Bone tissue content of TGF-β2 changes with time in human heterotopic ossification after total hip arthroplasty

Transforming growth factor beta isoforms (TGF-β₁, TGF-β₂, and TGF-β₃) most likely play a role in bone physiology, but little is known about their relative importance in normal as well as in heterotopic bone. This study focused on possible differences in the localization and relative content of different TGF beta isoforms in heterotopic ossifications (HO) by comparing HOs, which have developed less than 17 months (immature HOs) with those developed 3–9 years (mature HOs). The HOs were harvested after total hip arthroplasty (THA) during revision surgery. The HO samples were decalcified, embedded in paraffin and sectioned. Azan staining was used to evaluate histological structure of the ossifications and immunohistochemical analysis was performed to estimate the localization of three TGF beta isoforms in the HOs. Comparison of different TGF beta isoforms in the immature and the mature ossifications showed that the content of TGF-β₂ was decreased by almost three times in the mature HO as compared to the immature HO (p = 0.0064). The proportions of other isoforms in HOs did not differ significantly. This study shows that the relative importance of TGF betas change with HO development.     

Development and characterization of oxaceprol-loaded poly-lactide-co-glycolide nanoparticles for the treatment of osteoarthritis

Oxaceprol is well-defined therapeutic agent as an atypical inhibitor of inflammation in osteoarthritis. In the present study, we aimed to develop and characterize oxaceprol-loaded poly-lactide-co-glycolide (PLGA) nanoparticles for intra-articular administration in osteoarthritis. PLGA nanoparticles were prepared by double-emulsion solvent evaporation method. Meanwhile, a straightforward and generally applicable high performance liquid chromatography method was developed, and validated for the first time for the quantification of oxaceprol. To examine the drug carrying capacity of nanoparticles, varying amount of oxaceprol was entrapped into a constant amount of polymer matrix. Moreover, the efficacy of drug amount on nanoparticle characteristics such as particle size, zeta potential, morphology, drug entrapment, and in vitro drug release was investigated. Nanoparticle sizes were between 229 and 509 nm for different amount of oxaceprol with spherical smooth morphology. Encapsulation efficiency ranged between 39.73 and 63.83% by decreasing oxaceprol amount. The results of Fourier transform infrared and DSC showed absence of interaction between oxaceprol and PLGA. The in vitro drug release from these nanoparticles showed a sustained release of oxaceprol over 30 days. According to cell culture studies, oxaceprol-loaded nanoparticles had no cytotoxicity with high biocompatibility. This study was the first step of developing an intra-articular system in the treatment of osteoarthritis for the controlled release of oxaceprol. Our findings showed that these nanoparticles can be beneficial for an effective treatment of osteoarthritis avoiding side effects associated with oral administration.     

Targeted mesenchymal stem cell and vascular endothelial growth factor strategies for repair of nerve defects with nerve tissue implanted autogenous vein graft conduits

Peripheral nerve gaps exceeding 1 cm require a bridging repair strategy. Clinical feasibility of autogenous nerve grafting is limited by donor site comorbidity. In this study we investigated neuroregenerative efficacy of autogenous vein grafts implanted with tissue fragments from distal nerve in combination with vascular endothelial growth factor (VEGF) or mesenchymal stem cells (MSCs) in repair of rat peripheral nerve defects. Six‐groups of Sprague‐Dawley rats (n = 8 each) were evaluated in the autogenous setting using a 1.6 cm long peroneal nerve defect: Empty vein graft (group 1), Nerve graft (group 2), Vein graft and nerve fragments (group 3), Vein graft and nerve fragments and blank microspheres (group 4), Vein graft and nerve fragments and VEGF microspheres (group 5), Vein graft and nerve fragments and MSCs (group 6). Nerve fragments were derived from distal segment. Walking track analysis, electrophysiology and nerve histomorphometry were performed for assessment. Peroneal function indices (PFI), electrophysiology (amplitude) and axon count results for group 2 were ?9.12 ± 3.07, 12.81 ± 2.46 mV, and 1697.88 ± 166.18, whereas the results for group 5 were ?9.35 ± 2.55, 12.68 ± 1.78, and 1566 ± 131.44, respectively. The assessment results did not reveal statistical difference between groups 2 and 5 (P > 0.05). The best outcomes were seen in group 2 and 5 followed by group 6. Compared to other groups, poorest outcomes were seen in group 1 (P ≤ 0.05). PFI, electrophysiology (amplitude) and axon count results for group 1 were ?208.82 ± 110.69, 0.86 ± 0.52, and 444.50 ± 274.03, respectively. Vein conduits implanted with distal nerve‐derived nerve fragments improved axonal regeneration. VEGF was superior to MSCs in facilitating nerve regeneration. © 2015 Wiley Periodicals, Inc. Microsurgery 36:578–585, 2016.     

