Molecular Basis for Surface-Initiated Non-Thrombin-Generated Clot Formation Following Viral Infection.

In this paper, we propose a model that connects two standard inflammatory responses to viral infection, namely, elevation of fibrinogen and the lipid drop shower, to the initiation of non-thrombin-generated clot formation. In order to understand the molecular basis for the formation of non-thrombin-generated clots following viral infection, human epithelial and Madin-Darby Canine Kidney (MDCK, epithelial) cells were infected with H1N1, OC43, and adenovirus, and conditioned media was collected, which was later used to treat human umbilical vein endothelial cells and human lung microvascular endothelial cells. After direct infection or after exposure to conditioned media from infected cells, tissue surfaces of both epithelial and endothelial cells, exposed to 8 mg/mL fibrinogen, were observed to initiate fibrillogenesis in the absence of thrombin. No fibers were observed after direct viral exposure of the endothelium or when the epithelium cells were exposed to SARS-CoV-2 isolated spike proteins. Heating the conditioned media to 60 °C had no effect on fibrillogenesis, indicating that the effect was not enzymatic but rather associated with relatively thermally stable inflammatory factors released soon after viral infection. Spontaneous fibrillogenesis had previously been reported and interpreted as being due to the release of the alpha C domains due to strong interactions of the interior of the fibrinogen molecule in contact with hydrophobic material surfaces rather than cleavage of the fibrinopeptides. Contact angle goniometry and immunohistochemistry were used to demonstrate that the lipids produced within the epithelium and released in the conditioned media, probably after the death of infected epithelial cells, formed a hydrophobic residue responsible for fibrillogenesis. Hence, the standard inflammatory response constitutes the ideal conditions for surface-initiated clot formation.

Efficacy of hypochlorous acid (HOCl) fog in sanitizing surfaces against Enterococcus faecalis.

BACKGROUND: Fogging is an efficient method when disinfection of large areas is desired. METHODS: Two methods of ultrasonic fogging, pulsed and continuous, were compared on bacteria dried on either aluminum or polystyrene surfaces. We characterized commercial and home-made hypochlorous acid (HOCl) with respect to storage and means of production. RESULTS: We found that the initial chlorine concentration of the commercial solution was approximately 550 ppm, and when stored open under ambient conditions, the chlorine content decreased at a rate of 30% every 100 days. The HOCl produced using the home synthesizers had a maximum chlorine content of 257.6 ppm which decayed by 65% after 100 days. A second synthesizer produced a liquid with high chlorine content and pH, 750ppm and pH = 8.55. The anti-bacterial efficacy was probed using Enterococcus faecalis, a persistent source of infection in public and clinical spaces. Time course studies determined that E. faecalis could survive dry on surfaces for more than 12 weeks, but was easily eliminated in half the fogging time. CONCLUSIONS: The most effective mode of application was determined to be continuous fogging where a 6.59 log reduction was established in vertical geometry. The optimal pulsed fogging protocol produced a similar reduction, but required nearly 5 times as long. The home synthesized versions yielded much lower log bacterial reductions. No significant differences in outcome were determined between polymer or metal surfaces.

Engineering functional skin constructs: A quantitative comparison of three-dimensional bioprinting with traditional methods.

Tissue engineering has been successful in reproducing human skin equivalents while incorporating new approaches such as three-dimensional (3D) bioprinting. The latter method offers a plethora of advantages including increased production scale, ability to incorporate multiple cell types and printing on demand. However, the quality of printed skin equivalents compared to those developed manually has never been assessed. To leverage the benefits of this method, it is imperative that 3D-printed skin should be structurally and functionally similar to real human skin. Here, we developed four bilayered human skin epidermal-dermal equivalents: non-printed dermis and epidermis (NN), printed dermis and epidermis (PP), printed epidermis and non-printed dermis (PN), and non-printed epidermis and printed dermis (NP). The effects of printing induced shear stress [0.025 kPa (epidermis); 0.049 kPa (dermis)] were characterized both at the cellular and at the tissue level. At cellular level, no statistically significant differences in keratinocyte colony-forming efficiency (CFE) (p = 0.1641) were observed. In the case of fibroblasts, no significant differences in the cell alignment index (p < 0.1717) and their ability to contract collagen gel (p = 0.851) were detected. At the tissue levels, all the four skin equivalents were characterized using histological and immunohistochemical analysis with no significant differences found in either epidermal basal cell count, thickness of viable epidermis, and relative intensity of filaggrin and claudin-1. Our results demonstrated that 3D printing can achieve the same high-quality skin constructs as have been developed traditionally, thus opening new avenues for numerous high-throughput industrial and clinical applications.

