The impact of fabrication parameters and substrate stiffness in direct writing of living constructs

Biomolecules and living cells can be printed in high‐resolution patterns to fabricate living constructs for tissue engineering. To evaluate the impact of processing cells with rapid prototyping (RP) methods, we modeled the printing phase of two RP systems that use biomaterial inks containing living cells: a high‐resolution inkjet system (BioJet) and a lower‐resolution nozzle‐based contact printing system (PAM²). In the first fabrication method, we reasoned that cell damage occurs principally during drop collision on the printing surface, in the second we hypothesize that shear stresses act on cells during extrusion (within the printing nozzle). The two cases were modeled changing the printing conditions: biomaterial substrate stiffness and volumetric flow rate, respectively, in BioJet and PAM². Results show that during inkjet printing impact energies of about 10^?8 J are transmitted to cells, whereas extrusion energies of the order of 10^?11 J are exerted in direct printing. Viability tests of printed cells can be related to those numerical simulations, suggesting a threshold energy of 10^?9 J to avoid permanent cell damage. To obtain well‐defined living constructs, a combination of these methods is proposed for the fabrication of scaffolds with controlled 3D architecture and spatial distribution of biomolecules and cells. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Tissue engineering of cartilage using a mechanobioreactor exerting simultaneous mechanical shear and compression to simulate the rolling action of articular joints

The effect of dynamic mechanical shear and compression on the synthesis of human tissue‐engineered cartilage was investigated using a mechanobioreactor capable of simulating the rolling action of articular joints in a mixed fluid environment. Human chondrocytes seeded into polyglycolic acid (PGA) mesh or PGA–alginate scaffolds were precultured in shaking T‐flasks or recirculation perfusion bioreactors for 2.5 or 4 weeks prior to mechanical stimulation in the mechanobioreactor. Constructs were subjected to intermittent unconfined shear and compressive loading at a frequency of 0.05 Hz using a peak‐to‐peak compressive strain amplitude of 2.2% superimposed on a static axial compressive strain of 6.5%. The mechanical treatment was carried out for up to 2.5 weeks using a loading regime of 10 min duration each day with the direction of the shear forces reversed after 5 min and release of all loading at the end of the daily treatment period. Compared with shaking T‐flasks and mechanobioreactor control cultures without loading, mechanical treatment improved the amount and quality of cartilage produced. On a per cell basis, synthesis of both major structural components of cartilage, glycosaminoglycan (GAG) and collagen type II, was enhanced substantially by up to 5.3‐ and 10‐fold, respectively, depending on the scaffold type and seeding cell density. Levels of collagen type II as a percentage of total collagen were also increased after mechanical treatment by up to 3.4‐fold in PGA constructs. Mechanical treatment had a less pronounced effect on the composition of constructs precultured in perfusion bioreactors compared with perfusion culture controls. This work demonstrates that the quality of tissue‐engineered cartilage can be enhanced significantly by application of simultaneous dynamic mechanical shear and compression, with the greatest benefits evident for synthesis of collagen type II. Biotechnol. Bioeng. 2012; 109:1060–1073. © 2011 Wiley Periodicals, Inc.     

Bioassembly of three‐dimensional embryonic stem cell‐scaffold complexes using compressed gases

Tissues are composed of multiple cell types in a well‐organized three‐dimensional (3D) microenvironment. To faithfully mimic the tissue in vivo, tissue‐engineered constructs should have well‐defined 3D chemical and spatial control over cell behavior to recapitulate developmental processes in tissue‐ and organ‐specific differentiation and morphogenesis. It is a challenge to build a 3D complex from two‐dimensional (2D) patterned structures with the presence of cells. In this study, embryonic stem (ES) cells grown on polymeric scaffolds with well‐defined microstructure were constructed into a multilayer cell‐scaffold complex using low pressure carbon dioxide (CO₂) and nitrogen (N₂). The mouse ES cells in the assembled constructs were viable, retained the ES cell‐specific gene expression of Oct‐4, and maintained the formation of embryoid bodies (EBs). In particular, cell viability was increased from 80% to 90% when CO₂ was replaced with N₂. The compressed gas‐assisted bioassembly of stem cell‐polymer constructs opens up a new avenue for tissue engineering and cell therapy. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A simple cell transport device keeps culture alive and functional during shipping

