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Folding nanopatterned flat sheets into complex 3D structures enables the fabrication of meta-biomaterials that combine a rationally designed 3D architecture with nanoscale surface features. Self-folding is an attractive approach for realizing such materials. However, self-folded lattices are generally too compliant as there is an inherent competition between ease-of-folding requirements and final load-bearing characteristics. Inspired by sheet metal forming, an alternative route is proposed for the fabrication of origamilattices. This 'automated-folding' approach allows for the introduction of sharp folds into thick metal sheets, thereby enhancing their stiffness. The first time realization of automatically folded origami lattices with bone-mimicking mechanical properties is demonstrated. The proposed approach is highly scalable given that the unit cells making up the meta-biomaterial can be arbitrarily large in number and small in dimensions. To demonstrate the scalability and versatility of the proposed approach, it is fabricated origamilattices with > 100 unit cells, lattices with unit cells as small as 1.25 mm, and auxetic lattices. The nanopatterned the surface of the sheets prior to folding. Protected by a thin coating layer, these nanoscale features remained intact during the folding process. It is found that the nanopatterned folded specimens exhibits significantly increased mineralization as compared to their non-patterned counterparts., (© 2022 The Authors. Small published by Wiley-VCH GmbH.)

Black Ti (bTi) surfaces comprising high aspect ratio nanopillars exhibit a rare combination of bactericidal and osteogenic properties, framing them as cell-instructive meta-biomaterials. Despite the existing data indicating that bTi surfaces induce osteogenic differentiation in cells, the mechanisms by which this response is regulated are not fully understood. Here, we hypothesized that high aspect ratio bTi nanopillars regulate cell adhesion, contractility, and nuclear translocation of transcriptional factors, thereby inducing an osteogenic response in the cells. Upon the observation of significant changes in the morphological characteristics, nuclear localization of Yes-associated protein (YAP), and Runt-related transcription factor 2 (Runx2) expression in the human bone marrow-derived mesenchymal stem cells (hMSCs), we inhibited focal adhesion kinase (FAK), Rho-associated protein kinase (ROCK), and YAP in separate experiments to elucidate their effects on the subsequent expression of Runx2. Our findings indicated that the increased expression of Runx2 in the cells residing on the bTi nanopillars compared to the flat Ti is highly dependent on the activity of FAK and ROCK. A mechanotransduction pathway is then postulated in which the FAK-dependent adhesion of cells to the extreme topography of the surface is in close relation with ROCK to increase the endogenous forces within the cells, eventually determining the cell shape and area. The nuclear translocation of YAP may also enhance in response to the changes in cell shape and area, resulting in the translation of mechanical stimuli to biochemical factors such as Runx2., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Developing high-throughput nanopatterning techniques that also allow for precise control over the dimensions of the fabricated features is essential for the study of cell-nanopattern interactions. Here, we developed a process that fulfills both of these criteria. Firstly, we used electron-beam lithography (EBL) to fabricate precisely controlled arrays of submicron pillars with varying values of interspacing on a large area of fused silica. Two types of etching procedures with two different systems were developed to etch the fused silica and create the final desired height. We then studied the interactions of preosteoblasts (MC3T3-E1) with these pillars. Varying interspacing was observed to significantly affect the morphological characteristics of the cell, the organization of actin