Lung on a chip: Co-culture of alveolar epithelial cells and bone marrow derived stromal stem cells in a microfluidic system

Background: Microfluidics system are novel tools to study cell-cell interactions in vitro. This project focuses on the development of a new microfluidic device to co-culture alveolar epithelial cells and mesenchymal stem cells to study cellular interactions involved in healing the injured alveolar epithelium. Methods: Microfluidic systems in polydimethylsiloxane were fabricated by soft lithography. The alveolar A549 epithelial cells were seeded and injury tests were made on the cells by perfusion with media containing H2O2 or bleomycin during 6 or 18hrs. Rat Bone marrow derived stromal cells (BMSC) were then introduced into the system and cell-cell interaction was studied over 24 hrs. Results: A successful co-culture of A549 alveolar epithelial cells and BMS was achieved in the microfluidic system. The seeded alveolar epithelial cells and BMSC adhered to the bottom surface of the microfluidic device and proliferated under constant perfusion. Epithelial injury to mimic mechanisms seen in idiopathic pulmonary fibrosis was induced in the microchannels by perfusing with H2O2 or bleomycin. Migration of BMSC towards the injured epithelium was observed as well as cell-cell interaction between the two cell types was also seen. Conclusion: We demonstrate a novel microfluidic device aimed at showing interactions between different cell types on the basis of a changing microenvironment. Also we were able to confirm interaction between injured alvolar epithelium and BMSC, and showed that BMSC try to heal the injured epitelium.

Comparative analysis of canine monocyte- and bone-marrow-derived dendritic cells

Dendritic cells (DC) represent a heterogeneous cell family of major importance for innate immune responses against pathogens and antigen presentation during infection, cancer, allergy and autoimmunity. The aim of the present study was to characterize canine DC generated in vitro with respect to their phenotype, responsiveness to toll-like receptor (TLR) ligands and T-cell stimulatory capacity. DC were derived from monocytes (MoDC) and from bone marrow hematopoietic cells cultured with either Flt3-ligand (FL-BMDC) or with GM-CSF (GM-BMDC). All three methods generated cells with typical DC morphology that expressed CD1c, CD11c and CD14, similar to macrophages. However, CD40 was only found on DC, CD206 on MPhi and BMDC, but not on monocytes and MoDC. CD1c was not found on monocytes but on all in vitro differentiated cells. FL-BMDC and GM-BMDC were partially positive for CD4 and CD8. CD45RA was expressed on a subset of FL-BMDC but not on MoDC and GM-BMDC. MoDC and FL-DC responded well to TLR ligands including poly-IC (TLR2), Pam3Cys (TLR3), LPS (TLR4) and imiquimod (TLR7) by up-regulating MHC II and CD86. The generated DC and MPhi showed a stimulatory capacity for lymphocytes, which increased upon maturation with LPS. Taken together, our results are the basis for further characterization of canine DC subsets with respect to their role in inflammation and immune responses.

Functionalisation of PLLA nanofiber scaffolds using a possible cooperative effect between collagen type I and BMP-2: impact on colonization and bone formation in vivo

The reconstruction of large bone defects after injury or tumor resection often requires the use of bone substitution. Artificial scaffolds based on synthetic biomaterials can overcome disadvantages of autologous bone grafts, like limited availability and donor side morbidity. Among them, scaffolds based on nanofibers offer great advantages. They mimic the extracellular matrix, can be used as a carrier for growth factors and allow the differentiation of human mesenchymal stem cells. Differentiation is triggered by a series of signaling processes, including integrin and bone morphogenetic protein (BMP), which act in a cooperative manner. The aim of this study was to analyze whether these processes can be remodeled in artificial poly-(l)-lactide acid (PLLA) based nanofiber scaffolds in vivo. Electrospun matrices composed of PLLA-collagen type I or BMP-2 incorporated PLLA-collagen type I were implanted in calvarial critical size defects in rats. Cranial CT-scans were taken 4, 8 and 12 weeks after implantation. Specimens obtained after euthanasia were processed for histology and immunostainings on osteocalcin, BMP-2 and Smad5. After implantation the scaffolds were inhomogeneously colonized and cells were only present in wrinkle- or channel-like structures. Ossification was detected only in focal areas of the scaffold. This was independent of whether BMP-2 was incorporated in the scaffold. However, cells that migrated into the scaffold showed an increased ratio of osteocalcin and Smad5 positive cells compared to empty defects. Furthermore, in case of BMP-2 incorporated PLLA-collagen type I scaffolds, 4 weeks after implantation approximately 40 % of the cells stained positive for BMP-2 indicating an autocrine process of the ingrown cells. These findings indicate that a cooperative effect between BMP-2 and collagen type I can be transferred to PLLA nanofibers and furthermore, that this effect is active in vivo. However, this had no effect on bone formation. The reason for this seems to be an unbalanced colonization of the scaffolds with cells, due to insufficient pore size.

