Precise Micro-architecture Research Articles

Mechanical properties of a new 3D printed gradable porous cellular lattice structure considering surface-to-volume capability

In recent years, significant progress in additive manufacturing (AM) techniques has enabled the design and manufacturing of cellular structures with precise micro-architecture and complex cell topologies. This advancement has significantly bolstered the manufacturing of porous materials, leading to the enhancement of their mechanical properties without compromising their lightweight nature. In this paper, a new cellular structure with cell migration capability for the regeneration of bones is designed and introduced. Uniform and functional graded porous lattice structures are manufactured based on the new unit cell with AM technology. The mechanical properties of the unit cell and the cellular structures in two directions are derived analytically. To validate analytical mathematical equations, numerical modeling and experimental tests are conducted. The results of experimental, numerical, and analytical studies are compared to each other and show good agreement. The discrepancy between the yield stress values obtained from the analytical and experimental models of the functional specimen falls within an acceptable range of error regarding usual uncertainties in manufacturing process and surface quality besides the theoretical limitations. Finally, we utilize the genetic algorithm to perform single and multi-objective optimizations on the performance of the unit cell. The optimization results indicate that the new unit cell with optimized lengths sides can improve cell migration and bone regeneration according to the mentioned criteria for the optimization.

Injectable remote magnetic nanofiber/hydrogel multiscale scaffold for functional anisotropic skeletal muscle regeneration

Developing an injectable anisotropic scaffold with precisely topographic cues to induce 3D cellular organization plays a critical role in volumetric muscle loss (VML) repair in vivo. However, controlling aligned myofiber regeneration in vivo based on previous injectable scaffolds continues to prove challenging, especially in a 3D configuration. Herein, we prepare the monodisperse remote magnetic controlled short nanofibers (MSNFs) with a high yield using an advanced coaxial electrospinning-cyrocutting method. An injectable anisotropic MSNF/Gel nanofiber/hydrogel scaffold based on MSNFs within photocurable hydrogel is further designed, showing the ability to guide 3D cellular alignment and organization by the precise microarchitecture control via a remote magnetic field. MSNF/Gel anisotropic scaffolds were able to recreate the macroscale and microscale topographical features of orbicular muscle and bipennate muscle mimicking their anatomical locations. Furthermore, the resultant MSNF/Gel anisotropic scaffolds significantly enhanced aligned myofiber formation in vivo and improved functional recovery of injured muscles in animal VML models. In summary, this approach offers a new promising tissue engineering strategy not only for the aligned myofiber formation for enhancing skeletal muscle regeneration in vivo but also for other biofabrication of living constructs containing complex anisotropy in vitro.

Digital light processing mediated 3D printing of biocomposite bone scaffolds: Physico‐chemical interactions and in‐vitro biocompatibility

AbstractThis study reports development of digital light processing (DLP) mediated 3D printed customized bone scaffolds. Bioactive fluorcanasite glass ceramic was incorporated within photocurable resin matrix and the suspensions were 3D printed towards developing composite bone scaffolds. Physico‐chemical interaction in the biocomposite bone scaffolds were investigated using infrared spectroscopy, x‐ray diffraction, and field emission scanning electron microscopy. Further, the mechanical properties of the composite scaffold samples were also evaluated to understand the strengths of the samples in terms of its fracture toughness, flexural strength, and compressive strength. Infrared spectroscopy results demonstrated an active interaction between the acrylate functionalities of the polymer with the bioactive fluorcanasite glass ceramic reinforcement. This was further substantiated with x‐ray diffraction results, demonstrating rise in the bioactive crystalline peaks with corresponding increase in the fluorcanasite glass ceramic loading. Later, the samples were also evaluated for in‐vitro cellular response in terms of cell viability, proliferation, adhesion, and interaction. The surface hydrophilicity responsible for osteogenic interaction was also studied with contact angle goniometry. With increase in fluorcanasite loading in the formulation, the hydrophilicity was found to increase over the sample surface which in turn was found to enhance cell adhesion and proliferation, as revealed byin‐vitro MTT assay and also fluorescence microscopy. To establish the efficacy of DLP technique, two different porous architectural designs were 3D printed and investigated with synchrotron micro‐computed tomography. The microtomographs revealed precise microarchitecture and interconnected porosity within the scaffolds.

