Hybrid Polypyrrole and Polydopamine Nanosheets for Precise Raman/Photoacoustic Imaging and Photothermal Therapy.

The development of near-infrared light responsive conductive polymers provides a useful theranostic platform for malignant tumors by maximizing spatial resolution with deep tissue penetration for diagnosis and photothermal therapy. Herein, the self-assembly of ultrathin 2D polypyrrole nanosheets utilizing dopamine as a capping agent and a monolayer of octadecylamine as a template is demonstrated. The 2D polypyrrole-polydopamine nanostructure has tunable size distribution which shows strong absorption in the first and second near-infrared windows, enabling photoacoustic imaging and photothermal therapy. The hybrid double-layer is demonstrated to increase Raman intensity for 3D Raman imaging (up to two orders of magnitude enhancement and spatial resolution up to 1 µm). The acidic environment drives reversible doping of polypyrrole, which can be detected by Raman spectroscopy. The combined properties of the nanosheets can substantially enhance performance in dual-mode Raman and photoacoustic guided photothermal therapy, as shown by the 69% light to heat conversion efficiency and higher cytotoxicity against cancer spheroids. These pH-responsive features highlight the potential of 2D conductive polymers for applications in accurate, highly efficient theranostics.

Puffball-Inspired Microrobotic Systems with Robust Payload, Strong Protection, and Targeted Locomotion for On-Demand Drug Delivery.

Microrobots are recognized as transformative solutions for drug delivery systems (DDSs) because they can navigate through the body to specific locations and enable targeted drug release. However, their realization is substantially limited by insufficient payload capacity, unavoidable drug leakage/deactivation, and strict modification/stability criteria for drugs. Natural puffballs possess fascinating features that are highly desirable for DDSs, including a large fruitbody for storing spores, a flexible protective cap, and environmentally triggered release mechanisms. This report presents a puffball-inspired microrobotic system which incorporates an internal chamber for loading large drug quantities and spatial drug separation, and a near-infrared-responsive top-sealing layer offering strong drug protection and on-demand release. These puffball-inspired microrobots (PIMs) display tunable loading capacities up to high concentrations and enhanced drug protection with minimal drug leakage. Upon near-infrared laser irradiation, on-demand drug delivery with rapid release efficiency is achieved. The PIMs also demonstrate translational motion velocities, switchable motion modes, and precise locomotion under a rotating magnetic field. This work provides strong proof-of-concept for a DDS that combines the superior locomotion capability of microrobots with the unique characteristics of puffballs, thereby illustrating a versatile avenue for development of a new generation of microrobots for targeted drug delivery.

Nanoceria provides antioxidant and osteogenic properties to mesoporous silica nanoparticles for osteoporosis treatment.

Osteoporosis, a chronic metabolic bone disease, is the most common cause of fractures. Drugs for treating osteoporosis generally inhibit osteoclast (OC) activity, but are rarely aimed at encouraging new bone growth and often cause severe systemic side effects. Reactive oxygen species (ROS) are one of the key triggers of osteoporosis, by inducing osteoblast (OB) and osteocyte apoptosis and promoting osteoclastogenesis. Here we tested the capability of the ROS-scavenger nanoceria encapsulated within mesoporous silica nanoparticles (Ce@MSNs) to treat osteoporosis using a pre-osteoblast MC3T3-E1 cell monoculture in stressed and normal conditions. Ce@MSNs (diameter of 80 ± 10 nm) were synthesised following a scalable two-step process involving sol-gel and wet impregnation methods. The Ce@MSNs at concentration of 100 µg mL-1 induced a significant reduction in oxidative stress produced by t-butyl hydroperoxide and did not alter cell viability significantly. Confocal microscopy showed that MSNs and Ce@MsNs were internalised into the cytoplasm of the pre-osteoblasts after 24 h but were not in the nucleus, avoiding any DNA and RNA modifications. Ce@MSNs provoked mineralisation of the pre-osteoablasts without osteogenic supplements, which did not occur when the cells were exposed to MSN without nanoceria. In a co-culture system of MC3T3-E1 and RAW264.7 macrophages, the Ce@MSNs exhibited antioxidant capability and stimulated cell proliferation and osteogenic responses without adding osteogenic supplements to the culture. The work brings forward an effective platform based for facile synthesis of Ce@MSNs to interact with both OBs and OCs for treatment of osteoporosis.

