valuable reference for regulation of cell differentiation and fabrication of engineered tissues with complex hierarchical structures using the physical cues of scaffolds.The translational potential of this articleEffective AF repair is an essential need for treating degenerative disc disease. Tissue engineering is a promising approach to achieving tissue regeneration and restoring normal functions of tissues. By mimicking the key structural features of native AF tissue, including fiber size and alignment, this study deciphered the effect of scaffold materials on the cell differentiation and extracellular matrix deposition, which provides a solid basis for designing new strategies toward more effective AF repair and regeneration.

Chondrogenic differentiation of mesenchymal stem/stromal cells on 3D porous poly (ε-caprolactone) scaffolds: Effects of material alkaline treatment and chondroitin sulfate supplementation

Cartilage defects resultant from trauma or degenerative diseases (e.g., osteoarthritis) can potentially be repaired using tissue engineering (TE) strategies combining progenitor cells, biomaterial scaffolds and bio-physical/chemical cues. This work examines promoting chondrogenic differentiation of human bone marrow mesenchymal stem/stromal cells (BM-MSCs) by combining the effects of modified poly (ε-caprolactone) (PCL) scaffolds hydrophilicity and chondroitin sulfate (CS) supplementation in a hypoxic 5% oxygen atmosphere. 3D-extruded PCL scaffolds, characterized by μCT, featured a 21mm-1 surface area to volume ratio, 390μm pore size and approximately 100% pore interconnectivity. Scaffold immersion in sodium hydroxide solutions for different periods of time had major effects in scaffold surface morphology, wettability and mechanical properties, but without improvements on cell adhesion. In-situ chondrogenic differentiation of BM-MSC seeded in 3D-extruded PCL scaffolds resulted in higher cell populations and ECM deposition along all scaffold structure, when chondrogenesis was preceded by an expansion phase. Additionally, CS supplementation during BM-MSC expansion was crucial to enhance aggrecan gene expression, known as a hallmark of chondrogenesis. Overall, this study presents an approach to tailor the wettability and mechanical properties of PCL scaffolds and supports the use of CS-supplementation as a biochemical cue in integrated TE strategies for cartilage regeneration.

Nano and micro biomechanical analyses of the nucleus pulposus after in situ immobilization in rats

Immobilization can lead to intervertebral disc degeneration. The biomechanical characteristics of such discs have not so far been investigated at the micro- or nanoscale, the level at which cells sense and respond to the surrounding environment. This study aimed to characterize changes in the elastic modulus of the collagen fibrils in the nucleus pulposus at the nanoscale and correlate this with micro-biomechanical properties of the nucleus pulposus after immobilization, in addition to observation of tissue histology and its gene expressions. An immobilization system was used on the rat tail with an external fixation device. The elastic modulus was measured using both nano and micro probes for atomic force microscopy after 4 and 8 weeks of immobilization. Histology of the tissue was observed following hematoxylin and eosin staining. Gene expression in the annulus fibrosus tissue was quantified using real-time reverse transcription-polymerase chain reaction. The elastic modulus of the collagen fibrils in the nucleus pulposus at the nanoscale increased from 74.07 ± 17.06 MPa in the control to 90.06 ± 25.51 MPa after 8 weeks (P = 0.007), and from 33.51 ± 9.33 kPa to 43.18 ± 12.08 kPa at the microscale (P = 0.002). After immobilization for 8 weeks, a greater number of cells were observed by histology to be aggregated within the nucleus pulposus. Collagen II (P = 0.007) and aggrecan (P = 0.003) gene expression were downregulated whereas collagen I (P = 0.002), MMP-3 (P < 0.001), MMP-13 (P < 0.001) and ADAMTs-4 (P < 0.001) were upregulated. Immobilization not only influenced individual collagen fibrils at the nanoscale, but also altered the micro-biomechanics and cell response in the nucleus pulposus. These results suggest that significant changes occur in intervertebral discs at both scales after immobilization, a situation about which clinicians should be aware when immobilization has to be used clinically.

Description of the D4/D4 genotype in Miniature horses with dwarfism.