Spatial clustering of orientation preference in primary visual cortex of the large rodent agouti.

SummaryAll rodents investigated so far possess orientation-selective neurons in the primary visual cortex (V1) but – in contrast to carnivores and primates – no evidence of periodic maps with pinwheel-like structures. Theoretical studies debating whether phylogeny or universal principles determine development of pinwheels point to V1 size as a critical constraint. Thus, we set out to study maps of agouti, a big diurnal rodent with a V1 size comparable to cats'. In electrophysiology, we detected interspersed orientation and direction-selective neurons with a bias for horizontal contours, corroborated by homogeneous activation in optical imaging. Compatible with spatial clustering at short distance, nearby neurons tended to exhibit similar orientation preference. Our results argue against V1 size as a key parameter in determining the presence of periodic orientation maps. They are consistent with a phylogenetic influence on the map layout and development, potentially reflecting distinct retinal traits or interspecies differences in cortical circuitry.

ScafSLICR: A MATLAB-based slicing algorithm to enable 3D-printing of tissue engineering scaffolds with heterogeneous porous microarchitecture.

3D-printing is a powerful manufacturing tool that can create precise microscale architectures across macroscale geometries. Within biomedical research, 3D-printing of various materials has been used to fabricate rigid scaffolds for cell and tissue engineering constructs with precise microarchitecture to direct cell behavior and macroscale geometry provides patient specificity. While 3D-printing hardware has become low-cost due to modeling and rapid prototyping applications, there is no common paradigm or platform for the controlled design and manufacture of 3D-printed constructs for tissue engineering. Specifically, controlling the tissue engineering features of pore size, porosity, and pore arrangement is difficult using currently available software. We have developed a MATLAB approach termed scafSLICR to design and manufacture tissue-engineered scaffolds with precise microarchitecture and with simple options to enable spatially patterned pore properties. Using scafSLICR, we designed, manufactured, and characterized porous scaffolds in acrylonitrile butadiene styrene with a variety of pore sizes, porosities, and gradients. We found that transitions between different porous regions maintained an open, connected porous network without compromising mechanical integrity. Further, we demonstrated the usefulness of scafSLICR in patterning different porous designs throughout large anatomic shapes and in preparing craniofacial tissue engineering bone scaffolds. Finally, scafSLICR is distributed as open-source MATLAB scripts and as a stand-alone graphical interface.

Peripheral and Central Inputs Shape Network Dynamics in the Developing Visual Cortex In Vivo

Spontaneous network activity constitutes a central theme during the development of neuronal circuitry [1, 2]. Before the onset of vision, retinal neurons generate waves of spontaneous activity that are relayed along the ascending visual pathway [3, 4] and shape activity patterns in these regions [5, 6]. The spatiotemporal nature of retinal waves is required to establish precise functional maps in higher visual areas, and their disruption results in enlarged axonal projection areas (e.g., [7-10]). However, how retinal inputs shape network dynamics in the visual cortex on the cellular level is unknown. Using in vivo two-photon calcium imaging, we identified two independently occurring patterns of network activity in the mouse primary visual cortex (V1) before and at the onset of vision. Acute manipulations of spontaneous retinal activity revealed that one type of network activity largely originated in the retina and was characterized by low synchronicity (L-) events. In addition, we identified a type of high synchronicity (H-) events that required gap junction signaling but were independent of retinal input. Moreover, the patterns differed in wave progression and developmental profile. Our data suggest that different activity patterns have complementary functions during the formation of synaptic circuits in the developing visual cortex.