Bioenergetic-active materials enhance tissue regeneration by modulating cellular metabolic state.

Cellular bioenergetics (CBE) plays a critical role in tissue regeneration. Physiologically, an enhanced metabolic state facilitates anabolic biosynthesis and mitosis to accelerate regeneration. However, the development of approaches to reprogram CBE, toward the treatment of substantial tissue injuries, has been limited thus far. Here, we show that induced repair in a rabbit model of weight-bearing bone defects is greatly enhanced using a bioenergetic-active material (BAM) scaffold compared to commercialized poly(lactic acid) and calcium phosphate ceramic scaffolds. This material was composed of energy-active units that can be released in a sustained degradation-mediated fashion once implanted. By establishing an intramitochondrial metabolic bypass, the internalized energy-active units significantly elevate mitochondrial membrane potential (ΔΨm) to supply increased bioenergetic levels and accelerate bone formation. The ready-to-use material developed here represents a highly efficient and easy-to-implement therapeutic approach toward tissue regeneration, with promise for bench-to-bedside translation.

Fabrication of Hemin-Doped Serum Albumin-Based Fibrous Scaffolds for Neural Tissue Engineering Applications.

Neural tissue engineering (TE) represents a promising new avenue of therapy to support nerve recovery and regeneration. To recreate the complex environment in which neurons develop and mature, the ideal biomaterials for neural TE require a number of properties and capabilities including the appropriate biochemical and physical cues to adsorb and release specific growth factors. Here, we present neural TE constructs based on electrospun serum albumin (SA) fibrous scaffolds. We doped our SA scaffolds with an iron-containing porphyrin, hemin, to confer conductivity, and then functionalized them with different recombinant proteins and growth factors to ensure cell attachment and proliferation. We demonstrated the potential for these constructs combining topographical, biochemical, and electrical stimuli by testing them with clinically relevant neural populations derived from human induced pluripotent stem cells (hiPSCs). Our scaffolds could support the attachment, proliferation, and neuronal differentiation of hiPSC-derived neural stem cells (NSCs), and were also able to incorporate active growth factors and release them over time, which modified the behavior of cultured cells and substituted the need for growth factor supplementation by media change. Electrical stimulation on the doped SA scaffold positively influenced the maturation of neuronal populations, with neurons exhibiting more branched neurites compared to controls. Through promotion of cell proliferation, differentiation, and neurite branching of hiPSC-derived NSCs, these conductive SA fibrous scaffolds are of broad application in nerve regeneration strategies.

Selective etching of injection molded zirconia-toughened alumina: Towards osseointegrated and antibacterial ceramic implants.

Due to their outstanding mechanical properties and excellent biocompatibility, zirconia-toughened alumina (ZTA) ceramics have become the gold standard in orthopedics for the fabrication of ceramic bearing components over the last decade. However, ZTA is bioinert, which hampers its implantation in direct contact with bone. Furthermore, periprosthetic joint infections are now the leading cause of failure for joint arthroplasty prostheses. To address both issues, an improved surface design is required: a controlled micro- and nano-roughness can promote osseointegration and limit bacterial adhesion whereas surface porosity allows loading and delivery of antibacterial compounds. In this work, we developed an integrated strategy aiming to provide both osseointegrative and antibacterial properties to ZTA surfaces. The micro-topography was controlled by injection molding. Meanwhile a novel process involving the selective dissolution of zirconia (selective etching) was used to produce nano-roughness and interconnected nanoporosity. Potential utilization of the porosity for loading and delivery of antibiotic molecules was demonstrated, and the impact of selective etching on mechanical properties and hydrothermal stability was shown to be limited. The combination of injection molding and selective etching thus appears promising for fabricating a new generation of ZTA components implantable in direct contact with bone. STATEMENT OF SIGNIFICANCE: Zirconia-toughened alumina (ZTA) is the current gold standard for the fabrication of orthopedic ceramic components. In the present work, we propose an innovative strategy to provide both osseointegrative and antibacterial properties to ZTA surfaces: we demonstrate that injection molding allows a flexible design of surface micro-topography and can be combined with selective etching, a novel process that induces nano-roughness and surface interconnected porosity without the need for coating, avoiding reliability issues. These surface modifications have the potential to improve osseointegration. Furthermore, our results show that the porosity can be used for drug delivery and suggest that the etched surface could reduce bacterial adhesion.