Four causative mutations (D1, D2, D3*, and D4) of chondrodysplastic dwarfism have been described in the equine aggrecan (ACAN) gene. Homozygotes for one of these mutations and heterozygotes for any combination of these mutations exhibit the disproportionate dwarfism phenotype. However, no case description of homozygotes for D4 (D4/D4) has been reported in the literature, to our knowledge. We report 2 Miniature horses with the genotype D4/D4 in the ACAN gene. Clinically, the 2 dwarfs had a domed head that was large compared to the rest of the body, mandibular prognathism, and short and bowed limbs, mainly in the proximal region of the metatarsal bones. Radiographic examination revealed contour irregularities of the subchondral bone in the long bones and confirmed mandibular prognathism; histopathology revealed irregular chondrocyte organization. To determine the genotypes of the horses, we performed DNA extraction from white blood cells, PCR, and Sanger sequencing. Genotyping demonstrated that these 2 animals had the D4/D4 genotype in the ACAN gene. The D4/D4 dwarfs were clinically similar to animals with the other ACAN genotypes reported for this disease. Identification of heterozygous animals makes mating selection possible and is the most important control measure to minimize economic losses and casualties.

Research on the role and mechanism of indian hedgehog protein in abnormal calcification degeneration of chondrocytes

Objective To explore the role and mechanism of indian hedgehog (Ihh) in degenerative and abnormal calcification of chondrocyte. Methods Costal chondrocytes were extracted from C57 mice 6 days after birth, By HE staining, Saffron o solid green staining, collagen type Ⅱ immunohistochemical to identify the chondrocyte phenotype The experiment was divided into the ihh high expression group, and Ihh recombinant protein 5 mg/L was added. Ihh knockout group was added to cyclopamine 20 μmol/L. In the blank control group, phosphate buffer solution (PBS) was added. Using real-time quantitative polymerase chain reaction (Real-time PCR) to detect typeⅡcollagen (COLⅡ), type X collagen (COLX), Runt related transcription factor (Runx2), osteocalcin (OCN), alkaline phosphatase (ALP), aggrecan (AGG), progressive ankylosis (ANKH), extracellular nucleotide phosphatase 1 (ENPP1) factors mRNA expression level. Flow cytometry was used to detect the effect of inhibition of Ihh signaling pathway on chondrocyte apoptosis. Results Compared with the control group, the expression of ALP (3.450±1.357 vs. 1.223±0.740), OCN (2.410±0.395 vs. 1.093±0.453), Runx2 (3.057±1.477 vs. 1.144±0.574) and COLX (3.804±1.400 vs. 1.116±0.511) was increased in the high expression group, and the expression of ANKH (0.255±0.042 vs. 1.101±0.471), AGG (0.574±0.355 vs. 1.007±0.126) and COLII (0.670±0.065 vs. 1.027±0.236), ENPP1 (0.354±0.058 vs. 1.091±0.446) was decreased (P<0.05). When the cyclopamine is added, the opposite result appears(P<0.05). Flow cytometry results showed that Inhibition of Ihh signaling pathway can induce apoptosis (t=9.412, P<0.05). Conclusion Ihh can raise COLX, Runx2, OCN and ALP, inhibit the ANKH, ENPP1, AGG and COLⅡ gene expression, and further promote the cartilage cells anomaly calcified. Key words: Indian hedgehog; Chondrocytes; Calcification

Cytotoxic Properties of HT-2 Toxin in Human Chondrocytes: Could T3 Inhibit Toxicity of HT-2?

