Piezoelectric 3-D Fibrous Poly(3-hydroxybutyrate)-Based Scaffolds Ultrasound-Mineralized with Calcium Carbonate for Bone Tissue Engineering: Inorganic Phase Formation, Osteoblast Cell Adhesion, and Proliferation.

Elaboration of novel biocomposites providing simultaneously both biodegradability and stimulated bone tissue repair is essential for regenerative medicine. In particular, piezoelectric biocomposites are attractive because of a possibility to electrically stimulate cell response. In the present study, novel CaCO3-mineralized piezoelectric biodegradable scaffolds based on two polymers, poly[( R)3-hydroxybutyrate] (PHB) and poly[3-hydroxybutyrate- co-3-hydroxyvalerate] (PHBV), are presented. Mineralization of the scaffold surface is carried out by the in situ synthesis of CaCO3 in the vaterite and calcite polymorphs using ultrasound (U/S). Comparative characterization of PHB and PHBV scaffolds demonstrated an impact of the porosity and surface charge on the mineralization in a dynamic mechanical system, as no essential distinction was observed in wettability, structure, and surface chemical compositions. A significantly higher (4.3 times) piezoelectric charge and a higher porosity (∼15%) lead to a more homogenous CaCO3 growth in 3-D fibrous structures and result in a two times higher relative mass increase for PHB scaffolds compared to that for PHBV. This also increases the local ion concentration incurred upon mineralization under U/S-generated dynamic mechanical conditions. The modification of the wettability for PHB and PHBV scaffolds from hydrophobic (nonmineralized fibers) to superhydrophilic (mineralized fibers) led to a pronounced apatite-forming behavior of scaffolds in a simulated body fluid. In turn, this results in the formation of a dense monolayer of well-distributed and proliferated osteoblast cells along the fibers. CaCO3-mineralized PHBV surfaces had a higher osteoblast cell adhesion and proliferation assigned to a higher amount of CaCO3 on the surface compared to that on PHB scaffolds, as incurred from micro-computed tomography (µCT). Importantly, a cell viability study confirmed biocompatibility of all the scaffolds. Thus, hybrid biocomposites based on the piezoelectric PHB polymers represent an effective scaffold platform functionalized by an inorganic phase and stimulating the growth of the bone tissue.

Hybrids of Polymer Multilayers, Lipids, and Nanoparticles: Mimicking the Cellular Microenvironment.

Here we address research directions and trends developed following novel concepts in 2D/3D self-assembled polymer structures established in the department led by Helmuth Möhwald. These functional structures made of hybrids of polymer multilayers, lipids, and nanoparticles stimulated research in the design of the cellular microenvironment. The composition of the extracellular matrix (ECM) and dynamics of biofactor presentation in the ECM can be recapitulated by the hybrids. Proteins serve as models for protein-based biofactors such as growth factors, cytokines, hormones, and so forth. A fundamental understanding of complex intermolecular interactions and approaches developed for the externally IR-light-triggered release offers a powerful tool for controlling the biofactor presentation. Pure protein beads made via a mild templating on vaterite CaCO3 crystals can mimic cellular organelles in terms of the compartmentalization of active proteins. We believe that an integration of the approaches developed and described here offers a strong tool for engineering and mimicking both extra- and intracellular microenvironments.

The mechanism of catalase loading into porous vaterite CaCO3 crystals by co-synthesis.

Porous vaterite CaCO3 crystals are nowadays extensively used as high-capacity bio-friendly sacrificial templates for the fabrication of such protein-containing nano- and micro-particles as capsules and beads. The first step in the protein encapsulation is performed through loading of the protein molecules into the crystals. Co-synthesis is one of the most useful and simple methods proven to effectively load crystals with proteins; however, the loading mechanism is still unknown. To understand the mechanism, in this study, we focus on the loading of a model protein catalase into the crystals by means of adsorption into pre-formed crystals (ADS) and co-synthesis (COS). Analysis of the physico-chemical characteristics of the protein in solution and during the loading and simulation of the protein packing into the crystals are performed. COS provides more effective loading than ADS giving protein contents in the crystals of 20.3 and 3.5 w/w%, respectively. Extremely high loading for COS providing a local protein concentration of about 550 mg mL-1 is explained by intermolecular protein interactions, i.e. formation of protein aggregates induced by CaCl2 during the co-synthesis. This is supported by a lower equilibrium constant obtained for COS (5 × 105 M-1) than for ADS (23 × 105 M-1), indicating a higher affinity of single protein molecules rather than aggregates to the crystal surface. Fitting the adsorption isotherms by classical adsorption models has shown that the Langmuir and BET models describe the adsorption phenomenon better than the Freundlich model, proving the aggregation in solution followed by adsorption of the aggregates into the crystals. We believe that this study will be useful for protein encapsulation through CaCO3 crystals using the COS method.

Hybrid PCL/CaCO3 scaffolds with capabilities of carrying biologically active molecules: Synthesis, loading and in vivo applications.

Designing advanced biomaterials for tissue regeneration with drug delivery and release functionalities remains a challenge in regenerative medicine. In this research, we have developed novel composite scaffolds based on polymeric polycaprolactone fibers coated with porous calcium carbonate structures (PCL/CaCO3) for tissue engineering and have shown their drug delivery and release in rats. In vivo biocompatibility tests of PCL/CaCO3 scaffolds were complemented with in vivo drug release study, where tannic acid (TA) was used as a model drug. Release of TA from the scaffolds was realized by recrystallization of the porous vaterite phase of calcium carbonate into the crystalline calcite. Cell colonization and tissue vascularization as well as transplantability of developed PCL/CaCO3+TA scaffolds were observed. Detailed study of scaffold transformations during 21-day implantation period was followed by scanning electron microscopy and X-ray diffraction studies before and after in vivo implantation. The presented results demonstrate that PCL/CaCO3 scaffolds are attractive candidates for implants in bone regeneration and tissue engineering with a possibility of loading biologically active molecules and controlled release.

