The Use of Layered Freeform Fabrication Technologies to Produce Tissue Engineering Scaffolds for Skull Patches

Congenital skull defects in infants are difficult to correct using metal plates due to the growth of the skull. Tissue engineering of bone patches could be the answer to help such patients. Custom scaffolds have been designed based on Computed Tomography (CT) images of the patient’s skull. An in-house developed single screw extruder, casting and a commercial laser cutter has been evaluated in the fabrication of pure polycaprolactone (PCL) scaffolds as well as PCL mixed with hydroxyapatite (HA) scaffolds. Evaluation criteria for each process included the ability to maintain an optimal pore size for cells to proliferate, inclusion of micro surface properties for cell adhesion, incorporation of hydroxyapatite, and ability to maintain desired shape. The mechanical properties of the fabricated scaffolds will be presented in this paper as well as initial cell seeding results with human adipose-derived adult stem (hADAS) cells.Mechanical Engineerin

Gene expression of catabolic inflammatory cytokines peak before anabolic inflammatory cytokines after ACL injury in a preclinical model

Background: The response of the joint to anterior cruciate ligament (ACL) injury has not been fully characterized. In particular, the characterization of both catabolic factors, including interleukin-6 (IL-6), interleukin-8 (IL-8), and markers of ongoing tissue damage (CRP), and anabolic factors, including vascular endothelial growth factor (VEGF), transforming growth factor β-induced (TGFβI), and the presence of CD163+ macrophages, have not been well defined. In this study, we hypothesized ACL injury would catalyze both catabolic and anabolic processes and that these would have different temporal profiles of expression. Methods: Adolescent Yucatan minipigs were subjected to ACL transection. Within the joint, gene expression levels of IL-6, IL-8, VEGF, and TGFβI were quantified in the synovium, ligament, and provisional scaffold located between the torn ligament ends at days 1, 5, 9, and 14 post-injury. Macrophage infiltration was also assessed in the joint tissues over the two week period. Serum C-reactive protein (CRP) levels were measured at multiple time points between 1 hour to 14 days after injury. Results: Increases in IL-6 and IL-8 gene expression peaked at day 1 after injury in the synovium and ligament. CRP levels were significantly increased at day 3 before returning to pre-injury levels. VEGF and TGFβI gene expression did not significantly increase until day 9 in the synovium and were unchanged in the other tissues. CD163+ macrophages increased in the ligament and synovium until day 9. Conclusion: Taken together, these results suggest that the response within the joint is primarily catabolic in the first three days after injury, switching to a more anabolic phase by nine days after injury. The effect of medications which alter these processes may thus depend on the timing of administration after injury

A Normative Study of the Synovial Fluid Proteome from Healthy Porcine Knee Joints

Synovial fluid in an articulating joint contains proteins derived from the blood plasma and proteins that are produced by cells within the joint tissues, such as synovium, cartilage, ligament, and meniscus. The proteome composition of healthy synovial fluid and the cellular origins of many synovial fluid components are not fully understood. Here, we present a normative proteomics study using porcine synovial fluid. Using our optimized method, we identified 267 proteins with high confidence in healthy synovial fluid. We also evaluated mRNA expression data from tissues that can contribute to the synovial fluid proteome, including synovium, cartilage, blood, and liver, to better estimate the relative contributions from these sources to specific synovial fluid components. We identified 113 proteins in healthy synovial fluid that appear to be primarily derived from plasma transudates, 37 proteins primarily derived from synovium, and 11 proteins primarily derived from cartilage. Finally, we compared the identified synovial fluid proteome to the proteome of human plasma, and we found that the two body fluids share many similarities, underlining the detected plasma derived nature of many synovial fluid components. Knowing the synovial fluid proteome of a healthy joint will help to identify mechanisms that cause joint disease and pathways involved in disease progression

A normative study of the synovial fluid proteome from healthy porcine knee joints

