Rich Tissue Research Articles

Enabling second harmonic generation as a contrast mechanism for optical projection tomography (OPT) and scanning laser optical tomography (SLOT).

Volumetric imaging of connective tissue provides insights into the structure of biological tissue. Second harmonic generation (SHG) microscopy has become a standard method to image collagen rich tissue like skin or cornea. Due to the non-centrosymmetric architecture, no additional label is needed and tissue can be visualized noninvasively. Thus, SHG microscopy enables the investigation of collagen associated diseases, providing high resolution images and a field of view of several hundreds of μm. However, the in toto visualization of larger samples is limited to the working distance of the objective and the integration time of the microscope setup, which can sum up to several hours and days. A faster imaging technique for samples in the mesoscopic range is scanning laser optical tomography (SLOT), which provides linear fluorescence, scattering and absorption as intrinsic contrast mechanisms. Due to the advantages of SHG and the reduced measurement time of SLOT, the integration of SHG in SLOT would be a great extension. This way SHG measurements could be performed faster on large samples, providing isotropic resolution and simultaneous acquisition of all other contrast mechanisms available, such as fluorescence and absorption. SLOT is based on the principle of computed tomography, which requires the rotation of the sample. The SHG signal, however, depends strongly on the sample orientation and the polarization of the laser, which results in SHG intensity fluctuation during sample rotation and prevents successful 3D reconstruction. In this paper we investigate the angular dependence of the SHG signal by simulation and experiment and found a way to eliminate reconstruction artifacts caused by this angular dependence in SHG-SLOT data. This way, it is now possible to visualize samples in the mesoscopic range using SHG-SLOT, with isotropic resolution and in correlation to other contrast mechanisms as absorption, fluorescence and scattering.

Spatial map of human B cell compartmentalization

Abstract We have performed a comprehensive analysis of human lymphoid tissue from relatively inaccessible sites including SPL, BM and LN from normal donors. Most of what is known about human B cells comes from blood, a tissue that contains only ~2% of the immune cells in the body. Examination of different tissues from 14 independent donors reveals for the first time the global B cell population structure. We performed unbiased computational clustering on samples stained with antibodies to markers indicative of antigen experience, activation status, and tissue retention. We found that the population structure of splenic B cells is remarkably conserved across a large number of donors who vary by age, gender, environment and history of pathogen exposure. By contrast, blood B cell composition is highly individualized with respect to each donor while BM has a relatively uniform population structure with some individual tailoring. Comparison of paired tissues within individuals further demonstrates that SPL and blood contain largely non-overlapping populations, suggesting geographic segregation of B cell subsets within each person. Recently published Ig clonal tracking studies from this donor cohort demonstrates partitioning of B cell clonal sharing into two broad networks, one across blood rich tissues and the other restricted to portions of the GI tract. Knowledge about the mechanisms that direct the trafficking and retention of discrete B cell subsets to specific tissues has direct implication to how infections are controlled throughout the body. Our findings constitute the basis for a geographic understanding of B cell population structure that will inform studies on the human humoral immune response.