Thyroid hormone triiodothyronine (T3) plays an important role in coordinated endochondral ossification and hypertrophic differentiation of the growth plate, while aberrant thyroid hormone function appears to be related to skeletal malformations, osteoarthritis, and Kashin-Beck disease. The T-2 toxin, present extensively in cereal grains, and one of its main metabolites, HT-2 toxin, are hypothesized to be potential factors associated with hypertrophic chondrocyte-related osteochondropathy, known as the Kashin-Beck disease. In this study, we investigated the effects of T3 and HT-2 toxin on human chondrocytes. The immortalized human chondrocyte cell line, C-28/I2, was cultured in four different groups: controls, and cultures with T3, T3 plus HT-2 and HT-2 alone. Cytotoxicity was assessed using an MTT assay after 24-h-exposure. Quantitative RT-PCR was used to detect gene expression levels of collagen types II and X, aggrecan and runx2, and the differences in runx2 were confirmed with immunoblot analysis. T3 was only slightly cytotoxic, in contrast to the significant, dose-dependent cytotoxicity of HT-2 alone at concentrations ≥ 50 nM. T3, together with HT-2, significantly rescued the cytotoxic effect of HT-2. HT-2 induced significant increases in aggrecan and runx2 gene expression, while the hypertrophic differentiation marker, type X collagen, remained unchanged. Thus, T3 protected against HT-2 induced cytotoxicity, and HT-2 was an inducer of the pre-hypertrophic state of the chondrocytes.

Effects of combination TGF-B1 transfection and platelet rich plasma (PRP) on three-dimension chondrogenic differentiation of rabbit dental pulp-derived mesenchymal stem cells

ABSTRACT Aim: The aim of this study was to evaluate the effects of standard culture medium and chondrogenic differentiation medium with PRP on chondrogenic differentiation of rabbit dental pulp-derived mesenchymal stem cells (rabbit DPSCs) that are transfected with transforming growth factor-beta 1 (TGF-B1) gene, based on the hypothesis of TGF- B1 and PRP can be effective on the chondrogenesis of stem cells. Materials and Methods: Rabbit DPSCs were characterized by using flow cytometry, immunofluorescent staining, quantitative Real Time Polymerase Chain Reaction (qRT-PCR) and differentiation tests. For the characterization, CD29, CD44 and CD45 mesenchymal cell markers were used. Rabbit DPSCs were transfected with TGF-B1 gene using electroporation technique in group 1; with PRP 10% in group 2; with chondrogenic medium in group 3; with both chondrogenic medium and PRP in group 4. DPSCs were cultured in medium with 10% inactive PRP in group 5, chondrogenic medium in group 6, chondrogenic medium with PRP 10% in group 7. SOX9, MMP13 and Aggrecan gene expression levels were evaluated in 3, 6, 12. and 24. days by qRT-PCR. Results: The expression levels of SOX9, MMP13 and Aggrecan were higher in group 2, 3 and group 7 in 3th day however in 24th day group 7 and group 2 were found higher. The expression levels changed by time-dependent. The extracellular matrix of the cells in experimental groups were positively stained with safranin O and toluidine blue. Conclusion: The combination in culture medium of TGF-B1 gene transfection and 10% PRP accelerates the chondrogenic differentiation of DPSCs.

INTERVERTEBRAL DISC DEGENERATION LINKED TO STRUCTURAL GENE VARIATIONS

During the recent past focus has shifted from identifying intervertebral disc degeneration as being caused by physical exposure and strain to being linked with a variety of genetic variations. The objective of this review is to provide an up to date review of the existing research data regarding the relation of intervertebral disc degeneration to structural protein genes and their polymorphisms and thus help clearly establish further avenues where research into causation and treatment is needed. A comprehensive search using the keywords “Collagen”, “COL”, “Aggrecan”, “AGC”, “IVDD”, “intervertebral disc degeneration”, and “lumbar disc degeneration” from PubMed and Google Scholar, where literature in the English language was selected spanning from 1991 to 2019. There are many genes involved in the production of structural components of an intervertebral disc. The issues in production of these components involve the over-expression or under-expression of their genes, and single nucleotide polymorphisms and variable number of tandem repeats affecting their structures. These structural genes include primarily the collagen and the aggrecan genes. While genetic and environmental factors all come into play with a disease process like disc degeneration, the bulk of research now shows the significantly larger impact of hereditary over exposure. While further research is needed into some of the lesser studied genes linked to IVDD and also the racial variations in genetic makeup, the focus in the near future should be on establishment of genetic testing to identify individuals at greater risk of disease and deliberation regarding the use of gene therapy to prevent disc degeneration.