Characterization of Organosolv Lignins and Their Application in the Preparation of Aerogels

The production of novel materials and value-added chemicals from lignin has received considerable attention in recent years. Due to its abundant occurrence in nature, there is a growing interest in utilizing lignin as a feedstock for functional materials production, for example aerogels. Much like in the synthesis of phenol-based resins, the vacant ortho positions of the aromatic rings in lignin can crosslink with formaldehyde and form polymeric gels. After drying the hydrogels with supercritical CO₂, highly porous aerogels are obtained. Current study focuses on the preparation and thorough parametrization of organosolv lignins from different types of lignocellulosic biomass (aspen, pine, and barley straw) as well as their utilization for the preparation of lignin-5-methylresorcinol-formaldehyde aerogels. The thorough structural characterization of the obtained aerogels was carried out by gas adsorption, IR spectroscopy, and scanning electron microscopy. The obtained lignin-based monolithic mesoporous aerogels had specific surface areas and total pore volumes in the upward ranges of 450 m²/g and 1.4 cm³/g, respectively.     

Anti-myeloma activity of endogenous and adoptively transferred activated natural killer cells in experimental multiple myeloma model

OBJECTIVE: Despite advances in autologous stem cell transplantation and chemotherapy, multiple myeloma (MM) remains an incurable disease. Due to the role of natural killer (NK) cells in host resistance against several tumors, it is of interest to explore the anti-MM activity of NK cells. For this reason, we aimed to determine if NK cells provide anti-MM activity following interleukin-2 (IL-2) administration, and if ex vivo activated and intravenously administered NK cells prolong survival in MM-bearing C57BL/KaLwRij mice. METHODS: The anti-MM effect of IL-2 was tested by intraperitoneal injection into the 5T33MM-inoculated mice. Subsequently, in vivo effector cell depletions were performed by administration of anti-NK1.1 or anti-CD8 monoclonal antibodies. Finally, magnetically separated and activated NK cells from splenocytes of C57BL/KaLwRij mice were adoptively transferred to tumor-bearing mice in conjunction with IL-2 treatment. RESULTS: IL-2 administration into MM-bearing mice significantly prolonged their survival. This effect was diminished by in vivo depletion of NK cells. Adoptive transfer of activated NK cells showed a significant in vivo anti-MM effect that was dependent on cell dose. Biodistribution of the marked adoptively transferred NK cells correlated with MM cells' homing sites. CONCLUSION: These data suggest that activated NK cells have a promising potential in adoptive immunotherapy for MM.     

Enhanced ouabain resistance gene as a eukaryotic selection marker

Current selection markers allow selection by antibiotics or fluorescent/magnetic sorting by green fluorescent protein or membrane antigens. Antibiotic selection proceeds on a time scale of weeks, and flourescence-activated cell sorting requires complex equipment and may generate false-positive results when selection is performed too early after transduction with membrane markers. We have characterized an endogenous eukaryotic selection marker, the ouabain resistance gene (Oua(r)), which has the potential for quick and efficient in vitro selection of target cells. The Oua(r) used by us is derived from the rat alpha(1) isoform of Na(+),K(+)-ATPase, where leucine at position 799 is substituted for cysteine by targeted mutagenesis. This mutation confers resistance to more than 1 mM ouabain in vitro. We show that cells transfected with plasmid or transduced with a retrovirus vector encoding Oua(r) can be selected efficiently with ouabain in 48 hr and that a pure population of cells can be obtained. The ouabain resistance gene may be useful as a selection marker in general molecular biology, preclinical, and clinical applications because of its short selection time and also because of the safety of ouabain for human use.     

Response rate or time to progression as predictors of survival in trials of metastatic colorectal cancer or non-small-cell lung cancer: a meta-analysis

BACKGROUND: The duration and cost of cancer clinical trials could be reduced if a surrogate endpoint were used in place of survival. We did a meta-analysis to assess the extent to which two surrogates, tumour response and time to progression, are predictive of mortality in metastatic colorectal cancer and non-small-cell lung cancer. METHODS: Summary data (median time to progression, proportion of patients responding to treatment, and median overall survival) from randomised trials of first-line treatment in colorectal cancer (146 trials) and lung cancer (191 trials) were identified. Data were extracted and analysed by linear regression. We used prediction bands for trials with 250, 500, and 750 patients to identify the surrogate threshold effect that would predict a significant difference in survival. FINDINGS: Response to treatment and time to progression correlated with improvement in survival for both lung cancer (p<0.0001 and p=0.0003, respectively) and colorectal cancer (p<0.0001 for both). To predict a significant survival gain in colorectal cancer trials, an improvement of 20% in the number of patients responding to treatment was required in trials with 750 patients, increasing to 26% in trials with 500 patients and 38% in trials with 250 patients. In lung cancer trials, the same prediction required differences in response of 18% for 750 patients, 21% for 500 patients, and 30% for 250 patients. For time to progression for both cancer types, the incremental gain needed to predict a survival improvement was a median of 1.8 months for trials with 750 patients, 2.2 months for 500 patients, and 3.3 months for 250 patients. INTERPRETATION: Irrespective of trial size, large differences in tumour response rate are needed to predict a significant survival benefit. If surrogates are chosen as the primary endpoint in a clinical trial, time to progression is the preferred measure because more modest and achievable differences are needed for a significant survival benefit. Trials in metastatic lung cancer and colorectal cancer should measure survival as their primary outcome unless the surrogate outcome difference is anticipated to exceed the threshold effect size.