Formation and phenotype of cell clusters in osteoarthritic meniscus.
(65/1311)
OBJECTIVE: To determine the histologic changes that accompany the formation of cell clusters during the early stages of osteoarthritis development in the meniscus, and to characterize the expression phenotype of these cells. METHODS: Histologic sections of medial menisci from normal and anterior cruciate ligament (ACL)-deficient rabbit knees were immunolabeled with monoclonal antibodies for vimentin to highlight the cytoskeleton of meniscal cells, Ki-67 to identify proliferating cells, and type X collagen to evaluate changes in the cell expression phenotype. Tissue mineralization was assessed by specific staining with alizarin red. RESULTS: Following ACL transection, there was an alteration in the normal interconnected network of meniscal cells in the fibrocartilaginous region of the tissue. This led to isolation of islands of cells within the extracellular matrix of the meniscal tissue. These islands of cells displayed 3 different morphologies based on cell composition: 1) stellate cells, 2) stellate as well as round cells, and 3) round cells. Islands composed solely of round cells were more prominent in the latter stages following ACL transection, and the size of these islands increased with time, apparently as the result of cell proliferation. These islands of cells corresponded to the "clusters" previously described in osteoarthritic cartilage. Strong expression of type X collagen colocalized with the deposition of calcium within the meniscal regions enriched with cell clusters. CONCLUSION: Based on the observed changes in cell distribution, morphology, and cell proliferation as well as the previous detection of apoptosis in similar studies of rabbit knee joints, we propose a model for the development of cell clusters in the osteoarthritic meniscus. The morphologic appearance as well as the type X collagen expression phenotype of the meniscal cells forming the clusters is similar to that of hypertrophic chondrocytes. These findings provide a basis for understanding the origin of cell clusters in other joint connective tissues, such as osteoarthritic cartilage. (+info)
Non-enzymatic glycosylation of a type I collagen matrix: effects on osteoblastic development and oxidative stress.
(66/1311)
BACKGROUND: The tissue accumulation of protein-bound advanced glycation endproducts (AGE) may be involved in the etiology of diabetic chronic complications, including osteopenia. The aim of this study was to investigate the effect of an AGE-modified type I collagen substratum on the adhesion, spreading, proliferation and differentiation of rat osteosarcoma UMR106 and mouse non-transformed MC3T3E1 osteoblastic cells. We also studied the role of reactive oxygen species (ROS) and nitric oxide synthase (NOS) expression on these AGE-collagen mediated effects. RESULTS: AGE-collagen decreased the adhesion of UMR106 cells, but had no effect on the attachment of MC3T3E1 cells. In the UMR106 cell line, AGE-collagen also inhibited cellular proliferation, spreading and alkaline phosphatase (ALP) activity. In preosteoblastic MC3T3E1 cells (24-hour culture), proliferation and spreading were significantly increased by AGE-collagen. After one week of culture (differentiated MC3T3E1 osteoblasts) AGE-collagen inhibited ALP activity, but had no effect on cell number. In mineralizing MC3T3E1 cells (3-week culture) AGE-collagen induced a decrease in the number of surviving cells and of extracellular nodules of mineralization, without modifying their ALP activity. Intracellular ROS production, measured after a 48-hour culture, was decreased by AGE-collagen in MC3T3E1 cells, but was increased by AGE-collagen in UMR106 cells. After a 24-hour culture, AGE-collagen increased the expression of endothelial and inducible NOS, in both osteoblastic cell lines. CONCLUSIONS: These results suggest that the accumulation of AGE on bone extracellular matrix could regulate the proliferation and differentiation of osteoblastic cells. These effects appear to depend on the stage of osteoblastic development, and possibly involve the modulation of NOS expression and intracellular ROS pathways. (+info)
The inhibitory effects of colchicine on cell proliferation and mineralisation in culture.