fibers, and the formation of focal adhesions. The expression of osteopontin (OPN) significantly increased on the patterns, indicating the potential of the pillars for inducing osteogenic differentiation. The EBL pillars were thereafter used as master molds in two subsequent processing steps, namely soft lithography and thermal nanoimprint lithography for high-fidelity replication of the pillars on the substrates of interest. The molding parameters were optimized to maximize the fidelity of the generated patterns and minimize the wear and tear of the master mold. Comparing the replicated feature with those present on the original mold confirmed that the geometry and dimensions of the replicated pillars closely resemble those of the original ones. The method proposed in this study, therefore, enables the precise fabrication of submicron- and nanopatterns on a wide variety of materials that are relevant for systematic cell studies. STATEMENT OF SIGNIFICANCE: Submicron pillars with specific dimensions on the bone implants have been proven to be effective in controlling cell behaviors. Nowadays, numerous methods have been proposed to produce bio-instructive submicron-topographies. However, most of these techniques are suffering from being low-throughput, low-precision, and expensive. Here, we developed a high-throughput nanopatterning technique that allows for control over the dimensions of the features for the study of cell-nanotopography interactions. Assessing the adaptation of preosteoblast cells showed the potential of the pillars for inducing osteogenic differentiation. Afterward, the pillars were used for high-fidelity replication of the bio-instructive features on the substrates of interest. The results show the advantages of nanoimprint lithography as a unique technique for the patterning of large areas of bio-instructive surfaces., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The surface topography of implantable devices is of crucial importance for guiding the cascade of events that starts from the initial contact of the cells with the surface and continues until the complete integration of the device in its immediate environment. There is, however, limited quantitative information available regarding the relationships between the different stages of such cascade(s) and how the design of surface topography influences them. We, therefore, used direct laser writing to 3D-print submicron pillars with precisely controlled dimensions and spatial arrangements to perform a systematic study of such relationships. Using single-cell force spectroscopy, we measured the adhesion force and the work of adhesion of the preosteoblast cells residing on the different types of surfaces. Not only the adhesion parameters (after 2-60 s) but also the formation of focal adhesions was strongly dependent on the geometry and arrangement of the pillars: sufficiently tall and dense pillars enhanced both adhesion parameters and the formation of focal adhesions. Our morphological study of the cells (after 24 h) showed that those enhancements were associated with a specific way of cell settlement onto the surface (i.e., "top state"). The cells interacting with tall and dense pillars were also characterized by numerous thick actin stress fibers in the perinuclear region and possibly high internal stresses. Furthermore, living cells with highly organized cytoskeletal networks exhibited greater values of the elastic modulus. The early responses of the cells predicted their late response including matrix mineralization: tall and dense submicron pillars significantly upregulated the expression of osteopontin after 21 days of culture under both osteogenic and nonosteogenic conditions. Our findings paint a detailed picture of at least one possible cascade of events that starts from initial cell adhesion and continues to subsequent cellular functions and eventual matrix mineralization. These observations could inform the future developments of instructive surfaces for medical devices based on physical surface cues and early markers.

