mRNA Expression Levels Of Aggrecan Research Articles

Establishment of a rat caudal disc degeneration model by percutaneous fine-needle aspiration of the fibrous ring

Objective To compare the effect of different size needle gauges to the degenerative response in rat caudal discs. Methods A total of 40 Sprague Dawley (SD) rats, level 5/6, 7/8 and 9/10 interverbral discs of rat caudal spine were punctured with 18 or 21 or 25-gauge needles respectively. Radiographs and magnetic resonance imaging (MRI) were obtained at 1, 2, 4 and 6 weeks postsurgery. At each time point, ten rats from each group were sacrificed for histological analysis. Real-time fluorescent quantitative polymerase chain reaction (qPCR) was used to examine mRNA expression level. Results Significant differences were identified in the disc height index (DHI %) and MRI grade between 18 G and normal group, MRI grade, histological score between 21 G and normal group at 2, 4, and 6 weeks postsurgery. Significant differences were also identified in the histological score and mRNA expression levels between 18 G and normal group, alcian blue stain and hypoxia inducible factor-1α (HIF-1α) mRNA expression level between 21G and normal group at all time point postsurgery. Significant differences existed in DHI%, type Ⅱ collagen and aggrecan mRNA expression levels between 21 G and normal group, all type mRNA expression levels between 25 G and normal group at 4, 6 weeks. There were significant differences in MRI grade and histological score between 25 G and normal group at 6 weeks. Significant differences existed in almost all parameters compared between 18 G and 25 G at all time point. There were significant differences in DHI%, MRI grade, histological score and HIF-1α mRNA expression levels between 18 G and 21 G at 4, 6 weeks. There were significant differences in type Ⅱ collagen and aggrecan mRNA expression levels between 18 G and 21 G at all time point. Significant differences exist in DHI% and HIF-1α mRNA expression level between 21 G and 25 G at 6 weeks. Compared with the 25 G group, the DHI% and Pfirrmann scores and the pathological score of each time at 2, 4 and 6 weeks after operation in 18 G group have significant difference (P<0.05). Conclusions The middle size needle (21G) is better to induce disc degeneration. The 2-week time point may be the better time frame to further experimental treatments. Key words: Spinal puncture/MT/AE; Intervertebral disk degeneration/ET; Disease models, animal; Rats

Accumulated Spinal Axial Biomechanical Loading Induces Degeneration in Intervertebral Disc of Mice Lumbar Spine.

To investigate the effect of accumulated spinal axial biomechanical loading on mice lumbar disc and the feasibility of applying this method to establish a mice intervertebral disc degeneration model using a custom-made hot plate cage. In previous studies, we observed that the motion pattern of mice was greatly similar to that of humans when they were standing and jumping on their lower limbs. There is little data to demonstrate whether or not accumulated spinal axial biomechanical loading could induce intervertebral disc degeneration in vivo. Twenty-four 0-week-old mice were randomly divided into model 1-month and 3-month groups, and control 1-month and 3-month groups (n = 6 per group). The model groups was transferred into the custom-made hot plate cage three times per day for modeling. The control group was kept in a regular cage. The intervertebral disc samples of the L3 -L5 were harvested for histologic, molecular, and immunohistochemical studies after modeling for 1 and 3 months. Accumulated spinal axial biomechanical loading affects the histologic, molecular, and immunohistochemical changes of mice L3- L5 intervertebral discs. Decreased height of disc and endplate, fissures of annulus fibrosus, and ossification of cartilage endplate were found in morphological studies. Immunohistochemical studies of the protein level showed a similar expression of type II collagen at 1 month, but a slightly decreased expression at 3 months, and an increased expression level of type X collagen and matrix metalloproteinase 13 (MMP13). Molecular studies showed that ColIIa1 and aggrecan mRNA expression levels were slightly increased at 1 month (P > 0.05), but then decreased slightly (P > 0.05). ColXa1, ADAMTS-5, and MMP-13 expression levels werer increased both at 1 and 3 months (P < 0.05). In addition, increased expression of Runx2 was observed. Accumulated spinal axial loading provided by a custom-made hot plate accelerated mice lumbar disc and especially endplate degeneration. However, this method requires further development to establish a lumbar disc degeneration model.