Transporting living complex cellular constructs through the mail while retaining their full viability and functionality is challenging. During this process, cells often suffer from exposure to suboptimal life‐sustaining conditions (e.g. temperature, pH), as well as damage due to shear stress. We have developed a transport device for shipping intact cell/tissue constructs from one facility to another that overcomes these obstacles. Our transport device maintained three different cell lines (Caco2, A549, and HepG2 C3A) individually on transwell membranes with high viability (above 97%) for 48 h under simulated shipping conditions without an incubator. The device was also tested by actual overnight shipping of blood brain barrier constructs consisting of human induced pluripotent brain microvascular endothelial cells and rat astrocytes on transwell membranes to a remote facility (approximately 1200 miles away). The blood brain barrier constructs arrived with high cell viability and were able to regain full barrier integrity after equilibrating in the incubator for 24 h; this was assessed by the presence of continuous tight junction networks and in vivo‐like values for trans‐endothelial electrical resistance (TEER). These results demonstrated that our cell transport device could be a useful tool for long‐distance transport of membrane‐bound cell cultures and functional tissue constructs. Studies that involve various cell and tissue constructs, such as the “Multi‐Organ‐on‐Chip” devices (where multiple microscale tissue constructs are integrated on a single microfluidic device) and studies that involve microenvironments where multiple tissue interactions are of interest, would benefit from the ability to transport or receive these constructs. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1257–1266, 2017     

Development of large engineered cartilage constructs from a small population of cells

Confronted with articular cartilage's limited capacity for self‐repair, joint resurfacing techniques offer an attractive treatment for damaged or diseased tissue. Although tissue engineered cartilage constructs can be created, a substantial number of cells are required to generate sufficient quantities of tissue for the repair of large defects. As routine cell expansion methods tend to elicit negative effects on chondrocyte function, we have developed an approach to generate phenotypically stable, large‐sized engineered constructs (≥3 cm²) directly from a small amount of donor tissue or cells (as little as 20,000 cells to generate a 3 cm² tissue construct). Using rabbit donor tissue, the bioreactor‐cultivated constructs were hyaline‐like in appearance and possessed a biochemical composition similar to native articular cartilage. Longer bioreactor cultivation times resulted in increased matrix deposition and improved mechanical properties determined over a 4 week period. Additionally, as the anatomy of the joint will need to be taken in account to effectively resurface large affected areas, we have also explored the possibility of generating constructs matched to the shape and surface geometry of a defect site through the use of rapid‐prototyped defect tissue culture molds. Similar hyaline‐like tissue constructs were developed that also possessed a high degree of shape correlation to the original defect mold. Future studies will be aimed at determining the effectiveness of this approach to the repair of cartilage defects in an animal model and the creation of large‐sized osteochondral constructs. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Protein formulation and lyophilization cycle design: prevention of damage due to freeze-concentration induced phase separation

Isolated equine chondrocytes, from juveniles and adults, were cultured in resorbable polyglycolic acid meshes for up to 5 weeks with semicontinuous feeding using a custom-made system to intermittently compress the regenerating tissue. Assays of the tissue constructs indicate that intermittent compression at 500 and 1000 psi (3.44 and 6.87 MPa, respectively) stimulated the production of extracellular matrix, enhancing the rate of de novo chondrogenesis. Constructs derived from juvenile cells contained concentrations of extracellular matrix components at levels more like that of native tissue than did constructs derived from adult cells. With intermittent pressurization, however, even adult cells were induced to increase the production of extracellular matrix. At both levels of intermittent pressure, the concentration of sulfated glycosaminoglycan in constructs from juvenile cells was found to be up to ten times greater than concentrations in control (nonpressurized) and adult cell-derived constructs. Whereas collagen concentrations in the 500 psi and control constructs were not significantly different for either juvenile or adult cell-derived constructs, intermittent pressurization at 1000 psi enhanced the production of collagen, suggesting that there may be a minimum level of pressure necessary to stimulate collagen formation.     