Impact of estrogen replacement throughout childhood on growth, pituitary-gonadal axis and bone in a 46,XX patient with CYP19A1 deficiency

The adequate replacement dose of estrogens during infancy and childhood is still not known in girls. Aromatase deficiency offers an excellent model to study how much estrogens are needed during infancy, childhood and adulthood.

Genome-wide identification of quantitative trait loci in a cross between Hampshire and Landrace I: carcass traits

We report the identification of quantitative trait loci (QTL) affecting carcass composition, carcass length, fat deposition and lean meat content using a genome scan across 462 animals from a combined intercross and backcross between Hampshire and Landrace pigs. Data were analysed using multiple linear regression fitting additive and dominance effects. This model was compared with a model including a parent-of-origin effect to spot evidence of imprinting. Several precisely defined muscle phenotypes were measured in order to dissect body composition in more detail. Three significant QTL were detected in the study at the 1% genome-wide level, and twelve significant QTL were detected at the 5% genome-wide level. These QTL comprise loci affecting fat deposition and lean meat content on SSC1, 4, 9, 10, 13 and 16, a locus on SSC2 affecting the ratio between weight of meat and bone in back and weight of meat and bone in ham and two loci affecting carcass length on SSC12 and 17. The well-defined phenotypes in this study enabled us to detect QTL for sizes of individual muscles and to obtain information of relevance for the description of the complexity underlying other carcass traits.

Cyclooxygenase-2 inhibition selectively attenuates bone morphogenetic protein-6 synthesis and bone formation during guided tissue regeneration in a rat model

OBJECTIVES: Bone formation during guided tissue regeneration is a tightly regulated process involving cells, extracellular matrix and growth factors. The aims of this study were (i) to examine the expression of cyclooxygenase-2 (COX-2) during bone regeneration and (ii) the effects of selective COX-2 inhibition on osseous regeneration and growth factor expression in the rodent femur model. MATERIAL AND METHODS: A standardized transcortical defect of 5 x 1.5 mm was prepared in the femur of 12 male rats and a closed half-cylindrical titanium chamber was placed over the defect. The expression of COX-2 and of platelet-derived growth factor-B (PDGF-B), bone morphogenetic protein-6 (BMP-6) and insulin-like growth factor-I/II (IGF-I/II) was analyzed at Days 3, 7, 21 and 28 semiquantitatively by reverse transcriptase-polymerase chain reaction and immunohistochemistry. The effects of COX-2 inhibition by intraperitoneal injection of NS-398 (3 mg/kg/day) were analyzed in five additional animals sacrificed at Day 14. RESULTS: Histomorphometry revealed that new bone formation occurred in the cortical defect area as well as in the supracortical region, i.e. region within the chamber by Day 7 and increased through Day 28. Immunohistochemical evidence of COX-2 and PDGF-B levels were observed early (i.e. Day 3) and decreased rapidly by Day 7. BMP-6 expression was maximal at Day 3 and slowly declined by Day 28. In contrast, IGF-I/II expression gradually increased during the 28-day period. Systemic administration NS-398 caused a statistically significant reduction (P<0.05) in new bone formation (25-30%) and was associated with a statistically significant reduction in BMP-6 protein and mRNA expression (50% and 65% at P<0.05 and P<0.01, respectively). PDGF-B mRNA or protein expression was not affected by NS-398 treatment. CONCLUSION: COX-2 inhibition resulted in reduced BMP-6 expression and impaired osseous regeneration suggesting an important role for COX-2-induced signaling in BMP synthesis and new bone formation.