Effect of osteopontin on the mRNA expression of ADAMTS4 and ADAMTS5 in chondrocytes from patients with knee osteoarthritis.

Previous studies have demonstrated that osteopontin (OPN) levels are elevated in the synovial fluid and articular cartilage, and are associated with the severity of knee osteoarthritis (OA). However, the role of OPN in the pathogenesis of OA has yet to be elucidated. The present study aimed to investigate the effects of OPN on the expression of the aggrecanases, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4 and ADAMTS5, in human OA chondrocytes, as they serve a key function in aggrecan degradation. Human OA chondrocytes were obtained from the knees of 16 patients with OA, and subsequently cultured in a monolayer. The chondrocytes were divided into three groups, which included the control (no treatment), N-OPN (treated with 100 ng/ml OPN, the normal circulating OPN concentration) and the H-OPN groups (treated with 1 µg/ml OPN, a high OPN concentration). Reverse transcription-quantitative polymerase chain reaction was performed to quantify the relative mRNA expression levels of ADAMTS4, ADAMTS5 and aggrecan in the chondrocytes. The mRNA expression levels of ADAMTS4 were significantly reduced in the N-OPN and H-OPN groups when compared with the control group (P<0.0001). In addition, the mRNA expression levels of ADAMTS4 were lower in the H-OPN group when compared with the N-OPN group (P<0.001). However, no statistically significant difference was observed in the relative mRNA expression levels of ADAMTS5 among the three groups (P>0.05). Furthermore, the mRNA expression levels of aggrecan were higher in the N-OPN and H-OPN groups when compared with the control group (P<0.0001), and a statistically significant difference was observed between the N-OPN and H-OPN groups with regard to the mRNA expression of aggrecan (P<0.0001). These results demonstrated that OPN may exert a protective effect in human OA chondrocytes against aggrecan degradation by suppressing the expression of ADAMTS4.

Pulsed Electromagnetic Field (PEMF) Plus BMP-2 Upregulates Intervertebral Disc-Cell Matrix Synthesis More Than Either BMP-2 Alone or PEMF Alone

An in vitro study using human intervertebral disc (IVD) cells. To determine if pulsed electromagnetic field (PEMF) plus bone morphogenetic protein (BMP)-2 could upregulate IVD-cell matrix synthesis more than either BMP-2 alone or PEMF alone. BMP-7 and BMP-2 can both upregulate IVD-cell matrix synthesis. There are problems associated with using either BMP-2 or BMP-7. They can diffuse away rather quickly after injection into the IVD space, they cost a lot, and they have side effects such as soft-tissue inflammation and swelling. PEMF has been reported to stimulate various types of cells. PEMF is safe, inexpensive, and noninvasive, thus multiple use is possible. However, PEMF alone has a rather weak effect on disc cells. We decided to carry out an experiment whereby we combined PEMF with BMP-2. Our thoughts were that BMP-2 plus PEMF could be better than either alone. The PEMF signal used was similar to that used in the clinical treatment of fracture nonunions or delayed fracture healing. Human disc cells were treated with BMP-2 alone or PEMF alone or PEMF plus BMP-2. Quantitative real-time PCR was performed to determine mRNA expression levels of aggrecan, collagen-2, transforming growth factor (TGF)-β, BMP-2, and BMP-7. Sulfated glycosaminoglycansynthesis was analyzed using the dimethylmethylene blue method. Western blot analysis was performed to determine the protein levels of TGF-β, BMP-2, and BMP-7. PEMF plus BMP-2 upregulates IVD-cell matrix synthesis more than BMP-2 alone or PEMF alone, and the effect seems to be synergistic. Also, PEMF plus BMP-2 induces more endogenous BMP-7 and BMP-2 mRNA levels as well as protein levels, as compared with either PEMF alone or BMP-2 alone. PEMF plus BMP-2 acts in synergy to upregulate intervertebral disc-cell matrix synthesis more than either BMP-2 alone or PEMF alone.

