[show abstract][hide abstract] ABSTRACT: Substance P (SP) and norepinephrine (NE) containing sensory and sympathetic nerve fibers innervate bone and fracture callus. They are involved in controlling vascularization and matrix differentiation during skeletal growth. Both types of nerve fibers are known to modulate growth and metabolic activity of osteoblasts and osteoclasts. The aim of this study was to understand the roles of SP and NE in chondrocyte metabolism and their impact on chondrocyte proliferation, apoptosis, and cell adhesion.
Primary costal chondrocytes were isolated from newborn mice. Micromass and monolayer cell culture regimens were used to analyze the effects of SP and NE on matrix formation, as determined by quantitative polymerase chain reaction and immunohistochemistry. The effects of SP and NE on proliferation, adhesion, and apoptosis of chondrocytes were determined by enzyme-linked immunosorbent assay, bromodeoxyuridine, TUNEL, and morphometric analyses.
SP, neurokinin type 1 (NK-1) receptor, α-adrenergic receptor (α-AR), and β-AR were abundantly expressed in primary costal chondrocytes. Stimulation with SP or NE did not affect extracellular matrix formation with respect to types I, II, and IX collagen and aggrecan in micromass pellets. SP dose-dependently increased the rate of proliferation of chondrocytes via the NK-1 receptor, whereas NE decreased the apoptosis rate of chondrocytes by stimulating β-AR. Both neurotransmitters induced the formation of focal adhesion contacts.
Transmitters of sympathetic and sensory nerve fibers modulate the metabolic activity of chondrocytes. Endogenous SP, NK-1 receptor, and adrenoceptor expression in chondrocytes implicates as-yet-unknown, presumably trophic, functions of neurotransmitter for skeletal growth and might be of interest for use in cartilage regenerative medicine.
[show abstract][hide abstract] ABSTRACT: To determine the presence of mesenchymal progenitor cells (MPCs) in human articular cartilage.
Primary cell cultures established from normal and osteoarthritic (OA) human knee articular cartilage were analyzed for the expression of CD105 and CD166, cell surface markers whose coexpression defines mesenchymal stem cells (MSCs) in bone marrow and perichondrium. The potential of cartilage cells to differentiate to adipogenic, osteogenic, and chondrogenic lineages was analyzed after immunomagnetic selection for CD105+/CD166+ cells and was compared with bone marrow-derived MSCs (BM-MSCs).
Up to 95% of isolated cartilage cells were CD105+ and approximately 5% were CD166+. The mean +/- SEM percentage of CD105+/CD166+ cells in normal cartilage was 3.49 +/- 1.93%. Primary cell cultures from OA cartilage contained significantly increased numbers of CD105+/CD166+ cells. Confocal microscopy confirmed the coexpression of both markers in the majority of BM-MSCs and a subpopulation of cartilage cells. Differentiation to adipocytes occurred in cartilage-derived cell cultures, as indicated by characteristic cell morphology and oil red O staining of lipid vacuoles. Osteogenesis was observed in isolated CD105+/CD166+ cells as well as in primary chondrocytes cultured in the presence of osteogenic supplements. Purified cartilage-derived CD105+/CD166+ cells did not express markers of differentiated chondrocytes. However, the cells were capable of chondrocytic differentiation and formed cartilage tissue in micromass pellet cultures.
These findings indicate that multipotential MPCs are present in adult human articular cartilage and that their frequency is increased in OA cartilage. This observation has implications for understanding the intrinsic repair capacity of articular cartilage and raises the possibility that these progenitor cells might be involved in the pathogenesis of arthritis.
[show abstract][hide abstract] ABSTRACT: To examine the catabolic pathways mediated by Toll-like receptor (TLR) ligands in human osteoarthritic (OA) chondrocytes.
The presence of TLRs in OA and non-OA articular cartilage was analyzed by immunohistochemistry. The regulation of TLR messenger RNA (mRNA) by interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFalpha) was analyzed by reverse transcription-polymerase chain reaction. For stimulation of TLR-2 and TLR-4, chondrocytes were treated with Staphylococcus aureus peptidoglycan and lipopolysaccharides (LPS), respectively. Production of matrix metalloproteinases (MMPs) 1, 3, and 13 and prostaglandin E2 (PGE2) was evaluated by enzyme-linked immunosorbent assay. Production of nitric oxide (NO) was analyzed by the Griess reaction. Regulation of cyclooxygenase 2 protein and phosphorylation of MAPKs (p38, ERK, and JNK) were evaluated by Western blotting or solid-phase kinase assay. NF-kappaB activation was evaluated by electrophoretic mobility shift assay.
Expression of TLRs 2 and 4 was up-regulated in lesional areas of OA cartilage. Treatment with IL-1, TNFalpha, peptidoglycan, and LPS all significantly up-regulated TLR-2 mRNA expression in cultured chondrocytes. Production of MMPs 1, 3, and 13 and of NO and PGE2 was significantly increased after treating chondrocytes with either of the TLR ligands. Prolonged culture of cartilage explants with TLR ligands also led to a significant increase in the release of proteoglycan and type II collagen degradation product. Treatment with TLR ligands led to phosphorylation of all 3 MAPKs and activation of NF-kappaB.
We found that TLRs are increased in OA cartilage lesions. TLR-2 and TLR-4 ligands strongly induce catabolic responses in chondrocytes. Modulation of TLR-mediated signaling as a therapeutic strategy would require detailed elucidation of the signaling pathways involved.
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