Characterization of a three-dimensional mucosal equivalent: Similarities and differences with native oral mucosa

The aim of this study was to create and characterize a tissue-engineered mucosal equivalent (TEM) that closely resembles native mucosa. TEM consists of human primary keratinocytes and fibroblasts isolated from biopsies taken from healthy donors and seeded onto a de-epidermized dermis and cultured for 14 days at the air/liquid interface. The structure of TEM was examined and compared with native nonkeratinizing oral mucosa (NNOM). The various components of the newly formed epidermal layer, basement membrane and underlying connective tissue were analyzed using immunohistochemistry. The mucosal substitute presented in this study showed a mature stratified squamous epithelium that was similar to that of native oral mucosa, as demonstrated by K19, desmoglein-3 and involucrin staining. In addition, the expression of basement membrane components collagen type IV, laminin-5 and integrin α6 and β4 in TEM proved to be consistent with native oral mucosa. The expression of PAS, Ki67, K10 and K13, however, appeared to be different in TEM compared to NNOM. Nevertheless, the similarities with native oral mucosa makes TEM a promising tool for studying the biology of mucosal pathologies such as oral mucositis or fibrosis as well as the development of new therapies. Copyrigh

Influence of the cardiac cycle on velocity selective and acceleration selective arterial spin labeling

Purpose: In this study, the influence of the cardiac cycle on the amount of label produced by a velocity-selective (VSASL) and acceleration-selective arterial spin labeling (AccASL) module was investigated.Methods: A short-PLD sequence was developed where a single VSASL- or AccASL-module was preceded by pCASL labeling to isolate the arterial blood pool. ASL subtraction was performed with label/control images with similar cardiac phase and time-of-measurement, followed by retrospective binning in 10 cardiac phase bins. ASL signal variation over the heart cycle was evaluated and tested for significance using a permutation test.Results: VSASL and AccASL showed significant arterial signal fluctuations over the cardiac cycle of up to similar to 36% and similar to 64%, respectively, mainly in areas containing large arteries. pCASL also showed significant signal fluctuations, of up to similar to 25% in arteries. Raw label/control images confirmed that the observed signal fluctuations were caused by the amount of label produced during the cardiac cycle, rather than inflow-effects, because the raw images did not all show equal cardiac phase dependence. No significant effects of the cardiac cycle were found on the gray matter ASL-signal.Conclusion: Significant influence of the cardiac cycle on the generated label was found for spatially nonselective ASL-sequences. Hence, to become independent of the cardiac cycle, sufficient averages need to be taken. Alternatively, these findings could be highly interesting for the purpose of quantifying pulsatility more distally in the vascular tree.Cardiovascular Aspects of Radiolog

Combining T-2 measurements and crusher gradients into a single ASL sequence for comparison of the measurement of water transport across the blood-brain barrier

Purpose Arterial spin labeling can be used to assess the transition time of water molecules across the blood-brain barrier when combined with sequence modules, which allow a separation of intravascular from tissue signal. The bipolar gradient technique measures the intravascular fraction by removing flowing spins. The T-2-relaxation-under-spin-tagging (TRUST) technique modulates the TE to differentiate between intravascular and extravascular spins based on T-2. These modules were combined into a single time-encoded pseudo-continuous arterial spin labeling sequence to compare their mechanisms of action as well as their assessment of water transition across the blood-brain barrier.Methods This protocol was acquired on a scanner with 9 healthy volunteers who provided written, informed consent. The sequence consisted of a Hadamard-encoded pseudo-continuous arterial spin labeling module, followed by the TRUST module (effective TEs of 0, 40, and 80 ms) and bipolar flow-crushing gradients (2, 4, and infinity cm/s). An additional experiment was performed with TRUST and a 3D gradient and spin-echo readout.Results Gradients imperfectly canceled the intravascular signal, as evidenced by the presence of residual signal in the arteries at early postlabeling delays as well as the underestimation of the intravascular fraction as compared with the TRUST method. The TRUST module allowed us to detect the transport of water deeper into the vascular tree through changes in T-2 than the used crusher gradients could, with their limited b-value.Conclusion Of the implemented techniques, TRUST allowed us to follow intravascular signal deeper into the vascular tree than the approach with (relatively weak) crusher gradients when quantifying the transport time of water across the blood-brain barrier.Neuro Imaging Researc

Mechanical stress inhibits early stages of endogenous cell migration: A pilot study in an ex vivo osteocho