Visualizing feasible operating ranges within tissue engineering systems using a “windows of operation” approach: A perfusion‐scaffold bioreactor case study

Tissue engineering approaches to developing functional substitutes are often highly complex, multivariate systems where many aspects of the biomaterials, bio‐regulatory factors or cell sources may be controlled in an effort to enhance tissue formation. Furthermore, success is based on multiple performance criteria reflecting both the quantity and quality of the tissue produced. Managing the trade‐offs between different performance criteria is a challenge. A “windows of operation” tool that graphically represents feasible operating spaces to achieve user‐defined levels of performance has previously been described by researchers in the bio‐processing industry. This paper demonstrates the value of “windows of operation” to the tissue engineering field using a perfusion‐scaffold bioreactor system as a case study. In our laboratory, perfusion bioreactor systems are utilized in the context of bone tissue engineering to enhance the osteogenic differentiation of cell‐seeded scaffolds. A key challenge of such perfusion bioreactor systems is to maximize the induction of osteogenesis but minimize cell detachment from the scaffold. Two key operating variables that influence these performance criteria are the mean scaffold pore size and flow‐rate. Using cyclooxygenase‐2 and osteopontin gene expression levels as surrogate indicators of osteogenesis, we employed the “windows of operation” methodology to rapidly identify feasible operating ranges for the mean scaffold pore size and flow‐rate that achieved user‐defined levels of performance for cell detachment and differentiation. Incorporation of such tools into the tissue engineer's armory will hopefully yield a greater understanding of the highly complex systems used and help aid decision making in future translation of products from the bench top to the market place. Biotechnol. Bioeng. 2012; 109: 3161–3171. © 2012 Wiley Periodicals, Inc.     

Arresting mantle formation and redirecting embryonic shell gland tissue by platinum2+ leads to body plan modifications in Marisa cornuarietis (Gastropoda,Ampullariidae)

To evaluate the threat that anthropogenic substances pose to animals when they are emitted into the environment, tests like the invertebrate embryo toxicity test with the ramshorn snail Marisa cornuarietis have been developed. These tests are used to investigate substances like the heavy metal platinum (Pt) that is used in catalytic converters and is gradually released in car exhausts. In 2010, our group reported that high Pt concentrations cause body plan alterations in snails and prevent the formation of an external shell during M. cornuarietis embryogenesis. Now, this study presents scanning‐electron micrographs and histological sections of platinum²⁺ (Pt²⁺)‐treated and untreated M. cornuarietis embryos and compares “normally” developing and “shell‐less” embryos during embryogenesis, to reveal the exact course of events that lead to this body plan shift. Both groups showed similar development until the onset of torsion 70‐ to 82‐h postfertilization. In the Pt²⁺‐exposed embryos, the rudimentary shell gland (=anlage of both shell gland and mantle, which usually evaginates, grows, and eventually covers the visceral sac) does not spread across the visceral sac but remains on its ventral side. Without the excessive growth of the shell gland, a horizontal rotation of the visceral sac relative to head and foot does not occur, as being normal during the process of torsion. J. Morphol., 2012. © 2012 Wiley Periodicals, Inc.     

