Hong Tian Wang

The effect of Link N on differentiation of human bone marrow-derived mesenchymal stem cells

IntroductionWe previously showed that Link N can stimulate extracellular matrix biosynthesis by intervertebral disc (IVD) cells, both in vitro and in vivo, and is therefore a potential stimulator of IVD repair. The purpose of the present study was to determine how Link N may influence human mesenchymal stem cell (MSC) differentiation, as a prelude to using Link N and MSC supplementation in unison for optimal repair of the degenerated disc.MethodsMSCs isolated from the bone marrow of three osteoarthritis patients were cultured in chondrogenic or osteogenic differentiation medium without or with Link N for 21 days. Chondrogenic differentiation was monitored by proteoglycan staining and quantitation by using Alcian blue, and osteogenic differentiation was monitored by mineral staining and quantitation by using Alzarin red S. In addition, proteoglycan secretion was monitored with the sulfated glycosaminoglycan (GAG) content of the culture medium, and changes in gene expression were analyzed with real-time reverse transcription (RT) PCR.ResultsLink N alone did not promote MSC chondrogenesis. However, after MSCs were supplemented with Link N in chondrogenic differentiation medium, the quantity of GAG secreted into the culture medium, as well as aggrecan, COL2A1, and SOX9 gene expression, increased significantly. The gene expression of COL10A1 and osteocalcin (OC) were downregulated significantly. When MSCs were cultured in osteogenic differentiation medium, Link N supplementation led to a significant decrease in mineral deposition, and alkaline phosphatase (ALP), OC, and RUNX2 gene expression.ConclusionsLink N can enhance chondrogenic differentiation and downregulate hypertrophic and osteogenic differentiation of human MSCs. Therefore, in principle, Link N could be used to optimize MSC-mediated repair of the degenerated disc.

Effect of a Type II Collagen Fragment on the Expression of Genes of the Extracellular Matrix in Cells of the Intervertebral Disc.

Knowledge of factors regulating the turnover, repair, and degeneration of the intervertebral disc (IVD) is lacking. Although type II collagen (CII) fragments accumulate in the degenerative IVD, little is known of how they affect the degenerative process. A better understanding of the cellular interactions with fragments of matrix molecules are a key factor in promoting therapies for degenerative disc diseases. In the present study, we have investigated the effect of the CII (245-270) peptide on the expression of matrix molecules, proteinases, and interleukin genes in cells of the IVD. Cells isolated from the nucleus pulposus (NP) and annulus fibrosus (AF) of adult bovine tails were cultured up to 8 days in the absence (control) or presence of the CII (245-270) peptide. RT-PCR was used to analyze the expression of the different genes. Exposure of these cells to the CII (245-270) peptide led to a transient up-regulation of the aggrecan gene in AF cells while this up-regulation was maintained for a longer time in NP cells. The fragment also enhanced a transient up-regulation of the type II collagen gene in AF cells but had no effect in NP cells. The peptide enhanced transiently the expression of matrix metalloproteinase (MMP)-1 and cathepsin K genes in both AF and NP cells whereas it increased MMP-13 expression only in NP cells. The peptide up-regulated tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, and TIMP-3 gene expression on day 1 in AF cells but had very little effect on their expression in NP cells. Finally, the CII (245-270) peptide had no effect on IL-6 expression while IL-1α was not expressed in these cells. In conclusion, our results showed that the CII (245-270) peptide differentially alter the expression of genes in bovine AF and NP cells and suggest that degradation products of collagen may be involved in the regulation of IVD homeostasis.

Novel insights into the mechanism of decreased expression of type X collagen in human mesenchymal stem cells from patients with osteoarthritis cultured on nitrogen-rich plasma polymers: implication of cyclooxygenase-1.