Combination of 3D Printing and ALD for Dentin Fabrication from Dental Pulp Stem Cell Culture.

A combination of fused deposition modeling printing with atomic layer deposition (ALD) of titania was designed to achieve templated biomineralization and terminal odontogenic differentiation of dental pulp stem cells on three-dimensional (3D) printed polylactic acid (PLA) scaffolds. In the absence of the ALD-deposited titania coating, we had previously shown that both plating efficiency and differentiation are adversely impacted when scaffolds are produced by 3D printing rather than traditional polymer molding. These differences were removed when both printed and molded structures were coated with ALD of titania, which improved the outcomes regardless of the manufacturing method. In this case, on all titania-coated substrates, the plating efficiency increased, copious mineral deposition was observed, and RT-PCR indicated a significant upregulation of osteocalcin, a gene associated with mineral deposition. The influence of additional coatings of collagen, gelatin, or fibronectin on the ALD titania-coated and uncoated PLA-printed and molded scaffolds was also investigated. Upregulation of the odontogenic late-stage differentiation sibling protein, dentin sialoprotein, was observed on the collagen ALD-titania-coated scaffolds and to a lesser extent on the gelatin ALD-titania-coated scaffolds.

Regulating substrate mechanics to achieve odontogenic differentiation for dental pulp stem cells on TiO2 filled and unfilled polyisoprene.

We have shown that materials other than hydrogels commonly used in tissue engineering can be effective in enabling differentiation of dental pulp stem cells (DPSC). Here we demonstrate that a hydrophobic elastomer, polyisoprene (PI), a component of Gutta-percha, normally used to obturate the tooth canal, can also be used to initiate differentiation of the pulp. We showed that PI substrates without additional coating promote cell adhesion and differentiation, while their moduli can be easily adjusted either by varying the coating thickness or incorporation of inorganic particles. DPSC plated on those PI substrates were shown, using SPM and hysitron indentation, to adjust their moduli to conform to differentially small changes in the substrate modulus. In addition, optical tweezers were used to separately measure the membrane and cytoplasm moduli of DPSC, with and without Rho kinase inhibitor. The results indicated that the changes in modulus were attributed predominantly to changes within the cytoplasm, rather than the cell membrane. CLSM was used to identify cell morphology. Differentiation, as determined by qRT-PCR, of the upregulation of OCN, and COL1α1 as well as biomineralization, characterized by SEM/EDAX, was observed on hard PI substrates in the absence of induction factors, i.e. dexamethasone, with moduli 3-4 MPa, regardless of preparation. SEM showed that even though biomineralization was deposited on both spun cast thin PI and filled thick PI substrates, the minerals were aggregated into large clusters on thin PI, and uniformly distributed on filled thick PI, where it was templated within banded collagen fibers. STATEMENT OF SIGNIFICANCE: This manuscript demonstrates the potential of polyisoprene (PI), an elastomeric polymer, for use in tissue engineering. We show how dental pulp stem cells adjust their moduli continuously to match infinitesimally small changes in substrate mechanics, till a critical threshold is reached when they will differentiate. The lineage of differentiation then becomes a sensitive function of both mechanics and morphology for a given chemical composition. Since PI is a major component of Gutta-percha, the FDA approved material commonly used for obturating the root canal, this work suggests that it can easily be adapted for in vivo use in dental regeneration.

Effect of Graphene on Differentiation and Mineralization of Dental Pulp Stem Cells in Poly(4-vinylpyridine) Matrix in Vitro.

We have investigated the influence of graphene nanoplatelet scaffolds for dental pulp cells (DPSCs) made from poly(4-vinylpyridine) (P4VP) either via spin-casting flat films or electrospinning nano- and microscale fibers. We found that graphene predominated over other factors in promoting differentiation of DPSCs. In the absence of graphene, real-time-polymerase chain reaction (RT-PCR) and energy dispersive X-ray (EDX) analyses indicated that the DPSCs differentiated along odontogenic lineages only on the nano- and microelectrospun scaffolds. Closer scanning electron microscopy (SEM) imaging revealed formation of banded collagen structures, which nucleated on the electrospun fibers in the absence of graphene. Biomineral deposition was templated on these fibers, with mineral to protein ratios similar to dentin. In the microfibers, the graphene was completely encapsulated and appeared to hinder biomineralization. Previously minimal biomineralization and banded collagen were observed on flat spun cast substrates. Addition of graphene appeared to induce nucleation of banded collagen fibers and template biomineral deposition. Addition of graphene did not affect the outcome of the DPSCs cultured on the nanofibers, which biomineralized regardless of graphene inclusion. Based on these results, we hypothesize that direct contact with graphene is the primary factor determining differentiation of the DPSCs. On the flat surface and nanoscale electrospun fibers, the graphene protrudes from the sample enabling direct contact with the extracellular matrix (ECM) and cells, while on the microfibers, the graphene is fully encapsulated within the matrix. TUNA imaging with scanning force microscopy showed enhanced conductivity on fibers with encapsulated graphene, which we hypothesize may change the conformation of adsorbed ECM proteins, affecting DPSCs differentiation.