Spectral Analysis of Local Field Potentials from Rat Primary Visual Cortex (V1)

In primary visual cortex (V1) of rats, orientation selective neurons do not show topographic organization on a larger scale (Ohki et al., 2005), in contrast to the columnar organization of orientation selective cells in V1 of cat and monkey. Accordingly, while orientation tuning in mass signals like the local field potential (LFP) has been described in recordings from cat V1, it can be assumed to be weak or absent in rodent V1. However, in monkey motor cortex, which also shows no clear columnar organization, information about the direction of the monkey’s hand movement could be extracted from LFP power spectra with high predictive power (Rickert et al., 2005). To reveal in how far global network tuning properties could be extracted from mass signals of primary sensory areas which do not show clear topographic organization, we performed in vivo multi-electrode recordings in V1 of anesthetized rats while presenting visual stimuli with clear direction information (moving edges and gratings). Spectral analysis of LFP signals from multiple channels was performed using adaptive multivariate auto-regression (AMVAR; Ding et al., 2000) after removing the first two statistical moments (mean and standard deviation across trials) from the signal. This method allows high spectral estimation accuracy with high temporal resolution superior to other methods of time-dependent spectral analysis like multi-tapering (Nalatore & Rangarajan, 2009). Direction tuning was assessed using circular statistical measures. For moving edges, significant directional tuning in the LFP power spectrum can be found during the onset transient up to 500 ms after stimulus onset. Tuning strength was not distributed evenly across the whole observed spectrum (<200 Hz), however, but limited to mainly two distinct frequency bands centered around ~30 Hz and ~70 Hz. Preliminary analysis of moving grating stimuli did not reveal significant tuning either in the onset transient or the steady state during stimulus presentation. These results suggest that, even in the absence of coarse scale organization of cortical networks with respect to tuning properties, information about the stimulus properties can be inferred with appropriate methods.

A three-dimensional scaffold with precise micro-architecture and surface micro-textures

A three-dimensional (3D) structure comprising precisely defined micro-architecture and surface micro-textures, designed to present specific physical cues to cells and tissues, may provide an efficient scaffold in a variety of tissue engineering and regenerative medicine applications. We report a fabrication technique based on microfabrication and soft lithography that permits for the development of 3D scaffolds with both precisely engineered architecture and tailored surface topography. The scaffold fabrication technique consists of three key steps starting with microfabrication of a mold using an epoxy-based photoresist (SU-8), followed by dual-sided molding of a single layer of polydimethylsiloxane (PDMS) using a mechanical jig for precise motion control; and finally, alignment, stacking, and adhesion of multiple PDMS layers to achieve a 3D structure. This technique was used to produce 3D Texture and 3D Smooth PDMS scaffolds, where the surface topography comprised 10μm diameter/height posts and smooth surfaces, respectively. The potential utility of the 3D microfabricated scaffolds, and the role of surface topography, were subsequently investigated in vitro with a combined heterogeneous population of adult human stem cells and their resultant progenitor cells, collectively termed connective tissue progenitors (CTPs), under conditions promoting the osteoblastic phenotype. Examination of bone-marrow derived CTPs cultured on the 3D Texture scaffold for 9 days revealed cell growth in three dimensions and increased cell numbers compared to those on the 3D Smooth scaffold. Furthermore, expression of alkaline phosphatase mRNA was higher on the 3D Texture scaffold, while osteocalcin mRNA expression was comparable for both types of scaffolds.

Neuronal Selectivity and Local Map Structure in Visual Cortex

The organization of primary visual cortex (V1) into functional maps makes individual cells operate in a variety of contexts. For instance, some neurons lie in regions of fairly homogeneous orientation preference (iso-orientation domains), while others lie in regions with a variety of preferences (e.g., pinwheel centers). We asked whether this diversity in local map structure correlates with the degree of selectivity of spike responses. We used a combination of imaging and electrophysiology to reveal that neurons in regions of homogeneous orientation preference have much sharper tuning. Moreover, in both monkeys and cats, a common principle links the structure of the orientation map, on the spatial scale of dendritic integration, to the degree of selectivity of individual cells. We conclude that neural computation is not invariant across the cortical surface. This finding must factor into future theories of receptive field wiring and map development.

