Ashutosh Kumar Dubey

Optimization of electrical stimulation parameters for enhanced cell proliferation on biomaterial surfaces

From the point of view of biocompatibility of bone analog materials, cell-material interaction is of fundamental importance. In this article, we report the effect of pulse electric field stimulation on cell-material interaction by analyzing cellular functionality and viability. An in-house fabricated pulse electric field setup was used for the application of electric field during cell culture experiments. To optimize voltage/electric field, the first set of exploratory experiments was conducted with varying field strength at fixed frequency, and subsequently, the frequency of the electrical stimulation was varied to study its influence on the proliferation of L929 mouse fibroblast cells on gelatin-coated control disc. Subsequently, L929 cells were cultured on hydroxyapatite (HA) and HA-40 wt % BaTiO₃ composite. Cell-cultured samples were analyzed qualitatively as well as quantitatively using fluorescence microscope and scanning electron microscope. It has been demonstrated that due to the application of electric field during the cell culture experiment, the cell proliferation and the cell spreading on the surface of the biomaterials were enhanced within a narrow window of voltage/frequency of electrical stimulation. At lower field intensities, the energy density is quite low and increases parabolically with field strength. There is no significant increase in the temperature (ΔT ~10⁻⁵ K) of the medium due to the application of short duration pulse electric field. This led us to believe that electric field with appropriate strength and duration can enhance the cell-material interaction.

Cellular proliferation, cellular viability, and biocompatibility of HA‐ZnO composites

One of the important issues in the development of hydroxyapatite (HA)-based biomaterials is the prosthetic infection, which limits wider use of monolithic HA despite superior cellular response. Recently, we reported that ZnO addition to HA can induce bactericidal property. It is therefore important to assess how ZnO addition influences the cytotoxicity property and cell adhesion/proliferation on HA-ZnO composite surfaces in vitro. In the above perspective, the objective of this study is to investigate the cell type and material composition dependent cellular proliferation and viability of pressureless sintered HA-ZnO composites. The combination of cell viability data as well as morphological observations of cultured human osteoblast-like SaOS2 cells and mouse fibroblast L929 cells suggests that HA-ZnO composites containing 10 Wt % or lower ZnO exhibit the ability to support cell adhesion and proliferation. Both SaOS2 and L929 cells exhibit extensive multidirectional network of actin cytoskeleton and cell flattening on the lower ZnO containing (≤10 Wt %) HA-ZnO composites. The in vitro results illustrate how variation in ZnO content can influence significantly the cell vitality, as evaluated using MTT biochemical assay. Also, the critical statistical analysis reveals that ZnO addition needs to be carefully tailored to ensure good in vitro cytocompatibility. The underlying reasons for difference in biological properties are analyzed. It is suggested that surface wettability as well as dissolution of ZnO, both contribute to the observed differences in cellular viability and proliferation.

Characterization of hydroxyapatite-perovskite (CaTiO3) composites: Phase evaluation and cellular response

In this study, an attempt was made to develop an understanding of the densification behavior, phase stability, and biocompatibility property of HA-CaTiO(3) biocomposite. The composites with varying CaTiO(3) (40-80 wt %) content were sintered at temperatures ranging from 1200°C to 1500°C for 3-5 hr to establish optimum processing parameters. The phase analysis using spectral techniques indicate good thermochemical compatibility between HA and CaTiO(3). The microstructural observations reveal homogeneous distribution of finer CaTiO(3) phase (1-2 μm) along with coarser calcium phosphate phase. In vitro cell culture studies using L929 mouse fibroblast and SaOS2 human osteoblast cell lines provide clear evidence of cell adhesion, spreading, and proliferation as well as the formation of cellular bridges, and, hence, good in vitro biocompatibility of the developed composite can be realized. Also, the number of viable cells was found to increase with incubation period, as revealed by statistical analysis of the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay data.