[Image: see text] Synovial fluid in an articulating joint contains proteins derived from the blood plasma and proteins that are produced by cells within the joint tissues, such as synovium, cartilage, ligament, and meniscus. The proteome composition of healthy synovial fluid and the cellular origins of many synovial fluid components are not fully understood. Here, we present a normative proteomics study using porcine synovial fluid. Using our optimized method, we identified 267 proteins with high confidence in healthy synovial fluid. We also evaluated mRNA expression data from tissues that can contribute to the synovial fluid proteome, including synovium, cartilage, blood, and liver, to better estimate the relative contributions from these sources to specific synovial fluid components. We identified 113 proteins in healthy synovial fluid that appear to be primarily derived from plasma transudates, 37 proteins primarily derived from synovium, and 11 proteins primarily derived from cartilage. Finally, we compared the identified synovial fluid proteome to the proteome of human plasma, and we found that the two body fluids share many similarities, underlining the detected plasma derived nature of many synovial fluid components. Knowing the synovial fluid proteome of a healthy joint will help to identify mechanisms that cause joint disease and pathways involved in disease progression

A normative study of the synovial fluid proteome from healthy porcine knee joints

Description Synovial fluid in an articulating joint contains proteins derived from blood transudates, and proteins that are produced by cells within joint tissues, such as synovium, cartilage, ligament, and meniscus. The proteome composition of healthy synovial fluid is not fully understood. Also not fully delineated are the cellular origins of synovial fluid components. We here present a normative proteomics study for synovial fluid optimized in pig. Sample Processing Protocol Six adolescent Yucatan minipigs (Coyote CCI, Douglas, MA, USA), aged 12-15 months, were obtained for use in this study. All minipigs were housed and monitored by the Animal Resources at Children’s Hospital (ARCH, Boston, MA, USA) and handled according to approved Institutional Animal Care and Use Committee (IACUC) protocols. Minipigs were acclimated to the ARCH environment for a minimum of 3 days prior to experimental handling. At time of euthanasia, synovium tissue from both knee joints of each minipig was harvested. Each tissue specimen was submerged in a cryovial, then flash frozen in liquid nitrogen and stored at -80°C until gene expression analysis. Synovial fluid was also obtained from the joints, and centrifuged at 3,000 g for 10 min to remove any cells. In some cases, 3 mL sterile saline was injected to facilitate fluid extraction, after which the knee was bent 10 times to mix and the saline/synovial fluid mix was extracted and then centrifuged. The supernatant was removed, and approximately 240-500 μL sample was stored in 120 μL aliquots in cryovials at -80°C. Three protocols, employing different strategies were evaluated for the tryptic digestion of synovial fluid samples: urea filter assisted sample preparation (FASP), urea in-solution digestion, and in-gel digestion. 1) Urea FASP Digestion: Performed using the FASP Protein Digestion Kit (Protein Discovery, San Diego, CA, USA) according to manufacturer’s instructions using 30 kDa cutoff spin filters. 90 µg total synovial fluid protein was digested using two µg sequencing grade modified trypsin (Promega, Fitchburg, USA), and the samples were digested overnight at 37°C. To assess the need of glycan removal when working with synovial fluid, 500 U peptide-N4-(N-acetyl-beta-glucosaminyl)-asparagine amidase (PNGase F) (New England BioLabs Inc, Ipswich, USA) was added to these samples prior to the trypsin digestion step, and incubated overnight at 37°C, after which the normal FASP protocol was continued. After trypsin digestion, the samples were desalted with TAGRA C18 columns (Nest Group Inc, Southborough, MA, USA) and resuspended in 5% ACN 5% formic acid (FA) prior to analysis. 2) Urea in-solution Digestion: Performed according to Gallien et al. 90 µg total synovial fluid protein was diluted with 8 M urea in 100 mM ammonium bicarbonate to a final volume of 25 µL. The sample was reduced with DTT at a final concentration of 12 mM for 30 min at 37°C, and alkylated with iodoacetamide at a final concentration of 40 mM for 1 h at 25°C in the dark. The samples were diluted with 100 mM ammonium bicarbonate to a total volume of 100 µL, 2 µg trypsin was added and the sample was digested overnight at 37°C. The samples were desalted with Nest group TAGRA C18 columns and resuspended in 5% ACN 5% FA prior to analysis. 3) In-gel Digestion: Three gel-lanes, each loaded with 150 µg total synovial fluid protein, were divided into 10 sections each and subjected to in-gel tryptic digestion, followed by analysis. Two state of the art mass spectrometers were utilized: 1) A 5600 TripleTOF (AB Sciex, Framingham, USA) connected online by a nanoflow HPLC NanoFlex system (Exigent, Redwood, USA) by a nanospray ion source. The samples were loaded onto a 15 cm reversed phase C18 200 µm chip with 2 µL/min in 100% solvent A (0.1% FA). The samples were then separated using a 15 cm reversed phase C18 75 µm chip, and eluted with a linear gradient of 2% solvent B (0.1% FA in ACN) which was raised to 35% solvent B over 120 min (60 min for in-gel digested samples) at a constant flow rate of 500 nL/min. 2) The urea FASP digested synovial fluid samples were also analyzed on a Q Exactive (Thermo Scientific, Waltham, USA) connected online to an EASY-nLC 1000 (Thermo Scientific, Waltham, USA) by a nanospray ion source. The samples were loaded onto a 10.5 cm reversed phase C18 PicoChip with a flow rate of approximately 1 µL/min in 98% solvent A and 2% solvent B, and eluted with eluent B using a linear gradient which was raised to 35% over 120 min at a constant flow rate of 300 nL/min. Data Processing Protocol The AB Sciex .wiff data-files were analyzed using ProteinPilot 4.5 (Rev. 1656, Paragon Algorithm 4.5.0.0). To identify the most commonly single observed PTMs, data-files were searched in thorough-mode with a focus on biological modifications in ProteinPilot to include more than 300 different PTMs [ProteinPilot Online Help]. The Thermo Scientific .raw data-files were analyzed using MaxQuant 1.4.1.2. All standard settings were employed with carbamidomethyl(C) as a static modification and deamidation (NQ), and protein N-terminal acetylation was included as variable modifications. Label-free quantitation of all proteins was performed in MaxQuant based on integrated precursor intensities. Protein abundances are represented as protein intensity-based absolute quantitation values (iBAQ), and are reported for all proteins having at least two quantifiable unique peptides in at least three LC-MS runs. Contact Tue Bjerg Bennike, Aalborg University Hanno Steen, Department of Pathology and Proteomics Center, Boston Children's Hospital, Harvard Medical School, MA, USA (lab head) Submission Date 04/08/2014 Publication Date 09/09/2014 Project PXD00093

