Antifouling nanoplatform for controlled attachment ofE. coli.

Biofouling is the most common cause of bacterial contamination in implanted materials/devices resulting in severe inflammation, implant mobilization, and eventual failure. Since bacterial attachment represents the initial step toward biofouling, developing synthetic surfaces that prevent bacterial adhesion is of keen interest in biomaterials research. In this study, we develop antifouling nanoplatforms that effectively impede bacterial adhesion and the consequent biofilm formation. We synthesize the antifouling nanoplatform by introducing silicon (Si)/silica nanoassemblies to the surface through ultrafast ionization of Si substrates. We assess the effectiveness of these nanoplatforms in inhibitingEscherichia coli(E. coli) adhesion. The findings reveal a significant reduction in bacterial attachment on the nanoplatform compared to untreated silicon, with bacteria forming smaller colonies. By manipulating physicochemical characteristics such as nanoassembly size/concentration and nanovoid size, we further control bacterial attachment. These findings suggest the potential of our synthesized nanoplatform in developing biomedical implants/devices with improved antifouling properties.

Absence of E2f1 Negates Pro-osteogenic Impacts of p21 Absence.

Loss of p21 leads to increased bone formation post-injury; however, the mechanism(s) by which this occurs remains undetermined. E2f1 is downstream of p21 and as a transcription factor can act directly on gene expression; yet it is unknown if E2f1 plays a role in the osteogenic effects observed when p21 is differentially regulated. In this study we aimed to investigate the interplay between p21 and E2f1 and determine if the pro-regenerative osteogenic effects observed with the loss of p21 are E2f1 dependent. To accomplish this, we employed knockout p21 and E2f1 mice and additionally generated a p21/E2f1 double knockout. These mice underwent burr-hole injuries to their proximal tibiae and healing was assessed over 7 days via microCT imaging. We found that p21 and E2f1 play distinct roles in bone regeneration where the loss of p21 increased trabecular bone formation and loss of E2f1 increased cortical bone formation, yet loss of E2f1 led to poorer bone repair overall. Furthermore, when E2f1 was absent, either individually or simultaneously with p21, there was a dramatic decrease of the number of osteoblasts, osteoclasts, and chondrocytes at the site of injury compared to p21-/- and C57BL/6 mice. Together, these results suggest that E2f1 regulates the cell populations required for bone repair and has a distinct role in bone formation/repair compared to p21-/-E2f1-/-. These results highlight the possibility of cell cycle and/or p21/E2f1 being potential druggable targets that could be leveraged in clinical therapies to improve bone healing in pathologies such as osteoporosis.

Exogenously delivered iPSCs disrupt the natural repair response of endogenous MPCs after bone injury.

Promoting bone healing including fracture non-unions are promising targets for bone tissue engineering due to the limited success of current clinical treatment methods. There has been significant research on the use of stem cells with and without biomaterial scaffolds to treat bone fractures due to their promising regenerative capabilities. However, the relative roles of exogenous vs. endogenous stem cells and their overall contribution to in vivo fracture repair is not well understood. The purpose of this study was to determine the interaction between exogenous and endogenous stem cells during bone healing. This study was conducted using a standardized burr-hole bone injury model in a mesenchymal progenitor cell (MPC) lineage-tracing mouse under normal homeostatic and osteoporotic conditions. Burr-hole injuries were treated with a collagen-I biomaterial loaded with and without labelled induced pluripotent stem cells (iPSCs). Using lineage-tracing, the roles of exogenous and endogenous stem cells during bone healing were examined. It was observed that treatment with iPSCs resulted in muted healing compared to untreated controls in intact mice post-injury. When the cell populations were examined histologically, iPSC-treated burr-hole defects presented with a dramatic reduction in endogenous MPCs and cell proliferation throughout the injury site. However, when the ovaries were removed and an osteoporotic-like phenotype induced in the mice, iPSCs treatment resulted in increased bone formation relative to untreated controls. In the absence of iPSCs, endogenous MPCs demonstrated robust proliferative and osteogenic capacity to undertake repair and this behaviour was disrupted in the presence of iPSCs which instead took on an osteoblast fate but with little proliferation. This study clearly demonstrates that exogenously delivered cell populations can impact the normal function of endogenous stem/progenitor populations during the normal healing cascade. These interactions need to be better understood to inform cell and biomaterial therapies to treat fractures.

