Diabetes Causes the Accelerated Loss of Cartilage During Fracture Repair Which Is Reversed by Insulin Treatment

Fracture healing in diabetic individuals and in animal models of diabetes is impaired. To investigate mechanisms by which diabetes may affect fracture healing we focused on the transition from cartilage to bone, a midpoint in the fracture healing process. Femoral fractures were induced in mice rendered diabetic by multiple low dose streptozotocin treatment and compared to matching normoglycemic mice. One group of diabetic animals was treated with slow release insulin to maintain normal serum glucose levels. The results indicate that there was relatively little difference in the initial formation of the fracture callus on day 10. However, on day 16 the diabetic group had significantly smaller callus, greater loss of cartilage and enhanced osteoclastogenesis that was normalized by treatment with insulin when assessed by histomorphometric analysis. Chondrocyte apoptosis was significantly higher in diabetic mice and this increase was blocked by insulin. These changes were accompanied by diabetes-increased mRNA levels of RANKL, TNF-α, and ADAMTS-4 and -5 measured by real-time PCR, which was reversed by insulin treatment. On days 16 and 22 bone formation within the callus of diabetic mice was significantly less than the normoglycemic and brought to normal levels by insulin treatment. These results suggest that a significant effect of diabetes on fracture healing is increased chondrocyte apoptosis and osteoclastogenesis that accelerates the loss of cartilage and reduces the anlage for endochondral bone formation during fracture repair. That insulin reverses these effects demonstrates that they are directly related to the diabetic condition

Effect of platelet lysate on human cells involved in different phases of wound healing

Background Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization). Methodology/Principal Findings Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and keratinocytes, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/2) and inflammatory response evaluation (NFκB). Results were compared both with basal medium and with a positive control containing serum and growth factors. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and 20% v/v). Whereas both platelet lysate concentrations increased cell migration, only 20% platelet lysate was able to significantly promote angiogenic activity (p<0.05 vs. control), comparably to the positive control. Both platelet lysate concentrations activated important inflammatory pathways such as ERK1/2 and NFκB with the same early kinetics, whereas the effect was different for later time-points. Conclusion/Significance These data suggest the possibility of using allogeneic platelet lysate as both an alternative to growth factors commonly used for cell culture and as a tool for clinical regenerative application for wound healing

Chemokine Expression Is Upregulated in Chondrocytes in Diabetic Fracture Healing

Chemokines are thought to play an important role in several aspects of bone metabolism including the recruitment of leukocytes and the formation of osteoclasts. We investigated the impact of diabetes on chemokine expression in normal and diabetic fracture healing. Fracture of the femur was performed in streptozotocin-induced diabetic and matched normoglycemic control mice. Microarray analysis was carried out and chemokine mRNA levels in vivo were assessed. CCL4 were examined in fracture calluses by immunohistochemistry and the role of TNF in diabetes-enhanced expression was investigated by treatment of animals with the TNF-specific inhibitor, pegsunercept. In vitro studies were conducted with ATDC5 chondrocytes. Diabetes significantly upregulated mRNA levels of several chemokines in vivo including CCL4, CCL8, CCL6, CCL11, CCL20, CCL24, CXCL2, CXCL5 and chemokine receptors CCR5 and CXCR4. Chondrocytes were identified as a significant source of CCL4 and its expression in diabetic fractures was dependent on TNF (P \u3c 0.05). TNF-α significantly increased mRNA levels of several chemokines in vitro which were knocked down with FOXO1 siRNA (P \u3c 0.05). CCL4 expression at the mRNA and proteins levels was induced by FOXO1 over-expression and reduced by FOXO1 knockdown. The current studies point to the importance of TNF-α as a mechanism for diabetes enhanced chemokine expression by chondrocytes, which may contribute to the accelerated loss of cartilage observed in diabetic fracture healing. Moreover, in vitro results point to FOXO1 as a potentially important transcription factor in mediating this effect

Diabetes Reduces Mesenchymal Stem Cells in Fracture Healing Through a TNFα-Mediated Mechanism