Exostosin-1 enhances canonical Wnt signaling activity during chondrogenic differentiation

Exostosin-1 (Ext1) encodes a glycosyltransferase required for heparan sulfate (HS) chain elongation in HS-proteoglycan biosynthesis. HS chains serve as binding partners for signaling proteins, affecting their distribution and activity. The Wnt/β-catenin pathway emerged as critical regulator of chondrogenesis. Yet, how EXT1 and HS affect Wnt/β-catenin signaling during chondrogenesis remains unexplored. Ext1 was stably knocked-down or overexpressed in ATDC5 chondrogenic cells cultured as micromasses. HS content was determined using ELISA. Chondrogenic markers Sox9, Col2a1, Aggrecan, and Wnt direct target gene Axin2 were measured by RT-qPCR. Proteoglycan content was evaluated by Alcian blue and DMMB assay, canonical Wnt signaling activation by β-catenin Western blot and TOP/FOP assay. ATDC5 cells and human articular chondrocytes were treated with Wnt activators CHIR99021 and recombinant WNT3A. Ext1 knock-down reduced HS, and increased chondrogenic markers and proteoglycan accumulation. Ext1 knock-down reduced active Wnt/β-catenin signaling. Conversely, Ext1 overexpressing cells, with higher HS content, showed decreased chondrogenic differentiation and enhanced Wnt/β-catenin signaling. Wnt/β-catenin signaling activation led to a down-regulation of Ext1 expression in ATDC5 cells and in human articular chondrocytes. EXT1 affects chondrogenic differentiation of precursor cells, in part via changes in the activity of Wnt/β-catenin signaling. Wnt/β-catenin signaling controls Ext1 expression, suggesting a regulatory loop between EXT1 and Wnt/β-catenin signaling during chondrogenesis.

Effects of bio-mimic collagen II-g-hyaluronic acid copolymers on chondrocyte maintenance

Extracellular matrix has an important part of the role in tissue engineering and regenerative medicine, so it is necessary to understand the various interactions between cells and extracellular matrix. Type II collagen and hyaluronic acid are the major structural components of the extracellular matrix of articular cartilage, and they are involved in fibril formation, entanglement and binding. The aim of this study was to prepare type II collagen fibrils with surface grafted with hyaluronic acid modified at the reducing end. The topographic pattern of type II collagen fibrils showed a significant change after the surface coupling of hyaluronic acid according to atomic force microscopy scanning. The presence of hyaluronic acid on the type II collagen fibrillar surface was confirmed by the specific binding of nanogold labelled with lectin. No significant increase in cell proliferation was detected by a WST-1 assay. According to histochemical examination, the maintenance of the round shape of chondrocytes and increased glycosaminoglycan secretion revealed that these cell pellets with Col II- g-hyaluronic acid molecules contained un-dedifferentiated chondrocytes in vitro. In the mixture with the 220-kDa Col II- g-hyaluronic acid copolymer, the expression of type II collagen and aggrecan genes in chondrocytes increased as demonstrated by real-time polymerase chain reaction analysis. Experimental results show that the amount of hyaluronic acid added during culturing of chondrocytes can maintain the functionality of chondrocytes and thus allow for increased cell proliferation that is suitable for tissue repair of human cartilage.

The Combination of TGF-β3 and BMP-6 Synergistically Promotes the Chondrogenic Differentiation of Equine Bone Marrow-Derived Mesenchymal Stem Cells

Stem cell therapy is a new approach in veterinary medicine for the treatment of complicated disorders such as articular cartilage injuries in horses. Although the equine bone marrow-derived mesenchymal stem cells (eBMSCs) have a unique capacity in chondrogenic differentiation, it needs to be optimized. In the current study, we evaluated the enhancement of chondrogenic differentiation in eBMSCs using transforming growth factor beta-3 (TGF-β3) and bone morphogenic protein 6 (BMP-6) in vitro. The characterized eBMSCs at passage 3 (P3) were cultivated as micro-pellets for 21 days under different conditions: (1) basic culture medium (BCM), (2) chondrogenic differentiation medium (CDM), (3) CDM with BMP-6, (4) CDM with TGF-β3, and (5) CDM with BMP-6 and TGF-β3 to evaluate the chondrogenic differentiation by assessment of pellet size, histological characteristics, and gene expression profile. Pellet size in groups that contained growth factors had a significant difference in comparison with the control group or chondrogenic group without growth factors. The histological assessment showed that the cartilage progression score in groups containing TGF-β3, alone or combined with BMP-6, was significantly higher in comparison with that of the other groups (P < 0.05). Additionally, the aggrecan gene was expressed in all groups except the control group, while collagen type II was just expressed in groups containing TGF-β3, alone or combined with BMP-6. TGF-β3 may have more promising effects on the enhancement of chondrogenic differentiation of eBMSCs in comparison with BMP-6. However, the combination of TGF-β3 and BMP-6 promoted chondrogenic differentiation of eBMSCs in a synergistic manner.

