Yizhi Meng

The role of moderate static magnetic fields on biomineralization of osteoblasts on sulfonated polystyrene films

We have investigated the effects of moderate static magnetic fields (SMFs) on murine MC3T3-E1 osteoblasts, and found that they enhance proliferations and promote differentiation. The increase in proliferation rates in response to SMFs was greater in cultures grown on partially sulfonated polytstyrene (SPS, degree of sulfonation: 33%) than in cultures grown on tissue culture plastic. We have previously shown that when the degree of sulfonation exceeded a critical value (12%) [1], spontaneous fibrillogenesis occured which allowed for direct observation of the ECM fibrillar organization under the influence of external fields. We found that the ECM produced in cultures grown on the SPS in the presence of the SMFs assembled into a lattice with larger dimensions than the ECM of the cultures grown in the absence of SMFs. During the early stages of the biomineralization process (day 7), the SMF exposed cultures also templated mineral deposition more rapidly than the control cultures. The rapid response is attributed to orientation of diamagnetic ECM proteins already present in the serum, which could then initiate further cellular signaling. SMFs also influenced late stage osteoblast differentiation as measured by the increased rate of osteocalcin secretion and gene expression beginning 15 days after SFM exposure. This correlated with a large increase in mineral deposition, and in cell modulus. GIXD and EDXS analysis confirmed early deposition of crystalline hydroxyapatite. Previous studies on the effects of moderate SMF had focused on cellular gene and protein expression, but did not consider the organization of the ECM fibers. Our ability to form these fibers has allowed us explore this additional effect and highlight its significance in the initiation of the biomineralization process.

Biomineralization of a self-assembled extracellular matrix for bone tissue engineering.

Understanding how biomineralization occurs in the extracellular matrix (ECM) of bone cells is crucial to the understanding of bone formation and the development of a successfully engineered bone tissue scaffold. It is still unclear how ECM mechanical properties affect protein-mineral interactions in early stages of bone mineralization. We investigated the longitudinal mineralization properties of MC3T3-E1 cells and the elastic modulus of their ECM using shear modulation force microscopy, synchrotron grazing incidence X-ray diffraction (GIXD), scanning electron microscopy, energy dispersive X-ray spectroscopy, and confocal laser scanning microscopy (CLSM). The elastic modulus of the ECM fibers underwent significant changes for the mineralizing cells, which were not observed in the nonmineralizing cells. On substrates conducive to ECM network production, the elastic modulus of mineralizing cells increased at time points corresponding to mineral production, whereas that of the nonmineralizing cells did not vary over time. The presence of hydroxyapatite in mineralizing cells and the absence thereof in the nonmineralizing ones were confirmed by GIXD, and CLSM showed that a restructuring of actin occurred only for mineral-producing cells. These results show that the correct and complete development of the ECM network is required for osteoblasts to mineralize. This in turn requires a suitably prepared synthetic substrate for bone development to succeed in vitro.

Role of pH-responsiveness in the design of chitosan-based cancer nanotherapeutics: A review

There is a continuous demand for sensitive and efficient cancer drug delivery systems that, when administered at low concentrations, are capable of detecting early-stage pathological conditions and increasing patient survival without adverse side effects. Recent developments in the design of chitosan-based smart drug delivery nanocomplexes are able to respond to the distinctive features of the tumor microenvironment and have provided powerful tools for cancer targeted treatment. Due to its biocompatibility and pH-responsiveness, chitosan has emerged as a promising candidate for the formulation of novel, supramolecular multifunctional materials. This review will first present an overview of the characteristics of solid tumors and their microenvironment, with a particular emphasis on the role of pH as a key factor. In the second part of the review, the stimuli-responsive potential of chitosan-based micelles, current challenges in delivery, and strategies to improve therapeutic efficacy will be discussed.