(67/1311)
Colchicine is often used in the treatment of diseases such as familial Mediterranean fever (FMF) and gout. We have previously reported that patients with FMF who had colchicine on a daily basis and who had a total hip arthroplasty showed no heterotopic ossification after surgery. The mechanism by which colchicine causes this clinical phenomenon has never been elucidated. We therefore evaluated the effect of various concentrations of colchicine on cell proliferation and mineralisation in tissue culture, using rat and human cells with and without osteogenic potential. Cell proliferation was assessed by direct cell counts and uptake of (3H)thymidine, and mineralisation by measuring the amount of staining by Alizarin Red. Our findings indicate that concentrations of colchicine of up to 3 ng/ml did not affect cell proliferation but inhibition was observed at 10 to 30 ng/ml. Mineralisation decreased to almost 50%, which was the maximum inhibition observed, at concentrations of colchicine of 2.5 ng/ml. These results indicate that colchicine at low concentrations, of up to 3 ng/ml, has the capacity to inhibit selectively bone-like cell mineralisation in culture, without affecting cell proliferation. Further clinical and laboratory studies are necessary to evaluate the effects of colchicine on biological processes involving the proliferation of osteoblasts and tissue mineralisation in vivo, such as the healing of fractures, the formation of heterotopic bone and neoplastic bone growth. (+info)
Calcification in the planula and polyp of the hydroid Hydractinia symbiolongicarpus (Cnidaria, Hydrozoa).
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This study examines calcification in planulae and polyps of the hydroid Hydractinia symbiolongicarpus. We observed that established colonies produce a crystalline mat on their substratum and that crystals visible by polarized light microscopy occur in the vacuoles of the gastrodermal cells of both polyps and planulae. The crystalline mat was found by infrared spectroscopy to contain calcium carbonate in the form of aragonite. The composition of the vacuolar crystals and the cellular mechanisms for manufacturing them were explored by alteration of calcium levels in the environment and by the use of pharmacological agents (acetazolamide, caffeine, DIDS, diltiazem, nifedipine, procaine, Ruthenium Red, ryanodine and verapamil) that affect cellular uptake and transport of calcium and bicarbonate. The results indicated that the crystals in the vacuoles contained calcium carbonate. The gastrodermal cells are hypothesized to serve as a physiological sink for excess calcium that enters the organism during motility, secretion and metamorphosis of the planula, and to create a crystalline substratum for the colony of polyps. (+info)
Temporal and spatial mRNA expression of bone sialoprotein and type I collagen during rodent tooth movement.
(69/1311)
To investigate the mechanism of bone formation during tooth movement, in situ hybridization was performed with digoxigenin-labelled RNA probes to detect bone sialoprotein (BSP) and type I collagen mRNAs in the dentoalveolar tissue of 72 Sprague-Dawley rats. An elastic band was inserted between the first and second right maxillary molars, and the teeth experimentally moved for 1, 3, and 7 days. The left first maxillary molar was used as the control. For the untreated molars, osteoblasts and osteocytes near the distal surface of the interradicular septum (IRS) expressed a high level of both BSP and type I collagen mRNAs, while cells on the mesial side of the IRS showed a low level of these mRNAs. For the first molars subjected to experimental tooth movement, a high level of type I collagen mRNA expression was found in the osteoblasts on the tension side of the IRS after 1 day of experimental tooth movement. A high level of BSP mRNA was detected after 3 days of experimental tooth movement. However, a negligible amount of both mRNAs was found in cells on the compression side. These results support the hypothesis that BSP may be involved in mineralization during physiological bone remodelling. On application of orthodontic force, osteoblasts were activated and induced to express BSP mRNA, which is involved in bone remodelling due to orthodontic force. In addition, response to the orthodontic force was observed in osteocytes. (+info)
Keratin particle-induced osteolysis: a mouse model of inflammatory bone remodeling related to cholesteatoma.
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We implanted keratin and poly(methyl methacrylate) (PMMA) particles to the surface of mouse calvariae to produce a quantitative, localized, inflammatory bone remodeling similar to that seen in cholesteatoma. Both types of particles resulted in increased osteoclast density compared with controls. Osteoclasts infiltrated from marrow and vascular spaces and were active at the periphery of these spaces leading to significant bone remodeling, as demonstrated by the incorporation of bone-labelling fluorophores. Osteoclasts were rarely found on the surface of the calvariae, and mineral apposition rate at the ventral surface was not altered in keratin-implanted animals compared with nonoperated controls. While not useful for the study of the root cause of cholesteatoma, this model will allow the study ofpathologic bone remodeling related to cholesteatoma in a genetically defined animal. (+info)
Parathyroid hormone (PTH)-(1-14) and -(1-11) analogs conformationally constrained by alpha-aminoisobutyric acid mediate full agonist responses via the juxtamembrane region of the PTH-1 receptor.