Malignant melanoma and bone resorption

The cellular and humoral mechanisms accounting for osteolysis in skeletal metastases of malignant melanoma are uncertain. Osteoclasts, the specialised multinucleated cells that carry out bone resorption, are derived from monocyte/macrophage precursors. We isolated tumour-associated macrophages (TAMs) from metastatic (lymph node/skin) melanomas and cultured them in the presence and absence of osteoclastogenic cytokines and growth factors. The effect of tumour-derived fibroblasts and melanoma cells on osteoclast formation and resorption was also analysed. Melanoma TAMs (CD14+/CD51-) differentiated into osteoclasts (CD14-/CD51+) in the presence of receptor activator for nuclear factor kappaB ligand (RANKL) and macrophage-colony stimulating factor. Tumour-associated macrophage-osteoclast differentiation also occurred via a RANKL-independent pathway when TAMs were cultured with tumour necrosis factor-alpha and interleukin (IL)-1alpha. RT-PCR showed that fibroblasts isolated from metastatic melanomas expressed RANKL messenger RNA and the conditioned medium of cultured melanoma fibroblasts was found to be capable of inducing osteoclast formation in the absence of RANKL; this effect was inhibited by the addition of osteoprotegerin (OPG). We also found that cultured human SK-Mel-29 melanoma cells produce a soluble factor that induces osteoclast differentiation; this effect was not inhibited by OPG. Our findings indicate that TAMs in metastatic melanomas can differentiate into osteoclasts and that melanoma fibroblasts and melanoma tumour cells can induce osteoclast formation by RANKL-dependent and RANKL-independent mechanisms, respectively.

Reattachment of the tuberosities with cable wires and bone graft in hemiarthroplasties done for proximal humeral fractures with cable wire and bone graft: 58 patients with a 22-month minimum follow-up

OBJECTIVE: The stability of 2 fixation techniques for the tuberosities in patients with 3- or 4-part proximal humerus fractures treated with hemiarthroplasties was compared. DESIGN: Retrospective review of a nonrandomized sequential series of patients. SETTING: Level I university orthopaedic surgery department. PATIENTS: A consecutive series of 58 patients (average age, 64 years) from 1990 to 1999 with 3- and 4-part fractures of the proximal humerus. INTERVENTION: In group 1, 31 patients were treated with either a Neer or Aequalis shoulder prosthesis using nonabsorbable sutures and no bone graft for the reattachment of the tuberosities. In group 2, 27 patients were treated with either an Aequalis or Epoca shoulder prosthesis and a combination of cable fixation and bone grafting. MAIN OUTCOME MEASUREMENTS: At follow-up (average, 32 months), radiographs were taken to confirm tuberosity fixation or degree of displacement or resorption. Functional outcome was assessed by the Constant-Murley Score. RESULTS: Significantly more dislocated tuberosities were found radiographically in group 1 (10 of 13 in total, P = 0.011), and significantly more tuberosities were resorbed in group 1 (9 of 12 in total, P = 0.012). Significant differences in functional results among healed versus failed tuberosity fixation were observed for activity of daily living (P = 0.05), range of motion (P = 0.002), strength (P = 0.01), the total score (P = 0.008), and the passive rotation amplitude (P = 0.04). CONCLUSION: In hemiarthroplasties for proximal humeral fractures, the reattachment of the tuberosities with cable wire and bone grafting gives consistently better radiographic and functional results than with suture fixation alone.

Interventional management of hypervascular osseous metastasis: role of embolotherapy before orthopedic tumor resection and bone stabilization

