3D Computer-Aided Design and Manufacturing in Oromaxillofacial Surgery

Peer reviewe

Efficient ultrafiltration based protocol to deplete extracellular vesicles from fetal bovine serum

Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EVdepleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories.Peer reviewe

Extracellular small non-coding RNA contaminants in fetal bovine serum and serum-free media

In the research field of extracellular vesicles (EVs), the use of fetal bovine serum (FBS) depleted of EVs for in vitro studies is advocated to eliminate the confounding effects of media derived EVs. EV-depleted FBS may either be prepared by ultracentrifugation or purchased commercially. Nevertheless, these preparations do not guarantee an RNA-free FBS for in vitro use. In this study we address the RNA contamination issue, of small non-coding (nc)RNA in vesicular or non-vesicular fractions of FBS, ultracentrifugation EV-depleted FBS, commercial EV-depleted FBS, and in our recently developed filtration based EV-depleted FBS. Commercially available serum- and xeno-free defined media were also screened for small ncRNA contamination. Our small ncRNA sequencing data showed that all EV-depleted media and commercially available defined media contained small ncRNA contaminants. Out of the different FBS preparations studied, our ultrafiltration-based method for EV depletion performed the best in depleting miRNAs. Certain miRNAs such miR-122 and miR-203a proved difficult to remove completely and were found in all media. Compared to miRNAs, other small ncRNA (snRNA,Y RNA, snoRNA, and piRNA) were difficult to eliminate from all the studied media. Additionally, our tested defined media contained miRNAs and other small ncRNAs, albeit at a much lower level than in serum preparations. Our study showed that no media is free of small ncRNA contaminants. Therefore, in order to screen for baseline RNA contamination in culturing media, RNA sequencing data should be carefully controlled by adding a media sample as a control. This should be a mandatory step before performing cell culture experiments in order to eliminate the confounding effects of media.Peer reviewe

Proangiogenic Hypoxia-Mimicking Agents Attenuate Osteogenic Potential of Adipose Stem/Stromal Cells

BACKGROUND: Insufficient vascularization hampers bone tissue engineering strategies for reconstructing large bone defects. Delivery of prolyl-hydroxylase inhibitors (PHIs) is an interesting approach to upregulate vascular endothelial growth factor (VEGF) by mimicking hypoxic stabilization of hypoxia-inducible factor-1alpha (HIF-1α). This study assessed two PHIs: dimethyloxalylglycine (DMOG) and baicalein for their effects on human adipose tissue-derived mesenchymal stem/stromal cells (AT-MSCs). METHODS: Isolated AT-MSCs were characterized and treated with PHIs to assess the cellular proliferation response. Immunostaining and western-blots served to verify the HIF-1α stabilization response. The optimized concentrations for long-term treatment were tested for their effects on the cell cycle, apoptosis, cytokine secretion, and osteogenic differentiation of AT-MSCs. Gene expression levels were evaluated for alkaline phosphatase (ALPL), bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (RUNX2), vascular endothelial growth factor A (VEGFA), secreted phosphoprotein 1 (SPP1), and collagen type I alpha 1 (COL1A1). In addition, stemness-related genes Kruppel-like factor 4 (KLF4), Nanog homeobox (NANOG), and octamer-binding transcription factor 4 (OCT4) were assessed. RESULTS: PHIs stabilized HIF-1α in a dose-dependent manner and showed evident dose- and time dependent antiproliferative effects. With doses maintaining proliferation, DMOG and baicalein diminished the effect of osteogenic induction on the expression of RUNX2, ALPL, and COL1A1, and suppressed the formation of mineralized matrix. Suppressed osteogenic response of AT-MSCs was accompanied by an upregulation of stemness-related genes. CONCLUSION: PHIs significantly reduced the osteogenic differentiation of AT-MSCs and rather upregulated stemness-related genes. PHIs proangiogenic potential should be weighed against their longterm direct inhibitory effects on the osteogenic differentiation of AT-MSCs.Peer reviewe

Cranioplasty with Adipose-Derived Stem Cells, Beta-Tricalcium Phosphate Granules and Supporting Mesh : Six-Year Clinical Follow-Up Results