Patchiness of embedded particles and film stiffness control through concentration of gold nanoparticles.

Patchy particles are fabricated using a method of embedding-into and extracting-from thick, biocompatible, gel-like HA/PLL films. Control over the patchiness is achieved by adjusting the stiffness of films, which affects embedding and masking of particles. The stiffness is adjusted by the concentration of gold nanoparticles adsorbed onto the surface of the films.

Surface-supported multilayers decorated with bio-active material aimed at light-triggered drug delivery.

In this work, we report on the functionalization of layer-by-layer films with gold nanoparticles, microcapsules, and DNA molecules by spontaneous incorporation into the film. Exponentially growing films from biopolymers, namely, hyaluronic acid (HA) and poly-L-lysine (PLL), and linearly growing films from the synthetic polymers, namely, poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH), were examined for the embedding. The studied (PLL/HA)(24)/PLL and (PAH/PSS)(24)/PAH films are later named HA/PLL and PSS/PAH films, respectively. The HA/PLL film has been found to be more efficient for both particle and DNA embedding than PSS/PAH because of spontaneous PLL transport from the interior of the whole HA/PLL film to the surface in order to make additional contact with embedded particles or DNA. DNA and nanoparticles can be immobilized in HA/PLL films, reaching loading capacities of 1.5 and 100 microg/cm(2), respectively. The capacities of PSS/PAH films are 5 and 12 times lower than that for films made from biopolymers. Polyelectrolyte microcapsules adsorb irreversibly on the HA/PLL film surface as single particles whereas very poor interaction was observed for PSS/PAH. This intrinsic property of the HA/PLL film is due to the high mobility of PLL within the film whereas the structure of the PSS/PAH film is "frozen in". Gold nanoparticles and DNA form micrometer-sized aggregates or patches on the HA/PLL film surface. The diffusion of nanoparticles and DNA into the HA/PLL film is restricted at room temperature, but DNA diffusion is triggered by heating to 70 degrees C, leading to homogeneous filling of the film with DNA. The film has not only a high loading capacity but also can be activated by "biofriendly" near-infrared (IR) laser light, thanks to the gold nanoparticle aggregates on the film surface. Composite HA/PLL films with embedded gold nanoparticles and DNA can be activated by light, resulting in DNA release. We assume that the mechanism of the release is dependent on the disturbance in bonding between "doping" PLL and DNA, which is induced by local thermal decomposition of the HA/PLL network in the film when the film is exposed to IR light. Remote IR-light activation of dextran-filled microcapsules modified by gold nanoparticles and integrated into the HA/PLL film is also demonstrated, revealing an alternative release pathway using immobilized light-sensitive carriers (microcapsules).

Remote near-IR light activation of a hyaluronic acid/poly(l-lysine) multilayered film and film-entrapped microcapsules.

Spontaneous embedding of gold nanoparticle (NP) aggregates or polyelectrolyte microcapsules modified with NPs in biocompatible hyaluronic acid/poly(l-lysine) films is reported. The NPs were adsorbed in the aggregated state to induce near-IR light absorption. The films functionalized with gold NPs become active in response to a "biologically friendly" near-IR laser at a power of about 20 mW. The activation is characterized by a localized temperature increase in the film, allowing conversion of light energy to heat into confined volumes. Microcapsules adsorbed onto the film can release its cargo under stimulation with near-IR light because of localized permeability changes in their walls. This work is aimed at layer-by-layer film-based biomedical coatings and active surfaces with light-sensitive features wherein metal NPs and microcapsules are used as active centers or carriers with remote control of functionalities.

Photoactivated release of cargo from the cavity of polyelectrolyte capsules to the cytosol of cells.

Polyelectrolyte capsules with metal nanoparticles in their walls and fluorescently labeled polymers as cargo inside their cavity were prepared. Capsules were ingested by living cells with no uncontrolled release of the cargo upon the incorporation process. Photoinduced heating of the metal nanoparticles in the capsule walls lead to rupture of the capsule walls, and the polymeric cargo was released to the whole cytosol. Viability tests demonstrate that opening of capsules at moderate light intensities does not impair the cellular metabolism, whereas capsule opening at high light intensities ultimately leads to cell death.

Automated single-cell sorting system based on optical trapping.

We provide a basis for automated single-cell sorting based on optical trapping and manipulation using human peripheral blood as a model system. A counterpropagating dual-beam optical-trapping configuration is shown theoretically and experimentally to be preferred due to a greater ability to manipulate cells in three dimensions. Theoretical analysis performed by simulating the propagation of rays through the region containing an erythrocyte (red blood cell) divided into numerous elements confirms experimental results showing that a trapped erythrocyte orients with its longest axis in the direction of propagation of the beam. The single-cell sorting system includes an image-processing system using thresholding, background subtraction, and edge-enhancement algorithms, which allows for the identification of single cells. Erythrocytes have been identified and manipulated into designated volumes using the automated dual-beam trap. Potential applications of automated single-cell sorting, including the incorporation of molecular biology techniques, are discussed.

Amplification of a phase-conjugate signal in a nonlinear absorptive Kerr medium.

We present the results of amplification of the phase-conjugate reflectivity in an absorptive nonlinear Kerr medium by nondegenerate four-wave mixing. The influence of two-beam coupling on the phase-conjugate signal is observed and analyzed. The frequency domain response of nondegenerate four-wave mixing produces a tunable narrow-band optical filter.