Abstract 222: Translocator Protein as Novel Marker for Early Atherosclerosis

Atherosclerosis is characterized by lipid deposition, monocyte infiltration and foam cell formation in the artery wall. Translocator protein (TSPO) is abundantly expressed in lipid rich tissues. Recently, TSPO has been identified as a potential diagnostic tool in cardiovascular disease. Here, we aimed to determine if (i) the TSPO radioligand, 18 F-PBR111, can identify fatty streak formation that precedes atherosclerotic lesion development and (ii) TSPO expression can be used to identify distinct macrophage populations during different stages of lesion progression. Atherosclerosis-prone apoE -/- mice were maintained on a high-fat diet for 3, 9 and 12 weeks. C57BL6 mice maintained on chow diet served as controls. Mice were anesthetized, injected with 18 F-PBR111 (8-18 MBq, 0.1 mL, 0.2 nM) via the lateral tail vein and imaged using an Inveon PET/CT Scanner over 60 min. After euthanasia, aortas were isolated, fixed and optically cleared. Cleared aortas were immunostained with DAPI, fluorescently labelled anti-TSPO antibody and the macrophage markers F4/80 and CD11b. Tissues were visualized on a Lightsheet Z.1 microscope and analysed with 3D imaging software. A 3-fold increase in the uptake of 18 F-PBR111 was observed by PET/CT in the aortas of atherosclerotic mice relative to controls. Increased 18 F-PBR111 uptake was observed in the aortic arch and thoracic aorta, corresponding with sites of atherosclerotic lesion development. Light sheet microscopy revealed that TSPO expression increased correspondingly to atherosclerosis progression. The expression pattern of macrophage populations in early lesions differed from that in mature plaques. While F4/80-positive macrophages were evident uniformly throughout the aortas, CD11b-positive macrophages were increased in atherosclerotic areas. In conclusion, imaging TSPO expression is a new approach for studying atherosclerotic lesion progression and is associated with specific inflammatory cell infiltration. The TSPO ligand 18 F-PBR111 is a potential clinical diagnostic tool for the detection and quantification of atherosclerotic lesion progression in humans.

Pulp Regeneration by 3-dimensional Dental Pulp Stem Cell Constructs

Dental pulp regeneration therapy for the pulpless tooth has attracted recent attention, and clinical trial studies are underway with the tissue engineering approach. However, there remain many concerns, including the extended period for regenerating the dental pulp. In addition, the use of scaffolds increases the risk of inflammation and infection. To establish a basic technology for novel dental pulp regenerative therapy that allows transplant of pulp-like tissue, we attempted to fabricate scaffold-free 3-dimensional (3D) cell constructs composed of dental pulp stem cells (DPSCs). Furthermore, we assessed viability of these 3D DPSC constructs for dental pulp regeneration through in vitro and in vivo studies. For the in vitro study, we obtained 3D DPSC constructs by shaping sheet-like aggregates of DPSCs with a thermoresponsive hydrogel. DPSCs within constructs remained viable even after prolonged culture; furthermore, 3D DPSC constructs possessed a self-organization ability necessary to serve as a transplant tissue. For the in vivo study, we filled the human tooth root canal with DPSC constructs and implanted it subcutaneously into immunodeficient mice. We found that pulp-like tissues with rich blood vessels were formed within the human root canal 6 wk after implantation. Histologic analyses revealed that transplanted DPSCs differentiated into odontoblast-like mineralizing cells at sites in contact with dentin; furthermore, human CD31–positive endothelial cells were found at the center of regenerated tissue. Thus, the self-organizing ability of 3D DPSC constructs was active within the pulpless root canal in vivo. In addition, blood vessel–rich pulp-like tissues can be formed with DPSCs without requiring scaffolds or growth factors. The technology established in this study allows us to prepare DPSC constructs with variable sizes and shapes; therefore, transplantation of DPSC constructs shows promise for regeneration of pulpal tissue in the pulpless tooth.

Raman, AFM and SNOM high resolution imaging of carotene crystals in a model carrot cell system

Three non-destructive and complementary techniques, Raman imaging, Atomic Force Microscopy and Scanning Near-field Optical Microscopy were used simultaneously to show for the first time chemical and structural differences of carotenoid crystals. Spectroscopic and microscopic scanning probe measurements were applied to the released crystals or to crystals accumulated in a unique, carotenoids rich callus tissue growing in vitro that is considered as a new model system for plant carotenoid research. Three distinct morphological crystal types of various carotenoid composition were identified, a needle-like, rhomboidal and helical. Raman imaging using 532 and 488 nm excitation lines provided evidence that the needle-like and rhomboidal crystals had similar carotenoid composition and that they were composed mainly of β-carotene accompanied by α-carotene. However, the presence of α-carotene was not identified in the helical crystals, which had the characteristic spatial structure. AFM measurements of crystals identified by Raman imaging revealed the crystal topography and showed the needle-like and rhomboidal crystals were planar but they differed in all three dimensions. Combining SNOM and Raman imaging enabled indication of carotenoid rich structures and visualised their distribution in the cell. The morphology of identified subcellular structures was characteristic for crystalline, membraneous and tubular chromoplasts that are plant organelles responsible for carotenoid accumulation in cells.