Several alternative techniques exist to reconstruct skull defects. The complication rate of the cranioplasty procedure is high and the search for optimal materials and techniques continues. To report long-term results of patients who have received a cranioplasty using autologous adipose-derived stem cells (ASCs) seeded on beta-tricalcium phosphate (betaTCP) granules. Between 10/2008 and 3/2010, five cranioplasties were performed (four females, one male; average age 62.0 years) using ASCs, betaTCP granules and titanium or resorbable meshes. The average defect size was 8.1 x 6.7 cm(2). Patients were followed both clinically and radiologically. The initial results were promising, with no serious complications. Nevertheless, in the long-term follow-up, three of the five patients were re-operated due to graft related problems. Two patients showed marked resorption of the graft, which led to revision surgery. One patient developed a late infection (7.3 years post-operative) that required revision surgery and removal of the graft. One patient had a successfully ossified graft, but was re-operated due to recurrence of the meningioma 2.2 years post-operatively. One patient had an uneventful clinical follow-up, and the cosmetic result is satisfactory, even though skull x-rays show hypodensity in the borders of the graft. Albeit no serious adverse events occurred, the 6-year follow-up results of the five cases are unsatisfactory. The clinical results are not superior to results achieved by conventional cranial repair methods. The use of stem cells in combination with betaTCP granules and supporting meshes in cranial defect reconstruction need to be studied further before continuing with clinical trials.Peer reviewe

Monocyte-derived extracellular vesicles stimulate cytokinesecretion and gene expression of matrixmetalloproteinases by mesenchymal stem/stromal cells

Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. DatabaseGene expression data are available in the GEO database under the accession number .Peer reviewe

Effects of vatinoxan on xylazine-induced pulmonary alterations in sheep

It was hypothesized that premedication with vatinoxan, a peripheral alpha(2)-adrenoceptor antagonist, would mitigate xylazine-induced pulmonary alterations in sheep. Fourteen adult sheep were allotted into two equal groups and premedicated with either vatinoxan (750 mu g/kg IV) or saline and sedated 10 min later with xylazine (500 mu g/kg IV). Arterial oxygen saturation (SpO(2)) was measured and respiratory rate (RR) counted at intervals. The sheep were euthanized with IV pentobarbital 10 min after xylazine administration. The severity of pulmonary parenchymal alterations was assessed and graded grossly and histologically and correlations of the morphological changes with SpO(2) evaluated. Following xylazine injection, SpO(2) was significantly higher and RR significantly lower with vatinoxan than with saline and the sheep administered vatinoxan exhibited significantly smaller quantities of tracheal foam than those receiving saline. No significant differences in macroscopic oedema scores were detected between treatments. In contrast, the vatinoxan-treated animals exhibited significantly graver microscopic interstitial alveolar oedema and haemorrhage than saline-treated animals. The histological severity scores did not correlate with changes in SpO(2). In conclusion, xylazine induced a marked reduction in SpO(2) which was abolished by the prior administration of vatinoxan. The histologically detected alterations after pentobarbital euthanasia with vatinoxan premedication need to be studied further.Peer reviewe

Concentrations of vatinoxan and xylazine in plasma, cerebrospinal fluid and brain tissue following intravenous administration in sheep

Objectives To investigate the extent of vatinoxan distribution into sheep brain, and whether vatinoxan influences brain concentrations of xylazine; and to examine the utility of cerebrospinal fluid (CSF) as a surrogate of brain tissue concentrations for vatinoxan and xylazine. Study design Randomised, blinded, experimental study. Animals A total of 14 adult female sheep. Methods Sheep were randomly allocated into two equal groups and premeditated with either intravenous (IV) vatinoxan (750 mu g kg(-1), VX) or saline (SX) administered 10 minutes before IV xylazine (500 mu g kg(-1)). Sedation was subjectively assessed at selected intervals before and after treatments. At 10 minutes after xylazine administration, a venous blood sample was collected and the sheep were immediately euthanised with IV pentobarbital (100 mg kg(-1)). Plasma, CSF and brain tissues were harvested, and concentrations of vatinoxan and xylazine were quantified using liquid chromatography-tandem mass spectrometry. Drug ratios were then calculated and the data were analysed as appropriate. Results The brain-to-plasma and CSF-to-plasma ratios of vatinoxan were 0.06 +/- 0.013 and 0.05 +/- 0.01 (mean +/- standard deviation), respectively. Xylazine brain concentrations were not significantly different (835 +/- 262 versus 1029 +/- 297 ng g(-1) in groups VX and SX, respectively) and were approximately 15-fold higher than those in plasma. The CSF-to-brain ratio of vatinoxan was 0.8 +/- 0.2, whereas xylazine concentrations in the brain were approximately 17-fold greater than those in CSF, with and without vatinoxan. Conclusions and clinical relevance Vatinoxan did not significantly affect sedation with xylazine or the concentrations of xylazine in the brain. CSF is not a good predictor of xylazine concentrations in the brain, whereas vatinoxan concentrations were concordant between the brain and CSF, using the dosages in this study.Peer reviewe