Despite the potential of small-scale pillars of black titanium (bTi) for killing the bacteria and directing the fate of stem cells, not much is known about the effects of the pillars' design parameters on their biological properties. Here, three distinct bTi surfaces are designed and fabricated through dry etching of the titanium, each featuring different pillar designs. The interactions of the surfaces with MC3T3-E1 preosteoblast cells and Staphylococcus aureus bacteria are then investigated. Pillars with different heights and spatial organizations differently influence the morphological characteristics of the cells, including their spreading area, aspect ratio, nucleus area, and cytoskeletal organization. The preferential formation of focal adhesions (FAs) and their size variations also depend on the type of topography. When the pillars are neither fully separated nor extremely tall, the colocalization of actin fibers and FAs as well as an enhanced matrix mineralization are observed. However, the killing efficiency of these pillars against the bacteria is not as high as that of fully separated and tall pillars. This study provides a new perspective on the dual-functionality of bTi surfaces and elucidates how the surface design and fabrication parameters can be used to achieve a surface topography with balanced bactericidal and osteogenic properties., (© 2021 The Authors. Small published by Wiley-VCH GmbH.)

The recently developed additively manufacturing techniques have enabled the fabrication of porous biomaterials that mimic the characteristics of the native bone, thereby avoiding stress shielding and facilitating bony ingrowth. However, aseptic loosening and bacterial infection, as the leading causes of implant failure, need to be further addressed through surface biofunctionalization. Here, we used a combination of (1) plasma electrolytic oxidation (PEO) using Ca-, P-, and silver nanoparticle-rich electrolytes and (2) post-PEO hydrothermal treatments (HT) to furnish additively manufactured Ti-6Al-4V porous implants with a multi-functional surface. The applied HT led to the formation of hydroxyapatite (HA) nanocrystals throughout the oxide layer. This process was controlled by the supersaturation of Ca 2+ and PO 4 3- during the hydrothermal process. Initially, the high local supersaturation resulted in homogenous nucleation of spindle-like nanocrystals throughout the surface. As the process continued, the depletion of reactant ions in the outermost surface layer led to a remarkable decrease in the supersaturation degrees. High aspect-ratio nanorods and hexagonal nanopillars were, therefore, created. The unique hierarchical structure of the microporous PEO layer (pore size < 3 μm) and spindle-like HA nanocrystals (<150 nm) on the surface of macro-porous additively manufactured Ti-6Al-4V implants provided a favorable substrate for the anchorage of cytoplasmic extensions assisting cell attachment and migration on the surface. The results of our in vitro assays clearly showed the important benefits of the HT and the spindle-like HA nanocrystals including a significantly stronger and much more sustained antibacterial activity, significantly higher levels of pre-osteoblasts metabolic activity, and significantly higher levels of alkaline phosphatase activity as compared to similar PEO-treated implants lacking the HT., (Copyright © 2020 Elsevier B.V. All rights reserved.)

One of the methods to create sub-10 nm resolution metal-composed 3D nanopillars is electron beam-induced deposition (EBID). Surface nanotopographies (e.g., nanopillars) could play an important role in the design and fabrication of implantable medical devices by preventing the infections that are caused by the bacterial colonization of the implant surface. The mechanical properties of such nanoscale structures can influence their bactericidal efficiency. In addition, these properties are key factors in determining the fate of stem cells. In this study, we quantified the relevant mechanical properties of EBID nanopillars interacting with Staphylococcus aureus (S. aureus) using atomic force microscopy (AFM). We first determined the elastic modulus (17.7 GPa) and the fracture stress (3.0 ± 0.3 GPa) of the nanopillars using the quantitative imaging (QI) mode and contact mode (CM) of AFM. The displacement of the nanopillars interacting with the bacteria cells was measured by scanning electron microscopy (50.3 ± 9.0 nm). Finite element method based simulations were then applied to obtain the force-displacement curve of the nanopillars (considering the specified dimensions and the measured value of the elastic modulus) based on which an interaction force of 88.7 ± 36.1 nN was determined. The maximum von Mises stress of the nanopillars subjected to these forces was also determined (3.2 ± 0.3 GPa). These values were close to the maximum (i.e., fracture) stress of the pillars as measured by AFM, indicating that the nanopillars were close to their breaking point while interacting with S. aureus. These findings reveal unique quantitative data regarding the mechanical properties of nanopillars interacting with bacterial cells and highlight the possibilities of enhancing the bactericidal activity of the investigated EBID nanopillars by adjusting both their geometry and mechanical properties.

One of the major problems with the bone implant surfaces after surgery is the competition of host and bacterial cells to adhere to the implant surfaces. To keep the implants safe against implant-associated infections, the implant surface may be decorated with bactericidal nanostructures. Therefore, fabrication of nanostructures on biomaterials is of growing interest. Here, we systematically studied the effects of different processing parameters of inductively coupled plasma reactive ion etching (ICP RIE) on the Ti nanostructures. The resultant Ti surfaces were characterized by using scanning electron microscopy and contact angle measurements. The specimens etched using different chamber pressures were chosen for measurement of the mechanical properties using nanoindentation. The etched surfaces revealed various morphologies, from flat porous structures to relatively rough surfaces consisting of nanopillars with diameters between 26.4 ± 7.0 nm and 76.0 ± 24.4 nm and lengths between 0.5 ± 0.1 μm and 5.2 ± 0.3 μm. The wettability of the surfaces widely varied in the entire range of hydrophilicity. The structures obtained at higher chamber pressure showed enhanced mechanical properties. The bactericidal behavior of selected surfaces was assessed against Staphylococcus aureus and Escherichia coli bacteria while their cytocompatibility was evaluated with murine preosteoblasts. The findings indicated the potential of such ICP RIE Ti structures to incorporate both bactericidal and osteogenic activity, and pointed out that optimization of the process conditions is essential to maximize these biofunctionalities.