A randomized trial of planned cesarean or vaginal delivery for twin pregnancy

Background: Twin birth is associated with a higher risk of adverse perinatal outcomes than singleton birth. It is unclear whether planned cesarean section results in a lower risk of adverse outcomes than planned vaginal delivery in twin pregnancy.\ud \ud Methods: We randomly assigned women between 32 weeks 0 days and 38 weeks 6 days of gestation with twin pregnancy and with the first twin in the cephalic presentation to planned cesarean section or planned vaginal delivery with cesarean only if indicated. Elective delivery was planned between 37 weeks 5 days and 38 weeks 6 days of gestation. The primary outcome was a composite of fetal or neonatal death or serious neonatal morbidity, with the fetus or infant as the unit of analysis for the statistical comparison.\ud \ud Results: A total of 1398 women (2795 fetuses) were randomly assigned to planned cesarean delivery and 1406 women (2812 fetuses) to planned vaginal delivery. The rate of cesarean delivery was 90.7% in the planned-cesarean-delivery group and 43.8% in the planned-vaginal-delivery group. Women in the planned-cesarean-delivery group delivered earlier than did those in the planned-vaginal-delivery group (mean number of days from randomization to delivery, 12.4 vs. 13.3; P = 0.04). There was no significant difference in the composite primary outcome between the planned-cesarean-delivery group and the planned-vaginal-delivery group (2.2% and 1.9%, respectively; odds ratio with planned cesarean delivery, 1.16; 95% confidence interval, 0.77 to 1.74; P = 0.49).\ud \ud Conclusion: In twin pregnancy between 32 weeks 0 days and 38 weeks 6 days of gestation, with the first twin in the cephalic presentation, planned cesarean delivery did not significantly decrease or increase the risk of fetal or neonatal death or serious neonatal morbidity, as compared with planned vaginal delivery