Mechanical loading intensities affect the release of extracellular vesicles from mouse bone marrow-derived hematopoietic progenitor cells and change their osteoclast-modulating effect.

Low-intensity loading maintains or increases bone mass, whereas lack of mechanical loading and high-intensity loading decreases bone mass, possibly via the release of extracellular vesicles by mechanosensitive bone cells. How different loading intensities alter the biological effect of these vesicles is not fully understood. Dynamic fluid shear stress at low intensity (0.7 ± 0.3 Pa, 5 Hz) or high intensity (2.9 ± 0.2 Pa, 1 Hz) was used on mouse hematopoietic progenitor cells for 2 min in the presence or absence of chemical compounds that inhibit release or biogenesis of extracellular vesicles. We used a Receptor activator of nuclear factor kappa-Β ligand-induced osteoclastogenesis assay to evaluate the biological effect of different fractions of extracellular vesicles obtained through centrifugation of medium from hematopoietic stem cells. Osteoclast formation was reduced by microvesicles (10 000× g) obtained after low-intensity loading and induced by exosomes (100 000× g) obtained after high-intensity loading. These osteoclast-modulating effects could be diminished or eliminated by depletion of extracellular vesicles from the conditioned medium, inhibition of general extracellular vesicle release, inhibition of microvesicle biogenesis (low intensity), inhibition of ESCRT-independent exosome biogenesis (high intensity), as well as by inhibition of dynamin-dependent vesicle uptake in osteoclast progenitor cells. Taken together, the intensity of mechanical loading affects the release of extracellular vesicles and change their osteoclast-modulating effect.

Chronic colitis induces expression of ß-defensins in murine intestinal epithelial cells.

Anti-microbial peptides are important effectors in innate immunity. In the gut they defend against pathogens, shape the commensal microbiota and probably control intestinal homeostasis. Ulcerative colitis (UC), but not Crohn's disease, shows increased expression of inducible ß-defensins (hBD-2, hBD-3 and hBD-4) in colonic epithelial cells. Does inducible defensin production precede the chronic intestinal inflammation characteristic of UC, or is it a consequence of the T cell-driven chronic inflammation? The aim was to analyse defensin mRNA and protein expression in colonic epithelial cells in two colitis mouse models resembling UC, the interleukin (IL)-2(-/-) mouse and the dextran sulphate sodium (DSS)-induced colitis mouse. Defensin mRNA was assayed by in situ hybridization and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). Defensin peptide was assayed by immunohistochemistry. Mouse ß-defensin 3 (mBD-3, orthologue to hBD-2) was up-regulated strongly in colonic epithelium of 15-week-old IL-2(-/-) mice and DSS-induced colitis mice with chronic bowel inflammation, but not in apparently healthy IL-2(-/-) 5-week-old mice, IL-2(+/-) 15-week-old mice or in acute stage DSS mice. Up-regulation was seen both at the mRNA- and at the protein level (only mBD-3 investigated). IL-17, but not several other cytokines, including interferon (IFN)-γ, induced mBD-3 mRNA expression in mouse colon carcinoma cells. The mRNA expression level of the constitutively expressed α-defensin, cryptdin-4, was up-regulated marginally in acute stage DSS-colitis mice and in IL-2(-/-) mice before signs of colitis. Inducible ß-defensin expression in colonic epithelium is the consequence of the chronic bowel inflammation caused by activated T cells releasing cytokines including IL-17.

Radiographic joint space narrowing and histologic changes in a rabbit meniscectomy model of early knee osteoarthrosis.

The purpose of this study was to compare weightbearing radiographs with histologic cartilage evaluation in a rabbit meniscectomy model of the early stage of osteoarthrosis. Fifteen rabbits had a medial meniscectomy performed in one knee and a sham operation in the other knee. Five rabbits each were sacrificed at 13, 25, and 40 weeks after surgery. Radiographic joint space width and histologic cartilage changes of the medial knee compartment were quantified. Five non-operated knees and five knees in which the meniscus had been removed immediately before the evaluations served as control specimens. Overall, the joint space of the peripheral part of the medial knee compartment was narrower in knees operated on for meniscus removal than in sham-operated knees (P < 0.003). In the knees with the meniscus removed, more cartilage changes were seen at the joint surface area of contact on radiographs than in the sham-operated knees (P < 0.0015). Indeed, the area of contact had cartilage changes similar to those in the whole medial compartment. However, there was no correlation between the degree of histologic cartilage change and the corresponding joint space measurements. Joint space width as measured on weightbearing radiographs is reduced after meniscectomy in the rabbit, but it does not reflect the degree of cartilage damage of the loaded joint surfaces in early stages of osteoarthrosis.

A capsular incision leads to a fast osteoarthritic response, but also elevated levels of activated osteogenic protein-1 in rabbit knee joint cartilage.