Label-Free Saliva Test for Rapid Detection of Coronavirus Using Nanosensor-Enabled SERS.

The recent COVID-19 pandemic has highlighted the inadequacies of existing diagnostic techniques and the need for rapid and accurate diagnostic systems. Although molecular tests such as RT-PCR are the gold standard, they cannot be employed as point-of-care testing systems. Hence, a rapid, noninvasive diagnostic technique such as Surface-enhanced Raman scattering (SERS) is a promising analytical technique for rapid molecular or viral diagnosis. Here, we have designed a SERS- based test to rapidly diagnose SARS-CoV-2 from saliva. Physical methods synthesized the nanostructured sensor. It significantly increased the detection specificity and sensitivity by ~ten copies/mL of viral RNA (~femtomolar concentration of nucleic acids). Our technique combines the multiplexing capability of SERS with the sensitivity of novel nanostructures to detect whole virus particles and infection-associated antibodies. We have demonstrated the feasibility of the test with saliva samples from individuals who tested positive for SARS-CoV-2 with a specificity of 95%. The SERS-based test provides a promising breakthrough in detecting potential mutations that may come up with time while also preparing the world to deal with other pandemics in the future with rapid response and very accurate results.

Cell cycle regulators and bone: development and regeneration.

Cell cycle regulators act as inhibitors or activators to prevent cancerogenesis. It has also been established that they can play an active role in differentiation, apoptosis, senescence, and other cell processes. Emerging evidence has demonstrated a role for cell cycle regulators in bone healing/development cascade. We demonstrated that deletion of p21, a cell cycle regulator acting at the G1/S transition enhanced bone repair capacity after a burr-hole injury in the proximal tibia of mice. Similarly, another study has shown that inhibition of p27 can increase bone mineral density and bone formation. Here, we provide a concise review of cell cycle regulators that influence cells like osteoblasts, osteoclasts, and chondrocytes, during development and/or healing of bone. It is imperative to understand the regulatory processes that govern cell cycle during bone healing and development as this will pave the way to develop novel therapies to improve bone healing after injury in instances of aged or osteoporotic fractures.

A non-immunological role for γ-interferon-inducible lysosomal thiol reductase (GILT) in osteoclastic bone resorption.

The extracellular bone resorbing lacuna of the osteoclast shares many characteristics with the degradative lysosome of antigen-presenting cells. γ-Interferon-inducible lysosomal thiol reductase (GILT) enhances antigen processing within lysosomes through direct reduction of antigen disulfides and maintenance of cysteine protease activity. In this study, we found the osteoclastogenic cytokine RANKL drove expression of GILT in osteoclast precursors in a STAT1-dependent manner, resulting in high levels of GILT in mature osteoclasts, which could be further augmented by γ-interferon. GILT colocalized with the collagen-degrading cysteine protease, cathepsin K, suggesting a role for GILT inside the osteoclastic resorption lacuna. GILT-deficient osteoclasts had reduced bone-resorbing capacity, resulting in impaired bone turnover and an osteopetrotic phenotype in GILT-deficient mice. We demonstrated that GILT could directly reduce the noncollagenous bone matrix protein SPARC, and additionally, enhance collagen degradation by cathepsin K. Together, this work describes a previously unidentified, non-immunological role for GILT in osteoclast-mediated bone resorption.

Absence of Proteoglycan 4 (Prg4) Leads to Increased Subchondral Bone Porosity Which Can Be Mitigated Through Intra-Articular Injection of PRG4.