Relative Target Affinities of T-Cell-Dependent Bispecific Antibodies Determine Biodistribution in a Solid Tumor Mouse Model.

Anti-HER2/CD3, a T-cell-dependent bispecific antibody (TDB) construct, induces T-cell-mediated cell death in cancer cells expressing HER2 by cross-linking tumor HER2 with CD3 on cytotoxic T cells, thereby creating a functional cytolytic synapse. TDB design is a very challenging process that requires consideration of multiple parameters. Although therapeutic antibody design strategy is commonly driven by striving for the highest attainable antigen-binding affinity, little is known about how the affinity of each TDB arm can affect the targeting ability of the other arm and the consequent distribution and efficacy. To our knowledge, no distribution studies have been published using preclinical models wherein the T-cell-targeting arm of the TDB is actively bound to T cells. We used a combined approach involving radiochemistry, invasive biodistribution, and noninvasive single-photon emission tomographic (SPECT) imaging to measure TDB distribution and catabolism in transgenic mice with human CD3ε expression on T cells. Using CD3 affinity variants, we assessed the impact of CD3 affinity on short-term pharmacokinetics, tissue distribution, and cellular uptake. Our experimental approach determined the relative effects of (i) CD3 targeting to normal tissues, (ii) HER2 targeting to HER2-expressing tumors, and (iii) relative HER2/CD3 affinity, all as critical drivers for TDB distribution. We observed a strong correlation between CD3 affinity and distribution to T-cell-rich tissues, with higher CD3 affinity reducing systemic exposure and shifting TDB distribution away from tumor to T-cell-containing tissues. These observations have important implications for clinical translation of bispecific antibodies for cancer immunotherapy. Mol Cancer Ther; 17(4); 776-85. ©2018 AACR.

Molecular In Vivo Imaging of Bone Marrow Adipose Tissue

The in vivo study of molecular processes in human bone marrow is important for both diagnostics and understanding of disease pathophysiology. Traditionally, the hematopoietic component of the bone marrow has been a research focus, but recently, the role of bone marrow adipose tissue has been gaining interest in many applications. The purpose of the present review is to give an overview of existing imaging modalities allowing in vivo molecular imaging of bone marrow adipose tissue in humans with an emphasis on technical aspects: the characteristics of the extracted parameters and their application in bone marrow adipose tissue. Magnetic resonance (MR) imaging (MRI) and spectroscopy (MRS) are the most frequently used imaging methods for the examination of bone marrow adipose tissue as they provide rich soft tissue contrast and come without ionizing radiation. Existing MR methods allow the extraction of many different measures including proton density fat fraction, fatty acid characteristics, and diffusion and perfusion properties. However, many available techniques have to be carefully adjusted to be used in the investigation of the fat signal component, especially in the presence of trabecular bone. Dual-energy computed tomography (DECT) is an emerging technique—not yet widely available—which appears to be a promising alternative to MR for rapid fat fraction assessment. Positron emission tomography (PET) allows additional functional metabolic imaging and therefore provides valuable additional information (e.g., glucose uptake) to MR-based parameters at the cost of ionizing radiation. Bone marrow imaging still appears to be a niche with remaining technical challenges using existing imaging modalities. A good working knowledge of the underlying physical and technical principles is required as most techniques are yet not available out of the box and may need to be adjusted to fit the requirements for bone marrow adipose tissue imaging. In summary, MR, DECT, and PET enable the measurement of several inherently different parameters in in vivo molecular imaging of bone marrow adipose tissue. The growing interest for molecular imaging markers of bone marrow, thanks to its high metabolic and clinical significance, may eventually lead to new developments, as well as improvements of emerging techniques as soon as they become more broadly available.