We systematically reviewed the currently available evidence on how the design parameters of surface nanopatterns (e.g. height, diameter, and interspacing) relate to their bactericidal behavior. The systematic search of the literature resulted in 46 studies that satisfied the inclusion criteria of examining the bactericidal behavior of nanopatterns with known design parameters in absence of antibacterial agents. Twelve of the included studies also assessed the cytocompatibility of the nanopatterns. Natural and synthetic nanopatterns with a wide range of design parameters were reported in the included studies to exhibit bactericidal behavior. However, most design parameters were in the following ranges: heights of 100-1000 nm, diameters of 10-300 nm, and interspacings of  <500 nm. The most commonly used type of nanopatterns were nanopillars, which could kill bacteria in the following range of design parameters: heights of 100-900 nm, diameters of 20-207 nm, and interspacings of 9-380 nm. The vast majority of the cytocompatibility studies (11 out of 12) showed no adverse effects of bactericidal nanopatterns with the only exception being nanopatterns with extremely high aspect ratios. The paper concludes with a discussion on the evidence available in the literature regarding the killing mechanisms of nanopatterns and the effects of other parameters including surface affinity of bacteria, cell size, and extracellular polymeric substance (EPS) on the killing efficiency. STATEMENT OF SIGNIFICANCE: The use of nanopatterns to kill bacteria without the need for antibiotics represents a rapidly growing area of research. However, the optimum design parameters to maximize the bactericidal behavior of such physical features need to be fully identified. The present manuscript provides a systematic review of the bactericidal nanopatterned surfaces. Identifying the effective range of dimensions in terms of height, diameter, and interspacings, as well as covering their impact on mammalian cells, has enabled a comprehensive discussion including the bactericidal mechanisms and the factors controlling the bactericidal efficiency. Overall, this review helps the readers have a better understanding of the state-of-the-art in the design of bactericidal nanopatterns, serving as a design guideline and contributing to the design of future experimental studies., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The cellular microenvironment plays a crucial role in improving cell response and function of an engineered tissue. Scaffolds mimicking the native ECM and capable of releasing growth factors are great candidates for tissue engineering applications. Gelatin methacryloyl (GelMA) hydrogel, a photocrosslinkable biomaterial possessing tunable properties, has been widely used in tissue engineering. It has been suggested that incorporating micro/nano carriers in GelMA could provide a sustained release of growth factors. Specifically, chitosan nanoparticles can be used for growth factor delivery due to its biocompatibility, easy method of synthesis, and preventing the biomolecule from degradation. In this study, GelMA/chitosan nanoparticles composite hydrogel was developed to deliver an angiogenic growth factor (bFGF). The hydrogel was prepared by photopolymerization and its chemical and physical properties were characterized. Its degradation and swelling characteristics were also evaluated. The size of nanoparticles was evaluated and the profile of bFGF release from the hydrogel and its effect on the viability of fibroblast cells was studied. The results showed that GelMA/chitosan nanoparticles can significantly promote cell proliferation due to its biocompatible structure and providing a sustained profile of bFGF release. This hydrogel scaffold can be used for efficient delivery of bFGF in various applications and especially for angiogenesis.

Placenta-derived amniotic epithelial cells (AECs), a great cell source for tissue engineering and stem cell therapy, are immunologically inert in their native state; however, immunological changes in these cells after culture and differentiation have challenged their applications. The aim of this study was to investigate the effect of 2D and 3D scaffolds on human lymphocyte antigens (HLA) expression by AECs. The effect of different preparation parameters including pre-freezing time and temperature was evaluated on 3D chitosan-gelatine scaffolds properties. Evaluation of MHC class I, HLA-DR and HLA-G expression in AECs after 7 d culture on 2D bed and 3D scaffold of chitosan-gelatine showed that culture of AECs on the 2D substrate up-regulated MHC class I and HLA-DR protein markers on AECs surface and down-regulated HLA-G protein. In contrast, 3D scaffold did not increase protein expression of MHC class I and HLA-DR. Moreover, HLA-G protein expression remained unchanged in 3D culture. These results confirm that 3D scaffold can remain AECs in their native immunological state and modification of physical properties of the scaffold is a key regulator of immunological markers at the gene and protein expression levels; a strategy which circumvents rejection challenge of amniotic stem cells to be translated into the clinic.