OBJECTIVE: The purpose of this study was to evaluate, in relation to intraoperative estimated blood loss (EBL), the effectiveness of preoperative transcatheter arterial embolization of hypervascular osseous metastatic lesions before orthopedic resection and stabilization. MATERIALS AND METHODS: Between June 1987 and November 2007, 22 patients underwent transcatheter arterial embolization of tumors of the long bone, hip, or vertebrae before resection and stabilization. Osseous metastatic lesions from renal cell carcinoma, malignant melanoma, leiomyosarcoma, and prostate cancer were embolized. All patients were treated with a coaxial catheter technique with polyvinyl alcohol (PVA) particles alone or a combination of PVA particles and coils. After embolization, each tumor was angiographically graded according to devascularization (grades 1-3) based on tumor blush after contrast injection into the main tumor-feeding arteries. RESULTS: In patients with complete devascularization (grade 1), mean EBL was calculated to be 1,119 mL, whereas in patients with partial embolization (grades 2 and 3) EBL was 1,788 mL and 2,500 mL. With respect to intraoperative EBL, no significant difference between devascularization grades was found (p > 0.05). Moderate correlation (r = 0.51, p = 0.019) was observed between intraoperative EBL and tumor size before embolization. Only low correlation (r = 0.44, p = 0.046) was found between intraoperative EBL and operating time. Major complications included transient palsy of the sciatic nerve and gluteal abscess in one patient. CONCLUSION: The results of this study support the concept that there is no statistically significant difference among amounts of intraoperative EBL with varying degrees of embolization.

Compressive strength of interbody cages in the lumbar spine: the effect of cage shape, posterior instrumentation and bone density

One goal of interbody fusion is to increase the height of the degenerated disc space. Interbody cages in particular have been promoted with the claim that they can maintain the disc space better than other methods. There are many factors that can affect the disc height maintenance, including graft or cage design, the quality of the surrounding bone and the presence of supplementary posterior fixation. The present study is an in vitro biomechanical investigation of the compressive behaviour of three different interbody cage designs in a human cadaveric model. The effect of bone density and posterior instrumentation were assessed. Thirty-six lumbar functional spinal units were instrumented with one of three interbody cages: (1) a porous titanium implant with endplate fit (Stratec), (2) a porous, rectangular carbon-fibre implant (Brantigan) and (3) a porous, cylindrical threaded implant (Ray). Posterior instrumentation (USS) was applied to half of the specimens. All specimens were subjected to axial compression displacement until failure. Correlations between both the failure load and the load at 3 mm displacement with the bone density measurements were observed. Neither the cage design nor the presence of posterior instrumentation had a significant effect on the failure load. The loads at 3 mm were slightly less for the Stratec cage, implying lower axial stiffness, but were not different with posterior instrumentation. The large range of observed failure loads overlaps the potential in vivo compressive loads, implying that failure of the bone-implant interface may occur clinically. Preoperative measurements of bone density may be an effective tool to predict settling around interbody cages.

Aromatase deficiency in a female who is compound heterozygote for two new point mutations in the P450arom gene: impact of estrogens on hypergonadotropic hypogonadism, multicystic ovaries, and bone densitometry in childhood

We report on a female who is compound heterozygote for two new point mutations in the CYP19 gene. The allele inherited from her mother presented a base pair deletion (C) occurring at P408 (CCC, exon 9), causing a frameshift that results in a nonsense codon 111 bp (37 aa) further down in the CYP19 gene. The allele inherited from her father showed a point mutation from G-->A at the splicing point (canonical GT to mutational AT) between exon and intron 3. This mutation ignores the splice site and a stop codon 3 bp downstream occurs. Aromatase deficiency was already suspected because of the marked virilization occurring prepartum in the mother, and the diagnosis was confirmed shortly after birth. Extremely low levels of serum estrogens were found in contrast to high levels of androgens. Ultrasonographic follow-up studies revealed persistently enlarged ovaries (19.5-22 mL) during early childhood (2 to 4 yr) which contained numerous large cysts up to 4.8 x 3.7 cm and normal-appearing large tertiary follicles already at the age of 2 yr. In addition, both basal and GnRH-induced FSH levels remained consistently strikingly elevated. Low-dose estradiol (E2) (0.4 mg/day) given for 50 days at the age of 3 6/12 yr resulted in normalization of serum gonadotropin levels, regression of ovarian size, and increase of whole body and lumbar spine (L1-L4) bone mineral density. The FSH concentration and ovarian size returned to pretreatment levels shortly (150 days) after cessation of E2 therapy. Therefore, we recommend that affected females be treated with low-dose E2 in amounts sufficient to result in physiological prepubertal E2 concentrations using an ultrasensitive estrogen assay. However, E2 replacement needs to be adjusted throughout childhood and puberty to ensure normal skeletal maturation and adequate adolescent growth spurt, normal accretion of bone mineral density, and, at the appropriate age, female secondary sex maturation.