Aggrecan is required for chondrocyte differentiation in ATDC5 chondroprogenitor cells.

Aggrecan is an integral component of the extracellular matrix in cartilaginous tissues, including the growth plate. Heterozygous defects in the aggrecan gene have been identified as a cause of autosomal dominant short stature, bone age acceleration, and premature growth cessation. The mechanisms accounting for this phenotype remain unknown. We used ATDC5 cells, an established model of chondrogenesis, to evaluate the effects of aggrecan deficiency. ATDC5 aggrecan knockdown cell lines (AggKD) were generated using lentiviral shRNA transduction particles. Cells were stimulated with insulin/transferrin/selenium for up to 21 days to induce chondrogenesis. Control ATDC5 cells showed induction of Col2a1 starting at day 8 and induction of Col10a1 starting at day 12. AggKD cells had significantly reduced expression of Col2a1 and Col10a1 (p<0.0001) with only minimal increases in expression over time, indicating that chondrogenesis was markedly impaired. The induction of Col2a1 and Col10a1 was not rescued by culturing of AggKD cells in wells pre-conditioned with ATDC5 extracellular matrix or in co-culture with wild-type ATDC5 cells. We interpret our studies as indicating that aggrecan has an integral role in chondrogenesis that may be mediated through intracellular mechanisms.

Genetic Predisposition to Symptomatic Lumbar Disk Herniation in Pediatric and Young Adult Patients.

Case-control whole-genome sequencing analysis of a highly select, young cohort with symptomatic lumbar disk herniation (LDH) compared with the standard variation in a large reference population. To assess genetic influences predisposing pediatric and young adult patients to symptomatic LDH. LDH has traditionally been attributed to natural weakening or mechanical insult, but recent literature supports a potential genetic influence. Young patients with symptomatic, clinically confirmed LDH who underwent surgical treatment were included. Patients were younger than the average age of presentation, limiting the influence of environmental risks. DNA collected from these patients was compared with a reference genome (1000 Genomes Project). A genome-wide association study using whole-genome sequencing was used to characterize genetic mutations potentially associated with LDH. Among the 61 candidate genes flagged, 20 had missense mutations in 2 or more LDH cases. Missense mutations in collagen-encoding genes were observed in 12 of 15 patients (80%). A potential association with clinical presentation was indicated by odds ratios of key single-nucleotide polymorphism (SNP) variants in genes that encode collagen. Relative to the reference population, the LDH cohort demonstrated two statistically significant SNP variants in the gene encoding for aggrecan, a protein that facilitates load-bearing properties in the cartilaginous end plate. Aggrecan genes SNPs rs3817428 and rs11638262 were significantly associated with decreased odds of symptomatic LDH: odds ratio 0.05 (0.02-0.11) and 0.04 (0-0.26), respectively (P < 1 × 10 for both). These results suggest that collagen-encoding variants may be a genetic risk factor for LDH. They also shed new light on the role of variants that impact aggrecan, which sustains the cartilaginous end plate. Genetic predisposition to LDH may therefore be related to a multimodal combination of mutations that affect the nucleus pulposus, annulus fibrosus, and the cartilaginous end plates. 4.

Oral Administration of Strontium Gluconate Effectively Reduces Articular Cartilage Degeneration Through Enhanced Anabolic Activity of Chondrocytes and Chondrogenetic Differentiation of Mesenchymal Stromal Cells.