Perfusion seeding of channeled elastomeric scaffolds with myocytes and endothelial cells for cardiac tissue engineering

The requirements for engineering clinically sized cardiac constructs include medium perfusion (to maintain cell viability throughout the construct volume) and the protection of cardiac myocytes from hydrodynamic shear. To reconcile these conflicting requirements, we proposed the use of porous elastomeric scaffolds with an array of channels providing conduits for medium perfusion, and sized to provide efficient transport of oxygen to the cells, by a combination of convective flow and molecular diffusion over short distances between the channels. In this study, we investigate the conditions for perfusion seeding of channeled constructs with myocytes and endothelial cells without the gel carrier we previously used to lock the cells within the scaffold pores. We first established the flow parameters for perfusion seeding of porous elastomer scaffolds using the C2C12 myoblast line, and determined that a linear perfusion velocity of 1.0 mm/s resulted in seeding efficiency of 87% ± 26% within 2 hours. When applied to seeding of channeled scaffolds with neonatal rat cardiac myocytes, these conditions also resulted in high efficiency (77.2% ± 23.7%) of cell seeding. Uniform spatial cell distributions were obtained when scaffolds were stacked on top of one another in perfusion cartridges, effectively closing off the channels during perfusion seeding. Perfusion seeding of single scaffolds resulted in preferential cell attachment at the channel surfaces, and was employed for seeding scaffolds with rat aortic endothelial cells. We thus propose that these techniques can be utilized to engineer thick and compact cardiac constructs with parallel channels lined with endothelial cells. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Fluid-induced low shear stress improves cartilage like tissue fabrication by encapsulating chondrocytes

In the recent years, there has been considerable development in the regenerative medicine, which aims to repair, regenerate, and improve injured articular cartilage. The aim of the present study was to investigate the effect of flow-induced shear stress in perfusion bioreactor on alginate encapsulating chondrocytes. The shear stress imposed on the cells in the culture chamber of bioreactor was predicted with computational fluid dynamic. Bovine nasal chondrocytes were isolated and expanded to obtain a pellet. The cell pellet was resuspends in alginate solution, transferred to the culture chamber, and dynamically cultured under direct perfusion. At the end of culture, tissue constructs were examined histologically and by immunohistochemistry. The results of computational fluid dynamic modeling revealed that maximum wall shear stress was 4.820 × 10^?3 Pascal. Macroscopic views of the alginate/chondrocyte beads suggested that it possessed constant shape but were flexible. Under inverted microscope, round shape of chondrocyte observed. Cell distribution was homogeneous throughout the scaffold. Tissue construct subjected to shear showed morphological features, which are characteristic for natural cartilage. Immunohistochemistry results revealed immunopositivity for type II collagens in tissue constructs samples. Flow induced shear stress in the perfusion bioreactor and chnondrocyte encapsulation provide environment to support cell growth, and tissue regeneration and improve cartilage like tissue fabrication.     

Influence of intermittent pressure, fluid flow, and mixing on the regenerative properties of articular chondrocytes.

Equine articular chondrocytes, embedded within a polyglycolic acid nonwoven mesh, were cultured with various combinations of intermittent pressure, fluid flow, and mixing to examine the effects of different physical stimuli on neochondrogenesis from young cells. The cell/polymer constructs were cultured first in 125 ml spinner flasks for 1, 2, or 4 weeks and then in a perfusion system with intermittent pressure for a total of up to 6 weeks. Additional constructs were either cultured for all 6 weeks in the spinner flasks or for 1 week in spinners followed by 5 weeks in the perfusion system without intermittent pressure. Tissue constructs cultivated for 2 or 4 weeks in spinner flasks followed by perfusion with intermittent pressure had significantly higher concentrations of both sulfated glycosaminoglycan and collagen than constructs cultured entirely in spinners or almost entirely in the pressure/perfusion system. Initial cultivation in the spinner flasks, with turbulent mixing, enhanced both cell attachment and early development of the extracellular matrix. Subsequent culture with perfusion and intermittent pressure appeared to accelerate matrix formation. While the correlation was much stronger in the pressurized constructs, the compressive modulus was directly proportional to the concentration of sulfated glycosaminoglycan in all physically stressed constructs. Constructs that were not stressed beyond the 1-week seeding period lost mechanical integrity upon harvest, suggesting that physical stimulation, particularly with intermittent pressure, of immature tissue constructs during their development may contribute to their ultimate biomechanical functionality.     