95: Detecting plasma desmosine as a marker for elastic fiber degradation in pelvic floor connective tissue during the postpartum period

Elastic fibers in pelvic floor connective tissue are critical for pelvic organ support. Their breakdown and incomplete repair following parturition has been hypothesized as a mechanism to explain the association between childbirth and pelvic floor disorders. Desmosine and isodesmosine (desmosine) are amino acids detectable in bodily fluids, capable of reflecting elastic fiber degradation in elastin rich tissue. Their ability to reflect elastic fiber degradation in the pelvic floor is unknown. Therefore, we investigated whether the detection of plasma desmosine could reflect elastic fiber degradation in pelvic floor connective tissue. We hypothesized that direct trauma to pelvic floor tissue incurred during vaginal delivery will cause greater elastic fiber disruption resulting in an increased level of detectable plasma desmosine postpartum when compared to cesarean delivery. Forty-one primiparous women following uncomplicated pregnancies agreed to participate in this study, 24 following vaginal delivery and 17 following cesarean delivery. Twenty-three reproductive age nulliparous women also participated. Blood draws for plasma collection were performed early (1-4 days) and late (6-8 weeks) postpartum. Desmosine quantification was performed using liquid chromatography tandem mass spectroscopy. Our primary outcome was the comparison of desmosine concentration by mode of delivery early postpartum. The Mann Whitney U test was applied with p < 0.05 noting significance. Age, race, maternal weight, and gestational age were similar between postpartum groups. Median infant birth weight was greater in the cesarean delivery group (3538.8 grams vs. 3103.3 grams, p = 0.004). Median desmosine level was greater in the cesarean delivery group (0.51 ng/ml vs 0.40 ng/ml, p = 0.04). However, upon linear regression modeling controlling for age and infant birth weight, this difference did not remain significant (95% CI -0.01 to 0.20). The desmosine level in reproductive age nulliparous women, with a median age of 29.0 years, was 0.23 ng/ml. Counter to our hypothesis plasma desmosine concentration was higher following cesarean delivery. We suspect this finding reflects uterine elastic fiber remodeling after a hysterotomy, though this result may be confounded by infant birth weight. Antepartum plasma desmosine values would aid our understanding of this relationship. Another important observation was the low desmosine level seen in young, nulliparous women. Taken together, these findings confirm that plasma desmosine can reflect reproductive tract elastic fiber degradation. Further research is needed to determine if elastic fiber degradation occurring in pelvic floor tissue can be detected by plasma desmosine quantification.

King\u2019s Health Partners\u2019 Prostate Cancer Biobank (KHP PCaBB)

The KHP PCaBB was established in 2013 and recruits donors from the Urology or Oncology Departments at Guy’s Hospital in London (UK). Prostate cancer patients may be approached to give their consent for biobanking at any point in their treatment pathway, which allows residual material from their earlier diagnosis to be transferred and used by the Biobank. Currently, patients are specifically asked to donate samples of blood and surplus prostate tissue as well as permitting access to their clinical and pathological data that continues to be added throughout the course of their disease. Between 2013 and 2015, 549 prostate cancer patients gave their consent to the biobank and, the tissue repository collected 489 blood samples, 120 frozen prostate tissue samples and 1064 formalin fixed paraffin embedded diagnostic blocks.Prostate cancer has become a chronic disease in a large proportion of men, with many men receiving multiple subsequent treatments, and their treatment trajectory often spanning over decades. Therefore, this resource aims to provide an ideal research platform to explore potential variations in treatment response as well as disease markers in the different risk categories for prostate cancer.A recent audit of the KHP PCaBB revealed that between 2013 and 2015, 1796 patients were diagnosed with prostate cancer at King’s Health Partners (KHP), out of which 549 (30.6%) gave their consent to KHP PCaBB. Comparisons between demographic and clinical characteristics of patients who had consented compared to the total patient population revealed that the KHP PCaBB is demographically representative of the total prostate cancer patient population seen in Guy’s and St Thomas’ NHS Foundation Trust (GSTT). We observed no differences in distribution of ethnicity (p = 0.507) and socioeconomic status (p = 0.097). Some differences were observed in clinical characteristics, specifically with treatment type – which differed significantly between the patients who had given consent and total patient population.The KHP PCaBB has thereby amassed a rich data and tissue repository that is largely reflective of both the demographic and clinical diversity within the total prostate cancer patient population seen at KHP, making it an ideal platform for prostate cancer research.