Cell migration has a central role in osteochondral defect repair initiation and biomaterial-mediated regeneration. New advancements to reestablish tissue function include biomaterials and factors promoting cell recruitment, differentiation and tissue integration, but little is known about responses to mechanical stimuli. In the present pilot study, we tested the influence of extrinsic forces in combination with biomaterials releasing chemoattractant signals on cell migration. We used an ex vivo mechanically stimulated osteochondral defect explant filled with fibrin/hyaluronan hydrogel, in presence or absence of platelet-derived growth factor-BB or stromal cell-derived factor 1, to assess endogenous cell recruitment into the wound site. Periodic mechanical stress at early time point negatively influenced cell infiltration compared to unloaded samples, and the implementation of chemokines to increase cell migration was not efficient to overcome this negative effect. The gene expression at 15 days of culture indicated a marked downregulation of matrix metalloproteinase (MMP)13 and MMP3, a decrease of β1 integrin and increased mRNA levels of actin in osteochondral samples exposed to complex load. This work using an ex vivo osteochondral mechanically stimulated advanced platform demonstrated that recurrent mechanical stress at early time points impeded cell migration into the hydrogel, providing a unique opportunity to improve our understanding on management of joint injury

Sorting living mesenchymal stem cells using a TWIST1 RNA-based probe depends on incubation time and uptake capacity

Bone marrow derived mesenchymal stromal cells (BMSCs) are multipotent progenitors of particular interest for cell-based tissue engineering therapies. However, one disadvantage that limit their clinical use is their heterogeneity. In the last decades a great effort was made to select BMSC subpopulations based on cell surface markers, however there is still no general consensus on which markers to use to obtain the best BMSCs for tissue regeneration. Looking for alternatives we decided to focus on a probe-based method to detect intracellular mRNA in living cells, the SmartFlare technology. This technology does not require fixation of the cells and allows us to sort living cells based on gene expression into functionally different populations. However, since the technology is available it is debated whether the probes specifically recognize their target mRNAs. We validated the TWIST1 probe and demonstrated that it specifically recognizes TWIST1 in BMSCs. However, differences in probe concentration, incubation time and cellular uptake can strongly influence signal specificity. In addition we found that TWIST1high expressing cells have an increased expansion rate compared to TWIST1low expressing cells derivedfrom the same initial population of BMSCs. The SmartFlare probes recognize their target gene, however for each probe and cell type validation of the protocol is necessary

NELL-1, HMGB1, and CCN2 Enhance Migration and Vasculogenesis, But Not Osteogenic Differentiation Compared to BMP2

Currently, autografts still represent the gold standard treatment for the repair of large bone defects. However, these are associated with donor-site morbidity and increased pain, cost, and recovery time. The ideal therapy would use biomaterials combined with bone growth factors to induce and instruct bone defect repair without the need to harvest patient tissue. In this line, bone morphogenetic proteins (BMPs) have been the most extensively used agents for clinical bone repair, but at supraphysiological doses that are not without risk. Because of the need to eliminate the risks of BMP2 use in vivo, we assessed the ability of three putative osteogenic factors, nel-like molecule type 1 (NELL-1), high mobility group box 1 (HMGB1), and CCN2, to enhance the essential processes for bone defect repair in vitro and compared them to BMP2. Although it has been reported that NELL-1, HMGB1, and CCN2 play a role in bone formation, less is known about the contribution of these proteins to the different events involved, such as cell migration, osteogenesis, and vasculogenesis. In this study, we investigated the effects of different doses of NELL-1, HMGB, CCN2, and BMP2 on these three processes as a model for the recruitment and differentiation of resident cells in the in vivo bone defect repair situation, using cells of human origin. Our data demonstrated that NELL-1, HMGB1, and CCN2 significantly induced mesenchymal stem cell migration (from 1.58-fold increase compared to control), but BMP2 did not. Interestingly, only BMP2 increased osteogenesis in marrow stromal cells, whereas it inhibited osteogenesis in preosteoblasts. Moreover, the four proteins studied promoted significantly endothelial cell migration, reaching a maximum of 2.4-fold increase compared to control, and induced formation of tube-like structures. NELL-1, HMGB1, and CCN2 had these effects at relatively low doses compared to BMP2. This work indicates that NELL-1, HMGB1, and CCN2 might enhance bone defect healing via the recruitment of endogenous cells and induction of vascularization and act via different processes than BMP2