An architecturally rational hemostat for rapid stopping of massive bleeding on anticoagulation therapy.

Hemostatic devices are critical for managing emergent severe bleeding. With the increased use of anticoagulant therapy, there is a need for next-generation hemostats. We rationalized that a hemostat with an architecture designed to increase contact with blood, and engineered from a material that activates a distinct and undrugged coagulation pathway can address the emerging need. Inspired by lung alveolar architecture, here, we describe the engineering of a next-generation single-phase chitosan hemostat with a tortuous spherical microporous design that enables rapid blood absorption and concentrated platelets and fibrin microthrombi in localized regions, a phenomenon less observed with other classical hemostats without structural optimization. The interaction between blood components and the porous hemostat was further amplified based on the charged surface of chitosan. Contrary to the dogma that chitosan does not directly affect physiological clotting mechanism, the hemostat induced coagulation via a direct activation of platelet Toll-like receptor 2. Our engineered porous hemostat effectively stopped the bleeding from murine liver wounds, swine liver and carotid artery injuries, and the human radial artery puncture site within a few minutes with significantly reduced blood loss, even under the anticoagulant treatment. The integration of engineering design principles with an understanding of the molecular mechanisms can lead to hemostats with improved functions to address emerging medical needs.

Target volume coverage in clinically designed radiotherapy plan of post-mastectomy adjuvant radiotherapy of breast cancer patients in comparison with radiation therapy oncology group-based contoured plan: A dosimetric study.

Objective: Conventional field radiotherapy based on anatomical landmarks has been the traditional treatment for breast cancer. Having proven efficacy, it is still the current standard of treatment. The Radiation Therapy Oncology Group (RTOG) has published guidelines for contouring target volumes in postmastectomy patients. The impact of this guideline in the current clinical practice is less known; hence, we have analyzed dose-volume histograms (DVHs) for these plans and compared them with the proposed treatment plans to treat RTOG-defined targets. Subjects and Methods: RTOG consensus definitions were used to contour the target volumes in 20 previously treated postmastectomy patients. The prescription was 42.4 Gy in 16 fractions. DVHs were generated from clinically designed plans that had actually been delivered to each patient. For comparing dose to target volumes, new plans were generated with the goal of covering 95% of volume to 90% of prescribed dose. Results: In RTOG contoured Group, coverage improved for the supraclavicular (V90 = 83 vs. 94.9%, P < 0.05) and chest wall (V90 = 89.8 vs. 95.2%, P < 0.05). Axillary nodal coverage improved for Level-1(V90 = 80.35 vs. 96.40%, P < 0.05), Level-II (V90 = 85.93 vs. 97.09%, P < 0.05) and Level III (V90 = 86.67 vs. 98.6%, P < 0.05). The dose to the ipsilateral lung is increased (V20 = 23.87 vs. 28.73%, P < 0.05). Low dose to heart is increased in left-sided cases (V5 = 14.52 vs. 16.72%, P < 0.05) while same in right-sided cases. Conclusions: The study shows that radiotherapy using the RTOG consensus guidelines improves coverage to target volumes with a nonsignificant increase in normal organ dose compared to that based on anatomical landmarks.

Smooth Muscle Cell Alignment and Phenotype Control by Melt Spun Polycaprolactone Fibers for Seeding of Tissue Engineered Blood Vessels.

A method has been developed to induce and retain a contractile phenotype for vascular smooth muscle cells, as the first step towards the development of a biomimetic blood vessel construct with minimal compliance mismatch. Melt spun PCL fibers were deposited on a mandrel to form aligned fibers of 10 µm in diameter. The fibers were bonded into aligned arrangement through dip coating in chitosan solution. This formed a surface of parallel grooves, 10 µm deep by 10 µm across, presenting a surface layer of chitosan to promote cell surface interactions. The aligned fiber surface was used to culture cells present in the vascular wall, in particular fibroblasts and smooth muscle cells. This topography induced "surface guidance" over the orientation of the cells, which adopted an elongated spindle-like morphology, whereas cells on the unpatterned control surface did not show such orientation, assuming more rhomboid shapes. The preservation of VSMC contractile phenotype on the aligned scaffold was demonstrated by the retention of α-SMA expression after several days of culture. The effect was assessed on a prototype vascular graft prosthesis fabricated from polylactide caprolactone; VSMCs aligned longitudinally along a fiberless tube, whereas, for the aligned fiber coated tubes, the VSMCs aligned in the required circumferential orientation.

3D patterned substrates for bioartificial blood vessels - The effect of hydrogels on aligned cells on a biomaterial surface.