Three-Dimensional Mapping of Unitary Synaptic Connections by Two-Photon Macro Photolysis of Caged Glutamate

To understand the precise microarchitecture of the cortical circuitry, it is crucial to know the distribution of synaptic connections and their synaptic strengths at the level of a single cell, rather than a group of cells. Here, we describe a new application of two-photon photolysis of caged glutamate that enabled us to induce an action potential in only a small number (about five) of pyramidal neurons by increasing the volume of two-photon excitation by reducing the effective numerical aperture of the objective. We performed whole cell patch-clamp recordings from layer 2/3 pyramidal neurons in the rat visual cortex and stimulated many neurons in a large three-dimensional space (approximately 600 x 600 x 100 microm) including neurons in layers 2/3 and 4 using this new technique. We mapped the density and amplitude of unitary excitatory postsynaptic currents and found that the basic microarchitecture of excitatory synaptic connections consists of two regions: a columnar, dense core region with a radius of 150 microm and an outer, sparse region. The dense core region includes the majority of strong synaptic connections in layer 2/3. Our results reveal the columnar organization of synaptic connectivity in the rat visual cortex, where functional columns have not been clearly demonstrated. Thus this technique will be a uniquely powerful tool for quantifying synaptic connectivity and manipulating neural activity at the single-cell level.

Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex

Neurons in the cerebral cortex are organized into anatomical columns, with ensembles of cells arranged from the surface to the white matter. Within a column, neurons often share functional properties, such as selectivity for stimulus orientation; columns with distinct properties, such as different preferred orientations, tile the cortical surface in orderly patterns. This functional architecture was discovered with the relatively sparse sampling of microelectrode recordings. Optical imaging of membrane voltage or metabolic activity elucidated the overall geometry of functional maps, but is averaged over many cells (resolution >100 microm). Consequently, the purity of functional domains and the precision of the borders between them could not be resolved. Here, we labelled thousands of neurons of the visual cortex with a calcium-sensitive indicator in vivo. We then imaged the activity of neuronal populations at single-cell resolution with two-photon microscopy up to a depth of 400 microm. In rat primary visual cortex, neurons had robust orientation selectivity but there was no discernible local structure; neighbouring neurons often responded to different orientations. In area 18 of cat visual cortex, functional maps were organized at a fine scale. Neurons with opposite preferences for stimulus direction were segregated with extraordinary spatial precision in three dimensions, with columnar borders one to two cells wide. These results indicate that cortical maps can be built with single-cell precision.

Microfabricated 3D Scaffolds for Tissue Engineering Applications

ABSTRACTMicrofabrication and soft lithographic techniques are combined to develop three-dimensional (3D) polydimethylsiloxane (PDMS) scaffolds comprising multiple levels of meandering pore geometry textured with 10 μm posts. Both micro-architecture and surface micro-textures have been shown to selectively stimulate cell and tissue behavior. To achieve a 3D scaffold with precise micro-architecture and surface micro-textures, 100 μm thick PDMS films were manufactured using a stacking technique to realize a 66% porous 3D structure with 200 × 400 μm horizontal through holes, 300 μm diameter vertical through holes and 71% surface coverage with 10 μm diameter and 10 μm high posts. Each PDMS porous film level was manufactured by the dual-sided molding of uncured PDMS between a three level SU-8 photoresist mold (of 200, 10, and 100 μm thick features) and a PDMS mold with 10 μm deep micro-textures. Dual-sided molding was achieved using a custom motion control mechanical jig that allowed relative mold alignment to within ∼ ±10 μm.