Recent evidence indicates that a major drawback of current cartilage- and intervertebral disc (IVD) tissue engineering is that human mesenchymal stem cells (MSCs) from patients with osteoarthritis rapidly express type X collagen (COL10A1), a marker of late stage chondrocyte hypertrophy associated with endochondral ossification. We recently demonstrated that COL10A1 expression was inhibited in MSCs from patients with osteoarthritis cultured on nitrogen-rich plasma polymerized (PPE:N) coatings. Here, we sought to understand the mechanisms of action of this effect by culturing MSCs on PPE:N surfaces in the presence of different inhibitors of kinases and cyclooxygenases. The effect of PPE:N surfaces on COL10A1 expression was found to be mimicked by the cyclooxygenase inhibitor NPPB, but not by daphnetin (an inhibitor of protein kinases) nor by genistein (an inhibitor of tyrosine kinases). COL10A1 expression was also suppressed by the specific cyclooxygenase-1 (COX-1: SC-560) and 5-lipoxygenase (5-LOX: MK-866) inhibitors, but not by COX-2 (COX-2 inhibitor 2) and 12-LOX (baicalein) inhibitors. Finally, the incubation of MSCs on PPE:N surfaces inhibited the expression of COX-1 while 5-LOX was not expressed in these cells. Taken together, these results indicate that PPE:N surfaces inhibit COL10A1 expression via the suppression of COX-1.

The Potential of N-Rich Plasma-Polymerized Ethylene (PPE:N) Films for Regulating the Phenotype of the Nucleus Pulposus.

We recently developed a nitrogen-rich plasma-polymerized biomaterial, designated “PPE:N” (N-doped plasma-polymerized ethylene) that is capable of suppressing cellular hypertrophy while promoting type I collagen and aggrecan expression in mesenchymal stem cells from osteoarthritis patients. We then hypothesized that these surfaces would form an ideal substrate on which the nucleus pulposus (NP) phenotype would be maintained. Recent evidence using microarrays showed that in young rats, the relative mRNA levels of glypican-3 (GPC3) and pleiotrophin binding factor (PTN) were significantly higher in nucleus pulposus (NP) compared to annulus fibrosus (AF) and articular cartilage. Furthermore, vimentin (VIM) mRNA levels were higher in NP versus articular cartilage. In contrast, the levels of expression of cartilage oligomeric matrix protein (COMP) and matrix gla protein precursor (MGP) were lower in NP compared to articular cartilage. The objective of this study was to compare the expression profiles of these genes in NP cells from fetal bovine lumbar discs when cultured on either commercial polystyrene (PS) tissue culture dishes or on PPE:N with time. We found that the expression of these genes varies with the concentration of N ([N]). More specifically, the expression of several genes of NP was sensitive to [N], with a decrease of GPC3, VIM, PTN, and MGP in function of decreasing [N]. The expression of aggrecan, collagen type I, and collagen type II was also studied: no significant differences were observed in the cells on different surfaces with different culture time. The results support the concept that PPE:N may be a suitable scaffold for the culture of NP cells. Further studies are however necessary to better understand their effects on cellular phenotypes.

The Effects of Naproxen on Chondrogenesis of Human Mesenchymal Stem Cells.

Currently, there are no established treatments to prevent, stop, or even retard the degeneration of articular cartilage in osteoarthritis (OA). Biological repair of the degenerating articular cartilage would be preferable to surgery. There is no benign site where autologous chondrocytes can be harvested and used as a cell source for cartilage repair, leaving mesenchymal stem cells (MSCs) as an attractive option. However, MSCs from OA patients have been shown to constitutively express collagen type X (COL-X), a marker of late-stage chondrocyte hypertrophy. We recently found that naproxen (Npx), but not other nonsteroidal anti-inflammatory drugs, can induce collagen type X alpha 1 (COL10A1) gene expression in bone marrow-derived MSCs from healthy and OA donors. In this study, we determined the effect of Npx on COL10A1 expression and investigated the intracellular signaling pathways that mediate such effect in normal human MSCs during chondrogenesis. MSCs were cultured in standard chondrogenic differentiation media supplemented with or without Npx. Our results show that Npx can regulate chondrogenic differentiation by affecting the gene expression of both Indian hedgehog and parathyroid hormone/parathyroid hormone-related protein signaling pathways in a time-dependent manner, suggesting a complex interaction of different signaling pathways during the process.