Secondary osteon size and collagen/lamellar organization (“osteon morphotypes”) are not coupled,but potentially adapt independently for local strain mode or magnitude

In bone, matrix slippage that occurs at cement lines of secondary osteons during loading is an important toughening mechanism. Toughness can also be enhanced by modifications in osteon cross-sectional size (diameter) for specific load environments; for example, smaller osteons in more highly strained “compression” regions vs. larger osteons in less strained “tension” regions. Additional osteon characteristics that enhance toughness are distinctive variations in collagen/lamellar organization (i.e., “osteon morphotypes”). Interactions might exist between osteon diameter and morphotype that represent adaptations for resisting deleterious shear stresses that occur at the cement line. This may be why osteons often have a peripheral ring (or “hoop”) of highly oblique/transverse collagen. We hypothesized that well developed/distinct “hoops” are compensatory adaptations in cases where increased osteon diameter is mechanically advantageous (e.g., larger osteons in “tension” regions would have well developed/distinct “hoops” in order to resist deleterious consequences of co-existing localized shear stresses). We tested this hypothesis by determining if there are correlations between osteon diameters and strongly hooped morphotypes in “tension”, “compression”, and “neutral axis” regions of femora (chimpanzees, humans), radii (horse, sheep) and calcanei (horse, deer). The results reject the hypothesis—larger osteons are not associated with well developed/distinct “hoops”, even in “tension regions” where the effect was expected to be obvious. Although osteon diameter and morphotype are not coupled, osteon diameters seem to be associated with increased strain magnitudes in some cases, but this is inconsistent. By contrast, osteon morphotypes are more strongly correlated with the distribution of tension and compression.     

Regulation of autocrine fibroblast growth factor‐2 signaling by perfusion flow in 3D human mesenchymal stem cell constructs

Perfusion bioreactor systems play a crucial role in mitigating nutrient limitation as well as providing biomechanical stimuli and redistributing regulatory macromolecules that influence human mesenchymal stem cells (hMSC) fate in three‐dimensional (3D) scaffolds. As fibroblast growth factor‐2 (FGF‐2) is known to regulate hMSC phenotype, understanding the role of autocrine FGF‐2 signaling in the 3D construct under the different perfusion flow provides important insight into an optimal bioreactor design. To investigate FGF‐2 signaling inhibition in hMSC cultured in the porous poly(ethylene terephthalate) (PET) scaffolds perfused under two flow configurations, PD173074, an FGFR1 inhibitor, was added in growth media after 7 day of pre‐culture and its impact on hMSC proliferation and clonogenicity during the subsequent 7 days of cultivation was analyzed. Compared with control constructs in growth media, the addition of PD173074 resulted in significant reduction in hMSC proliferation and colony formation in both constructs with a more dramatic reduction in the parallel flow constructs. The results demonstrate that autocrine FGF‐2 plays a significant role in 3D scaffold and suggest modulation of the perfusion flow in the bioreactor as a strategy to influence autocrine actions and cell fate in the 3D scaffold. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

In vitro bone growth responds to local mechanical strain in three-dimensional polymer scaffolds

Mechanical stimulation plays a key role in healing and remodelling of bone tissue in vivo, and is used in bone tissue regeneration strategies in vitro. Although macroscopic compression of three-dimensional (3-D) seeded constructs can increase bone formation, it is not yet reported how this response is related to differences in local mechanical strains inside the scaffolds. In this study, we experimentally test the hypothesis that differences in local average of heterogeneous strains in a polymer scaffold will correlate with induced differences in the local biological response.Twenty-four poly(l-lactic acid) porous scaffolds seeded with rat bone cells were cultured first for 2 and 3 weeks under static conditions, respectively. Then for 1 week, half of the scaffolds were cyclically compressed (1.5%, 1 Hz), 1 h daily, with continuous perfusion (0.1 ml/min). The remaining half was kept under static conditions. The pore-surface strains in the scaffolds at the start of culture were calculated with micro-finite element modelling based on micro-Computed Tomography (μCT) images. The locations of mineralized nodules were determined from μCT images and coupled to the calculated strains.Detectable mineralized nodules (>10³ μm³) were only present in the loaded samples. Averages of absolute principal strains at the start of culture were significantly higher at nodule sites than at sites without a nodule.The results support the hypothesis that regenerating bone tissue in a 3-D porous scaffold responds to local mechanical strain. The methodology presented in this study can contribute design optimisation of tissue regeneration strategies relying on mechanical stimulation.     