Periodic acid-Schiff (PAS) reaction and plastination in whole body slices. A novel technique to identify fascial tissue structures

Since collagen rich fascial tissue is often very delicate and difficult to discern on native tissue slices, we have developed a method for staining full-body slices using the periodic acid-Schiff (PAS) reaction with subsequent plastination. Since the PAS reaction primarily stains carbohydrates, we could exploit the circumstance that different collagen types vary in carbohydrate content. Contrary to fasciae, tissues such as muscle, bone, nerves and blood vessels exhibit significantly less staining or remain unstained. We have validated the whole-body slice staining results in microscopic tissue slides which were stained with standard extracellular matrix stains such as Masson-Goldner trichrome stain and van-Gieson stain. Furthermore, we have performed immunofluorescence imaging to confirm the presence of collagen in the stained tissue. We achieved very good staining and plastination results and were able to clearly identify even very thin fascia in transversal body slices. This technique may prove useful in advancing our knowledge on the complex topography of fascial structures.

A histological study of tail width variation in the greater Japanese shrew-mole,Urotrichus talpoides

The greater Japanese shrew-mole, Urotrichus talpoides, has a wide and thick tail like a baseball bat with bristles like bottle brushes. It is known that not only length variations but also the width variations are observed in the tails of U. talpoides. To understand such width variations of the tail, we examined morphological and histological characteristics. The width variation was not related to the season when captured or aging, as indicated by the skull sizes. In addition, tail vertebra width was not correlated with tail width. On the other hand, according to histological observation of tail skin, the ratio of subcutaneous tissue/corium indicating the thickness of the subcutaneous tissue layer was positively correlated with the tail width (p < 0.05). The thickness of the subcutaneous tissue layer means that the rich adipose tissue and cells observed were unilocular, then identified as white adipose cells. It is well known that one of the functions of white adipose cells is to store excess energy as neutral fat. Thus, it is suggested that U. talpoides stores an energy source as adipose cells in the skin of the tail, demonstrating that tail width variations are caused by the thickness of the subcutaneous tissue layer.

Physiochemical and nutritional characterization of coconut (Cocos nucifera L.) haustorium based extrudates

Rice and maize are commonly used for the production of extrudates and incorporation of nutritionally rich materials is prime important to make extrudates nutritionally superior. Coconut haustorium, a nutrient rich spongy tissue formed during coconut germination, was added to rice and maize (1:1) flour at levels of 0%, 10%, 20% and 30% (w/w) by replacing the maize and extruded in twin-screw extruder. Effect of addition of haustorium on the physico-chemical parameters like bulk density, expansion ratio, water absorption index, water solubility index, hardness, breaking force, breaking strength, compressive strength, color parameters like L*, hue, and chroma value, sensory characteristics and biochemical parameters like proximates, minerals, phenolics, flavonoids and antioxidant potential in the extrudates were studied. Results indicated that the addition of haustorium had a significant effect on parameters studied. Incorporation of haustorium resulted in less expanded, lengthy, dark colored, hardened, protein, minerals, phenolics and flavonoids rich extrudates with high anti-oxidant activity. A combination of 50% rice, 30% maize and 20% haustorium was found suitable for healthy and nutritious extrudates preparation based on the biochemical constituents and overall sensory acceptability.