MEMS-Based Technologies for Cellular Encapsulation

In the past, significant progress has been made in applying micro-electromechanical based systems to biomedical applications (BioMEMS). While the focus of such research has primarily been on diagnostic tools, more recently therapeutic applications have been explored. This paper discusses the current state of BioMEMS for therapeutic applications, particularly with respect to diabetes mellitus. A novel therapeutic application of microfabrication technology, a micromachined membrane-based biocapsule, has been developed for the transplantation of protein-secreting cells without the need for immunosuppression. Conventional lithographic techniques can be manipulated to produce monodisperse, nanoporous, biocompatible, silicon membranes. This new approach to cell encapsulation is based on microfabrication technology whereby immunoisolation membranes are bulk and surface micromachined to present uniform and well-controlled pore sizes as small as 10nm, tailored surface chemistries, and precise microarchitecture. Through its ability to achieve highly controlled microarchitectures on size scales relevant to living systems (from μm to nm), microfabrication technology offers unique opportunities to more precisely engineer biocapsules that allow free exchange of the nutrients, waste products, and secreted therapeutic proteins between the host (patient) and implanted cells, but exclude lymphocytes and antibodies that may attack foreign cells. Results indicate that both primary pancreatic cells and insulinoma cells can remain viable and functional within nanoporous silicon biocapsular environments. The cells within such environments can secrete insulin at rates comparable to unencapsulated cells. Moreover, these membranes are able to allow insulin and glucose diffusion, while hindering antibody diffusion. Implanted microfabricated biocapsules show in vivo biocompatibility and vascular ingrowth, particularly when modified with polyethylene oxide surface coating. Thus, microfabricated inorganic encapsulation devices may provide biocompatibility, in vivo chemical and mechanical stability, tailored pore geometries, and superior immunoisolation for encapsulated cells over conventional encapsulation approaches. By using microfabrication techniques, structures can be fabricated with spatial features from the sub-micron range up to several millimeters. These multi-scale structures correspond well with hierarchical biologic structures, from proteins and sub-cellular organelles to the tissue and organ levels.

Nanoporous biocapsules for the encapsulation of insulinoma cells: biotransport and biocompatibility considerations.

This study investigates whether nanoporous micromachined biocapsules, with uniform membrane pore sizes of 24.5-nm, can be used to encapsulate insulin-secreting cells in vitro. This approach to cell encapsulation is based on microfabrication technology whereby immunoisolation membranes are bulk and surface micromachined to present uniform and well-controlled pore sizes as small as 10 nm, tailored surface chemistries, and precise microarchitectures. This study evaluates the behavior of insulinoma cells with micromachined membranes, the effect of matrix configurations within the biocapsule on cell behavior, as well as insulin and glucose transport through the biocapsule membranes.

Micromachined interfaces: new approaches in cell immunoisolation and biomolecular separation

As a novel therapeutic application of microfabrication technology, a micromachined membrane-based biocapsule is described for the transplantation of protein-secreting cells without the need for immunosuppression. This new approach to cell encapsulation is based on microfabrication technology whereby immunoisolation membranes are bulk and surface micromachined to present uniform and well-controlled pore sizes as small as 10 nm, tailored surface chemistries, and precise microarchitecture. Through its ability to achieve highly controlled microarchitectures on size scales relevant to living systems (from μm to nm), microfabrication technology offers unique opportunities to more precisely engineer biocapsules that allow free exchange of the nutrients, waste products, and secreted therapeutic proteins between the host (patient) and implanted cells, but exclude lymphocytes and antibodies that may attack foreign cells. Microfabricated inorganic encapsulation devices may provide biocompatibility, in vivo chemical and mechanical stability, tailored pore geometries, and superior immunoisolation for encapsulated cells over conventional encapsulation approaches. By using microfabrication techniques, structures can be fabricated with spatial features from the sub-micron range up to several millimeters. These multi-scale structures correspond well with hierarchical biological structures, from proteins and sub-cellular organelles to the tissue and organ levels.