Dielectric and Pyroelectric Properties of HAp-BaTiO3 Composites

In order to mimic the electrical properties of natural bone, the present work investigated the dielectric, AC conductivity, pyroelectric and piezoelectric properties of HA-40 wt% BaTiO3 (HA-26 vol% BaTiO3) and HA-60 wt% BaTiO3 (HA-44 vol% BaTiO3) composites. Multistage spark plasma sintering was used to achieve the desired combination of properties. The electrical parameters were measured as a function of temperature and frequency. The values of dielectric constant and loss for both the developed composites, measured at room temperature and at 1 KHz frequency was 21, 38 and 0.01 and 0.02, respectively. The AC conductivity for both the composites is found to be of the order of 10−10 and 10−9 (Ωcm)−1, measured under similar conditions. Activation energy calculated from σac vs. temperature plot for HA-40 wt% BaTiO3 is 0.50 eV. The room temperature pyroelectric coefficients for both the compositions are 2.35 and 21 μC/m2K, respectively. The piezoelectric coefficient values (d33) for both the compositions are 0.9 and 1 pC/N, respectively. The observed values of electrical parameters closely resemble with that of the natural human bone.

Mechanical properties of novel calcium phosphate–mullite biocomposites

Herein, the results of systematic mechanical property measurements of pressureless sintered calcium phosphate (CaP)–mullite composites are discussed. Our experimental results demonstrated how the mullite addition (upto 30 wt%) influenced hardness, elastic modulus, strength and toughness properties of the composites. In assessing each of these fundamental material properties, either a range of load or a number of complimentary techniques were used to obtain reliable measure of mechanical properties. Importantly, the results of single edge V notch beam measurements revealed that a reliable toughness value of ∼1.5 MPa m0.5 could be obtained in composites containing 20 or 30 wt% mullite. Our results clearly illustrated that a combination of elastic modulus (∼80 GPa), compressive strength of more than 350 MPa, three-point flexural strength of 70–80 MPa, hardness of 4–5 GPa were achievable with the investigated composites. Such a combination of material properties, in addition to modest toughness property appeared to indicate that CaP–mullite composites could be used as a biomaterial for hard tissue replacement.

Multifunctionality of Perovskites BaTiO 3 and CaTiO 3 in a Composite with Hydroxyapatite as Orthopedic Implant Materials

In the present article, the multifunctional behavior of perovskites BaTiO3 and CaTiO3 has been studied for their potential orthopedic applications. The biocompatibility and electrical conductivity properties of HA-40 wt%BaTiO3 (25.9 vol% BaTiO3) and HA-40 wt% CaTiO3 (35.3 wt% CaTiO3) composites were investigated to verify its suitability as hard tissue replacement materials. L929 mouse fibroblasts and Human osteoblasts (SaOS2) cells were used to study the cell adhesion and proliferation behavior on the composite surfaces. It has been observed that the developed composites are biocompatible and the conductivity and dielectric behavior of the composite is comparable to dry human bone.

Time constant determination for electrical equivalent of biological cells

The electric field interactions with biological cells are of significant interest in various biophysical and biomedical applications. In order to study such important aspect, it is necessary to evaluate the time constant in order to estimate the response time of living cells in the electric field (E-field). In the present study, the time constant is evaluated by considering the hypothesis of electrical analog of spherical shaped cells and assuming realistic values for capacitance and resistivity properties of cell/nuclear membrane, cytoplasm, and nucleus. In addition, the resistance of cytoplasm and nucleoplasm was computed based on simple geometrical considerations. Importantly, the analysis on the basis of first principles shows that the average values of time constant would be around 2–3 μs, assuming the theoretical capacitance values and the analytically computed resistance values. The implication of our analytical solution has been discussed in reference to the cellular adaptation processes such as a...

Impedance spectroscopy and mechanical response of porous nanophase hydroxyapatite-barium titanate composite.