Determination of extracellular matrix collagen fibril architectures and pathological remodeling by polarization dependent second harmonic microscopy

Polarization dependence second harmonic generation (P-SHG) microscopy is gaining increase popularity for in situ quantification of fibrillar protein architectures. In this report, we combine P-SHG microscopy, new linear least square (LLS) fitting and modeling to determine and convert the complex second-order non-linear optical anisotropy parameter ρ of several collagen rich tissues into a simple geometric organization of collagen fibrils. Modeling integrates a priori knowledge of polyhelical organization of collagen molecule polymers forming fibrils and bundles of fibrils as well as Poisson photonic shot noise of the detection system. The results, which accurately predict the known sub-microscopic hierarchical organization of collagen fibrils in several tissues, suggest that they can be subdivided into three classes according to their microscopic and macroscopic hierarchical organization of collagen fibrils. They also show, for the first time to our knowledge, intrahepatic spatial discrimination between genuine fibrotic and non-fibrotic vessels. CCl4-treated livers are characterized by an increase in the percentage of fibrotic vessels and their remodeling involves peri-portal compaction and alignment of collagen fibrils that should contribute to portal hypertension. This integrated P-SHG image analysis method is a powerful tool that should open new avenue for the determination of pathophysiological and chemo-mechanical cues impacting collagen fibrils organization.

Toxicokinetics, disposition and metabolism of fluoxetine in crabs

The disposition and metabolism of fluoxetine in the European shore crab and the Dungeness crab were assessed. Crabs received intracardiac doses of either 0.13 μg/kg or 0.5 mg/kg fluoxetine, respectively. In addition, fluoxetine was administered to Metacarcinus cancer by oral gavage at 7.8 mg/kg. The distribution of fluoxetine was quantified in haemolymph and digestive gland for both crabs, as well as brain, muscle, and testis of Carcinus maenas, over 12 days. The metabolite norfluoxetine, was also measured in C. maenas. Fluoxetine was mainly found in lipid rich tissues. Distribution coefficients increased for digestive gland until three days after fluoxetine administration and then decreased until the end of the observations. The highest distribution coefficients were obtained for brain. Norfluoxetine displayed continuously high levels in digestive gland and brain. The strong decrease in fluoxetine and the concomitant increase in norfluoxetine demonstrates that decapod crustaceans metabolise fluoxetine into the more biologically active norfluoxetine. Fluoxetine levels in the haemolymph of M. cancer declined within 20 h, but showed a second peak 25 h later, suggesting remobilisation from tissues sequestering the compound. The steady state volume distribution and the total body clearance of fluoxetine were high, consistent with high diffusion of fluoxetine into the peripheral tissues and biotransformation as an important elimination pathway. Oral administration of fluoxetine prolonged its half-life in M. cancer, but bioavailability was low. These results confirm the high distribution into nervous tissue, extensive biotransformation into the highly active norfluoxetine and a half-life similar to that observed in vertebrates.

COMPARISON OF ORGANOCHLORINE PESTICIDE LEVELS BETWEEN HUMAN BLOOD SERUM AND ADIPOSE TISSUE

The organochlorine pesticides, being lipophilic compounds, show extremely high thermodynamic stability and resistance to degradation processes in the environment and in living organisms. They accumulate in lipid rich tissues and distribute between transport compartment (blood) and store compartment (adipose tissue). The purpose of this study is to make a comparison between organochlorine pesticide levels in blood serum and adipose tissue and calculate the differences in their concentrations based on routine biomonitoring study. One hundred and twenty six pair samples of adipose tissue and blood serum during autopsies as a case study were taken and analyzed in Los Mochis Sinaloa, Mexico. Among organochlorine pesticides, higher concentrations correspond to b-HCH, pp’DDE and op’DDT in blood serum lipids; and pp’DDT shows higher concentrations in adipose tissue. Using statistical comparisons, we found a significant linear association of lipid serum organochlorine pesticide concentrations with that in adipose tissue. This concludes that serum lipid organochlorine pesticide concentrations represent an important indicator for both human biological matrices.