Proteoglycan 4 (PRG4) is a mucin-like glycoprotein important for joint health. Mice lacking Prg4 demonstrate degeneration of the cartilage and altered skeletal morphology. The purpose of this study was to examine if Prg4 deficiency leads to subchondral bone defects and if these defects could be mitigated through intra-articular injection of recombinant human PRG4 (rhPRG4). Mice deficient in Prg4 expression demonstrated increased cartilage thickness and increased subchondral bone porosity compared with C57BL/6 controls. While the porosity of the subchondral bone of Prg4-/- mice decreased over time with maturation, intra-articular injection of rhPRG4 was able to forestall the increase in porosity. In contrast, neither hyaluronan (HA) nor methylprednisolone injections had beneficial effects on the subchondral bone porosity in the Prg4 knockout mice. Bone marrow progenitor cells from Prg4-/- mice demonstrated reduced osteogenic differentiation capacity at 4 weeks of age, but not at 16 weeks of age. While most studies on PRG4/lubricin focus on the health of the cartilage, this study demonstrates that PRG4 plays a role in the maturation of the subchondral bone. Furthermore, increasing joint lubrication/viscosupplementation through injection of HA or controlling joint inflammation through injection of methylprednisolone may help maintain the cartilage surface, but had no positive effect on the subchondral bone in animals lacking Prg4. Therefore, alterations in the subchondral bone in models with absent or diminished Prg4 expression should not be overlooked when investigating changes within the articular cartilage regarding the pathogenesis of osteoarthritis/arthrosis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2077-2088, 2019.

Absence of p21(WAF1/CIP1/SDI1) protects against osteopenia and minimizes bone loss after ovariectomy in a mouse model.

p21(WAF1/CIP1/SDI1) is a critical sentinel of the cell cycle that plays an important role in determining cell fate with respect to proliferation, differentiation and apoptosis. Recent studies have demonstrated that inhibition/loss of p21 promotes osteo-chondro differentiation in progenitor/stem cells, and that p21 knockout (p21-/-) mice demonstrate enhanced bone regeneration compared to wild-type controls after a non-critical size defect. It was therefore hypothesized that the absence of p21 may also protect against bone loss through enhancing bone formation, tilting the balance away from bone resorption, in an ovariectomy-induced osteopenia mouse model, investigated via microCT imaging. While p21-/- mice demonstrated significantly less bone loss after ovariectomy compared to wild-type controls, no increase in the number osteoclasts or osteoblasts in the bone or bone marrow was observed, nor was there an increase in osteoclast activity. Therefore, while the absence of p21 protected mice against estrogen mediated bone loss, the mechanisms/pathways responsible remained elusive. This study demonstrates that p21 may play a significant role in bone remodeling, and a better understanding of how the p21 pathway regulates bone anabolism and catabolism could lead to novel therapies for osteoporosis in the future.

p21-/- mice exhibit enhanced bone regeneration after injury.

BACKGROUND: p21(WAF1/CIP1/SDI1), a cyclin dependent kinase inhibitor has been shown to influence cell proliferation, differentiation and apoptosis; but more recently, p21 has been implicated in tissue repair. Studies on p21(-/-) knockout mice have demonstrated results that vary from complete regeneration and healing of tissue to attenuated healing. There have however been no studies that have evaluated the role of p21 inhibition in bone healing and remodeling. METHODS: The current study employs a burr-hole fracture model to investigate bone regeneration subsequent to an injury in a p21-/- mouse model. p21-/- and C57BL/6 mice were subjected to a burr-hole fracture on their proximal tibia, and their bony parameters were measured over 4 weeks via in vivo µCT scanning. RESULTS: p21-/- mice present with enhanced healing from week 1 through week 4. Differences in bone formation and resorption potential between the two mouse models are assessed via quantitative and functional assays. While the µCT analysis indicates that p21-/- mice have enhanced bone healing capabilities, it appears that the differences observed may not be due to the function of osteoblasts or osteoclasts. Furthermore, no differences were observed in the differentiation of progenitor cells (mesenchymal or monocytic) into osteoblasts or osteoclasts respectively. CONCLUSIONS: Therefore, it remains unknown how p21 is regulating enhanced fracture repair and further studies are required to determine which cell type(s) are responsible for this regenerative phenotype.

Noble Hybrid Nanostructures as Efficient Anti-Proliferative Platforms for Human Breast Cancer Cell.