Gold-silica quantum rattles for multimodal imaging and therapy.

Gold quantum dots exhibit distinctive optical and magnetic behaviors compared with larger gold nanoparticles. However, their unfavorable interaction with living systems and lack of stability in aqueous solvents has so far prevented their adoption in biology and medicine. Here, a simple synthetic pathway integrates gold quantum dots within a mesoporous silica shell, alongside larger gold nanoparticles within the shell's central cavity. This "quantum rattle" structure is stable in aqueous solutions, does not elicit cell toxicity, preserves the attractive near-infrared photonics and paramagnetism of gold quantum dots, and enhances the drug-carrier performance of the silica shell. In vivo, the quantum rattles reduced tumor burden in a single course of photothermal therapy while coupling three complementary imaging modalities: near-infrared fluorescence, photoacoustic, and magnetic resonance imaging. The incorporation of gold within the quantum rattles significantly enhanced the drug-carrier performance of the silica shell. This innovative material design based on the mutually beneficial interaction of gold and silica introduces the use of gold quantum dots for imaging and therapeutic applications.

Monodispersed bioactive glass submicron particles and their effect on bone marrow and adipose tissue-derived stem cells.

Spherical monodispersed bioactive particles are potential candidates for nanocomposite synthesis or as injectable particles that could be internalized by cells for the local sustained delivery of inorganic therapeutic ions (e.g., calcium or strontium). Particles are also likely to be released from porous bioactive glass and sol-gel hybrid scaffolds as they degrade; thus, it is vital to investigate their interaction with cells. Spherical monodispersed bioactive glass particles (mono-SMBG), with diameters of 215 ± 20 nm are synthesized using a modified Stöber process. Confocal and transmission electron microscopy demonstrate that mono-SMBGs are internalized by human bone marrow (MSCs) and adipose-derived stem cells (ADSCs) and located within cell vesicles and in the cytoplasm. Particle dissolution inside the cells is observed. Alamar Blue, MTT and Cyquant assays demonstrate that 50 µg mL(-1) of mono-SMBGs did not inhibit significantly MSC or ADSC metabolic activity. However, at higher concentrations (100 and 200 µg mL(-1)) small decrease in metabolic activity and total DNA is observed. Mono-SMBG did not induce ALPase activity, an early marker of osteogenic differentiation, without osteogenic supplements; however, in their presence osteogenic differentiation is achieved. Additionally, large numbers of particles are internalized by the cells but have little effect on cell behavior.

Correlative spectroscopy of silicates in mineralised nodules formed from osteoblasts.