Aims/hypothesis Diabetes interferes with bone formation and impairs fracture healing, an important complication in humans and animal models. The aim of this study was to examine the impact of diabetes on mesenchymal stem cells (MSCs) during fracture repair. Methods Fracture of the long bones was induced in a streptozotocin-induced type 1 diabetic mouse model with or without insulin or a specific TNFα inhibitor, pegsunercept. MSCs were detected with cluster designation-271 (also known as p75 neurotrophin receptor) or stem cell antigen-1 (Sca-1) antibodies in areas of new endochondral bone formation in the calluses. MSC apoptosis was measured by TUNEL assay and proliferation was measured by Ki67 antibody. In vitro apoptosis and proliferation were examined in C3H10T1/2 and human-bone-marrow-derived MSCs following transfection with FOXO1 small interfering (si)RNA. Results Diabetes significantly increased TNFα levels and reduced MSC numbers in new bone area. MSC numbers were restored to normal levels with insulin or pegsunercept treatment. Inhibition of TNFα significantly reduced MSC loss by increasing MSC proliferation and decreasing MSC apoptosis in diabetic animals, but had no effect on MSCs in normoglycaemic animals. In vitro experiments established that TNFα alone was sufficient to induce apoptosis and inhibit proliferation of MSCs. Furthermore, silencing forkhead box protein O1 (FOXO1) prevented TNFα-induced MSC apoptosis and reduced proliferation by regulating apoptotic and cell cycle genes. Conclusions/interpretation Diabetes-enhanced TNFα significantly reduced MSC numbers in new bone areas during fracture healing. Mechanistically, diabetes-enhanced TNFα reduced MSC proliferation and increased MSC apoptosis. Reducing the activity of TNFα in vivo may help to preserve endogenous MSCs and maximise regenerative potential in diabetic patients

Diminished Bone Formation During Diabetic Fracture Healing Is Related to the Premature Resorption of Cartilage Associated with Increased Osteoclast Activity

Histological and molecular analysis of fracture healing in normal and diabetic animals showed significantly enhanced removal of cartilage in diabetic animals. Increased cartilage turnover was associated with elevated osteoclast numbers, a higher expression of genes that promote osteoclastogenesis, and diminished primary bone formation. Introduction Diminished bone formation, an increased incidence of nonunions, and delayed fracture healing have been observed in animal models and in patients with diabetes. Fracture healing is characterized by the formation of a stabilizing callus in which cartilage is formed and then resorbed and replaced by bone. To gain insight into how diabetes affects fracture healing, studies were carried out focusing on the impact of diabetes on the transition from cartilage to bone. Materials and Methods A low-dose treatment protocol of streptozotocin in CD-1 mice was used to induce a type 1 diabetic condition. After mice were hyperglycemic for 3 weeks, controlled closed simple transverse fractures of the tibia were induced and fixed by intramedullary pins. Histomorphometric analysis of the tibias obtained 12, 16, and 22 days after fracture was performed across the fracture callus at 0.5 mm proximal and distal increments using computer-assisted image analysis. Another group of 16-day samples were examined by μCT. RNA was isolated from a separate set of animals, and the expression of genes that reflect the formation and removal of cartilage and bone was measured by real-time PCR. Results Molecular analysis of collagen types II and X mRNA expression showed that cartilage formation was the same during the initial period of callus formation. Histomorphometric analysis of day 12 fracture calluses showed that callus size and cartilage area were also similar in normoglycemic and diabetic mice. In contrast, on day 16, callus size, cartilage tissue, and new bone area were 2.0-, 4.4-, and 1.5-fold larger, respectively, in the normoglycemic compared with the diabetic group (p \u3c 0.05). Analysis of μCT images indicated that the bone volume in the normoglycemic animals was 38% larger than in diabetic animals. There were 78% more osteoclasts in the diabetic group compared with the normoglycemic group (p \u3c 0.05) on day 16, consistent with the reduction in cartilage. Real-time PCR showed significantly elevated levels of mRNA expression for TNF-α, macrophage-colony stimulating factor, RANKL, and vascular endothelial growth factor-A in the diabetic group. Similarly, the mRNA encoding ADAMTS 4 and 5, major aggrecanases that degrade cartilage, was also elevated in diabetic animals. Conclusions These results suggest that impaired fracture healing in diabetes is characterized by increased rates of cartilage resorption. This premature loss of cartilage leads to a reduction in callus size and contributes to decreased bone formation and mechanical strength frequently reported in diabetic fracture healing