Nanomaterials have proven to possess great potential in biomaterials research. Recently, they have suggested considerable promise in cancer diagnosis and therapy. Among others, silicon (Si) nanomaterials have been extensively employed for various biomedical applications; however, the utilization of Si for cancer therapy has been limited to nanoparticles, and its potential as anticancer substrates has not been fully explored. Noble nanoparticles have also received considerable attention owing to unique anticancer properties to improve the efficiency of biomaterials for numerous biological applications. Nevertheless, immobilization and control over delivery of the nanoparticles have been challenge. Here, we develop hybrid nanoplatforms to efficiently hamper breast cancer cell adhesion and proliferation. Platforms are synthesized by femtosecond laser processing of Si into multiphase nanostructures, followed by sputter-coating with gold (Au)/gold-palladium (Au-Pd) nanoparticles. The performance of the developed platforms was then examined by exploring the response of normal fibroblast and metastatic breast cancer cells. Our results from the quantitative and qualitative analyses show a dramatic decrease in the number of breast cancer cells on the hybrid platform compared to untreated substrates. Whereas, fibroblast cells form stable adhesion with stretched and elongated cytoskeleton and actin filaments. The hybrid platforms perform as dual-acting cytophobic/cytostatic stages where Si nanostructures depress breast cancer cell adhesion while immobilized Au/Au-Pd nanoparticles are gradually released to affect any surviving cell on the nanostructures. The nanoparticles are believed to be taken up by breast cancer cells via endocytosis, which subsequently alter the cell nucleus and may cause cell death. The findings suggest that the density of nanostructures and concentration of coated nanoparticles play critical roles on cytophobic/cytostatic properties of the platforms on human breast cancer cells while having no or even cytophilic effects on fibroblast cells. Because of the remarkable contrary responses of normal and cancer cells to the proposed platform, we envision that it will provide novel applications in cancer research.

Tuning cell adhesion by direct nanostructuring silicon into cell repulsive/adhesive patterns.

Developing platforms that allow tuning cell functionality through incorporating physical, chemical, or mechanical cues onto the material surfaces is one of the key challenges in research in the field of biomaterials. In this respect, various approaches have been proposed and numerous structures have been developed on a variety of materials. Most of these approaches, however, demand a multistep process or post-chemical treatment. Therefore, a simple approach would be desirable to develop bio-functionalized platforms for effectively modulating cell adhesion and consequently programming cell functionality without requiring any chemical or biological surface treatment. This study introduces a versatile yet simple laser approach to structure silicon (Si) chips into cytophobic/cytophilic patterns in order to modulate cell adhesion and proliferation. These patterns are fabricated on platforms through direct laser processing of Si substrates, which renders a desired computer-generated configuration into patterns. We investigate the morphology, chemistry, and wettability of the platform surfaces. Subsequently, we study the functionality of the fabricated platforms on modulating cervical cancer cells (HeLa) behaviour. The results from in vitro studies suggest that the nanostructures efficiently repel HeLa cells and drive them to migrate onto untreated sites. The study of the morphology of the cells reveals that cells evade the cytophobic area by bending and changing direction. Additionally, cell patterning, cell directionality, cell channelling, and cell trapping are achieved by developing different platforms with specific patterns. The flexibility and controllability of this approach to effectively structure Si substrates to cell-repulsive and cell-adhesive patterns offer perceptible outlook for developing bio-functionalized platforms for a variety of biomedical devices. Moreover, this approach could pave the way for developing anti-cancer platforms that selectively repel cancer cells while favoring the adhesion of normal cells.

Programming cancer through phase-functionalized silicon based biomaterials.

Applications of biomaterials in cancer therapy has been limited to drug delivery systems and markers in radiation therapy. In this article, we introduce the concept of phase-functionalization of silicon to preferentially select cancer cell populations for survival in a catalyst and additive free approach. Silicon is phase-functionalized by the interaction of ultrafast laser pulses, resulting in the formation of rare phases of SiO2 in conjunction with differing silicon crystal lattices. The degree of phase-functionalization is programmed to dictate the degree of repulsion of cancer cells. Unstable phases of silicon oxides are synthesized during phase-functionalization and remain stable at ambient conditions. This change in phase of silicon as well as formation of oxides contributes to changes in surface chemistry as well as surface energy. These material properties elicit in precise control of migration, cytoskeleton shape, direction and population. To the best of our knowledge, phase-functionalized silicon without any changes in topology or additive layers and its applications in cancer therapy has not been reported before. This unique programmable phase-functionalized silicon has the potential to change current trends in cancer research and generate focus on biomaterials as cancer repelling or potentially cancer killing surfaces.

Programming cell fate on bio-functionalized silicon.