Silicon supplementation has been shown to play an important role in skeleton development, however, the potential role that silicon plays in mediating bone formation, and an understanding of where it might localise in the resulting bone tissue remain elusive. An improved understanding of these processes could have important implications for treating pathological mineralisation. A key aspect of defining the role of silicon in bone is to characterise its distribution and coordination environment, however, there is currently almost no information available on either. We have combined a sample-preparation method that simultaneously preserved mineral, ions, and the extracellular matrix (ECM) with secondary ion mass spectroscopy (SIMS) and electron energy-loss spectroscopy (EELS) to examine the distribution and coordination environment of silicon in murine osteoblasts (OBs) in an in vitro model of bone formation. SIMS analysis showed a high level of surface contamination from polydimethysiloxane (PDMS) resulting from sample preparation. When the PDMS was removed, silicon compounds could not be detected within the nodules either by SIMS or by energy dispersive X-ray spectroscopy (EDX) analysis. In comparison, electron energy-loss spectroscopy (EELS) provided a powerful and potentially widely applicable means to define the coordination environment and localisation of silicon in mineralising tissues. We show that trace levels of silicon were only detectable from the mineral deposits located on the collagen and in the peripheral region of mineralised matrix, possibly the newly mineralised regions of the OB nodules. Taken together our results suggest that silicon plays a biological role in bone formation, however, the precise mechanism by which silicon exerts its physicochemical effects remains uncertain. Our analytical results open the door for compelling new sets of EELS experiments that can provide detailed and specific information about the role that silicates play in bone formation and disease.

Characterization of porcine aortic valvular interstitial cell 'calcified' nodules.

Valve interstitial cells populate aortic valve cusps and have been implicated in aortic valve calcification. Here we investigate a common in vitro model for aortic valve calcification by characterizing nodule formation in porcine aortic valve interstitial cells (PAVICs) cultured in osteogenic (OST) medium supplemented with transforming growth factor beta 1 (TGF-ß1). Using a combination of materials science and biological techniques, we investigate the relevance of PAVICs nodules in modeling the mineralised material produced in calcified aortic valve disease. PAVICs were grown in OST medium supplemented with TGF-ß1 (OST+TGF-ß1) or basal (CTL) medium for up to 21 days. Murine calvarial osteoblasts (MOBs) were grown in OST medium for 28 days as a known mineralizing model for comparison. PAVICs grown in OST+TGF-ß1 produced nodular structures staining positive for calcium content; however, micro-Raman spectroscopy allowed live, noninvasive imaging that showed an absence of mineralized material, which was readily identified in nodules formed by MOBs and has been identified in human valves. Gene expression analysis, immunostaining, and transmission electron microscopy imaging revealed that PAVICs grown in OST+TGF-ß1 medium produced abundant extracellular matrix via the upregulation of the gene for Type I Collagen. PAVICs, nevertheless, did not appear to further transdifferentiate to osteoblasts. Our results demonstrate that 'calcified' nodules formed from PAVICs grown in OST+TGF-ß1 medium do not mineralize after 21 days in culture, but rather they express a myofibroblast-like phenotype and produce a collagen-rich extracellular matrix. This study clarifies further the role of PAVICs as a model of calcification of the human aortic valve.

Gene-expression analysis reveals that embryonic stem cells cultured under osteogenic conditions produce mineral non-specifically compared to marrow stromal cells or osteoblasts.

Pluripotent cells, such as embryonic stem cells (ESCs), divide indefinitely and can differentiate to form mineralised nodules in response to osteogenic supplements. This suggests that they may be used as a cell source for bone replacement strategies. Here, we related the expression of osteogenic and chondrogenic genes in cultures of murine ESCs, marrow stromal cells (MSCs) and calvarial osteoblasts (OBs) cultured under osteogenic conditions to the biochemical composition and quantity of mineral formed. Mineralisation, measured by calcium sequestration, was >2-fold greater in ESC cultures than in either MSCs or OBs. Micro-Raman spectroscopy and spectral mapping revealed a lower mineral-to-matrix ratio and confirmed a more diffuse pattern of mineralisation in ESCs compared to MSCs and OBs. Baseline expression of chondrogenic and osteogenic genes was between 1 and 4 orders of magnitude greater in MSCs and OBs than in ESCs. Osteogenic culture of MSCs and OBs was accompanied by increases in osteogenic gene expression by factors of ~100 compared to only ~10 in ESCs. Consequentially, peak expression of osteogenic and chondrogenic genes was greater in MSCs and OBs than ESCs by factors of 100-1000, despite the fact that mineralisation was more extensive in ESCs than either MSCs or OBs. We also observed significant cell death in ESC nodules. We conclude that the mineralised material observed in cultures of murine ESCs during osteogenic differentiation may accumulate non-specifically, perhaps in necrotic cell layers, and that thorough characterisation of the tissue formed by ESCs must be achieved before these cells can be considered as a cell source for clinical applications.