Osteoarthritis (OA), a common degenerative disease affecting articular cartilage, is caused by multiple factors, and currently, there are few approaches to effectively delay its progression. This study aimed to evaluate whether a strontium compound (in the form of strontium gluconate, Glu-Sr) could reduce OA pathology severity in osteoarthritic rat models by directly targeting chondrocytes, including catabolic/anabolic activities and/or chondrogenic differentiation. Glu-Sr was administered to OA rats by oral gavage beginning during OA induction and continuing for 8weeks. Glu-Sr treatment was found to significantly reduce cartilage degeneration and delay OA progression. Further examination showed that collagen II, Sox9, and aggrecan (ACAN) genes were up-regulated whereas IL-1β was down-regulated in chondrocytes isolated from Glu-Sr-treated rats. Glu-Sr also antagonized the catabolic effects of IL-1β on chondrocytes. Furthermore, Glu-Sr was shown to promote the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs), possibly through promoting chondrogenic gene expression, including CTGF and FGF1, as revealed by RNA-sequencing (RNA-seq). These results suggest that systemic administration of Glu-Sr may be useful in prophylactic and therapeutic treatment of chronic cartilage degradation through affecting multiple steps from chondrogenic differentiation of progenitors to matrix formation in mature chondrocytes.

MON-262 Aggrecan Is Required for Chondrocyte Differentiation in ATDC5 Chondroprogenitor Cells

Background Aggrecan is an integral part of the extracellular matrix in cartilaginous tissue, including that of the growth plate. Heterozygous defects in the aggrecan gene have been identified as a cause of autosomal dominant short stature, bone age acceleration, and premature growth cessation, all of which are consistent with a role for aggrecan in the cartilaginous growth plate. Previous animal studies of aggrecanopathies have shown disruption of growth plate architecture and acceleration of chondrocyte hypertrophy, although the mechanisms accounting for this phenotype remains unknown. We undertook studies to elucidate the mechanism by which aggrecan loss-of-function mutations lead to impaired long bone growth in concert with bone age acceleration. Methods We used ATDC5 chondroprogenitor cells, an established model of chondrogenesis, to evaluate the effects of aggrecan deficiency. ATDC5 aggrecan knock down cell lines (AggKD) were generated using shRNA lentiviral transduction particles. Cells were stimulated with insulin to induce chondrogenesis for 21 days and studied. Aggrecan qPCR and Alcian blue staining were used to assess levels of aggrecan knock down. Control and AggKD cells were cultured in media containing 10 μg/ml insulin to stimulate chondrogenesis. Cells were studied at multiple time points over 21 days using qPCR, hemocytometer counting, and flow cytometry with propidium iodide staining for cell cycle analysis. Extracellular matrix (ECM) pre-conditioning experiments and co-culture experiments of AggKD cells with wild-type (WT) ATDC5 cells were also used to evaluate effects on chondrogenesis. Results Control ATDC5 cells showed induction of col2a1 starting at day 8 and induction of col10a1 starting at day 12. AggKD cells had significantly reduced expression of col2a1 and col10a1 (p<0.0001) with only minimal increases in expression over time, indicating that chondrogenesis was markedly impaired. This reduction of col2a1 and col10a1 was not rescued by culturing AggKD cells in wells pre-conditioned with WT ATDC5 ECM, or even by co-culturing with WT ATDC5 cells. Sox9 and ihh levels were similar between control and AggKD cell lines. Hemocytometer counts showed increased cells in the control line compared to AggKD lines at Day 8 (p<0.0005), but there was no difference in cell cycle analysis using flow cytometry. Conclusion Our studies indicate that AggKD cells do not undergo normal chondrogenesis, suggesting for the first time that aggrecan has an integral role in this cell development process. Experiments that reintroduced aggrecan via extracelluar and pericellular matrices from pre-conditioning wells and co-culturing cells were unsuccessful at rescuing chondrogenesis in AggKD cells. This suggests that the role of aggrecan in chondrogenesis appears to be mediated through intracellular mechanisms.

