Results of an interlaboratory study on the working curve in vat photopolymerization.

The working curve informs resin properties and print parameters for stereolithography, digital light processing, and other photopolymer additive manufacturing (PAM) technologies. First demonstrated in 1992, the working curve measurement of cure depth vs radiant exposure of light is now a foundational measurement in the field of PAM. Despite its widespread use in industry and academia, there is no formal method or procedure for performing the working curve measurement, raising questions about the utility of reported working curve parameters. Here, an interlaboratory study (ILS) is described in which 24 individual laboratories performed a working curve measurement on an aliquot from a single batch of PAM resin. The ILS reveals that there is enormous scatter in the working curve data and the key fit parameters derived from it. The measured depth of light penetration Dp varied by as much as 7x between participants, while the critical radiant exposure for gelation Ec varied by as much as 70x. This significant scatter is attributed to a lack of common procedure, variation in light engines, epistemic uncertainties from the Jacobs equation, and the use of measurement tools with insufficient precision. The ILS findings highlight an urgent need for procedural standardization and better hardware characterization in this rapidly growing field.

Versatile and inexpensive Hall-Effect force sensor for mechanical characterization of soft biological materials.

Mismatch of hierarchical structure and mechanical properties between tissue-engineered implants and native tissue may result in signal cues that negatively impact repair and remodeling. With bottom-up tissue engineering approaches, designing tissue components with proper microscale mechanical properties is crucial to achieve necessary macroscale properties in the final implant. However, characterizing microscale mechanical properties is challenging, and current methods do not provide the versatility and sensitivity required to measure these fragile, soft biological materials. Here, we developed a novel, highly sensitive Hall-Effect based force sensor that is capable of measuring mechanical properties of biological materials over wide force ranges (µN to N), allowing its use at all steps in layer-by-layer fabrication of engineered tissues. The force sensor design can be easily customized to measure specific force ranges, while remaining easy to fabricate using inexpensive, commercial materials. Although we used the force sensor to characterize mechanics of single-layer cell sheets and silk fibers, the design can be easily adapted for different applications spanning larger force ranges (>N). This platform is thus a novel, versatile, and practical tool for mechanically characterizing biological and biomimetic materials.

A Robust Method to Generate Mechanically Anisotropic Vascular Smooth Muscle Cell Sheets for Vascular Tissue Engineering.

In arterial tissue engineering, mimicking native structure and mechanical properties is essential because compliance mismatch can lead to graft failure and further disease. With bottom-up tissue engineering approaches, designing tissue components with proper microscale mechanical properties is crucial to achieve the necessary macroscale properties in the final implant. This study develops a thermoresponsive cell culture platform for growing aligned vascular smooth muscle cell (VSMC) sheets by photografting N-isopropylacrylamide (NIPAAm) onto micropatterned poly(dimethysiloxane) (PDMS). The grafting process is experimentally and computationally optimized to produce PNIPAAm-PDMS substrates optimal for VSMC attachment. To allow long-term VSMC sheet culture and increase the rate of VSMC sheet formation, PNIPAAm-PDMS surfaces were further modified with 3-aminopropyltriethoxysilane yielding a robust, thermoresponsive cell culture platform for culturing VSMC sheets. VSMC cell sheets cultured on patterned thermoresponsive substrates exhibit cellular and collagen alignment in the direction of the micropattern. Mechanical characterization of patterned, single-layer VSMC sheets reveals increased stiffness in the aligned direction compared to the perpendicular direction whereas nonpatterned cell sheets exhibit no directional dependence. Structural and mechanical anisotropy of aligned, single-layer VSMC sheets makes this platform an attractive microstructural building block for engineering a vascular graft to match the in vivo mechanical properties of native arterial tissue.

Silk-fibronectin protein alloy fibres support cell adhesion and viability as a high strength, matrix fibre analogue.

Silk is a natural polymer with broad utility in biomedical applications because it exhibits general biocompatibility and high tensile material properties. While mechanical integrity is important for most biomaterial applications, proper function and integration also requires biomaterial incorporation into complex surrounding tissues for many physiologically relevant processes such as wound healing. In this study, we spin silk fibroin into a protein alloy fibre with whole fibronectin using wet spinning approaches in order to synergize their respective strength and cell interaction capabilities. Results demonstrate that silk fibroin alone is a poor adhesive surface for fibroblasts, endothelial cells, and vascular smooth muscle cells in the absence of serum. However, significantly improved cell attachment is observed to silk-fibronectin alloy fibres without serum present while not compromising the fibres' mechanical integrity. Additionally, cell viability is improved up to six fold on alloy fibres when serum is present while migration and spreading generally increase as well. These findings demonstrate the utility of composite protein alloys as inexpensive and effective means to create durable, biologically active biomaterials.

