Improvement of bone allograft recolonization by adipose stem cells: impact of bone graft demineralization

Bone allografts combined with mesenchymal stem cells are proposed for bone tissue engineering but remains limited by a low degree of stem cells spreading and cellular recolonization. We postulate to demineralize bone allografts in view to improve adipose stem cells (ASCs) colonization and the bioactivity of the graft. Bone allografts (n=16) were treated for decellularization (4 groups of demineralization time: 0, 4, 8, 12 hours). The implants were compared in terms of residual calcium, mineral density and bioactivity (for BMP-2/VEGF contents). Each implant was scanned by microtomography to analyze macroporosity and open porosity. Helium pycnometry and Hg porosimetry were performed to assess the absolute density and microporosity. Bone surface analysis was assessed by X-Ray photoelectron spectroscopy and SEM. The bone graft recolonization by ASCs was studied in vitro by SEM, histology and DNA extraction at 24 hours/day 15 post-cellular seeding. Finally, ASCs combined with non-/demineralized bone matrix were implanted into the lumbar muscles of 10 nude rats (in comparison to bone grafts w/o cells) to study the osteoinductivity/angiogenicity by imagery/histology 29 days after implantation. A significant reduction of the calcium concentration (>-90%) was found in demineralized bone in comparison to native grafts as revealed by ionometry (0.27 vs. 4.1 g/L) and pQCT (0 vs. 0.39 g/ cm?). Demineralization significantly increase the macroporosity (>100μm by +13%) and the open porosity (>4 cm?/g vs. 2.1±1.0 cm?/g in comparison to the native graft,p<0.05). A significant increase of microporosity (>10 μm by +158% and <100 nm by 558%) was also found after demineralization. Helium pycnometry confirmed the correlation between the decrease of absolute density and demineralization of the bone graft (R?=0.81). A positive linear correlation between the decrease of calcium/increase of nitrogen atoms (at the bone surface) and the time of demineralization was found (R?=0.99,p<0.001). At day 15 post-incubation, a significant higher ASCs colonization of the bone graft was found for tissue demineralized during 12 hours (p<0.05). In conclusion, the demineralization of cancellous bones significantly improves the colonization by ASCs in view to return the bioactivity for bone regeneration

Expiratory flow limitation during sleep in heavy snorers and obstructive sleep apnoea patients

Inspiratory flow limitation (FL) during sleep has previously been reported both in obstructive sleep apnoea (OSA) patients and heavy snorers (HS), Recent results from this laboratory have shown the occurrence both of inspiratory and expiratory flow limitation during muscular relaxation in awake healthy subjects and OSA patients. In this study, we have investigated whether expiratory flow limitation occurs during sleep in heavy snorers and in patients with OSA, We studied four nonapnoeic, heavy snorers and five OSA patients, Airflow was measured with a pneumotachograph attached to a tight-fitting mask, and supraglottic pressure with a catheter placed at the supraglottic level, Scoring for flow limitation was achieved by visual inspection of 200 breaths recorded during sleep. About 20% of the respiratory cycles presented isolated inspiratory flow limitation, Approximately the same percentage was observed in heavy snorers and OSA patients, Isolated expiratory flow limitation was less frequently recorded, Coupled inspiratory and expiratory flow limitations were more numerous, especially in heavy snorers. We conclude that both in heavy snorers and obstructive sleep apnoea patients, inspiratory flow limitation is associated with expiratory now limitation, This suggests that upper airway obstruction during sleep is both an inspiratory and expiratory event

Bioactivity of a tissue-engineered product: bridging the gap between academic and clinical studies

The translation of cell-based therapeutics from academic and fundamental sciences to clinical trial settings follows a preclinical pathway with rigorous regulatory oversights to ensure the cellmediated therapeutic effect. Although international guidelines thoroughly describe single-dose toxicity and biodistribution studies, the quantitative evaluation of the biological activity remains a major challenge for biotechnology companies and authorities. Here we discuss a strategy to demonstrate the bioactivity of an osteogenic tissue-engineered product intended to promote angiogenesis and osteogenesis in a bone defect. Osteogenic 3-dimensional (3D) grafts were manufactured as a pharmaceutical batch. A sequence of chemical treatment was applied on the native graft in view to obtained a decellularized 3D-matrix as confirmed by a reduction of >90% and 40-80% of the cellular and growth factors (VEGF, IGF1) contents, respectively. Decellularized and native grafts were implanted intra-muscularly (after cauterization of the lumbar region, n=10 nude rats) to assess the mineralization (X-ray microCT and histomorphometry on Von Kossa staining) and angiogenesis (histomorphometry on Masson’s Trichrome and Von Willebrand Factor immunostaining) at day 29 postimplantation. Human cells detection (in the explanted tissue) were also quantified after KU80/HLA type I immunostaining at the implantation site. A significant higher mineralization was found for explanted samples from the native osteogenic 3D grafts in comparison to the decellularized tissues (Bone volume/tissue volume of 2.92 ± 1.12% vs. 0.38 ± 0.59%, respectively

Airways obstruction, chronic expectoration, and rapid decline of FEV1 in smokers are associated with increased levels of sputum neutrophils.

BACKGROUND: Smoking may cause inflammation of the airways and impairment of lung function. To determine the relationship between the type and degree of airways inflammation and the decline in lung function, leucocytes in the sputum of smokers and ex-smokers were examined. METHODS: Forty six smokers and ex-smokers of median age 64 years (25%; 75% percentiles 62;66) with a smoking history of 40.1 (31.7;53) pack years were studied with lung function tests and a questionnaire at the end of a 15 year follow up period. Sputum was induced by inhalation of hypertonic saline and differential leucocyte counts were performed on cytospin preparations. RESULTS: Adequate sputum samples were obtained in 38 subjects (78%). The ratio of forced expiratory volume in one second (FEV1) to vital capacity (VC) was 67.1 (60; 72)% and the annual decline in FEV1 was 19.4 (12;30) ml/year. Subjects with airways obstruction (FEV1/VC < 63%) had more neutrophils (77 (50;86)%) than those without airways obstruction (60 (43;73)%). The percentage of neutrophils was also significantly greater (77 (62;85)%) in those with chronic expectoration than in those without expectoration (57 (45;75)%. Increased levels of neutrophils in the sputum were correlated with a rapid decline in FEV1 over the 15 year follow up period. CONCLUSIONS: Airways obstruction and chronic expectoration, as well as accelerated decline in lung function, are associated with increased numbers of neutrophils in the sputum of smokers and ex-smokers which suggests that neutrophilic inflammation of the airways may be involved in the pathogenesis of chronic obstructive pulmonary disease