Upregulation of Intervertebral Disc-Cell Matrix Synthesis by Pulsed Electromagnetic Field Is Mediated by Bone Morphogenetic Proteins

An in vitro study on the effects of pulsed electromagnetic field (PEMF) on intervertebral disc-cell matrix synthesis. The objective of the study was to determine whether (1) PEMF can upregulate intervertebral disc-cell matrix synthesis and (2) any upregulation obtained is through transforming growth factor (TGF)-β or bone morphogenetic proteins (BMPs). PEMF has been reported to produce cell proliferation, enhance cell function, and upregulate matrix synthesis in cell types such as osteoblasts, chondroblasts, endothelial cells, and fibroblasts through the upregulation of several growth factors. PEMF has been used clinically in the treatment of delayed bone union. However, PEMF has never been tested on human intervertebral disc cells. The PEMF signal used was similar to that used in the clinical treatment of delayed fracture healing. Human disc cells were treated with PEMF for 8 hours per day for 3 days. Quantitative real-time polymerase chain reaction was performed to determine mRNA expression levels of aggrecan, collagen-2, TGF-β, BMP-2, and BMP-7. Sulfated glycosaminoglycan synthesis was analyzed using the dimethylmethylene blue (DMMB) method. Western blot analysis was performed to determine the protein levels of TGF-β, BMP-2, and BMP-7. To determine whether any action of PEMF was through BMP, recombinant human Noggin was used at a dose of 100 ng/mL to block BMP. PEMF could upregulate intervertebral disc-cell matrix synthesis. BMP-7 was markedly upregulated by PEMF and was upregulated much more than BMP-2. TGF-β was not upregulated by PEMF. The effect of PEMF on disc-cell matrix was entirely inhibited in the presence of Noggin. PEMF acts through BMPs to upregulate intervertebral disc-cell matrix synthesis.

An evaluation of chondrocyte morphology and gene expression on superhydrophilic vertically-aligned multi-walled carbon nanotube films

Cartilage serves as a low-friction and wear-resistant articulating surface in diarthrodial joints and is also important during early stages of bone remodeling. Recently, regenerative cartilage research has focused on combinations of cells paired with scaffolds. Superhydrophilic vertically aligned carbon nanotubes (VACNTs) are of particular interest in regenerative medicine. The aim of this study is to evaluate cell expansion of human articular chondrocytes on superhydrophilic VACNTs, as well as their morphology and gene expression. VACNT films were produced using a microwave plasma chamber on Ti substrates and submitted to an O2 plasma treatment to make them superhydrophilic. Human chondrocytes were cultivated on superhydrophilic VACNTs up to five days. Quantitative RT-PCR was performed to measure type I and type II Collagen, Sox9, and Aggrecan mRNA expression levels. The morphology was analyzed by scanning electron microscopy (SEM) and confocal microscopy. SEM images demonstrated that superhydrophilic VACNTs permit cell growth and adhesion of human chondrocytes. The chondrocytes had an elongated morphology with some prolongations. Chondrocytes cultivated on superhydrophilic VACNTs maintain the level expression of Aggrecan, Sox9, and Collagen II determined by qPCR. This study was the first to indicate that superhydrophilic VACNTs may be used as an efficient scaffold for cartilage or bone repair.

Konjac glucomannan-based hydrogel with hyaluronic acid as a candidate for a novel scaffold for chondrocyte culture

Chondrocytes were cultured using konjac glucomannan (KGM) and hyaluronic acid (HA) as a scaffold for cartilage regeneration. They were subsequently compared with scaffolds produced using agarose hydrogels. Chondrocytes derived from Japanese white rabbits were cultured: 2.0 x 10(5) cells were seeded on KGM containing hyaluronic acid (KGM/HA) and agarose and cultured for 5 days. Their viability was assayed using WST-8 procedures; the ultimate stress and modulus of elasticity of each construct was calculated. After 3 days of cultivation, mRNA in chondrocytes, such as collagen types I and II and aggrecan, were measured using RT-PCR. Both chondrocyte-seeded constructs were stained with safranin O/fast green and were evaluated histologically. Chondrocyte viability decreased concomitantly with increasing KGM/HA or agarose concentration and with culture time. Cell viability in 2% agarose was significantly lower than that in 2% KGM/HA on the third and fifth days (p < 0.05). The primary elastic modulus increased concomitantly with increasing polysaccharide concentration. Elastic moduli of 2% KGM/HA with chondrocytes (0.389 +/- 0.119 N/mm(2)) showed little difference from those without chondrocytes (0.283 +/- 0.243 N/mm(2)), although those of 2% agarose with chondrocytes (0.403 +/- 0.094 N/mm(2)) were significantly lower than those without chondrocytes (0.736 +/- 0.227 N/mm(2); p < 0.05). Collagen type II mRNA expression was higher in KGM/HA and agarose than in monolayer cultures, although KGM/HA had lower aggrecan mRNA expression levels than did agarose. Histological tests of KGM/HA-chondrocyte constructs revealed chondrocyte aggregation and proteoglycan production in the pericellular region. The results show that KGM/HA might be useful for chondrocyte culture.