We studied whether a small capsular incision alone, or combined with meniscectomy could induce early osteoarthritic changes in the rabbit knee. Thirty-one rabbits were operated on with a capsular incision in the left knee and meniscectomy in the right knee. Another 12 rabbits were used as controls. The rabbits were killed 3, 6 and 12 weeks after surgery. Osteoarthritic changes in the articular cartilage were evaluated by the modified Mankin score. The subchondral bone was evaluated by scintimetry ((99m)Tc-HDP) and semiquantitative grading of histological changes. Osteogenic protein (OP-1) in its mature and pro-form was examined by immunohistochemistry. Both a capsular incision and meniscectomy induced articular cartilage fibrillation and increased bone metabolic activity during the initial weeks after surgery. Capsular incision led to lesser changes than meniscectomy. Mature OP-1 was elevated, and its pro-form reduced, in meniscectomized knees. A similar pattern was observed in knees with capsular incision. Already 3 weeks after surgery, the articular cartilage and subchondral bone showed typical signs of early osteoarthritis (OA), and a reparative response was suggested by increased intensity of OP-1 staining. As these signs were also found in knees with capsular incision only, it appears that trauma-related factors such as increased bleeding and inflammation are critical for the development of OA.

CDMP-2 injection improves early tendon healing in a rabbit model for surgical repair.

This study examines the hypothesis that cartilage-derived morphogenic protein-2 (CDMP-2) can improve tendon healing after surgical repair. We have previously found improved tendon healing by applying CDMP-2 in models for conservative treatment with mechanically loaded Achilles tendon defects in rats and rabbits. In this study, the patellar tendon was unloaded by patello- tibial cerclage and cut transversely in 40 rabbits. Two hours post-operative, the rabbits received a dose of 20 microg of CDMP-2 or vehicle injected into the hematoma. Specimens were harvested after 14 and 28 days and evaluated by biomechanical testing, radiography and histology. At 14 days, CDMP-2 caused a 65% increase in force at failure, a 50% increase in ultimate stress and a 57% increase in stiffness, as compared with controls. There was no effect on callus size. At 28 days, no differences between the treatment groups could be demonstrated. No bone or cartilage was found in any tendon or regenerated tissue at any time point. Thus, early tendon repair can be stimulated by CDMP-2 in an unloaded model. These results suggest that CDMP-2 might be of interest for clinical use as a complement to surgical treatment of tendon ruptures.

TGF-beta1 as a prognostic factor in the process of early osteoarthrosis in the rabbit knee.

OBJECTIVE: To assess changes in knee joint fluid concentrations of transforming growth factor-beta1 (TGF-beta1) and proteoglycan (PG) fragments during the early course of post-traumatic osteoarthrosis (OA) after meniscectomy in the rabbit knee, and to ascertain whether the concentrations of these substances shortly after operation could be used as prognostic markers for the OA process. DESIGN: In 15 rabbits with medial meniscectomy in one knee and a sham operation in the other knee, synovial lavage fluid samples were taken repeatedly, before operation, every third week post-operatively until 12 weeks, thereafter every sixth week, and at death. Five rabbits each were killed at 13, 25 and 40 weeks. Synovial lavage fluid samples from five non-operated rabbits served as controls. At death, two histological scores were formed that characterized the highest (MAX) and the overall (ALL) degree of OA changes in each joint. RESULTS: TGF-beta1 and PG fragment concentrations in synovial lavage fluid correlated highly (R=0.81, P< 0.001). Both OA scores were higher in meniscectomized than controls (P< 0.05). The synovial lavage fluid concentration of TGF-beta1 at 3 weeks, but no other time point, correlated to the histological scores (ALL, R=0.58; MAX, R=0.52;P< 0.001). CONCLUSION: Higher concentrations of TGF-beta1 in synovial lavage fluid early after surgery seemed indicative for the later development of more severe OA changes in contrast to lower concentrations. The association between TGF-beta1 and the changes found later in the cartilage was underlined by the high correlations between this substance and PG fragment concentrations in synovial lavage fluid at all time points.

Increased expression of antimicrobial peptides and lysozyme in colonic epithelial cells of patients with ulcerative colitis.

The impact of chronic inflammation on the expression of human alpha-defensins 5 and 6 (HD-5, HD-6), beta-defensins 1 and 2 (hBD-1, hBD-2) and lysozyme in epithelial cells of small and large intestine was investigated. Intestinal specimens from 16 patients with ulcerative colitis (UC), 14 patients with Crohn's disease (CD) and 40 controls with no history of inflammatory bowel disease were studied. mRNA expression levels of the five defence molecules were determined in freshly isolated epithelial cells by real-time quantitative RT-PCR. Specific copy standards were used allowing comparison between the expression levels of the different defensins. HD-5 and lysozyme protein expression was also studied by immunohistochemistry. Colonic epithelial cells from patients with UC displayed a significant increase of hBD-2, HD-5, HD-6 and lysozyme mRNA as compared to epithelial cells in controls. Lysozyme mRNA was expressed at very high average copy numbers followed by HD-5, HD-6, hBD-1 and hBD-2 mRNA. HD-5 and lysozyme protein was demonstrated in metaplastic Paneth-like cells in UC colon. There was no correlation between hBD-2 mRNA levels and HD-5 or HD-6 mRNA levels in colon epithelial cells of UC patients. Colonic epithelial cells of Crohn's colitis patients showed increased mRNA levels of HD-5 and lysozyme mRNA whereas ileal epithelial cells of Crohn's patients with ileo-caecal inflammation did not. Chronic inflammation in colon results in induction of hBD-2 and alpha-defensins and increased lysozyme expression. hBD-1 expression levels in colon remain unchanged in colitis. The high antimicrobial activity of epithelial cells in chronic colitis may be a consequence of changes in the epithelial lining, permitting adherence of both pathogenic bacteria and commensals directly to the epithelial cell surface.