Complementary effects of multi-protein components on biomineralization in vitro

The extracellular matrix (ECM) is composed of mixed protein fibers whose precise composition affects biomineralization. New methods are needed to probe the interactions of these proteins with calcium phosphate mineral and with each other. Here we follow calcium phosphate mineralization on protein fibers self-assembled in vitro from solutions of fibronectin, elastin and their mixture. We probe the surface morphology and mechanical properties of the protein fibers during the early stages. The development of mineral crystals on the protein matrices is also investigated. In physiological mineralization solution, the elastic modulus of the fibers in the fibronectin-elastin mixture increases to a greater extent than that of the fibers from either pure protein. In the presence of fibronectin, longer exposure in the mineral solution leads to the formation of amorphous calcium phosphate particles templated along the self-assembled fibers, while elastin fibers only collect calcium without any mineral observed during early stage. TEM images confirm that small needle-shape crystals are confined inside elastin fibers which suppress the release of mineral outside the fibers during late stage, while hydroxyapatite crystals form when fibronectin is present. These results demonstrate complementary actions of the two ECM proteins fibronectin and elastin to collect cations and template mineral, respectively.

The Effect of Exogenous Zinc Concentration on the Responsiveness of MC3T3-E1 Pre-Osteoblasts to Surface Microtopography: Part II (Differentiation)

Osseointegration of bone implants is a vital part of the recovery process. Numerous studies have shown that micropatterned geometries can promote cell-substrate associations and strengthen the bond between tissue and the implanted material. As demonstrated previously, exogenous zinc levels can influence the responsiveness of pre-osteoblasts to micropatterns and modify their migratory behavior. In this study, we sought to determine the effect of exogenous zinc on differentiation of osteoblasts cultured on micropatterned vs. planar substrates. Levels of activated metalloproteinase-2 (MMP-2) and transforming growth factor-beta 1 (TGF-β1), as well as early stage differentiation marker alkaline phosphatase, were altered with the addition of zinc. These results suggest that exogenous zinc concentration and micropatterning may interdependently modulate osteoblast differentiation.

Effect of low-intensity pulsed ultrasound on biocompatibility and cellular uptake of chitosan-tripolyphosphate nanoparticles

Using low molecular weight chitosan nanoparticles (CNPs) prepared by an ionic gelation method, the authors report the effect of low-intensity pulsed ultrasound (US) on cell viability and nanoparticle uptake in cultured murine preosteoblasts. Particle size and zeta potential are measured using dynamic light scattering, and cell viability is evaluated using the of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] assay. Results show that 30 min delivery of CNPs at 0.5 mg/mL is able to prevent loss of cell viability due to either serum starvation or subsequent exposure to US (1 W/cm(2) or 2 W/cm(2), up to 1 min). Additionally, flow cytometry data suggest that there is a close association between cellular membrane integrity and the presence of CNPs when US at 2 W/cm(2) is administered.

Photolithography-Based Substrate Microfabrication for Patterning Semaphorin 3A to Study Neuronal Development

Protein micropatterning techniques, including microfluidic devices and protein micro-contact printing, enable the generation of highly controllable substrates for spatial manipulation of intracellular and extracellular signaling determinants to examine the development of cultured dissociated neurons in vitro. In particular, culture substrates coated with proteins of interest in defined stripes, including cell adhesion molecules and secreted proteins, have been successfully used to study neuronal polarization, a process in which the neuron establishes axon and dendrite identities, a critical architecture for the input/output functions of the neuron. We have recently used this methodology to pattern the extracellular protein Semaphorin 3A (Sema3A), a secreted factor known to control neuronal development in the mammalian embryonic cortex. We showed that stripe-patterned Sema3A regulates axon and dendrite formation during the early phase of neuronal polarization in cultured rat hippocampal neurons. Here, we describe microfabrication and substrate stripe micropatterning of Sema3A. We note that same methodologies can be applied to pattern other extracellular proteins that regulate neuronal development in the embryonic brain, as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and Netrin-1. We describe modifications of these methodologies for stripe micropatterning of membrane-permeable analog of the second messengers cyclic AMP (cAMP) and cyclic GMP (cGMP), intracellular regulators of neuronal polarization that might act downstream of Sema3A.