One of the main goals of tissue engineering and regenerative medicine is to develop skin substitutes for treating deep dermal and full thickness wounds. In this regard, both scaffold and cell source have a fundamental role to achieve exactly the same histological and physiological analog of skin. Amnion epithelial and mesenchymal cells possess the characteristics of pluripotent stem cells which have the capability to differentiate into all three germ layers and can be obtained without any ethical concern. Amniotic cells also produce different growth factors, angio-modulatory cytokines, anti-bacterial peptides and a wide range of anti-inflammatory agents which eventually cause acceleration in wound healing. In addition, amniotic membrane matrix exhibits characteristics of an ideal scaffold and skin substitute through various types of extracellular proteins such as collagens, laminins and fibronectins which serve as an anchor for cell attachment and proliferation, a bed for cell delivery and a reservoir of drugs and growth factors involved in wound healing process. Recently, isolation of amniotic cells exosomes, surface modification and cross-linking approaches, construction of amnion based nanocomposites and impregnation of amnion with nanoparticles, construction of amnion hydrogel and micronizing process promoted its properties for tissue engineering. In this manuscript, the recent progress was reviewed which approve that amnion-derived cells and matrix have potential to be involved in skin substitutes; an enriched cell containing scaffold which has a great capability to be translated into the clinic.

Due to antibacterial characteristic, amnion has been frequently used in different clinical situations. Developing an in vitro method to augment endogenous antibacterial ingredient of amniotic epithelial and mesenchymal stem cells is desirable for a higher efficacy of this promising biomaterial. In this study, epithelial or mesenchymal side dependent effect of amniotic membrane (AM) on antibacterial activity against some laboratory and clinical isolated strains was investigated by modified disk diffusion method and colony count assay. The effect of exposure to IL-1β in production and release of antibacterial ingredients was investigated by ELISA assay. The results showed that there is no significant difference between epithelial and mesenchymal sides of amnion in inhibition of bacterial growth. Although the results of disk diffusion showed that the AM inhibitory effect depends on bacterial genus and strain, colony count assay showed that the extract of AM inhibits all investigated bacterial strains. The exposure of AM to IL-1β leads to a higher level of antibacterial peptides secretion including elafin, HBD-2, HBD-3 and cathelicidic LL-37. Based on these results, amniotic cells possess antibacterial activity which can be augmented by inflammatory signal inducers; a process which make amnion and its epithelial and mesenchymal stem cells more suitable for tissue engineering and regenerative medicine.

Human amniotic membrane (AM) is an appropriate candidate for treatment of cancer due to special properties, such as inhibition of angiogenesis and secretion of pro-apoptotic factors. This research was designed to evaluate the impact of cryopreservation on cancer cell death induction and anti-angiogenic properties of the AM. Cancer cells were treated with fresh and cryopreserved amniotic condition medium during 24 h and cancer cell viability was determined by MTT assay. To evaluate angiogenesis, the rat aorta ring assay was performed for both fresh and cryopreserved AM within 7 days. In addition, four anti-angiogenic factors Tissue Inhibitor of Matrix Metalloproteinase-1 and 2 (TIMP-1 and TIMP-2), Thrombospondin, and Endostatin were measured by ELISA assay before and after cryopreservation. The results showed that the viability of cultured cancer cells dose-dependently decreased after treatment with condition medium of fresh and cryopreserved tissue and no significant difference was observed between the fresh and cryopreserved AM. The results revealed that the amniotic epithelial stem cells inhibit the penetration of fibroblast-like cells and angiogenesis. Moreover, the penetration of fibroblast-like cells in both epithelial and mesenchymal sides of fresh and cryopreserved AM was observed after removing of epithelial cells. The cryopreservation procedure significantly decreased anti-angiogenic factors TIMP-1, TIMP-2, Thrombospondin, and Endostatin which shows that angio-modulatory property is not fully dependent on proteomic and metabolomic profiles of the AM. These promising results demonstrate that cancer cell death induction and anti-angiogenic properties of the AM were maintained within cryopreservation; a procedure which can circumvent limitations of the fresh AM., (Copyright © 2016 Elsevier Inc. All rights reserved.)