Simultaneous changes in bone mineral density and articular cartilage in a rabbit meniscectomy model of knee osteoarthrosis.

OBJECTIVE: It was hypothesized that increased bone mineral density of the medial proximal tibia would precede or coincide with the development of more severe cartilage changes after meniscectomy. METHODS: In a rabbit knee model, mineral density of subchondral bone and changes of articular cartilage were monitored 13 to 40 weeks after medial meniscectomy or a sham operation. RESULTS: Both procedures resulted in a decrease of bone mineral density, especially of the medial proximal tibia, which persisted up to 40 weeks (P< 0.02-0.0007). Meniscectomy induced cartilage changes typical for osteoarthrosis (P< 0.009), which progressed over time on the posterior aspect of the medial tibial plateau (P< 0.009), which is physiologically covered by the meniscus, but the procedure also induced iatrogenic changes which were located mainly on the anterior aspect of the concerned compartment, and which did not progress or develop to osteoarthrosis. CONCLUSIONS: The data suggest that the cartilage changes after meniscectomy in this animal model are caused by the surgical trauma, subsequent limb misuse, and altered load distribution, and initially associated by a decrease not an increase in bone mineral density of the proximal tibia. Moreover, the cartilage changes progressed without a simultaneous increase of the bone mineral density at corresponding sites.

beta-Defensin-3 and -4 in intestinal epithelial cells display increased mRNA expression in ulcerative colitis.

mRNA expression of two recently described human beta-defensins (hBD-3 and hBD-4) in epithelial cells of normal small and large intestine and the impact of chronic intestinal inflammation on their expression levels was investigated. Intestinal specimens from patients with ulcerative colitis (UC), Crohn's disease (CD) and controls with no history of inflammatory bowel disease were studied. hBD-3 and hBD-4 mRNAs were determined in freshly isolated epithelial cells by real-time quantitative reverse transcription-polymerase chain reaction (QRT-PCR) and by in situ hybridization. The effect of proinflammatory cytokines on hBD-3 and hBD-4 mRNA expression in colon carcinoma cells was also investigated. Purified epithelial cells of normal small and large intestine expressed both hBD-3 and hBD-4 mRNA, with higher expression levels of hBD-3 mRNA. In situ hybridization revealed higher levels of mRNA expression in the crypt- compared to the villus/luminal-compartment. Interferon (IFN)-gamma, but not tumour necrosis factor (TNF)-alpha or IL-1beta, augmented hBD-3 mRNA expression. None of these agents stimulated hBD-4 expression. Colonic epithelial cells from patients with UC displayed a significant increase in hBD-3 and hBD-4 mRNA compared to epithelial cells of controls. In contrast, small intestinal epithelial cells from CD patients did not show increased expression levels compared to the corresponding control cells. Moreover, Crohn's colitis did not show increased expression of hBD-4 mRNA, while the data are inconclusive for hBD-3 mRNA. We conclude that the chronic inflammatory reaction induced in the colon of UC patients enhances hBD-3 and hBD-4 mRNA expression in the epithelium, whereas in CD this is less evident.

Interferon-gamma tempers the expression of carcinoembryonic antigen family molecules in human colon cells: a possible role in innate mucosal defence.

Four carcinoembryonic antigen-related cell adhesion molecule (CEACAM)s, i.e. CEA, CEACAM1, CEACAM6 and CEACAM7, are localized to the apical glycocalyx of normal colonic epithelium and have been suggested to play a role in innate immunity. The expression of these molecules in colon carcinoma cells was studied at the mRNA and protein levels after treatment with interferon-gamma (IFN-gamma), interleukin-1beta, live bacteria or lipopolysaccharide. The colon carcinoma cell lines LS174T and HT-29 were studied in detail using real-time quantitative reverse transcriptase-polymerase chain reaction, immunoflow cytometry and immunoelectron microscopy. IFN-gamma, but not the other agents, modified expression of CEA, CEACAM1 and CEACAM6. None of the agents upregulated CEACAM7 expression. Two expression patterns were seen. HT-29 cells, which initially showed low quantities of mRNAs and proteins, displayed marked upregulation of both mRNAs and proteins. LS174T cells transcribed stable high levels of mRNA before and after treatment. Additionally, IFN-gamma induced increased cell surface expression of CEA, CEACAM1 and CECAM6. IFN-gamma has two important effects on the expression levels of the CEA family molecules in colon epithelial cells: direct upregulation of CEACAM1 and promotion of cell differentiation resulting in increased expression of CEA and CEACAM6 and decreased expression of CEACAM7.