Controlling the growth of cells on the surface of silicon without an additive layer or topographical modification is unexplored. This research article delineates the discovery of unique properties of a bio-functionalized silicon substrate, programmed to repel or control cells, generated by ultrafast femtosecond pulse interaction with silicon. Remarkably, bio-functionalization in any shape or size without change in topology or morphology is observed indicating only sub-surface phase transformations. Material characterization reveals the presence of a unique mixture of phases of SiO2 and Si. Consequently, these variations in phase alter the physicochemical characteristics on the surface of silicon resulting in its bio-functionalization. The culture of mouse embryonic fibroblasts shows unique adhesion characteristics on these bio-functionalized silicon surfaces that include cell controlling, cell trapping, and cell shaping. Furthermore, the directionality of fibroblasts is restrained parallel to bio-functionalized zones as evidenced by changes in cytoskeleton. The controlling of proliferation, migration and adhesion of cells is attributed to unique phase bio-functionalization. This method presents considerable promise in a myriad of applications such as tissue engineering, MEMS, and lab-on-a-chip devices.

Nanostructured hybrid of immiscible gold and silicon and its effect on proliferation and adhesion of fibroblasts and osteoblasts.

Hybrid biomaterials are a combination of two or more different materials that work synergistically to produce superior properties. Nano structuring of such hybrid materials has also posed complications. In this study, we present, for the first time a nanofibrous hybrid of gold and silicon fabricated by femtosecond laser synthesis for tissue engineering applications. The formation of a completely new phase, Au3Si (212) is reported. The formation mechanism is explained by vapor condensation. Particle sizes of 2-10 nm and 37-49 nm for gold and gold concentrations of 35-78% are achieved. The effect of this hybrid on cell growth was assessed using fibroblasts and osteoblasts. There was a significant decrease in both osteoblast and fibroblast proliferation with the increase of gold in the hybrid nanostructure. This novel hybrid nanofibrous matrix provides a method to effectively control the proliferation and adhesion of cells. Femtosecond laser synthesis presents a new standard by which not only a single element biomaterial but also multiple immiscible element hybrid biomaterials can be fabricated. This technique provides a paradigm shift in the fabrication of novel nanostructured immiscible hybrid biomaterials.

Direct patterning of free standing three dimensional silicon nanofibrous network to facilitate multi-dimensional growth of fibroblasts and osteoblasts.

The advent of tissue engineering has invigorated interest in novel tissue regeneration matrices. An ideal matrix that simulates the natural extra cellular matrix (ECM) should be nanoscale, with three dimensionally interconnected nanofibers which cannot be generated by current methods such as electrospinning. Furthermore, certain biocompatible materials like silicon cannot be electrospun. We present a novel MHz laser synthesis method that permits sub-100 nm scale structures on any material, including silicon, that mimic the natural ECM. Owing to its three dimensional interlinked nature, the nanofibrous substrate is shown to guide the osteoblasts and fibroblasts to grow not only planarly to the surface, as is true for conventional scaffolds, but also expand and grow upward vertically. This method of synthesis demonstrates promise for novel three dimensional (3D) scaffolds that can assist in tissue and bone regeneration and a myriad of other applications such as drug delivery and biosensing.

Bioactive interlinked extracellular matrix-like silicon nano-network fabricated by femtosecond laser synthesis.

Nanostructured silicon has proven to be a promising candidate in tissue engineering. However, recent research on fabrication of silicon scaffolds has been limited to expensive, complex, and time-consuming lithographic techniques that require the addition of caustic chemicals. Moreover, these techniques generate structures that do not truly mimic the extracellular matrix (ECM). Therefore, we introduce a novel, interlinked, silicon nano-network fabricated by MHz ultrafast laser synthesis. We demonstrate that ultrafast laser synthesis is simple, rapid, free of any chemical additions, and can be carried out under ambient conditions. Variation in laser parameters resulted in an alteration in the pore size and density of the silicon fibrous network. Microscopic analysis revealed a highly charged silicon network with elevated adhesion forces. In vitro bioactivity tests indicate the precipitation of bone-like apatite in just 3 days. Cell proliferation studies on the silicon nano-network present a 300% increase in comparison to its bulk counterpart. Scanning electron microscopy analysis shows healthy migration and attachment of cells on the silicon nano-network. This study points to a correlation between elevated cell proliferation and the ECM-like structure of the silicon nano-network. This ECM-like silicon nano-network suggests significant potential not only in tissue engineering and regeneration but also in other biomedical applications such as biosensor detection.