Search for Neutral Higgs Bosons in e+e- Collisions at sqrt(s) ~189GeV

A search for neutral Higgs bosons has been performed with the OPAL detector at LEP, using approximately 170 pb-1 of e+e- collision data collected at sqrt(s)~189GeV. Searches have been performed for the Standard Model (SM) process e+e- to H0Z0 and the MSSM processes e+e- to H0Z0, A0h0. The searches are sensitive to the b b-bar and tau antitau decay modes of the Higgs bosons, and also to the MSSM decay mode h0 to A0A0. OPAL search results at lower centre-of-mass energies have been incorporated in the limits we set, which are valid at the 95% confidence level. For the SM Higgs boson, we obtain a lower mass bound of 91.0 GeV. In the MSSM, our limits are mh>74.8GeV and mA>76.5GeV, assuming tan(beta)>1, that the mixing of the scalar top quarks is either zero or maximal, and that the soft SUSY-breaking masses are 1 TeV. For the case of zero scalar top mixing, we exclude values of tan(beta) between 0.72 and 2.19.Comment: 38 pages, 15 figures, submitted Euro. Phys. J.

Bose-Einstein Correlations of Three Charged Pions in Hadronic Z^0 Decays

Bose-Einstein Correlations (BEC) of three identical charged pions were studied in 4 x 10^6 hadronic Z^0 decays recorded with the OPAL detector at LEP. The genuine three-pion correlations, corrected for the Coulomb effect, were separated from the known two-pion correlations by a new subtraction procedure. A significant genuine three-pion BEC enhancement near threshold was observed having an emitter source radius of r_3 = 0.580 +/- 0.004 (stat.) +/- 0.029 (syst.) fm and a strength of \lambda_3 = 0.504 +/- 0.010 (stat.) +/- 0.041 (syst.). The Coulomb correction was found to increase the \lambda_3 value by \~9% and to reduce r_3 by ~6%. The measured \lambda_3 corresponds to a value of 0.707 +/- 0.014 (stat.) +/- 0.078 (syst.) when one takes into account the three-pion sample purity. A relation between the two-pion and the three-pion source parameters is discussed.Comment: 19 pages, LaTeX, 5 eps figures included, accepted by Eur. Phys. J.

Selective masking and demasking for the stepwise complexometric determination of aluminium, lead and zinc from the same solution

Background: A complexometric method based on selective masking and de-masking has been developed for the rapid determination of aluminium, lead and zinc from the same solution in glass and glass frit samples. The determination is carried out using potassium cyanide to mask zinc, and excess disodium salt of EDTA to mask lead and aluminium. The excess EDTA was titrated with standard Mn(II)SO(4) solution using Erichrome Black-T as the indicator. Subsequently selective de-masking agents - triethanolamine, 2,3-dimercaptopropanol and a formaldehyde/acetone mixture - were used to determine quantities of aluminium, lead and zinc in a stepwise and selective manner. Results: The accuracy of the method was established by analysing glass certified reference material NBS 1412. The standard deviation of the measurements, calculated by analysing five replicates of each sample, was found to be less than 1.5% for the method proposed. Conclusion: The novelty of the method lies in its simplicity and accuracy afforded by there not being a need for a prior separation or instrumentation. The proposed method was found to be highly selective for the precise determination of aluminum, zinc and lead in the routine analysis of glass batch and allied materials

Measurements of Flavour Dependent Fragmentation Functions in Z^0 -> qq(bar) Events

Fragmentation functions for charged particles in Z -> qq(bar) events have been measured for bottom (b), charm (c) and light (uds) quarks as well as for all flavours together. The results are based on data recorded between 1990 and 1995 using the OPAL detector at LEP. Event samples with different flavour compositions were formed using reconstructed D* mesons and secondary vertices. The \xi_p = ln(1/x_E) distributions and the position of their maxima \xi_max are also presented separately for uds, c and b quark events. The fragmentation function for b quarks is significantly softer than for uds quarks.Comment: 29 pages, LaTeX, 5 eps figures (and colour figs) included, submitted to Eur. Phys. J.