Predicting multicellular function through multi-layer tissue networks.

MotivationUnderstanding functions of proteins in specific human tissues is essential for insights into disease diagnostics and therapeutics, yet prediction of tissue-specific cellular function remains a critical challenge for biomedicine.ResultsHere, we present OhmNet, a hierarchy-aware unsupervised node feature learning approach for multi-layer networks. We build a multi-layer network, where each layer represents molecular interactions in a different human tissue. OhmNet then automatically learns a mapping of proteins, represented as nodes, to a neural embedding-based low-dimensional space of features. OhmNet encourages sharing of similar features among proteins with similar network neighborhoods and among proteins activated in similar tissues. The algorithm generalizes prior work, which generally ignores relationships between tissues, by modeling tissue organization with a rich multiscale tissue hierarchy. We use OhmNet to study multicellular function in a multi-layer protein interaction network of 107 human tissues. In 48 tissues with known tissue-specific cellular functions, OhmNet provides more accurate predictions of cellular function than alternative approaches, and also generates more accurate hypotheses about tissue-specific protein actions. We show that taking into account the tissue hierarchy leads to improved predictive power. Remarkably, we also demonstrate that it is possible to leverage the tissue hierarchy in order to effectively transfer cellular functions to a functionally uncharacterized tissue. Overall, OhmNet moves from flat networks to multiscale models able to predict a range of phenotypes spanning cellular subsystems. Availability and implementationSource code and datasets are available at http://snap.stanford.edu/ohmnet.

Microstructure dependent binding of pigment epithelium derived factor (PEDF) to type I collagen fibrils.

Pigment epithelium derived factor (PEDF) is a multifunctional extracellular protein. In addition to its known anti-angiogenic and neurotrophic roles in collagen rich tissues, PEDF is thought to be involved in collagen fibril assembly due to its sequence specific binding to the collagen fibril and high expression in regions of active bone formation. In order to image the presence of the protein on the fibrils, PEDF was recombinantly made with a strep tag (strep-PEDF) and then gold nanoparticles conjugated to streptavidin (AuNP) were used as a secondary tag. The gold nanoparticles were detected using phase imaging in tapping mode AFM to image where exogenous PEDF bound in rabbit femur. These findings demonstrate that PEDF binds heterogeneously in cortical rabbit femur. Exogenous PEDF binding was concentrated at areas between microstructures with highly aligned collagen fibrils. Binding was not observed on or within the collagen fibrils themselves.

Biological activity evaluation of magnesium fluoride coated Mg-Zn-Zr alloy in vivo

AimTo explore the biodegradable characteristics and biological properties, which could promote new bone formation, of MgF2 coated magnesium alloy (Mg-3wt%Zn-0.5wt%Zr) in rabbits. MethodsMagnesium alloy with MgF2 coating was made and the MgF2/Mg-Zn-Zr was implanted in the femoral condyle of rabbits. Twelve healthy adult Japanese white rabbits in weight of 2.8–3.2kg were averagely divided into A(Mg-Zn-Zr) group and B(MgF2/MgZn-Zr) group. Indexes such as microstructural evolution, SEM scan, X-ray, Micro-CT and mechanical properties were observed and detected at 1th day, 2th, 4th, 8th, 12th, 24th week after implantation. ResultsLow-density regions occurred around the cancellous bone, and the regions gradually expanded during the 12weeks after implantation. The implant was gradually absorbed from 12 to 24weeks. The density of surrounding cancellous bone increased compared with the 12th week data. The degradation rate of B group was lower than that of A group (P<0.01), while the density of the surrounding cancellous bone increased more evenly. In B group, SEM images after 12weeks showed the rich bone tissues on the alloy surface that were attached by active fibers. Micro-CT also presented alloy residue potholes on the surfaces of alloy combinated with bone tissues. Additionally, the trabecular bone had relatively integrated structures with surrounding cavities. ConclusionsMgF2 can effectively decrease the degradation rate of Mg-Zn-Zr in vivo. Mg-Zn-Zr coated with MgF2 can effectively inhibit the corrosion, and delay the release of magnesium ions. The biological properties of the coating itself presented good biocompatibility and bioactivity.