The Effect of Electrical Fields on Gene and Protein Expression in Human Osteoarthritic Cartilage Explants

The destruction of cartilage in patients with osteoarthritis is a consequence of an imbalance between matrix synthesis and degradation. The purpose of the present study was to determine the effects of electrical stimulation on these processes in full-thickness osteoarthritic adult human articular cartilage explants. Full-thickness articular cartilage explants from osteoarthritic adult human knee joints were cultured in the absence or presence of interleukin-1beta (IL-1beta) and in the absence or presence of a specifically defined capacitively coupled electrical signal for seven or fourteen days. Total collagen and proteoglycan production were assessed by means of hydroxyproline and hexosamine analyses, respectively. Quantitative real-time polymerase chain reaction assays were used to measure mRNA expression levels of aggrecan, type-II collagen, collagenase-1 (MMP-1), collagenase-3 (MMP-13), stromelysin-1 (MMP-3), aggrecanase-1 (ADAM-TS4), and aggrecanase-2 (ADAM-TS5). Electrical stimulation of cultured explants for seven or fourteen days resulted in significant increases (p < 0.007) in proteoglycan and collagen production and a highly significant upregulation (p <or= 0.005) of aggrecan and type-II collagen mRNA expression. This occurred even in the presence of IL-1beta. In the absence of IL-1beta, the expression of metalloproteinases was at barely detectable levels in these explants. Treatment with IL-1beta led to the significant upregulation of metalloproteinase expression (p < 0.03), but simultaneous administration of the capacitively coupled electrical signal dramatically inhibited this stimulation. The data show that, even in the presence of IL-1beta, a specific, defined capacitively coupled electrical signal can result in significant upregulation of cartilage matrix protein expression and production while simultaneously significantly attenuating the upregulation of metalloproteinase expression. These results support the contention that delivery of a specific, defined electrical field to articular cartilage could result in matrix preservation.

Effects of glucosamine sulfate on intracellular UDP-hexosamine and UDP-glucuronic acid levels in bovine primary chondrocytes

To analyze the effects of exogenously added glucose (Glc), glucosamine (GlcN) and glucosamine sulfate (GS) on the intracellular UDP-hexoses (UDP-Hex), UDP-N-acetylhexosamines (UDP-HexN) and UDP-glucuronic acid (UDP-GlcA) levels in bovine primary chondrocytes. Chondrocytes were incubated with different concentrations of Glc, GlcN and GS either in high- or low-glucose DMEM for up to 120min to analyze the intracellular levels of UDP-Hex, UDP-GlcA and UDP-HexN by a reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry analysis. Glycosaminoglycan (GAG) synthesis rate and aggrecan mRNA expression levels were quantified using (35)S-sulfate incorporation assay and quantitative real-time RT-PCR, respectively. The cells were cultivated for 2 days or 8 days before UDP-sugar analysis. Levels of UDP-HexN and UDP-GlcA were unchanged at 10microM concentration of GS in low-glucose DMEM, while addition of 1mM GlcN or GS in low-glucose DMEM for 10min increased UDP-HexN level. The highest intracellular level of UDP-HexN was reached at 30min after addition of 1mM GS to the cells. The intracellular contents of UDP-HexN and UDP-GlcA related to UDP-Hex were higher after prolonged cultivation of chondrocytes for 8 days compared with 2-day-old cultures. Aggrecan mRNA expression and GAG synthesis remained at control level after the cells were treated with 10, 100microM or 1mM of GS for 24h. Physiologically relevant level of GS could not increase the intracellular UDP-HexN and UDP-GlcA levels in bovine primary chondrocyte, while longer-time culture itself appeared to increase the intracellular UDP-HexN and UDP-GlcA levels.