Risk scoring for setting priorities in a monitoring of antimicrobial resistance in meat and meat products

Meat and meat products can be contaminated with different species of bacteria resistant to various antimicrobials. The human health risk of a type of meat or meat product carry by emerging antimicrobial resistance depends on (i) the prevalence of contamination with resistant bacteria, (ii) the human health consequences of an infection with a specific bacterium resistant to a specific antimicrobial and (iii) the consumption volume of a specific product. The objective of this study was to compare the risk for consumers arising from their exposure to antibiotic resistant bacteria from meat of four different types (chicken, pork, beef and veal), distributed in four different product categories (fresh meat, frozen meat, dried raw meat products and heat-treated meat products). A semi-quantitative risk assessment model, evaluating each food chain step, was built in order to get an estimated score for the prevalence of Campylobacter spp., Enterococcus spp. and Escherichia coli in each product category. To assess human health impact, nine combinations of bacterial species and antimicrobial agents were considered based on a published risk profile. The combination of the prevalence at retail, the human health impact and the amount of meat or product consumed, provided the relative proportion of total risk attributed to each category of product, resulting in a high, medium or low human health risk. According to the results of the model, chicken (mostly fresh and frozen meat) contributed 6.7% of the overall risk in the highest category and pork (mostly fresh meat and dried raw meat products) contributed 4.0%. The contribution of beef and veal was of 0.4% and 0.1% respectively. The results were tested and discussed for single parameter changes of the model. This risk assessment was a useful tool for targeting antimicrobial resistance monitoring to those meat product categories where the expected risk for public health was greater.

Neospora caninum and bone marrow-derived dendritic cells: parasite survival, proliferation, and induction of cytokine expression

Dendritic cells (DCs) represent the first line defence of the innate immune system following infection with pathogens. We exploratively addressed invasion and survival ability of Neospora caninum, a parasite causing abortion in cattle, in mouse bone marrow DCs (BMDCs), and respective cytokine expression patterns. Immature BMDCs were exposed to viable (untreated) and nonviable parasites that had been inactivated by different means. Invasion and/or internalization, as well as intracellular survival and proliferation of tachyzoites were determined by NcGRA2-RT-PCR and transmission electron microscopy (TEM). Cytokine expression was evaluated by reverse transcription (RT)-PCR and cytokine ELISA. Transmission electron microscopy of DCs stimulated with untreated viable parasites revealed that N. caninum was able to invade and proliferate within BMDCs. This was confirmed by NcGRA2-RT-PCR. On the other hand, no viable parasite organisms were revealed by TEM when exposing BMDCs to inactivated parasites (nonviability demonstrated by NcGRA2-RT-PCR). Cytokine expression analysis (as assessed by both RT-PCR and ELISA) demonstrated that both viable and nonviable parasites stimulated mBMDCs to express IL-12p40, IL-10 and TNF-alpha, whereas IL-4 RNA expression was not detected. Thus, exposure of mBMDCs to both viable and nonviable parasites results in the expression of cytokines that are relevant for a mixed Th1/Th2 immune response.

Comparison of genotypes and antibiotic resistances of Campylobacter jejuni and Campylobacter coli on chicken retail meat and at slaughter

Multilocus sequence typing (MLST) and antibiotic resistance patterns of Campylobacter jejuni and Campylobacter coli from retail chicken meat showed high overlap with isolates collected at slaughterhouses, indicating little selection along the production chain. They also showed significant common sequence types with human clinical isolates, revealing chicken meat as a likely source for human infection.

Responses to Adding Fat to Corn-Based Diets Supplemented with Urea and Soybean Meal for Finishing Steers

Yearling steers were fed corn-based diets supplemented with urea or soybean meal plus urea, and none, 2%, or 4% fat. All steers were implanted with Revalorâ-S and fed for 118 days. Adding fat did not improve performance of the steers in the feedlot or improve carcass characteristics. Feeding soybean meal increased rate of gain, improved feed efficiency, increased carcass weight, and tended to improve carcass quality grades compared with feeding urea. Adding 4% fat decreased feed intake, suggesting that corn-based diets may contain enough oil to approach the quantity of fat that can be utilized effectively in a ruminant diet.