In situ examination of osteoblast biomineralization on sulfonated polystyrene-modified substrates using Fourier transform infrared microspectroscopy

Osteoporosis is a skeletal disorder that is characterized by the loss of bone mineral density (BMD) resulting in increased risk of fracture. However, it has been shown that BMD is not the only indicator of fracture risk, as the strength of bone depends on a number of factors, including bone mass, architecture and material properties. Physiological mineral deposition requires the formation of a properly developed extracellular matrix (ECM), which recruits calcium and phosphate ions into the synthesis of apatite crystals. Temporal and spatial compositional and structural changes of biological apatite greatly depend on the properties of the crystals initially formed. As such, Fourier-transform infrared microspectroscopy (FTIRM) is capable of examining adaptive remodeling by providing compositional information such as the level of mineralization and carbonate substitution, as well as quality and perfection of the mineral phase. The objective of this study was to evaluate the in vitro mineralization development of MC3T3-E1 murine calvarial preosteoblasts cultured on different substrata by comparing FTIRM measurements from two subclones (mineralizing subclone 4 and nonmineralizing subclone 24) maintained in culture for up to 21 days. The results showed that modulation of the substrate surface using a thin coating of sulfonated polystyrene (SPS) provided favorable conditions for the development of a mineralizable ECM and that the mineral formed by the osteoblasts was similar to that of fully mineralized bone tissue. Specifically, the mineralizing subclone produced significantly more mineral phosphate when cultured on SPS-coated substrates for 21 days, compared to the same culture on bare substrates. In contrast, the level of mineralization in nonmineralizing subclone was low on both SPS-coated and uncoated substrates. The mineralizing subclone also produced comparable amounts of collagen on both substrates; however, mineralization was significantly higher in the SPS culture. The nonmineralizing subclone produced comparable amounts of collagen on day 1 but much less on day 21. Collagen maturity ratio increased in the mineralizing subclone from day 1 to day 21, but remained unchanged in the nonmineralizing subclone. These results suggest that SPS-treatment of the substrate surface may alter collagen remodeling; however, other factors may also influence osteoblast mineralization in the long term.

Apatitic Deposition in Osteoblasts Cultured on Micropatterned Silicon

In this study, the osteogenic differentiation of murine pre-osteoblasts cultured on patterned silicon was investigated. Specifically, micropatterns were fabricated in orientation silicon wafers to create linear gratings with a width of either 2 μm (2 μm depth, 10 μm pitch) or 20 μm (2 μm depth, 30 μm pitch). Two subclones (4, 24) of the MC3T3-E1 osteoblast-like cell line were seeded on the micropatterns and cultured in osteogenic induction medium for 28 days. Cells cultured on planar silicon served as the controls. Apatite formation was examined using synchrotron X-ray diffraction. Deposition of [002] and [211]/[112] apatite-like material was clearly observed in subclone 4 (strongly mineralizing) osteoblasts cultured on both micro patterns.

Nanometer resolution hard X-ray microscopy of bone and mineralized tissue

Full field hard X-ray microscopy has recently been developed utilizing synchrotron light, enabling absorption and phase contrast imaging with a spatial resolution of 50 -100 nm. This technique has allowed us to image bone tissue at the nanoscale level, viewing the substructure of mineralized collagen fibers. Thin sections of tissue (5 and 50 mum thick) from the L3 vertebrae of Beagles and mineralizing MC3T3-E1 mouse osteoblasts were imaged at beamline 01B1 at the National Synchrotron Radiation Research Center (Hsinchu, Taiwan). Collagen fiber orientation was observed in the lamellar structure of an osteon. Mineralizing osteoblasts were found to produce disordered mineralized nodules with varying density and structure.