The present study aims to develop the porous nanophase hydroxyapatite (HA)-barium titanate (BT) composite with reasonable mechanical and electrical properties as an electrically-active prosthetic orthopedic implant alternate. The porous samples (densification ~40-70%) with varying amounts of BT (0, 25, 35 and 100 vol.%) in HA were synthesized using optimal spark plasma sintering conditions, which revealed the thermochemical stability between both the phases. The reasonably good combination of functional properties such as compressive [(236.00 ± 44.90)MPa] and flexural [(56.18 ± 5.82) MPa] strengths, AC conductivity [7.62 × 10(-9)(ohm-cm)(-1) at 10 kHz] and relative permittivity [15.20 at 10 kHz] have been achieved with nanostructured HA-25 vol.% BT composite as far as significant sample porosity (~30%) is concerned. Detailed impedance spectroscopic analysis was performed to reveal the electrical microstructure of developed porous samples. The resistance and capacitance values (at 500 °C) of grain (RG, CG) and grain boundary (RGB, CGB) for the porous HA-25 vol.% BT composite are (1.3 × 10(7) ohm, 3.1 × 10(-11)F) and (1.6 × 10(7) ohm, 5.9 × 10(-10)F), respectively. Almost similar value of activation energy (~1-1.5 eV) for grain and grain boundary has been observed for all the samples. The mechanism of conduction is found to be same for porous monolithic HA as well as composite samples. Relaxation spectroscopic analyses suggest that both the localized as well as long range charge carrier translocations are responsible for conduction in these samples. The degree of polarization of porous samples has been assessed by measuring thermally stimulated depolarization current of the poled samples. The depolarization current is observed to depend on the heating rate. The maximum current density, measured for HA-25 vol.% BT sample at a heating rate of 1 °C/min is 2.7 nA/cm(2). Formation of oxygen vacancies due to the reduced atmosphere sintering contribute to the space charge polarization, which is obtained as the dominant polarization mechanism in the developed porous samples. Overall, such integrated functional responses do establish spark plasma sintered porous HA-BaTiO3 nanocomposite as potential alternative for electroactive prosthetic orthopedic implants.

Thermal Expansion Behavior of Biocompatible Hydroxyapatite-BaTiO3 Composites for Bone Substitutes

In the present study, the thermal expansion behavior of biocompatible pure Hydroxyapatite (HA) and HA-BaTiO3 composites were studied in the temperature range of −150 to 300°C. The composites were optimally processed by conventional pressure-less sintering at 1250°C for 2 hrs. The measured coefficients of thermal expansion (CTE) values in the temperature range close to the human body temperature were ranging from 11 × 10−6/°C to 12 × 10−6/°C. It was observed that the CTE values are almost stable in the entire temperature range of measurement and closely resembled with the values for natural human bone.

Enhanced polarization of hydroxyapatite using the design concept of functionally graded materials with sodium potassium niobate

The present work aims to enhance the electrical activities of hydroxyapatite (HA) without affecting its bioactivity through the development of functionally graded materials (FGM) using biocompatible sodium potassium niobate (NKN) piezoelectrics as an intermediary layer. The NKN layer was sandwiched between HA layers via buffer interlayers (abbreviated as HA–NKN–HA) and optimally processed using the spark plasma sintering route. The dielectric and electrical properties were studied over a wide range of temperatures (25–500 °C) and frequencies (10−1 to 106 Hz). In vitro cellular response in terms of initial cell adhesion and proliferation on the FGM as well as the corresponding monoliths was assessed using human osteoblast-like SaOS2 cells. A reasonably good combination of dielectric and electrical properties, such as dielectric constant (38), AC conductivity [5.5 × 10−9 (ohm cm)−1], piezoelectric strain coefficient (4.2 pC N−1), electromechanical coupling coefficient (0.17), mechanical quality factor (81) and remnant polarization (0.06 μC cm−2) in reference to natural bone has been achieved with the developed FGM. The mechanism of conduction remains similar in the FGM to that in pure HA. Impedance analyses suggest the occurrence of two polarization processes in HA and NKN monoliths, whereas more than two polarization processes are observed in the FGM. The significant increase in cell proliferation with culture duration of up to 5 days suggests that the developed FGM favor the cell growth and proliferation. In addition, the present study also establishes the superior cytocompatibility of the perovskite NKN phase. The developed FGM can be a potential substitute for electro-active orthopedic prosthetic implant applications.