Introducing biomimetic shear and ion gradients to microfluidic spinning improves silk fiber strength.

Silkworm silk is an attractive biopolymer for biomedical applications due to its high mechanical strength and biocompatibility; as a result, there is increasing interest in scalable devices to spin silk and recombinant silk so as to improve and customize their properties for diverse biomedical purposes (Vepari and Kaplan 2007 Prog. Polym. Sci. 32 ). While artificial spinning of regenerated silk fibroins adds tunability to properties such as degradation rate and surface functionalization, the resulting fibers do not yet approach the mechanical strength of native silkworm silk. These drawbacks reduce the applicability and attractiveness of artificial silk (Kinahan et al 2011 Biomacromolecules 12 ). Here, we used computational fluid dynamic simulations to incorporate shear in tandem with biomimetic ion gradients by coupling a modular novel glass microfluidic device to our previous co-axial flow device. Fibers spun with this combined apparatus demonstrated a significant increase in mechanical strength compared to fibers spun with the basic apparatus alone, with a three-fold increase in Young's modulus and extensibility and a twelve-fold increase in toughness. These results thus demonstrate the critical importance of ionic milieu and shear stress in spinning strong fibers from solubilized silk fibroin.

Primordial soup was edible: abiotically produced Miller-Urey mixture supports bacterial growth.

Sixty years after the seminal Miller-Urey experiment that abiotically produced a mixture of racemized amino acids, we provide a definite proof that this primordial soup, when properly cooked, was edible for primitive organisms. Direct admixture of even small amounts of Miller-Urey mixture strongly inhibits E. coli bacteria growth due to the toxicity of abundant components, such as cyanides. However, these toxic compounds are both volatile and extremely reactive, while bacteria are highly capable of adaptation. Consequently, after bacterial adaptation to a mixture of the two most abundant abiotic amino acids, glycine and racemized alanine, dried and reconstituted MU soup was found to support bacterial growth and even accelerate it compared to a simple mixture of the two amino acids. Therefore, primordial Miller-Urey soup was perfectly suitable as a growth media for early life forms.

Engineering myocardial tissue patches with hierarchical structure-function.

Complex hierarchical organization is a hallmark of tissues and their subsequent integration into organs. A major challenge in tissue engineering is to generate arrays of cells with defined structural organization that display appropriate functional properties. Given what is known about cellular responses to physiochemical cues from the surrounding environment, we can build tissue structures that mimic these microenvironments and validate these platforms using both experimental and computational approaches. Tissue generation encompasses many methods and tissue types, but here we review layering cell sheets to create scaffold-less myocardial patches. We discuss surgical criteria that can drive the design of myocardial cell sheets and the methods used to fabricate, mechanically condition, and functionally test them. We also focus on how computational and experimental approaches could be integrated to optimize tissue mechanical properties by using measurements of biomechanical properties and tissue anisotropy to create predictive computational models. Tissue anisotropy and dynamic mechanical stimuli affect cell phenotype in terms of protein expression and secretion, which in turn, leads to compositional and structural changes that ultimately impact tissue function. Therefore, a combinatorial approach of design, fabrication, testing, and modeling can be carried out iteratively to optimize engineered tissue function.

Micropatterned cell sheets with defined cell and extracellular matrix orientation exhibit anisotropic mechanical properties.

For an arterial replacement graft to be effective, it must possess the appropriate strength in order to withstand long-term hemodynamic stress without failure, yet be compliant enough that the mismatch between the stiffness of the graft and the native vessel wall is minimized. The native vessel wall is a structurally complex tissue characterized by circumferentially oriented collagen fibers/cells and lamellar elastin. Besides the biochemical composition, the functional properties of the wall, including stiffness, depend critically on the structural organization. Therefore, it will be crucial to develop methods of producing tissues with defined structures in order to more closely mimic the properties of a native vessel. To this end, we sought to generate cell sheets that have specific ECM/cell organization using micropatterned polydimethylsiloxane (PDMS) substrates to guide cell organization and tissue growth. The patterns consisted of large arrays of alternating grooves and ridges. Adult bovine aortic smooth muscle cells cultured on these substrates in the presence of ascorbic acid produced ECM-rich sheets several cell layers thick in which both the cells and ECM exhibited strong alignment in the direction of the micropattern. Moreover, mechanical testing revealed that the sheets exhibited mechanical anisotropy similar to that of native vessels with both the stiffness and strength being significantly larger in the direction of alignment, demonstrating that the microscale control of ECM organization results in functional changes in macroscale material behavior.