A novel model to characterize structure and function of BRCA1

BRCA1 plays a central role in DNA repair. Although N‐terminal RING and C‐terminal BRCT domains are studied well, the functions of the central region of BRCA1 are poorly characterized. Here, we report a structural and functional analysis of BRCA1 alleles and functional human BRCA1 in chicken B‐lymphocyte cell line DT40. The combination of “homologous recombineering” and “RT‐cassette” enables modifications of chicken BRCA1 gene in Escherichia coli. Mutant BRCA1 knock‐in DT40 cell lines were generated using BRCA1 mutation constructs by homologous recombination with a targeting efficiency of up to 100%. Our study demonstrated that deletion of motifs 2–9 BRCA1^{Δ/Δ181‐1415} (Caenorhabditis elegans BRCA1 mimic) or deletion of motif 1 BRCA1^{Δ/Δ126‐136} decreased cell viability following cisplatin treatment. Furthermore, deletion of motifs 5 and 6 BRCA1^{Δ/Δ525‐881} within DNA‐binding region, even the conserved 7‐amino acid deletion BRCA1^{Δ/Δ872‐878} within motif 6, caused a decreased cell viability upon cisplatin treatment. Surprisingly, human BRCA1 is functional in DT40 cells as indicated by DNA damage‐induced Rad 51 foci formation in human BRCA1 knock‐in DT40 cells. These results demonstrate that those conserved motifs within the central region are essential for DNA repair functions of BRCA1. These findings provide a valuable tool for the development of new therapeutic modalities of breast cancer linked to BRCA1.     

Design and characterization of a protein superagonist of IL‐15 fused with IL‐15Rα and a high‐affinity T cell receptor

To avoid high systemic doses, strategies involving antigen‐specific delivery of cytokine via linked antibodies or antibody fragments have been used. Targeting cancer‐associated peptides presented by major histocompatibility complex (MHC) molecules (pepMHC) increases the number of potential target antigens and takes advantage of cross‐presentation on tumor stroma and in draining lymph nodes. Here, we use a soluble, high‐affinity single‐chain T cell receptor Vα‐Vβ (scTv), to deliver cytokines to intracellular tumor‐associated antigens presented as pepMHC. As typical wild‐type T cell receptors (TCRs) exhibit low affinity (K_d = 1–100 μM or more), we used an engineered TCR, m33, that binds its antigenic peptide SIYRYYGL (SIY) bound to the murine class I major histocompatability complex protein H2‐K^b (SIY/K^b) with nanomolar affinity (K_d = 30 nM). We generated constructs consisting of m33 scTv fused to murine interleukin 2 (IL‐2), interleukin 15 (IL‐15), or IL‐15/IL‐15Rα (IL‐15 linked to IL‐15Rα sushi domain, called “superfusion”). The fusions were purified with good yields and bound specifically to SIY/K^b with high affinity. Proper cytokine folding and binding were confirmed, and the fusions were capable of stimulating proliferation of cytokine‐dependent cells, both when added directly and when presented in trans, bound to cells with the target pepMHC. The m33 superfusion was particularly potent and stable and represents a promising design for targeted antitumor immunomodulation. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Vascular engineering using human embryonic stem cells