Evidence of stress in β cells obtained with laser capture microdissection from pancreases of brain dead donors

ABSTRACTIsolated islets used for transplantation are known to be stressed, which can result from the circumstances of death, in particular brain death, the preservation of the pancreas with its warm and cold ischemia, from the trauma of the isolation process, and the complex events that occur during tissue culture. The current study focused upon the events that occur before the islet isolation procedure. Pancreases were obtained from brain dead donors (n = 7) with mean age 50 (11) and normal pancreatic tissue obtained at surgery done for pancreatic neoplasms (n = 7), mean age 69 (9). Frozen sections were subjected to laser capture microdissection (LCM) to obtain β-cell rich islet tissue, from which extracted RNA was analyzed with microarrays. Gene expression of the 2 groups was evaluated with differential expression analysis for genes and pathways. Marked changes were found in pathways concerned with endoplasmic reticulum stress with its unfolded protein response (UPR), apoptotic pathways and components of inflammation. In addition, there were changes in genes important for islet cell identity. These findings advance our understanding of why islets are stressed before transplantation, which may lead to strategies to reduce this stress and lead to better clinical outcomes.

Repair mechanisms of the wounds with exposed bone structures using an artificial dermis

Objective: To investigate the repair mechanisms of the wounds with bone exposed by artificial dermis transplantation. Methods: Seventy two rabbits were randomly divided into 3 groups of 24 rabbits, the wound model was made on the top of head. In the skin defect group (SD group), skin was removed and skull periosteum was retained. In the periosteal defect group (PD group), the skin and skull periosteum were both removed, and the skull was exposed. In the skull burn group (SB group), after the periosteum was removed, the exposed skull bone was burned out with electric iron to cause skull surface necrosis, then the artificial dermis transplantation were performed in each group. In 1, 2, 3 and 4 weeks after transplantation, 2 specimens including artificial dermis and the underlying tissue in each group were cut for biopsy with HE staining to observe the angiogenesis in artificial dermis. Evans Blue perfusion was performed in four animals from each group to quantify angiogenesis in artificial dermis. Results: One week after transplantation, in SD group, a few microvessels can be observed in artificial dermis, but in the rest of the two groups, only a small amount of inflammatory cells can be seen. Two weeks after transplantation, in SD group, a large number of new blood vessels and fibroblasts can be seen filling in the artificial dermis, but angiogenesis delayed obviously in the PD and SB group. Three weeks after transplantation, the angiogenesis of artificial dermis in the PD and SB group accelerated obviously, and a thin layer of blood rich tissue membrane can be seen over the burned skull. The Evans Blue perfusion showed that the content of dye perfusion in the artificial dermis was closed to the peek value at 2 weeks after transplantation in the SD group, which was significantly higher than that in PD and SB group [(2 741±976) vs (1 039±590) and (403±209) μg/g, P<0.01]. Three weeks after transplantation, the content of Evans Blue in artificial dermis reached the peek value in SD and PD group, no significant difference was found between this two groups, but both significantly higher than that in SB group [(2 943±793) and (2 255±316) vs (1 125±404) μg/g, P<0.01]. Four weeks after transplantation, the content of Evans Blue in artificial dermis reached the peek value in SB group, although the value was still lower than that in SD and PD group, the difference was not significant [(1 609±787) vs (2 298±778) and (2 141±385) μg/g, P>0.05]. Conclusions: Lack or injury of periosteum can cause vascularization delay after artificial dermis transplantation. The vascularization of artificial dermis mainly originates from the normal tissues surrounding the wound when artificial dermis is transplanted on the wound with periosteum defect or bone exposed.