Emerging techniques for submicrometer particle sizing applied to Stöber silica.

The accurate characterization of submicrometer and nanometer sized particles presents a major challenge in the diverse applications envisaged for them including cosmetics, biosensors, renewable energy, and electronics. Size is one of the principal parameters for classifying particles and understanding their behavior, with other particle characteristics usually only quantifiable when size is accounted for. We present a comparative study of emerging and established techniques to size submicrometer particles, evaluating their sizing precision and relative resolution, and demonstrating the variety of physical principles upon which they are based, with the aim of developing a framework in which they can be compared. We used in-house synthesized Stöber silica particles between 100 and 400 nm in diameter as reference materials for this study. The emerging techniques of scanning ion occlusion sensing (SIOS), differential centrifugal sedimentation (DCS), and nanoparticle tracking analysis (NTA) were compared to the established techniques of transmission electron microscopy (TEM), scanning mobility particle sizing (SMPS), and dynamic light scattering (DLS). The size distributions were described using the mode, arithmetic mean, and standard deviation. Uncertainties associated with the six techniques were evaluated, including the statistical uncertainties in the mean sizes measured by the single-particle counting techniques. Q-Q plots were used to analyze the shapes of the size distributions. Through the use of complementary techniques for particle sizing, a more complete characterization of the particles was achieved, with additional information on their density and porosity attained.

Human periosteum is a source of cells for orthopaedic tissue engineering: a pilot study.

BACKGROUND: Periosteal cells are important in embryogenesis, fracture healing, and cartilage repair and could provide cells for osteochondral tissue engineering. QUESTIONS/PURPOSE: We determined whether a population of cells isolated from human periosteal tissue contains cells with a mesenchymal stem cell (MSC) phenotype and whether these cells can be expanded in culture and used to form tissue in vitro. METHODS: We obtained periosteal tissue from six patients. Initial expression of cell surface markers was assessed using flow cytometry. Cells were cultured over 10 generations and changes in gene expression evaluated to assess phenotypic stability. Phenotype was confirmed using flow cytometry and colony-forming ability assays. Mineral formation was assessed by culturing Stro-1(-) and unsorted cells with osteogenic supplements. Three cell culture samples were used for a reverse transcription-polymerase chain reaction, four for flow cytometry, three for colony-forming assay, and three for mineralization. RESULTS: Primary cultures, containing large numbers of hematopoietic cells were replaced initially by Stro-1 and ALP-expressing immature osteoblastic cell types and later by ALP-expressing cells, which lacked Stro-1 and which became the predominant cell population during subculture. Approximately 10% of the total cell population continued to express markers for Stro1(+)/ALP(-) cells throughout. CONCLUSIONS: These data suggest periosteum contains a large number of undifferentiated cells that can differentiate into neotissue and persist despite culture in noncell-specific media for over 10 passages. CLINICAL RELEVANCE: Cultured periosteal cells may contribute to tissue formation and may be applicable for tissue engineering applications.

Differentiation of fetal osteoblasts and formation of mineralized bone nodules by 45S5 Bioglass conditioned medium in the absence of osteogenic supplements.