Engineering vascularized tissue constructs remains a major problem in regenerative medicine. The formation of such a microvasculature—like the vasculogenesis in early embryogenesis that it closely resembles—is guided by biochemical and biophysical cues, such as growth factors, extracellular matrix proteins, hypoxia, and hydrodynamic shear. As they undergo spontaneous and directed vascular differentiation, human embryonic stem cells can be used as a model system to explore central issues in engineering vascularized tissue constructs and, potentially, to elucidate vasculogenic and angiogenic mechanisms involved in such vascular diseases as limb and cardiac ischemia. Because the conventional spontaneous differentiation approach can only isolate small quantities of vascular cells, recent efforts have sought to develop controlled approaches, including the development of three‐dimensional scaffolds to reengineer the microenvironments of early embryogenesis. This review focuses on emerging approaches to deriving and directing vasculatures from human embryonic stem cells and efforts to engineer 3D vasculatures from such derivatives. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Midgut pseudotumors and the maintenance of tissue homeostasis: Studies on aging and manipulated stick insects

Stick insects (Carausius morosus) develop pseudotumors in aging adults. Pseudotumor formation starts at the M2 midgut region where an accumulation of stomatogastric nerve terminals is observed. Pseudotumors arise from dying columnar cells whose basal parts form an “amorphous substance” at the basement membrane whereas the apical parts, including the nucleus, are expelled into the gut lumen. The “amorphous substance” is ensheathed by hemocytes. These nodules, which do not melanize, characterize the phenotype of the pseudotumors. With age, cell death and pseudotumor infestation increases. It is shown that the maintenance of midgut tissue homoeostasis is disturbed and becomes more serious with growing pseudotumor incidence. The increased death rate of differentiated columnar cells is no longer compensated by the proliferation of regenerative cells, i.e., intestinal stem cells, in the midgut nidi. The appearance of “holes” in the intestinal wall is evidently a causative factor of premature death. Extirpation of the hypocerebral ganglion in young adults of the stick insect (before the onset of spontaneous pseudotumor formation) provokes the apoptosis of a large number of columnar cells within 24 h and the formation of pseudotumors that are histologically identical with spontaneous ones. We conclude that the stomatogastric nervous system plays a decisive role in the regulatory mechanism maintaining midgut tissue homeostasis. The possibility of experimentally manipulating the regulatory system provides a valuable tool for the exploration of extrinsic factors involved into the feedback circuitry of tissue homeostasis. The fact that comparable pseudotumors were observed in a number of orthopteromorphan species, where they could also be induced by the interruption of the stomatogastric nervous system, indicates that its role in tissue homoeostasis may be widespread in insects and possibly represent a general principle. J. Morphol., 2009. © 2008 Wiley‐Liss, Inc.     

Weight‐Related Terms Differentially Affect Self‐Efficacy and Perception of Obesity

Objective

Little work has explored the effect of weight‐related terms on treatment initiation; only one study has investigated weight‐related terms and the psychological constructs associated with treatment uptake. The present study examines the effects of four common weight‐related terms on treatment initiation and the moderating effect of weight bias internalization.

Methods

Adult participants with overweight and obesity (n = 436) were recruited online and asked to read three vignettes describing clinical encounters; the weight‐related term (i.e., “weight,” “BMI,” “obesity,” or “fat”) was varied randomly. Participants then reported self‐efficacy, cognitive and emotional illness beliefs about obesity (i.e., illness perception), and interest in a weight loss program.

Results

The term “obesity” resulted in the greatest self‐efficacy and perceived control over obesity. “Fat” resulted in the least illness coherence (i.e., understanding of obesity). Weight bias internalization did not moderate the effect of term on self‐efficacy, nor did it moderate illness perception. No differences in weight loss program enrollment were observed.

Conclusions

Use of the term “obesity” may promote patients’ perceived control and self‐efficacy. Use of “fat” should be avoided. Results suggest that, despite patient and clinician preference for euphemistic weight terms, use of clinical language such as “obesity” may perform better in provider intervention.

     

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions ¹. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA ² and PLGA ³ or a number of naturally occurring proteins such as collagen ⁴, hyaluronic acid ⁵ or fibrin ^6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body''s natural wound healing processes ⁸. Fibrin is cell-degradable and potentially autologous ⁹, making it an ideal temporary scaffold for tissue engineering.Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering ⁴. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers ¹⁰. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries ^11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture ¹³.After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions ⁶. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.