The optimal bio-artificial blood vessel construct is one that has a compliant tubular core with circumferentially aligned smooth muscle cells (SMCs). Obtaining this well-aligned pattern of SMCs on a scaffold is highly beneficial as this cellular orientation preserves the SMC contractile phenotype. We used 3D patterning to create channels on a polycaprolactone (PCL) scaffold; SMCs were then found to be aligned within the microchannels. To preserve this alignment, and to provide a protective coating that could further incorporate cells, we evaluated the use of two hydrogels, one based on poly(ethylene glycol) diacrylate (PEGDA) and the other based on gelatin. Hydrogels were either physically coated on the PCL surfaces or covalently linked via suitable surface modification of PCL. For covalent immobilization of PEGDA hydrogel, alkene groups were introduced on PCL, while for gelatin covalent linkage, serum proteins were introduced. It is, however, crucial that the hydrogel coating does not disrupt the cellular patterning and distribution. We show in this work that both the process of coating as well as the nature of the coating are critical to preservation of the aligned SMCs. The covalent coating methods involving the crosslinking of hydrogels with the surface of PCL films promoted hydrogel retention time on the film as compared with physical deposition. Furthermore, subsequent hydrogel degradation is affected by the components of the cell culture medium, hinting at a possible route to in vivo biodegradation. STATEMENT OF SIGNIFICANCE: Surface features control cellular orientation and subsequently influence their functionality, a useful effect for cellularized biomedical devices. Such devices also can benefit from protective and cell friendly hydrogel coatings. However, literature is lacking on the fate of cells that have endured hydrogel coating whilst orientated on a biomaterial surface. In particular, elucidation of the cells ability to remain adherent and orientated post hydrogel addition. Coating requires two procedures that may be deleterious to the orientated cells: the surface pretreatment for gel binding and the hydrogel crosslinking reaction. We compare transglutaminase gelatin crosslinking and UV initiated PEGDA crosslinking, coated onto smooth muscle cells orientated on patterned PCL surfaces. This original study will be of considerable use to the wider biomaterials community.

Printing cell-laden gelatin constructs by free-form fabrication and enzymatic protein crosslinking.

Considerable interest has arisen in precision fabrication of cell bearing scaffolds and structures by free form fabrication. Gelatin is an ideal material for creating cell entrapping constructs, yet its application in free form fabrication remains challenging. We demonstrate the use of gelatin, crosslinked with microbial transglutaminase (mTgase), as a material to print cell bearing hydrogels for both 2-dimensional (2-D) precision patterns and 3-dimensional (3-D) constructs. The precision patterning was attained with 3 % gelatin and 2 % high molecular weight poly (ethylene oxide) (PEO) whereas 3-D constructs were obtained using a 5 % gelatin solution. These hydrogels, referred to as "bioinks" supported entrapped cell growth, allowing cell spreading and proliferation for both HEK293 cells and Human Umbilical Vein Endothelial Cells (HUVECs). These bioinks were shown to be dispensable by robotic precision, forming patterns and constructs that were insoluble and of suitable stiffness to endure post gelation handling. The two bioinks were further characterized for fabrication parameters and mechanical properties.

Strong fiber-reinforced hydrogel.

In biological hydrogels, the gel matrix is usually reinforced with micro- or nanofibers, and the resulting composite is tough and strong. In contrast, synthetic hydrogels are weak and brittle, although they are highly elastic. The are many potential applications for strong synthetic hydrogels in medical devices, including as scaffolds for tissue growth. This work describes a new class of hydrogel composites reinforced with elastic fibers, giving them a cartilage-like structure. A three-dimensional rapid prototyping technique was used to form crossed "log-piles" of elastic fibers that are then impregnated with an epoxy-based hydrogel in order to form the fiber-reinforced gel. The fibrous construct improves the strength, modulus and toughness of the hydrogel, and also constrains the swelling. By altering the construct geometry and studying the effect on mechanical properties, we will develop the understanding needed to design strong hydrogels for biomedical devices and soft machines.

Expression profiling of genes regulated by TGF-beta: differential regulation in normal and tumour cells.

BACKGROUND: TGF-beta is one of the key cytokines implicated in various disease processes including cancer. TGF-beta inhibits growth and promotes apoptosis in normal epithelial cells and in contrast, acts as a pro-tumour cytokine by promoting tumour angiogenesis, immune-escape and metastasis. It is not clear if various actions of TGF-beta on normal and tumour cells are due to differential gene regulations. Hence we studied the regulation of gene expression by TGF-beta in normal and cancer cells. RESULTS: Using human 19 K cDNA microarrays, we show that 1757 genes are exclusively regulated by TGF-beta in A549 cells in contrast to 733 genes exclusively regulated in HPL1D cells. In addition, 267 genes are commonly regulated in both the cell-lines. Semi-quantitative and real-time qRT-PCR analysis of some genes agrees with the microarray data. In order to identify the signalling pathways that influence TGF-beta mediated gene regulation, we used specific inhibitors of p38 MAP kinase, ERK kinase, JNK kinase and integrin signalling pathways. The data suggest that regulation of majority of the selected genes is dependent on at least one of these pathways and this dependence is cell-type specific. Interestingly, an integrin pathway inhibitor, RGD peptide, significantly affected TGF-beta regulation of Thrombospondin 1 in A549 cells. CONCLUSION: These data suggest major differences with respect to TGF-beta mediated gene regulation in normal and transformed cells and significant role of non-canonical TGF-beta pathways in the regulation of many genes by TGF-beta.