The influence of roughness on stem cell differentiation using 3D printed polylactic acid scaffolds.

With the increase in popularity of 3D printing, an important question arises as to the equivalence between devices manufactured by standard methods vs. those presenting with identical bulk specifications, but manufactured via fused deposition modeling (FDM) printing. Using thermal imaging in conjunction with electron and atomic force microscopy, we demonstrate that large thermal gradients, whose distribution is difficult to predict, are associated with FDM printing and result in incomplete fusion and sharkskin of the printing filament. Even though these features are micro or submicron scale, and hence may not interfere with the intended function of the device, they can have a profound influence if the device comes in contact with living tissue. Dental pulp stem cells were cultured on substrates of identical dimensions, which were either printed or molded from the same PLA stock material. The cultures exhibited significant differences in plating efficiency, migration trajectory, and morphology at early times stemming from attempts by the cells to minimize cytoplasm deformation as they attempt to adhere on the printed surfaces. Even though biomineralization without dexamethasone induction was observed in all cultures at later times, different gene expression patterns were observed on the two surfaces. (Osteogenic markers were upregulated on molded substrates, while odontogenic markers were upregulated on the FDM printed surfaces.) Our results clearly indicate that the method of manufacturing is an important consideration in comparing devices, which come in contact with living tissues.

Templated dentin formation by dental pulp stem cells on banded collagen bundles nucleated on electrospun poly (4-vinyl pyridine) fibers in vitro.

Eventhough it is well established that materials can promote stem cell differentiation, hard tissue formation is a templated process for which little is known regarding the in vitro process. We have found that surface curvature enables self-assembly of triple helical collagen fibrils into banded bundle structures from rat tail and human collagen secreted by dental pulp stem cells. Collagen fibrils were adsorbed at 4 °C on spun cast flat P4VP films and electrospun fibers. Protein adsorption was observed on both surfaces, but large banded bundles with a uniform spacing of approximately 55 nm were present only on the fiber surfaces. SEM/EDS mapping showed that dental pulp stem cells plated on the same surfaces biomineralized copiously only along the electrospun fibers. Raman spectroscopy indicated that despite the presence of adsorbed collagen on the flat surfaces, only the deposits present on the fibrous surface had a protein to hydroxyl apatite ratio similar to natural dentin from human teeth. RT-PCR indicated up regulation of collagen, osteocalcin and dental sialophosphate protein, confirming that odontogenic differentiation is promoted only on the fiber scaffolds. Taken together the results indicate that, in addition to surface chemistry, the supermolecular structure of ECM collagen, which is essential in directing DPSCs differentiation and templating biomineralization, can be modified by the underlying surface morphology. STATEMENT OF SIGNIFICANCE: The past decade has been focused efforts in the use of dental pulp stem cells (DPSC) for dental regeneration. Eventhough the factors required for DPSCs differentiation have been well studied, actual mineral deposition, positively identified as dentin, has not been achieved in vitro. Hard tissue is known to be a templated process in vivo where the mineral to protein ratio is tightly controlled via proteins which aid in collagen conformation and mineral sequestration. Here we show that one can mimic this process in vitro via the combination of materials selection and morphology. The material chemistry is shown to induce genetic upregulation the genes responsible for collagen and osteocalcin, while Raman spectroscopy confirms the translation and adsorption the proteins on the substrate. But, we show that the simple presence of collagen is not enough to template actual biomineral deposition similar to that found in vivo. Mineral deposition is a complicated process templated on collagen bundles and mediated by specific sibling proteins that determine the protein to mineral ratio. Here we show that surface curvature can reduce the barrier to collagen bundle formation, directing DPSC differentiation along odontogenic lineage, and subsequently templating actual dentin, comparable to that found in vivo in human teeth.