Expression of type II collagen and aggrecan genes is regulated through distinct epigenetic modifications of their multiple enhancer elements

To maintain normal function of cartilage tissue normally, the presence of a sufficient amount of type II collagen and aggrecan is essential, and their synthesis is tightly regulated. Therefore, understanding the mechanisms that control the expression of type II collagen and aggrecan would be useful for understanding gene expression changes in diseases such as osteoarthritis. Recently, we have identified two pairs of enhancer elements, termed E1 and E2 in the type II collagen gene and Ea and Eb in the aggrecan gene. However, their different mechanisms of action remained unclear. Thus, the central aim of this study was to clarify the different transcriptional regulation mediated through each enhancer element. To this end, we established different stable reporter cell lines that express a reporter gene under the control of different enhancer elements using a silent reporter system we previously constructed. Using these cell lines, we found that dexamethasone, forskolin, and trichostatin A affect the gene expression of type II collagen and aggrecan via different enhancer elements. Moreover, we clarified that E1 and E2 enhancer activities are regulated through distinct epigenetic modifications by histone deacetylase 10 and sirtuin 6.

Differential transactivation of the upstream aggrecan enhancer regulated by PAX1/9 depends on SOX9-driven transactivation

A previously identified enhancer 10 kb upstream of the Aggrecan (Acan) gene (UE) can drive cartilage specific reporter expression in vivo. Here, we report that the paralogous transcription factors PAX1 and PAX9 differentially drive UE, depending on the presence or absence of SOX9-driven transactivation. In the developing vertebral column, PAX1/9 expression was inversely correlated with Acan expression. Moreover, PAX1/9 was co-expressed with SOX9/5/6 in the intervertebral mesenchyme and the inner annulus fibrosus (AF), and with SOX9 in the outer AF. Significant Acan upregulation was observed during chondrification of Pax1-silenced AF cells, while, Acan was significantly downregulated by persistent expression of Pax1 in cartilage. Deletion of UE using CRISPR/Cas9 resulted in ~30% and ~40% reduction of Acan expression in cartilage and the AF, respectively. In the UE, PAX1/9 acts as weak transactivators through a PAX1/9-binding site partially overlapped with a SOX9-binding site. In the presence of SOX9, which otherwise drives robust Acan expression along with SOX5/6, PAX1/9 competes with SOX9 for occupancy of the binding site, resulting in reduced transactivation of Acan. Coimmunoprecipitation revealed the physical interaction of Pax1 with SOX9. Thus, transactivation of the UE is differentially regulated by concerted action of PAX1/9, SOX9, and SOX5/6 in a context-dependent manner.

Nutrient supply and nucleus pulposus cell function: effects of the transport properties of the cartilage endplate and potential implications for intradiscal biologic therapy

Intradiscal biologic therapy is a promising strategy for managing intervertebral disc degeneration. However, these therapies require a rich nutrient supply, which may be limited by the transport properties of the cartilage endplate (CEP). This study investigated how fluctuations in CEP transport properties impact nutrient diffusion and disc cell survival and function. Human CEP tissues harvested from six fresh cadaveric lumbar spines (38-66 years old) were placed at the open sides of diffusion chambers. Bovine nucleus pulposus (NP) cells cultured inside the chambers were nourished exclusively by nutrients diffusing through the CEP tissues. After 72h in culture, depth-dependent NP cell viability and gene expression were measured, and related to CEP transport properties and biochemical composition determined using fluorescence recovery after photobleaching and Fourier transform infrared (FTIR) spectroscopy. Solute diffusivity varied nearly 4-fold amongst the CEPs studied, and chambers with the least permeable CEPs appeared to have lower aggrecan, collagen-2, and matrix metalloproteinase-2 gene expression, as well as a significantly shorter viable distance from the CEP/nutrient interface. Increasing chamber cell density shortened the viable distance; however, this effect was lost for low-diffusivity CEPs, which suggests that these CEPs may not provide enough nutrient diffusion to satisfy cell demands. Solute diffusivity in the CEP was associated with biochemical composition: low-diffusivity CEPs had greater amounts of collagen and aggrecan, more mineral, and lower cross-link maturity. CEP transport properties dramatically affect NP cell survival/function. Degeneration-related CEP matrix changes could hinder the success of biologic therapies that require increased nutrient supply.