Bioactive glasses bond strongly to bone in vivo and their ionic dissolution products have previously been shown to have stimulatory properties on adult and fetal osteoblasts and to induce the differentiation of embryonic stem cells towards the osteoblastic lineage in vitro. In the present study, the effect of 45S5 Bioglass conditioned medium with two different Si concentrations (15 microg/ml (BGCM/15) and 20 microg/ml (BGCM/20)) on human fetal osteoblast growth, differentiation and extracellular matrix production and mineralization was investigated. In the first instance, primary fetal osteoblasts were examined for the osteoblast phenotypic markers alkaline phosphatase (ALP), collagen type I (Col I) and OB Cadherin (Cadherin 11) (OB Cad) as well as for the mesenchymal stem cell markers CD105 and CD166. At passage 0 more than 50% of the population was positive for Col I and ALP, but at passage 2, the proportion of cells expressing ALP increased. In addition at passage 0 more than 50% of the fetal osteoblasts expressed the mesenchymal stem cell surface markers CD105 and CD166. Treatment with BGCM/15 and BGCM/20 in the absence of osteogenic supplements increased the gene expression of the bone extracellular matrix proteins alkaline phosphatase, osteonectin and bone sialoprotein as determined by quantitative real time reverse transcriptase-polymerase chain reaction (rt RT-PCR) analysis. Extracellular matrix production was also enhanced in the absence of osteogenic supplements by the 45S5 Bioglass conditioned medium as demonstrated by ALP enzymatic activity, osteocalcin and Col I protein synthesis. Furthermore, BGCM/15 and BGCM/20 significantly enhanced the formation of mineralized nodules, based on alizarin red histochemical staining, without necessitating the addition of beta-glycerophosphate, l-ascorbate-2-phosphate or dexamethasone (commonly used osteogenic supplements).

Extracellular matrix formation and mineralization on a phosphate-free porous bioactive glass scaffold using primary human osteoblast (HOB) cells.

Sol-gel derived bioactive glasses of the 70S30C (70mol% SiO2, 30mol% CaO) composition have been foamed to produce 3D bioactive scaffolds with hierarchical interconnected pore morphologies similar to trabecular bone. The aim of this study was to investigate primary human osteoblast response to porous bioactive glass scaffolds. The scaffolds supported osteoblast growth and induced differentiation, within the 3-week culture period, as depicted by enhanced ALPase enzymatic activity, without the addition of supplementary factors such as ascorbic acid, beta-glycerophosphate and dexamethasone. This is the first time this has been observed on a bioactive glass that does not contain phosphate. Deposition of extracellular matrix was also confirmed by enhanced production of the extracellular matrix protein collagen type I. SEM showed indications of mineralized bone nodule formation without the addition of growth factors. The 70S30C bioactive glass scaffolds therefore fulfil many of the criteria for an ideal scaffold for bone tissue engineering applications.

FGF-2 enhances TGF-beta1-induced periosteal chondrogenesis.

The use of periosteum as a cell source for the in vitro engineering of grafts for articular cartilage repair requires the development of methods to obtain high viable cell numbers in the early stages of culture. In this study, we demonstrate that the addition of a mitogen, fibroblast growth factor-2 (FGF-2), during the early stage of the in vitro culture of periosteum in the presence of transforming growth factor-beta1 (TGF-beta1), significantly enhances cell proliferation, which results in increased neo-cartilage formation at later stages. Periosteal explants were cultured in vitro within alginate or agarose based gels in the presence of either FGF-2 for the first week, TGF-beta1 for the first 2 weeks, FGF-2 and TGF-beta1 for the first week and first 2 weeks respectively, or no added factors. Consistent with previous studies, periosteum derived neo-chondrogenesis occurred only in the presence of TGF-beta1. The neo-cartilage was found to contain cartilage specific proteoglycans and Type-II collagen as determined by safranin-O and immunohistochemical staining respectively. Further medium supplementation with FGF-2 stimulated early cell proliferation (>3 fold higher total DNA content per explant at day 10). This resulted in a marked increase in the size of the cultured explants and in the total area of the explant staining positive for safranin-O (from around 50% to 85%, (p<0.05)) after 6 weeks culture. The ability to generate significant quantities of neo-cartilage within a biocompatible and biodegradable matrix such as alginate, which lacks the immunogenicity of agarose, could open new pathways to utilizing such constructs in articular cartilage tissue engineering applications.