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The N-terminal portion of parathyroid hormone is critical for PTH-1 receptor (P1R) activation and has been postulated to be alpha-helical when bound to the receptor. We investigated whether substitution of the sterically hindered and helix-promoting amino acid alpha-aminoisobutyric acid (Aib) in N-terminal PTH oligopeptides would improve the capacity of the peptide to activate the P1R. Analysis of the effects of individual Aib substitutions at each position in [Ala(3,12),Gln(10),Har(11),Trp(14)]PTH(1-14)NH(2) ([M]PTH(1-14)) on cAMP-stimulating potency in HKRK-B28 cells revealed that Aib at most positions diminished potency; however, Aib at positions 1 and 3 enhanced potency. Thus [Aib(1,3),M]PTH(1-14) was approximately 100-fold more potent than [M]PTH(1-14) (EC(50) = 1.1 +/- 0.1 and 100 +/- 20 nm, respectively), approximately 100,000-fold more potent than native PTH(1-14), and 2-fold more potent than PTH(1-34). The shorter peptide, [Aib(1,3),M]PTH(1-11), was also fully efficacious and 1,000-fold more potent than [M]PTH(1-11) (EC(50) 4 +/- 1 nm versus 3 +/- 1 microm). In cAMP stimulation assays performed in COS-7 cells expressing P1R-delNt, a receptor that lacks most of the N-terminal extracellular domain, [Aib(1,3),M]PTH(1-14) was 50-fold more potent than [M]PTH(1-14) (EC(50) = 0.7 +/- 0.2 versus 40 +/- 2 nm) and 1,000-fold more potent than PTH(1-34) (EC(50) = 700 nm). [Aib(1,3),M]PTH(1-14), but not PTH(1-34), inhibited the binding of (125)I-[Aib(1,3),Nle(8),Gln(10),Har(11),Ala(12),Trp(14),Arg(19),Tyr(21)]PTH(1-21) NH(2) to hP1R-delNt (IC(50) = 1,600 +/- 200 nm). The Aib(1,3) substitutions in otherwise unmodified PTH(1-34) enhanced potency and binding affinity on hP1R-delNt, but they had no effect for this peptide on hP1R-WT. Circular dichroism spectroscopy demonstrated that the Aib-1,3 substitutions increased helicity in all peptides tested, including PTH(1-34). The overall data thus suggest that the N-terminal residues of PTH are intrinsically disordered but become conformationally constrained, possibly as an alpha-helix, upon interaction with the activation domain of the PTH-1 receptor. (+info)
The anabolic activity of bone tissue, suppressed by disuse, is normalized by brief exposure to extremely low-magnitude mechanical stimuli.
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It is generally believed that mechanical signals must be large in order to be anabolic to bone tissue. Recent evidence indicates, however, that extremely low-magnitude (<10 microstrain) mechanical signals readily stimulate bone formation if induced at a high frequency. We examined the ability of extremely low-magnitude, high-frequency mechanical signals to restore anabolic bone cell activity inhibited by disuse. Adult female rats were randomly assigned to six groups: baseline control, age-matched control, mechanically stimulated for 10 min/day, disuse (hind limb suspension), disuse interrupted by 10 min/day of weight bearing, and disuse interrupted by 10 min/day of mechanical stimulation. After a 28 day protocol, bone formation rates (BFR) in the proximal tibia of mechanically stimulated rats increased compared with age-matched control (+97%). Disuse alone reduced BFR (-92%), a suppression only slightly curbed when disuse was interrupted by 10 min of weight bearing (-61%). In contrast, disuse interrupted by 10 min per day of low-level mechanical intervention normalized BFR to values seen in age-matched controls. This work indicates that this noninvasive, extremely low-level stimulus may provide an effective biomechanical intervention for the bone loss that plagues long-term space flight, bed rest, or immobilization caused by paralysis. (+info)